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Reply to the reviewers

We appreciate the positive feedback from both reviewers and their critical comments, which will help us to improve the manuscript. Below, we provide a point-by-point response and how we propose to address their queries and comments.

As the laboratory is currently undergoing a major transition, we propose essential experiments that are realistic to perform under these circumstances. We are positive that we can address all the most critical points identified by the reviewers.

Suggestions for minor changes to the figures are already included.

We also include responses to questions the reviewers raise.

Reviewer #1 (Evidence, reproducibility and clarity):

Major comments:

1 - The authors assessed acridine orange incorporation in BECs upon LiverZap and concluded that LiverZap triggers hepatocyte-specific cell death without a bystander effect in adjacent cells (Figure 1 D-E). What happened to endothelial cells, which could also be affected either directly by ROS production in hepatocytes or indirectly by gross morphological changes in tissue organization?

Response:

The reviewer raises two excellent points:

(i) Bystander effect of hepatocyte-produced ROS on endothelial cells: the cell death analysis included in the manuscript, shows that Acridine Orange staining overlaps with hepatocyte _tg(fabp10a:dsRed) expression, but not with biliary tg(tp1:H2B-mCherry) indicating cell death is specific to hepatocytes. Moreover, singlet oxygen species as produced by LiverZap have been shown to have a very short half-life and short range of action, suggesting that neighbouring cells are unlikely affected (Liang et al., 2020).

PLAN: Investigate potential bystander effect in endothelial cells, by activating the LiverZap tool in livers expressing transgenic tg(kdrl:mcherry) marking the vascular network followed by live staining with Acridine Orange at 8 hours post illumination.

(ii) indirect effects of morphological tissue changes on endothelial cells: studying the tissue response of the vascular network to hepatocyte ablation would be very interesting. A separate and detailed study would be required to generate meaningful data and insights into said process. This could encompass for instance the use of the transgenic endothelial _tg(kdrl:mcherry) line for LiverZap experiments and parallel those in Figure 4A-S. Thus, it seems beyond the scope of this work.

NO CHANGES PROPOSED.

2 - The evaluation criteria for distinguishing mCherry and cells in imaging experiments should be clearly described in the methods section. The authors should also provide some quantitative data regarding the level of correlation between the mCherry hepatocytes and the BEC-derived hepatocytes strictly defined based on the TP1-H2B-EGFP lineage tracing, as the former was used as a surrogate marker for the latter in some experiments.

Response:

Here, we believe the reviewer refers to the Tp1:H2B-mCherry-based lineage tracing, since the tg(tp1:egfp) line has not been used for this purpose. Similar to previous studies in the regeneration field (e.g. Choi et al., 2014; He at al., 2014), we have used histone inheritance of Tp1:H2B-mCherry for short-term lineage tracing. Tp1:H2B-mCherry-based lineage tracing was assessed on the whole organ level, for which we will describe the quantification pipeline. Tp1:H2B-mCherrylow cells were identified as BEC-derived hepatocytes after severe hepatocyte ablation, as shown in Fig. 2A,C, correlating with hepatocyte marker tg(fabp10a:GFP) expression. Tp1high and Tp1low cell numbers were quantified for 12, 24, 48 and 72 hpi and can be added as supplementary information.

PLAN: Update the material and methods section and produce a more detailed description. This would include the following information: Whole-mounted livers of tg(tp1:H2B-mCherry) fish were stained for mCherry and imaged using an Leica SP8 confocal microscope. Image processing was carried out using the Imaris software. All mCherry-expressing cells in the liver were masked using the “spots” function, which allows quantification of signal intensity of all cells, represented by a sphere. Tp1high and Tp1low cells were identified using an automatically generated intensity threshold. Due to intensity differences with increasing imaging depth/z-position, segmented Tp1high cells were manually curated.

To showcase the analysis strategy, we propose to include an example showing original image data, semi-automated quantification at the surface and deep tissue levels, as well as the overall Tp1:H2B-mCherry intensities for all positive cells and specifically Tp1high cells for all z-positions of an entire liver (see data figure below). This example could be included as supplementary data. Likewise, cell number quantification for Tp1high and Tp1low across regeneration can be added to Fig. S2.

Fig. Quantification of Tp1:H2B-mCherryhigh cells. (A,B) 10 µm maximum intensity projections from whole mount stained tg(LiverZap);tg(tp1:H2B-mCherry) livers at 48 hpi: at the surface (A-A’) and deep in the liver (B-B’). Tp1high cells are identified by fluorescence intensity of segmented nuclei, outlined in yellow (A’ and B’). Graphs showing distribution of all Tp1:H2B-mCherry nuclei (C) and Tp1high nuclei (D) by fluorescence intensity and z-position (C). The intensity of all mCherry+ nuclei decreases with increasing z-position (C-D). The dotted line outlines the liver in A-B’.

3 - OPTIONAL: In the locally restricted ablation model, do hepatocytes located adjacent to the ROI proliferate and/or contribute to the regeneration of the injured region?

Response:

An important consideration, as highlighted by the reviewer, is whether neighbouring hepatocytes also contribute to regeneration following ROI ablation.

PLAN: To address this point, LiverZap ROI ablation will be followed by cell proliferation analysis using an EdU incorporation assay at 24 and 72 hpi. These time points are selected based on the proliferation results following global LiverZap ablation; see Fig. 2D-F. The experiment will be performed in a tg(tp1:H2B-mcherry); _tg(fabp10a:gfp)_background to distinguish proliferating GFP-positive hepatocytes, which are H2B-mCherry-negative, from LPC-derived hepatocytes that have inherited H2B-mCherry (Tp1low). The resulting insights may help to refine hypotheses regarding the process(es) stimulating the formation of new hepatocytes adjacent to the ablated region.

4 - OPTIONAL: Figure 4, A-S. It should be of significant interest if the authors could also analyze the BEC dynamics using the locally restricted hepatocyte ablation model, comparing those in the injured region (ROI) and the outside of the ROI.

Response:

We agree with the reviewer that this is the exciting next question, as it likely would provide insights into the cellular mechanism by which the biliary network is de- and re-constructed, as well as the mechanism by which BECs outside the ROI may initiate the LPC response to give rise to hepatocytes in a semi-systemic response. For this, the experimental set-up introduced in Fig.4J-P, in which BECs in the ROI are distinguished from adjacent ones by photoconversion, would be followed by extended live light-sheet microscopy of the regenerating liver. Due to the complexity, extent of the experiments and current unavailability of a light-sheet microscope, we would address this optional comment in future investigations.

NO CHANGES PROPOSED.

5A- Figure 4, T-V'. The data shown here for the changes in E-cadherin distribution is difficult to understand and interpret. The authors should provide magnified images and better description on how to distinguish the membranous (spotted signals?) and intracellular localization. Quantitative assessment should certainly be a plus, if possible.

Response:

We appreciate that it may be difficult to recognize the changes in E-Cadherin localisation, in particular at BEC membranes, given that there are intracellular puncta, and that E-Cadherin is expressed both in BECs and hepatocytes. We are convinced of the related data described in Figures 4 and S4, because the first experiment allowed quantification of the staining using both Tp1:H2B-mCherry to identify BECs and intestinal E-Cadherin for normalisation, which revealed a 51% E-Cadherin reduction at BEC cell membranes following injury. Unfortunately, the signal-to-noise ratio declined in consecutive experiments precluding further quantification although we could still observe a change in localisation. We tested alternative antibodies against E-Cadherin as well as optimized staining protocols, yet without success.

5B - OPTIONAL: In relation to the above point, it is this reviewer's candid impression that the very last part regarding the possible role of E-cadherin dynamics in regulating the biliary network remodeling is still preliminary compared to the remaining parts, thereby rather depreciating the value of the entire manuscript. Perhaps this part could be published separately, together with more functional evidence regarding the causal relationship between them (e.g., showing the effect of Ecadherin knockdown in hepatocytes on the biliary remodeling and the induction of the BECdependent regeneration program)

Response:

PLAN: Following this reviewer’s and reviewer 2’s comments and suggestions, we agree to remove the data on E-Cadherin. Loss of adhesion as a mechanism for adopting an LPC-state remains very exciting, future investigations with novel tools to monitor and modulate E‑Cadherin expression in BECs would thus be needed.

6 - Do zebrafish livers possess lobular structures with the portal-to-central vein axis and the metabolic zonation as typically observed in mammalian livers? As has been described in the manuscript, the "localized" injury patters in the mammalian livers usually occur at the sub-lobular structure levels (i.e., peri-portal region-restricted vs. peri-central region-restricted). Although the "localized" injury model described in this study using the zebrafish livers was indeed localized from the viewpoint of the entire organ (or the lobe), it still seemed much more "global" when considering those situations in the mammalian livers, so that the authors' claim that the former recapitulating the latter might be too exaggerated and somehow misleading. The authors should clarify and discuss this point in the manuscript.

Response:

The reviewer raises an important point, and it seems that our wording might not have been clear. In mammals, boundaries between injured and healthy tissue arise, because liver injuries frequently occur at the sub-lobular level. Although zebrafish livers are composed of metabolically diverse hepatocytes, a spatial arrangement comparable to mammalian zonation has so far not been identified (Morrison et al. 2022; Oderberg and Goessling, 2023). Yet, the liver lobes in the adult zebrafish have a central vein and periportal veins at the periphery of the organ, similar to the mammalian lobular organisation (Ota et al. 2022). Therefore, the scale of injury in the mammalian setting and the ROI-ablation model introduced in the current work differs. It, nevertheless, creates boundaries of healthy and injured liver tissue relevant for uncovering dynamic cellular processes mediating tissue repair in chronic liver disease. Importantly, with its suitability for advanced live imaging and optogenetic methods (e.g. photoconversion), LiverZap, complements mammalian models, in which this is still challenging. This offers therefore the powerful opportunity to employ LiverZap to screen for dynamic repair behaviours, which subsequently can be validated in a target approach in mammalian injury models.

PLAN: To describe the relevance of our ROI ablation paradigm for elucidating repair processes at the interface of injured and healthy tissue more precisely. We will further edit and clarify text to place the ROI ablation into the context of hepatic injuries at the sub-lobular level throughout the mammalian liver.

Minor comments:

7 - Figure 4. Panels D and G should correspond to the same one image and the way of labeling be changed (as in Figure 1G). Likewise, in panel J, the bars shown separately as "M" and "S" at 12 dpi should correspond to the same data, so that they should be unified as one bar.

Response:

Thank you for pointing this out, this is changed in the updated figures; panels Fig. 4D and I.

8 - Figure S3L. How was the ROI border defined? Perhaps the shape of the ROI should change significantly during regeneration due to dynamic tissue remodeling processes, thereby moving the position of the border as well.

Response:

The ROI border was defined as the interface between photoconverted and non-converted BECs. We concur with the reviewer’s notion that cell movement and rearrangement may occur during the regeneration process (see Fig. 4A-J), and the initially straight ROI border could consequently change during the regeneration process. Nevertheless, the border between photoconverted and non-converted BECs persists, serving as a landmark for the measurements shown in Figure S3L.

Fig.: Quantification strategy for determining the region exhibiting an LPC-response outside the ROI ablation region. The dashed line of the ROI indicates morphogenetic changes of the interface between photoconverted and nonconverted cells over time due to repair-related cell rearrangement.

PLAN: In the revised manuscript, we propose to include the below schematic as panel J to Figure S3. Moreover, we also suggest to change the solid line of the squares indicating the ROI area in figure panels 3C,G,O,P and S3D,H,K into a dashed line at the interface between photoconverted and non-converted tissue (see below figure as an example).

9 - The authors should comment in the manuscript as to whether the system can be applicable for induction of more restricted areas (e.g., at a single hepatocyte level; in particular metabolic zones, if existing), as well as for ablation of other hepatic cell types such as BECs and endothelial cells.

Response:

Indeed, the optogenetic nature of the LiverZap system allows to induce hepatocyte death at the single cell level, as well as any defined region of interest that can be generated by the light source (e.g. confocal microscope software).

Likewise, the FAP-TAP system can be easily applied to BECs or endothelial cells, or any cell type for which a specific promoter has been identified to drive the genetic FAP component fluorogen-activating protein dL5**.

Response:

PLAN: Both points will be included in the discussion section of the manuscript.

Reviewer #2 (Evidence, reproducibility and clarity):

MAJOR COMMENTS:

1 - The LiverZap is an elegant new tool to induce localized ablation of hepatocytes. It is not as claimed by the authors a real breakthrough: (1) While localized ablation is nice compared to NTR-MTZ model in zebrafish, mice model such as CCl4 chronic injury can also study the interaction between healthy and injured tissue. (2) Although not using MTZ, the system still requires injection or exposure to malachite green derivate dye MG-2I. A few searches suggest that this compound could induce toxicity. Can the authors study and compare the toxicity of malachite green derivate dye MG-2I to the toxicity of MTZ? This is important as this would be indeed a strong argument in favor of the presented tool.

Response:

Point 1 – studying interactions between healthy and injured liver tissue: The reviewer is of course correct that interactions between healthy and injured tissue can also be studied in the mouse. However, ROI ablation with the LiverZap system can be combined with live imaging, thereby enabling the observation of cellular responses of the same sample over time, at a resolution currently difficult to achieve in mammals. Moreover, the possibility to induce cell death in a defined ROI, also allows to simultaneously employ other genetic tools, including cell-type specific lineage tracing by photoconversion, which is difficult to achieve in mammalian systems. The finding that BECs beyond the ROI of hepatocyte ablation produce new hepatocytes by a LPC response, illustrates the power of this approach. The optogenetic LiverZap ablation system would therefore complement existing mammalian and zebrafish liver regeneration models.

PLAN: to include a more detailed discussion of this point and the complementary knowledge that can be gained in the discussion section.

Point 2 – MG-2I toxicity__: Indeed, as described in the manuscript, the FAP-TAP system, underlying LiverZap hepatocyte ablation, requires MG-2I incubation for the formation of the photosensitiser. Compared to the NTR/MTZ system, incubation with MG2I is short, requiring <3 hours in contrast to more than 24hours MTZ incubation. The system, including MG-2I has also been employed in cells, as well as in the zebrafish heart and nervous system without reported adverse effects (He et al., 2016; Xie et al., 2020). Consistently, we have not observed any apparent adverse effects between 0-72 hpi following 3-18 hour MG-2I incubation (unpublished). Nevertheless, toxicity studies evaluating survival upon MG-2I incubation have not yet been carried out and may be required for comparison with MTZ.

PLAN: To perform toxicity studies for MG-2I, similar to those previously performed for MTZ (e.g. Mathias et al. 2014), in which larval survival after 3, 24 and 48 hour MG-2I exposure starting at 4 dpf will be assessed daily until 8 days post fertilisation.

2 -The term ablation is choose because it is anticipated that it induces heaptospecific death. However, the consequences of cell death is not shown. In particular, the inflammatory immune response is not shown nor discussed.

Response:

The reviewer raises an interesting point, namely the inflammatory immune response, which is not the focus of this manuscript. Acridine Orange- and TUNEL-positive cells during the ablation process indicate that the reactive oxygen species produced by the FAP-TAP system cause hepatocyte apoptosis. We predict that this would recruit and be cleared by macrophages with little or no inflammatory response, like findings for the NTR-MTZ system (Stoddard et al., 2019). However, the role of neutrophils is unclear due to a possible direct effect of MTZ on this cell type.

PLAN: We will include this point in the discussion.

Future in-depth live imaging of transgenic reporters will be required for detailed studies of macrophage and neutrophil recruitment and their role in efferocytosis, including transcriptome analysis of specific gene signatures to detect an inflammatory response.

3 - The difference between mild and severe ablation is hard to grasp. Can the authors explain more clearly the differences between mild and severe: what are the criteria as there is no difference in liver volume between mild and severe ablation? How do you achieve mild or severe ablation? It appears that the severity of the ablation is judged a posteriori and not decided per the experiment.

Response:

Concerning the first point, there must be a misunderstanding. Mild and severe hepatocyte ablation result in clearly different liver sizes, for instance at 30 hpi, the end of ablation, liver volumes are reduced by 23 % for mild or 64 % for severe cases (Fig. 1Q). This is supported by representative image data in Figs. 1F-P and S1A-C. Nevertheless, for consistency, we had represented the 12 hpi volume data as the same two data bars, although we cannot distinguish them yet at that timepoint of the experiment, as shown by images in Fig. 1F-G.

PLAN: Adjust Fig.1Q and represent the 12 hpi liver volume data as a shared graph for mild/severe ablation, see included figure 1Q. We propose to similarly represent all 12 hpi quantifications, as represented in Figs. S1F, 2D-F and S2A.

For the second point, the reviewer is correct that ablation severity is evaluated and determined between 24-30 hpi, at the end of hepatocyte ablation, given there is some variability in the response. Nonetheless, both length of 660nm illumination and oxygen availability can be used to shift the proportion of mild and severe ablation, depending on the desired outcome (Figs. 1Q, S1G-H).

NO CHANGES PLANNED.

4 - The work supports that biliary-driven regeneration also occurs when hepatocyte ablation happens in a small area of interest. Our knowledge is that you need a large defect in hepatocyte or a chronic liver injury ro activate the BDC-driven auxiliary process for regeneration. Could this be a specificity of the fish model?

Response:

Like the reviewer, our understanding is that severe hepatocyte loss, senescence or chronic liver injury activate BEC-derived regeneration in mammals and in zebrafish. All these cases are characterised by substantial reduction of local hepatocyte density or loss of function (in senescence). Given the overall hepatocyte loss is only 10-20% in the ROI model, the induction of the local LPC response was very surprising, on the other hand it corresponds to a near complete local hepatocyte depletion. The hepatobiliary architecture in zebrafish is similar to that of the mammalian ductular reaction, an adaptation of the biliary network to severe hepatocyte loss. In both cases, the majority of hepatocytes connect directly via their apical canaliculi to biliary ductules to ensure physiologic transport of hepatocyte products, often preceding the LPC response (Sato et al., 2019; Caviglia et al., 2022). Therefore, we propose that the LPC response following ROI hepatocyte ablation is not specific to the zebrafish model, but a common mechanism elicited across species and related to the severity of the injury and the configuration of the hepatobiliary network at the time of injury, such as the ductular reaction.

PLAN: To edit the text and discuss this point clearly.

5 - Pathways revealed to control liver regeneration or BEC-driven regeneration in fish have not be found to have a similar drastic predominance in rodents. This mitigate perhaps the use of fish for this type of research?

Response:

On the contrary, zebrafish has been established and validated as a model to investigate and elucidate developmental hepatic programs as well as regeneration (Goessling and Sadler, 2015; Wang et al., 2017). However, we acknowledge that more comparative studies are needed to understand the molecular pathways driving regeneration both in zebrafish and mammals and their similarity.

Specifically, zebrafish and mammals display high conservation in the parenchymal and non-parenchymal cell types of the liver as well as their developmental programs (Goessling and Sadler, 2015; Wang et al., 2017). Using different injury paradigms in zebrafish, including ethanol, acetaminophen toxicity and the pharmacogenetic NTR-MTZ model, it has been shown that cellular responses to liver injury are also remarkably conserved with mammals where hepatocyte proliferation governs repair after mild injury while severe injury repair is driven by conversion of BECs into LPCs (So, et al., 2020; Forbes and Newsome, 2019). Major pathways, such as Wnt, FGF and BMP signaling show conserved functions in restorative hepatocyte proliferation (Goessling et al., 2008; Kan et al. 2009, Böhm et al 2010). At present, only very little is known about the molecular mechanisms controlling the BEC/LPC to hepatocyte conversion particularly in rodent models (Kim et al., 2023), while a number of zebrafish studies have started to elucidate the signals governing the different steps of this process (Kim et al., 2023), due to the relative ease of using the larval zebrafish model for this work. Notably, the Notch pathway plays multiple roles in both mouse and zebrafish LPC-mediated repair (Minnis-Lyons et al., 2021; Huang et al 2014; Russel et al.,2019), however further work will be necessary to determine the detailed corresponding functions. Therefore, future work in both rodents and zebrafish will be essential to uncover the molecular mechanisms of this repair process relevant for chronic injury. Given the large conservation of developmental and repair mechanism between mammals and zebrafish observed so far, it is highly likely that this will also apply to LPC-mediated repair. Studies promise to uncover even greater similarity between zebrafish and human (e.g. Fang et al 2011), underscoring the power of using complementary vertebrate models.

PLAN: To edit the text in the introduction and discussion to clarify and highlight the similarities, differences, and opportunities the zebrafish model offers for understanding the mechanisms of vertebrate liver regeneration in general and in particular by using the LiverZap system.

6 - The authors show that in the case of mild ablation, hepatocytes are responsible for replenishment of the parenchyma, but in the context of severe ablation, LPC-mediated regeneration takes control. However, when the authors perform localized and controlled ablation, which is small (around 10-20%) and, to my understanding, a mild / local ablation, however the authors show that LPC mediates the regeneration. Can the authors explain the discrepancy between their results?

Response:

We agree with the reviewer that the LPC response in the smaller, local ROI ablation was unexpected. However, it could be explained by the following: while such ROI hepatocyte ablation represents only a 10-20% ablation of the total hepatocyte population, by sheer numbers comparable to a mild global ablation, the near-complete local hepatocyte loss however makes it more similar to a severe or chronic global injury. Notably, the zebrafish hepatobiliary architecture in zebrafish is similar to that of the mammalian ductular reaction, an adaptation of the biliary network to severe hepatocyte loss. In both cases, the majority of hepatocytes connect directly via their apical canaliculi to biliary ductules to ensure physiologic transport of hepatocyte products, often preceding the LPC response (Sato et al., 2019; Caviglia et al., 2022). We hypothesize that if a similar local, near complete hepatocyte loss would be induced in a mammalian liver exhibiting a ductular reaction, it would similarly induce local LPC-mediated repair. Since this is, to our knowledge not possible, the LiverZap model represents a unique opportunity to induce the LPC-response in a controlled manner and in addition investigate the underlying cellular and molecular processes of injured and adjacent healthy tissues at high resolution in an in vivo context.

PLAN: We will edit the discussion to clarify this important point.

7 - The last part of the paper about E-Cadherin expression is not convincing. I am not sure about the quality of the IF stainings of E-Cadherin, and it is not helping proving the point of the authors. Can the authors provide better stainings for this figure?

Response:

(Same response as to point 5A+B of reviewer 1). We appreciate that it may be difficult to recognize the changes in E-Cadherin localisation, in particular at BEC membranes, given that there are intracellular puncta and that E-Cadherin is expressed both in BECs and hepatocytes. We are convinced of the related data described in Figures 4 and S4, because the first experiment allowed quantification of the staining using both Tp1:H2B-mCherry to identify BECs and intestinal E-Cadherin for normalisation, which revealed a 51 % E-Cadherin reduction at BEC cell membranes following injury. Unfortunately, the signal to noise ratio declined in consecutive experiments, while we could still observe a change in localisation, it challenged a meaningful quantification. We tested alternative antibodies against E-Cadherin, yet without success.

PLAN: Following both reviewers’ comments and suggestions, we agree to remove the data on E-Cadherin.

8 - Could the authors provide a bit more information on the live imaging. Exactly how do they achieve imaging for such a long time?

Response:

Thank you for pointing this out, the information was not very detailed. We used relatively standard mounting conditions (low-melting point agarose and Tricaine anaesthesia, see below for details), combined with light-sheet microscopy, which was the key to achieving the long imaging. We believe that in addition to the known gentle imaging condition, the mounting set-up is critical as the fish is completely suspended in a very low-percentage, low melting point agarose within a large volume of embryo medium.

PLAN: Update the material and methods section with the following details: Long-term live imaging was performed using a LS1 Live light sheet microscopy system (Viventis Microscopy Sàrl). Larvae were with anesthetized with 0.4% Tricaine and mounted ventrally in 0.8% low melting point agarose in E3/PTU media supplemented with 0.16% tricaine. Once the agarose solidified, the chamber was filled with E3/PTU with 0.16% Tricaine to maintain anaesthesia. A 25X objective was used and acquisition was performed every 20 minutes.

MINOR COMMENTS:

9 - It is hard to imagine the full-size liver in Figure 1, bad contrast. Can the authors manually delineate it?

Response:

The livers in this figure are now outlined in the updated figures, see new Figure 1.

10 - "This finding is very surprising, since current understanding in the field links the generation of new hepatocytes from BECs/LPCs with global hepatocyte death." This statement lacks references.

Response:

PLAN: To add the following primary references to the above sentence: (Choi et al., 2014; He et al., 2014; Manco et al., 2019; Raven et al., 2017) and recent review (Kim et al_._, 2023).

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Reviewer #2 (Public Review):

Tejeda Muñoz et al. investigate the intersection of Wnt signaling, macropinocytosis, lysosomes, focal adhesions and membrane trafficking in embryogenesis and cancer. Following up on their previous papers, the authors present evidence that PMA enhances Wnt signaling and embryonic patterning through macropinocytosis. Proteins that are associated with the endo-lysosomal pathway and Wnt signaling are co-increased in colorectal cancer samples, consistent with their pro-tumorigenic action. The function of macropinocytosis is not well understood in most physiological contexts, and its role in Wnt signaling is intriguing. The authors use a wide range of models - Xenopus embryos, cancer cells in culture and in xenografts and patient samples to investigate several endolysosomal processes that appear to act upstream or downstream of Wnt. A downside of this broad approach is a lack of mechanistic depth. In particular, few experiments monitor macropinocytosis directly, and macropinocytosis manipulations have pleiotropic effects that are open alternative interpretations. Several experiments are confirmatory of previous findings; the manuscript could be improved by focusing on the novel relationship between PMA-induced macropinocytosis and better support these conclusions with additional experiments.

The authors use a range of inhibitors that suppress macropinosome formation (EIPA, Bafilomycin A1, Rac1 inhibition). However, these are not specific macropinocytosis inhibitors (EIPA blocks an Na+/H+ exchanger, which is highly toxic and perturbs cellular pH balance; Bafilomycin blocks the V-ATPase, which has essential functions in the Golgi, endosomes and lysosomes; Rac1 signals through multiple downstream pathways). A specific macropinocytosis inhibitor does not exist, and it is thus important to support key conclusions with dextran uptake experiments.

The title states that PMA increases Wnt signaling through macropinocytosis. However, the mechanistic relationship between PMA-induced macropinocytosis and Wnt signaling is not well supported. The authors refer to a classical paper that demonstrates macropinocytosis induction by PMA in macrophages (PMID: 2613767). Unlike most cell types, macrophages display growth factor-induced and constitutive macropinocytic pathways (PMID: 30967001). It would thus be important to demonstrate macropinocytosis induction by PMA experimentally in Xenopus embryos / cancer cells. Does treatment with EIPA / Bafilomycin / Rac1i decrease the dextran signal in embryos? In macrophages, the PKC inhibitor Calphostin C blocks macropinocytosis induction by PMA (PMID: 25688212). Does Calphostin C block macropinocytosis in embryos / cancer cells? Do the various combinations of Wnts / Wnt agonists and PMA have additive or synergistic effects on dextran uptake? If the authors want to conclude that PMA activates Wnt signaling, it would also be important to demonstrate the effect of PMA on Wnt target gene expression.

The experiments concerning macropinosome formation in Xenopus embryos are not very convincing. Macropinosomes are circular vesicles whose size in mammalian cells ranges from 0.2 - 10 µM (PMID: 18612320). The TMR-dextran signal in Fig. 1A does not obviously label structures that look like macropinosomes; rather the signal is diffusely localized throughout the dorsal compartment, which could be extracellular (or perhaps cytosolic). I have similar concerns for the cell culture experiments, where dextran uptake is only shown for SW480 spheroids in Fig. S2. It would be helpful to quantify size of the circular structures (is this consistent with macropinosomes?).

In Fig. 4I - J, the dramatic decrease in b-catenin and especially in Rac1 after overnight EIPA treatment is rather surprising. How do the authors explain these findings? Is there any evidence that macropinocytosis stabilizes Rac1? Could this be another effect of EIPA or general toxicity?

On a similar note, Fig. 6 K - L the FAK staining in control cells appears to localize to focal adhesions, but in PMA-treated cells is strongly localized throughout the cell. Do the authors have any thoughts on how PMA stabilizes FAK and where the kinase localizes under these conditions? Does PMA treatment increase FAK signaling activity?

The tumor stainings in Figure 5 are interesting but correlative. Pak1 functions in multiple cellular processes and Pak1 levels are not a direct marker for macropinocytosis. In the discussion, the authors discuss evidence that the V-ATPase translocates to the plasma membrane in cancer to drive extracellular acidification. To which extent does the Voa3 staining reflect lysosomal V-ATPase? Do the authors have controls for antibody specificity?

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

Please find our point-to-point response to the reviewer’s comments below, where we marked all changes implemented in the manuscript in italics.

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

With the emergence and spread of resistance to Artemisinin (ART), a key component of current frontline malaria combination therapies, there is a growing effort to understand the mechanisms that lead to ART resistance. Previous work has shown that ART resistant parasites harbour mutations in the Kelch13 protein, which in turn leads to reduced endocytosis of host haemoglobin. The digestion of haemoglobin is thought to be critical for the activation of the artemisinin endoperoxide bridge, leading to the production of free radicals and parasite death. However, the mechanisms by which the parasites endocytose host cell haemoglobin remain poorly understood.

Previous work by the authors identified several proteins in the proximity of K13 using proximity-based labelling (BioID) (Birnbaum et al. 2020). The authors then went on to characterise several of these proteins, showing that when proteins including EPS15, AP2mu, UBP1 and KIC7 are disrupted, this leads to ART resistance and defects in endocytosis leading to the hypothesis that these two processes are inextricably linked.

In this manuscript, Schmidt et al. set themselves the task of characterising more K13 component candidates identified in their previous work (Birnbaum et al. 2020) that were not previously validated or characterised. They chose 10 candidates and investigated their localisations, and colocalisation with K13, and their involvement in endocytosis and in vitro ART resistance, 2 processes mediated by K13 and some members of the K13 compartments

The authors show that of their 10 candidates, only 4 can be co-localised with K13. Then, using a combination of targeted gene disruption (TGD) as well as knock sideways (KS), they characterised these 4 proteins found in the K13 compartment. They show that MyoF and KIC12 are involved in endocytosis and are important for parasite growth, however their disruption does not lead to a change in ART sensitivity. The authors also confirm the findings of their previous publication (Birnbaum et al. 2020), using a slightly different TGD

(note from the authors: we apologise if this has not properly transpired from the manuscript but the difference between the TGDs is substantial and relevant: one has less than 3% of the protein left and hence can be considered to fully inactivate MCA2 and has a growth defect whereas the other contains about two thirds of the protein (1344 amino acids/~66% are left), has no growth defect, although it lacks the MCA2 domain (hence that domain can not be critical for the growth defect)),

that MCA2 is involved in ART resistance, however they did not check whether its disruption impacts haemoglobin uptake. They also show that KIC11 is not involved in mediating haemoglobin uptake or ART resistance. To finish, the authors used AlphaFold to identify new domains in the proteins of the K13 compartment. This led them to the conclusion that vesicle trafficking domains are enriched in proteins of the K13 compartment involved in endocytosis and in vitro ART resistance.

The majority of the experiments conducted by the authors are performed to a good standard in biological and technical replicates, with the correct controls. Their findings provide confirmation that their 4 candidate genes seem to be important for parasite growth, and show that some of their candidates are involved in endocytosis. While the KD and KS approaches employed by the authors to study their candidate genes each have their own advantages and can be excellent tools for studying a large sets or genes, this manuscript highlights the many limitations of these approaches. For example, the large tag used for the KS approach can mislocalise proteins or disrupt their function (as is the case for MyoF), resulting in spurious results, or indeed the inability to generate the tagged line (as is the case for MCA2). The KS approach also makes the results of a protein with a dual localisation, like KIC12, extremely difficult to interpret.

We thank the reviewer for this thorough and insightful review.

The limitations mentioned above were addressed in the response to the main points and a general detailed response in regards to the systems used for this research are added at the end of this rebuttal. Briefly summarised here: while we agree that there are limitations of the system used, we are convinced that

• the advantages of using a large tag in most cases outweighs the drawbacks as it permits to track the inactivation of the target, if need be on the individual cell level

• while not optimal for MyoF, the partial inactivation actually helps in its functional study as detailed in major point 23&28 or reviewer#3 major point 11: it shows a consistent correlation of the phenotype with different causes and degrees of inactivation (this is now better illustrated in Figure 1L1M). Further, regarding the concern of the large tag: the effect of the tag based on localisation was overestimated in the review by what seems to have been a mix up comparing numbers from MyoF with a number from MCA2 (there is a difference, but it is only small) (see reviewer#1 major point #23).

• KS is the optimal method for most of the assays in this work (e.g. bloated food vacuole assays and RSAs); these assays would be impossible or difficult to use with other inactivation systems currently used in P. falciparum research (see details in the response to the specific points and after the rebuttal)

In regards to the difficulty to interpret KIC12 data: this is only true for measuring absolute essentiality, everything else we believe we actually have the optimal method. If not KS, which method targets a specific pool of a protein with a dual localisastion? Again, our assays targeting the K13 pool and revealing the specific function would have been difficult or impossible with any other system.

Ultimately the question is whether any other system would have resulted in a different conclusion on the function of the proteins studied. At present we are confident this would not be the case and other systems probably would not have delivered the specific functional data shown in this work. Clearly, more in depth work will provide more nuanced and detailed insights into the proteins analysed in this work and this likely will also include the use of other systems for specific aspects they are most suitable for. However, this (e.g. different complementations in a diCre cKO) is complex and therefore beyond what fits into this work which had the goal to assess which proteins are true positives for the K13 compartment and to place them into functional groups in regards to endocytosis.

Moreover, the manuscript is disjointed at times, with the authors choosing to conduct certain experiments for only a subset of genes, but not for others. For example, considering that the aim of this paper was to identify more proteins involved in ART resistance and endocytosis, it is confusing why the authors do not perform the endocytosis assays for all their selected proteins, and why they do not do this for the proteins they identify in their domain search. There is significant room for improvement for this manuscript, and a generally interesting question.

The reviewer remarks that not every experiment was done for every target. Based on the rebuttal we tried to amend this but also note that there was some sentiment by the reviewers to better stick to the point and not make the manuscript more disjointed. We attempted to balance that as much as possible and hope we were able to honour both aspects (amendments were done as detailed in the point by point response below).

In regards to endocytosis and choice of targets: We did do endocytosis assays for all proteins that showed a growth phenotype upon inactivation in this work. We therefore assume the reviewer here refers to major point #40 asking for endocytosis assays with KIC4 and KIC5 (which were not studied in this manuscript) as well as MCA2 (point 17). We fully agree with the reviewer that this would fill a gap in the work on K13 compartment proteins but such assays are difficult with TGDs (there are issues with non-comparable samples and compensatory effects) and proteins that are not essential (and hence likely have a smaller impact on endocytosis when truncated). We nevertheless now carried them out, but due to the limitations to do this with these lines would be hesitant to draw definite conclusions (see major point 17 and 40 for details and outcomes).

But in it's current format, other than confirming that MCA2 is involved in ART resistance (which was already known from the Birnbaum paper), the authors do not further expand our understanding of the link between ART resistance and endocytosis in this manuscript.

We would like to point out that the importance of the K13 compartment and endocytosis goes beyond ART resistance (see e.g. also newly published papers on the K13 compartment in Toxoplasma, (Wan et al., 2023; Koreny et al., 2023)). Endocytosis is an essential and prominent process in blood stages. However, in contrast to processes such as invasion, our understanding about endocytosis is only rudimentary. Hence, this manuscript provides important insights on an emerging topic that in our opinion deserves more attention:

• it identifies novel proteins at the K13 compartment and provides 2 new proteins in endocytosis (MyoF and KIC12); getting an as complete as possible list of proteins involved in the process will be critical to study and understand it

• it leads to the realisation that not all growth-relevant proteins detected at the K13 compartment are needed for endocytosis

• it provides domains and stage specificity of function for several K13 compartment proteins, overall bolstering the model of endocytosis in ART resistance and providing a framework critical to direct future studies on endocytosis and their detailed mechanistic function at the cytostome

• the identified vesicle trafficking domains (for instance now also found in UBP1) are expected to strengthen the support for the role of endocytosis of the K13 compartment; this and also the above points are important as (based on the current literature) there still seems to be prominent sentiment in the field that (in part due to the involvement of UBP1 and K13) the cause of ART resistance is due to various unclearly defined stress response pathways

• with MyoF it also shows the first protein in connection with the K13 compartment that acts downstream of the generation of hemoglobin-filled containers in the parasite and provides the first protein that explains the suspected involvement of actin in endocytosis (so far this was only based on CytD studies)

Overall we therefore believe this manuscript contains critical information and a framework for future studies on endocytosis and the K13 compartment. We hope the relevance of endocytosis as one of the most prominent and essential processes in the parasites and the connection to various aspects linked with many commercial drugs (in addition to the role of endocytosis in ART resistance), is adequately explained in the introduction. We also would like to mention that the main focus of the work is reflected in the title of the manuscript which does not mention ART susceptibility.

Major Comments

1) line 31: please change defined to characterised - defined suggests that novel proteins were identified in this study, which is not the case.

We apologise, but we do not fully understand this comment. We did identify novel proteins not before known to be at the K13 compartment (MCA2 (admittedly this one was likely but had not previously been verified), MyoF, KIC11 and KIC12). In our view "further defining the composition of the K13 compartment" therefore is an accurate statement. Additionally, the identification of previously not-discovered domains, the stage-specificity and function of these proteins helped to further define the K13 compartment.

If the reviewer is referring to the fact that the proteins analysed in this study were taken from a previously generated list of hits, we would like to stress that the presence in such a list (obtained from a BioID, but also if from an IP etc) can not be equalled for them to be true positives, they are merely candidates that still need to be experimentally validated. This is what we did in this work to find out which further proteins from the list can be classified as K13 compartment proteins (for hits with lower FDRs this is even more relevant as illustrated by the fact that 6 of the here analysed hits were not at the K13 compartment). In an attempt to address this comment in the manuscript, we changed the wording of this sentence to (line 31): "Here we further defined the composition of the K13 compartment by analysing more hits from a previous BioID, showing that MyoF and MCA2 as well as Kelch13 interaction candidate (KIC) 11 and 12 are found at this site."

2) line 37: please change 'second' to "another". As explained further below, the authors identified 3 classes of proteins (confer ART resistance + involved in HCCU, involved in HCCU only, or involved in neither).

We realized that the groups description wasn’t clear in the abstract. Please see response to major comment #41 for a detailed answer to this (endocytosis is an overarching criterion, ART resistance is a subgroup and applies only to those proteins with a function in endocytosis in ring stages). To clarify this (see also major point #8) we added an explanation on the influence of stage-specificity of endocytosis on ART susceptibility to the introduction (line 76): “In contrast to K13 which is only needed for endocytosis in ring stages (the stage relevant for in vitro ART resistance), some of these proteins (AP2µ and UBP1) are also needed for endocytosis in later stage parasites (Birnbaum et al., 2020). At least in the case of UBP1, this is associated with a higher fitness cost but lower resistance compared to K13 mutations (Behrens et al., 2021; Behrens et al., 2023). Hence, the stage-specificity of endocytosis functions is relevant for in vitro ART resistance: proteins influencing endocytosis in trophozoites are expected to have a high fitness cost whereas proteins not needed for endocytosis in rings would not be expected to influence resistance.” The abstract was changed in response to this and other comments and hope it is now clearer in regards to the groups.

3) Line 40: You define KIC11 as essential but according to your data some parasites are still alive and replicating 2 cycles after induction of the knock sideways. Please consider changing "essential" to "important for asexual parasite growth".

We fully agree with the reviewer, we reworded the sentence as suggested.

4) Line 40: please change 'second group' to 'this group'

We reworded this part of the abstract and it know reads: (line 38): “While this strengthened the link of the K13 compartment to endocytosis, many proteins of this group showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation of this process.”

5) line 41: state here that despite it being essential, it is unknown what it is involved in.

With the newly added data we show that this protein either has a function in invasion or very early ring development although we did not see any evidence for the latter. We therefore changed the sentence to (line 43): “We here identified the first protein of this group that is important for asexual blood stage development and showed that it likely is involved in invasion*..” *

6) Line 50: the authors should state here that there is actually a reversal in this trend over the last few years.

Done as suggested.

7) Line 54: please separate out the references for each of the two statements made in this line (a: that ART resistance is widespread in SEA, and b: that ART resistance is now in Africa) Reference 14 also seems to reference ART resistance in Amazonia - which is not covered by the statement made by the authors (in which case the authors should state ART is now present in Africa and South America). The authors should also reference PMID: 34279219 for their statement that ART resistance is now found in Africa (albeit a different mutation to the one found in SEA).

Done as suggested.

8) Line 65: it is also worth mentioning here that there are other mutations in proteins other than K13, such as AP2mu and UBP1 (PMID: 24994911;24270944) that can lead to ART resistance.

As suggested by the reviewer, we included a sentence about non-K13 mutations linked with reduced ART susceptibility in the introduction (line 74): “Beside K13 mutations in other genes, such as Coronin (Demas et al., 2018) UBP1 (Borrmann et al., 2013; Henrici et al., 2020b; Birnbaum et al., 2020; Simwela et al., 2020) or AP2µ (Henriques et al., 2014; Henrici et al., 2020b)* have also been linked with reduced ART susceptibility." *

We here also added data on fitness cost that is related to this and is also relevant for the issue of proteins with a stage-specific function in endocytosis, making a transition for this statement which might help clarifying the grouping of K13 compartment proteins (see also major point #2).

9) Line 80, 86: ref 43 is misused. Reference 43 refers to Maurer's clefts trafficking which takes place in the erythrocyte cytosol and is not involved in haemoglobin uptake as far as I know. Please replace ref 43 with one showing the role of actin in haemoglobin uptake.

We thank the reviewer for pointing this out, Ref 43 was removed from the manuscript.

10) Line 98: the authors state here that they 'identified' further candidates from the K13 proxiome. This suggests that they identified new proteins in this paper, when in fact the list was already generated in ref 26. All they did was characterise proteins from that list that were not previously characterised. The authors should therefore remove identified from this statement.

We agree with the reviewer that we did not identify further candidates, we identified new K13 compartment proteins from the list of potential K13 compartment proteins. We therefore changed “identified further candidates” into “identified further K13 compartment proteins” (line 116). Please see also response to major comment #1.

11) Line 107-108: it is not clear from this sentence why these proteins were left out of the initial analysis in Ref 26. A sentence here explaining this would be valuable for the reader.

This is a good point. One reason why we did not analyse more in our previous publication was that we had to stop somewhere and adding more would have been very difficult to fit into what was already a packed paper. However, as shown in this work, the list does contain further interesting candidates (e.g. K13 compartment proteins that are involved in endocytosis).

We altered the relevant part of the introduction to highlight that we previously analysed the top hits, clarifying that the 'remaining' hits analysed in this work were further down in the list. This now reads: (line 113)“We reasoned that due to the high number of proteins that turned out to belong to the K13 compartment when validating the top hits of the K13 BioID (Birnbaum et al., 2020), the remaining hits of these experiments might contain further proteins belonging to the K13 compartment.” We hope this clarifies that we simply moved further down in the candidate list.

12) Line 117-123: The authors say that PF3D7_0204300, PF3D7_1117900 and PF3D7_1016200 were not studied because they were not in the top 10 hits. However, the current organisation of Supplementary Table 1 shows all 3 proteins among the top 10 hits (MyoF, KIC12, UIS14 and 0907200 being after them). I think the authors should reorganise their table. It is also unclear according to what the proteins in the table are ranked. Could the authors indicate the metric used for the ranking?

We thank the reviewer for alerting us to this. The issue here is that the 3 non-analysed proteins belong to a 'lower stringency' group comprising hits significant with FDRThe information about ranking is now also included as “Table legend” in the revised manuscript and the Table heading has been changed to: “List of putative K13 compartment proteins, proteins selected for further characterization in this manuscript are highlighted.”

13) Line 129-141: Can the authors be clearer with their explanations of the identification of mutation Y1344Stop? One dataset (ref 61) shows that 52% of African parasites have a mutation in MCA2 in position 1344 leading to a STOP codon. But another dataset (ref 62) shows that the next base is also mutated, reverting the stop codon. That should have been seen in the first dataset as well. Could the authors please clarify.

This mutation was first spotted in the MalariaGEN database (https://www.malariagen.net) (MalariaGEN et al., 2021), which allows online accessing of the data by using the “variant catalogue” tool, which is in a table format of frequency rather than in a sequence context. Hence, only after further research later on it became evident to us, that this mutation does not occur alone when looking at individual MCA2 sequences from patient samples in (Wichers et al., 2021b). We hope this is accurately reflected in our results section.

14) Line 147: the authors say that MCA2 is expressed throughout the intraerythrocytic cycle as shown by live cell imaging. In Birnbaum et al 2020 fig 4I, the authors show that MCA2 is mainly expressed between 4 and 16hpi. But in Figure 1B of this manuscript there is a clear multiplication of MCA2 signal between trophozoite and schizont. How do the authors explain this discrepancy? Could expression of the truncated MCA2 be different than the full length? This cannot be assessed as expression and localisation of the full-length HA tag MCA2 is not shown in Schizonts.

The key difference lies in transcription vs protein expression (usually protein levels peak after mRNA levels peak and - depending on turnover - protein levels can stay high even after mRNA levels have declined). Figure 4 of the Birnbaum et al paper presents transcriptomic data, but with a peak in trophozoites (The axis label in Fig. 4l of that publication is a bit confusing, as hour 0 is at the top, 48 h at the bottom; it is clearer in Fig. S13 of that paper) which would fit very well with the multiplication of the signal between trophozoites and schizonts mentioned by the reviewer. So, overall, the temporal peaks of transcripts and protein of that protein fit well.

For the signal in rings: Likely the protein has a turnover rate that is sufficiently low for some protein to be taken into the new cycle after re-invasion. Also different transcriptomic datasets e.g. (Otto et al., 2010; Wichers et al., 2019; Subudhi et al., 2020) available on plasmoDB show some mRNA present across the complete asexual development cycle, with each dataset showing maximum peak at a slightly different stage.

Even when located in foci and hence aiding detection of small amounts of protein (as is the case for MCA2-Y1344-GFP), the MCA2 signal in rings is not strong. For MCA2-TGD, the GFP signal is dispersed and therefore likely below our detection limit, while the same amount of protein concentrated at the K13 compartment is visible as foci in the MCA2-Y1344 cell line. Please note that MCA2-TGD has only 2.8% of the protein left whereas MCA2-Y1344 has 66.5% left and based on our manuscript is almost fully functional, hence fitting the different locations between the two versions.

Overall we believe this shows that there are actually no significant discrepancies of the expression of the different MCA2 versions.

15) Line 158: would it not have been more useful for the authors to have episomally expressed MCA2-3xHA in their MCA2Y1344STOP-GFPENDO line to make sure that the truncated protein is indeed going to the correct compartment? The experiments done by the authors suggests that the MCA2Y1344STOP goes to the right location but does not really confirm it.

We appreciate the reviewers caution here. However, considering that MCA2Y1344STOP-GFPendo co-locates with mCherryK13 and endogenously HA-tagged full length MCA2 does the same to a similar extent, there is in our opinion little doubt that MCA2 is found at the K13 compartment and that this is similar with both constructs. If there are minor differences, these might as well occur if MCA2 is episomally (as suggested in the comment) instead of endogenously expressed. Given the limited insight, we therefore decided against the episomal overexpression (which due to its size of > 6000bp may also be somewhat less straight forward than it may sound).

16) Line 191: it is stated that MCA2 confers resistance independently of the MCA domain, however in both the MCA2-TGD and MCA2Y1344STOP-GFPENDO parasites, the MCA domain is deleted, and for both parasites, there is resistance (albeit to a lower level in the MCA2Y1344STOP-GFPENDO line). Therefore, how can the authors state that the ART resistance is independent of the MCA domain? This statement should be that resistance is dependent on the loss of the MCA domain.

We agree that this can’t be categorically excluded. However, a ~5 fold difference in ART sensitivity was observed between the parasites with MCA2 truncated at amino acid 57 compared to those with MCA at amino acid 1344 even though both do not contain the MCA2 domain. Hence, at least this difference is not dependent on the MCA2 domain. The larger construct missing the MCA domain shows only a very moderate reduction in RSA survival, again suggesting the MCA domain is not the main factor. We amended our statement in an attempt to more accurately reflect the data (line 487): “This considerable reduction in ART susceptibility in the parasites with the truncation at MCA2 position 57 compared to the parasites still expressing 1344 amino acids of MCA2, despite both versions of the protein lacking the MCA domain, indicates that the influence on ART resistance is not, or only partially due to the MCA domain.” We would be hesitant to state the reviewer's conclusion that “resistance is dependent on the loss of the MCA domain”, as the larger construct missing the MCA2 domain has a milder RSA effect compared to MCA2-TGD, which suggests the reduction in ART susceptibility is independent of the MCA domain. These considerations also agree with the fact that the parasites with the longer MCA2 version (in contrast to the MCA2-TGD) do not have any detectable growth defect which indicates that the protein can fulfil its function without the MCA2 domain.

17) Line 192: Why did the authors not check if MCA2 is involved in endocytosis? They state later on in the manuscript that they did not do endocytosis assays with TGD lines, however if the authors include the correct controls, this could be easily done. It would also be really interesting to see whether endocytosis gets progressively worse going from WT to MCA2Y1344STOP to MAC2TGD. This experiment (as well as doing endocytosis assays for KIC4 and KIC5 TGD lines) would drastically increase the impact of this study. These experiments would not take more than 3 weeks to perform, and would not require the generation of new lines.

So far were very hesitant to do bloated FV assays with TGDs (even though TGDs were available for the genes encoding MCA2 and KIC4 and KIC5). The reason for this was:

1. the fact that these proteins could be disrupted indicated either redundancy or only a partial effect on endocytosis which might lead to only small effects that likely are difficult to pick up in an assay scoring for the rather absolute phenotype of bloated vs non-bloated. Using the refined assay measuring FV size could partly amend this but we note that also FV without hemoglobin have a certain size, reducing the relative effect if there are smaller differences.
2. a TGD line does not permit tightly controlled inactivation of the target which makes comparing the outcome of bloated food vacuole assays difficult if there are smaller growth and stage differences to the 3D7 control.
3. in contrast to conditional inactivation parasites, the TGD lines had ample times to adapt to loss of the target protein (compensatory mechanisms are well known for endocytosis, for instance in clathrin mediated endocytosis loss of individual components can be compensated (Chen and Schmid, 2020)). We nevertheless see the reviewer's point that this should at least be attempted and now conducted these assays (see also major point 40). For MCA2 (as requested in this point), the data is shown in Figure S5C-E. This assay showed that in MCA2-TGD, MCA2Y1344STOP-GFPendo (similar to the 3D7 control) >95% of parasites developed bloated food vacuoles. Additionally, we also measured the parasite and food vacuole size of individual cells in an attempt to solve some of the problems with TGDs with such assays. In order to specifically solve problem 2 mentioned above, we analysed the food vacuoles of similarly sized parasites, however, they were non-distinguishable between the three lines. Of note, in agreement with the reduced parasite proliferation rate (Birnbaum et al., 2020) a general effect on parasite and food vacuole size was observed for MCA2-TGD parasites, indicating reduced development speed in these parasites. Hence, it is possible that a potential endocytosis reduction was accompanied by a slowed growth, and the comparison of similarly sized parasites may have obscured the effect. It is therefore not sure if there indeed is no endocytosis phenotype, although we can exclude a strong effect in trophozoites.

Based on the RSA results at least rings can be expected to have a reduced endocytosis in the MCA2-TGD. Apart from options 1-3 mentioned above, it is therefore possible there is an effect restricted to rings, although in that case the reduced growth in trophozoites would be due to other functions of MCA2. Overall, we can conclude that the MCA2-TGD parasites do not have a strongly reduced endocytosis, but given the fact that the parasites are viable, this is not surprising. Whether the MCA2-TGD has no effect at all on endocytosis we would be very hesitant to postulate based on these results.

18) The authors should consider re-organising the MCA2 section, first showing that the 3xHA tagged line colocalises with K13, then performing the new truncation.

We attempted to re-organise as suggested but because we now included additional fluorescence microscopy images of schizont and merozoites (in response to reviewer 2 major comment 3) the main figure would become even larger. To prevent this, we kept the 3xHA data in the supplement.

19) Line 197: Once again ref 43 is not correct to illustrate that actin/myosin is involved in endocytosis

We thank the reviewer for pointing this out – we removed Ref 43.

20) Line 202: the authors state that MyoF localises near the food vacuole from ring stage/trophs onwards. However, how can this statement be made in schizonts based on these images (Fig. 2A), where it doesn't look like MyoF is anywhere near the FV? This statement can only be made for schizonts if co-localised with a FV marker (which is done in Fig. 2B), however, based on the number of MyoF foci, it appears that this was not done for schizonts. Please either remove the statement that MyoF is near the food vacuole from trophs onwards (because it is only seen near the FV up until trophs) or show the data in Fig. 2B of schizonts to substantiate these claims.

This is a valid point. We originally did not focus on schizonts because most markers end up in some focal area in the forming merozoite but other proteins (such as e.g. K13) also have one or more additional foci at the FV, making interpretation unclear, particularly if the schizont is still organizing to become fully segmented. This is why we generally focused the K13 co-localisations on the trophozoite stage to obtain the clearest information on endocytosis. However, given the fact that this manuscript gives the first localization of MyoF in P. falciparum parasites, we now provide a comprehensive time course (Figure 1C, S1A) including schizonts, which show quite a complex pattern: while the MyoF-GFP localization in trophozoites appeared as multiple foci close to K13 and also the FV, the MyoF-GFP pattern changes in late schizonts (fully segmented) and merozoites, appearing as elongated foci no longer close to K13 or the FV. Of note, this pattern has been previously reported for MyoE in P. berghei (Wall et al., 2019).

We therefore revised the statement about MyoF localization in schizont to better reflect the observed localization: (line 175): “In late schizonts and merozoite the MyoF-GFP signal was not associated with K13, but showed elongated GFP foci (Figure 1C, S2A) reminiscent of the MyoE signal previously reported in P. berghei schizonts (Wall et al., 2019).”

21) Line 204-206: what does this statement bring to the paper? Is it to show that it is the real localisation of MyoF because 2 tag cell line show the same localisation? I don't think this is needed, especially as later in the manuscript an HA-tag MyoF line is used and show similar localisation.

We see the reviewers point, but prefer to keep this data included in the supplement, particularly because potential differences in the location of tagged MyoF were a major concern.

Related to the tag issue: in order to get a better understanding of the effect of C-terminally tagging with different sized tags we now performed a more detailed analysis of the MyoF-3xHA cell line (Figure S2F-G), showing that this cell line shows a growth rate similar to the 3D7 wild type parasites, and has less vesicles than the 2x-FKBP-GFP-2xFKBP cell line, but still slightly, but significantly more than 3D7 parasites. Overall, this indicates that the smaller 3xHA tag has less effect on the parasite, than the larger 2x-FKBP-GFP-2xFKBP tag (see also new Figure 1L, showing a correlation of level of inactivation and the endocytosis phenotype for MyoF).

22) Line 212: The overlap of K13 with MyoF in Figure 2C 3rd panel (1st trophozoite panel) is not obvious, especially as the MyoF signal seems inexistant. I would advise the authors to replace with a better image. Also, why are there no images of schizonts shown in Figure 2C?

As suggested we exchanged the trophozoite image of panel Figure 2 C (now Figure 1C) and expanded this panel with images covering the complete asexual development cycle including schizonts in response to this and the previous points. As indicated above (point 20), schizont stages are complex to interpret. While late schizonts likely are not very relevant for endocytosis this is the first description of the location of the protein in this parasite and we therefore now provide a more thorough representation of the MyoF location across asexual stages in Figure1C and S2A.

23) Line 217: the spatial association of MyoF with K13 is very different when it is tagged with GFP and when it is tagged with 3xHA. The way the authors word it here, it seems that there is agreement with the two datasets, when this is not in fact the case (59% overlap for MyoF-GFP and only 16% overlap with MyoF-3xHA). These data suggest that the GFP and the multiple FKBP tags are doing something to the protein and therefore maybe the ensuing results using this line should not be trusted or be taken with a pinch of salt.

We agree with the reviewer that the location of this MyoF-GFP in the cell might differ due to the partial inactivation but in contrast to this comment, the data does not indicate any large differences. It seems the reviewer mixed something up (the 59% mentioned might come from the MCA2 figure?). The data with the two lines with differently tagged MyoF co-localised with K13 are actually quite comparable: GFP-tagged vs HA-tagged MyoF overlapping with K13 was 8% vs 16% full overlap, 12% vs 19% partially overlapping foci, 36% vs 63% foci that were touching but not overlapping (compare what now is Figure 1D and Figure S2C). Only in the 'no overlap' there is a much smaller proportion in the HA-tagged line. However, given that these are IFAs which on the one hand are more sensitive to see small protein pools but on the other hand also have pitfalls due to fixing of the cells (e.g. tiny increase in focus size due to fixing could increase the number of touching foci that in live cells might be close but did not touch), some variation can be expected to the live cells. We agree though that the partly reduced functionality of MyoF might be the reason for the consistent tendency of a lower overlap even though the difference is much less than indicated in the comment. We added "with a tendency for higher overlap with K13 which might be due to the partial inactivation of the GFP-tagged MyoF" to the sentence "IFA confirmed the focal localisation of MyoF and its spatial association with mCherry-K13 foci"

While we expect the fact that the difference between these parasites is only small somewhat reduces the "pinch of salt" with the MyoF line, we do agree that the partial functional inactivation of the GFP-tagged MyoF line may have some impact. However, we do not think that this means the results with the MyoF-GFP line are untrustworthy. On the contrary, it provides insights into its function that in some ways is equivalent to a knock down or TGD. Overall all the MyoF lines show: few vesicles occur in the MyoF-HA-line, more in the MyoF-GFP line and even more after knock sideways of MyoF-GFP. Importantly the severity of this phenotype correlates with the growth rates in these lines. Hence, together with the bloated food vacuole assays, this provides consistent data indicating that MyoF has a role in the transport of HCC to the FV and its level of activity correlates with the number of vesicles and growth. To better highlight this, it is now summarised in Figure 1M.

24) Line 219: the authors state here that they could not detect MyoF-GFP in rings, when in Figure 2C they show MyoF-GFP in rings, and also show that they could detect MyoF in Sup Fig. 3B with the 3xHA tagged line. Is this a labelling mistake in Figure 2C? If the authors could indeed not see MoyF-GFP in rings, this statement should have been made when Figure 2A was presented, and not so late in the manuscript, which causes confusion.

We thank the reviewer for pointing this out. We now provide a detailed time course (see also previous points) which shows that there is no detectable MyoF-GFP signal during ring stage development until the stage where the parasites starts the transition to trophozoites (i.e. MyoF-GFP signal could only be observed in parasites already containing hemozoin). In addition to the extended time course in Figure 1C (previously 2C) we included a panel of example ring stage images below to further highlight this. We also changed the labelling of the parasite with MyoF-GFP signal the reviewer mentions in Figure 1C to “late ring stage” (it already contains hemozoin) to clarify this.

The description of Figure 1A is now changed to: (line 153) *“The tagged MyoF was detectable as foci close to the food vacuole from the stage parasites turned from late rings to young trophozoite stage onwards, while in schizonts multiple MyoF foci were visible (Figure 1A, S2A).” *

Please see our answer to major comment #45 where we provide an explanation for the difference between MyoF-3xHA and MyoF-GFP signal in ring stage parasites.

[Figure MyoF]

25) Line 237: Showing a DNA marker (DAPI, Hoecht) for Figure 2E, and subsequent figures using mislocalisation to the nucleus, would help the reader assess efficiency of the mislocalisation.

Please see response to major comment #64 for a detailed answer on why we did not include DNA staining in the imaging used to assess mislocalization upon knock-sideways.

26) Line 254-256: authors should show the results of the bloating assay for parental 3D7 parasites (+ and - rapalog) to see whether the MyoF line - rapalog has increased baseline bloating. This applies to all subsequent FV bloating assays.

We did do several controls for bloated assays (including +/- rapalog of an irrelevant knock sideways line as well as using a chemical insult for which the control was 3D7 without treatment) in previous work (Birnbaum et al., 2020), which indicated that there is no effect of rapalog to reduce bloating. Although these controls are more stringent, we nevertheless did a 3D7 +/- rapalog control and added this to the manuscript (Figure S2I). As it is not possible to do this side by side with the assays that are already in the manuscript and the +/- rapalog 3D7 cells consistently showed no or very low numbers of cells without bloating (and stringent controls in the past equally did not show an effect), we believe adding this control once suffices.

27) Line 254-257: The authors say that because fewer parasites show a bloated food vacuole upon inactivation of MyoF it means that less hemoglobin reached the food vacuole. I understand the authors statement, however, shouldn't they look at the size of the food vacuole, instead of the number of parasites with bloated FV, to make such a statement? This has been done for KIC12 so why not doing it for MyoF?

This was now done and is provided as Figure 1J-K, S2J. The results confirm the assessment scoring bloated vs non-boated food vacuoles.

28) Line 259-261: these results would be difficult to interpret namely because the authors have dying parasites, which is exacerbated with the protein being knocked sideways. The authors should mention the pitfalls their knock sideways and tagging design here. Line 260-261: RSA is an assay relying on measuring parasite growth 1 cycle after a challenge with ART for 6 hours.

Fortunately, this concern is unfounded, as the survival (measured by parasitemia after one cycle) of the same sample + and - DHA is assessed, isolating the DHA effect independent of potential growth defects which are cancelled out. Hence, if there were parasites dying in the MyoF line (please note that they might not actually die, but simply grow more slowly), this factor applies for both the + and - ART condition. As we are testing for a decreased susceptibility to ART which would manifest as an increased survival in RSA surfacing above 1%, antagonistic effects of reduced MyoF function and ART treatment would not result in detectable differences as without effect, the RSA survival is always close to zero.

The same applies for the knock sideways where we assess the survival of +rapalog between +ART and -ART. If the reduced MyoF activity of the knock sideways leads to a decreased survival, this applies to both +ART and -ART. Please also note that rapalog was lifted after the DHA pulse (see e.g. Figure S2K).

That effects on growth are cancelled out is nicely illustrated for proteins where there is a stronger and more rapid effect on growth upon their conditional inactivation. For instance when KIC7 is knocked aside, there is a considerable increased of RSA survival, even though continued inactivation of KIC7 would have a severe growth defect (Birnbaum et al., 2020). Vice versa, a growth defect alone does not result in reduced RSA susceptibility as evident from knock sideways of an unrelated protein or using a chemical insult (Figure 4H in (Birnbaum et al., 2020) or simply slowing the ring stage by e.g. reducing EXP1 levels (Mesén-Ramírez et al., 2019). Hence, a growth reduction is not expected to alter the RSA outcome. And even if it did, it would only lead to an underestimation of the readout if growth is too severely affected (which would be obvious in the + rapalog without DHA sample, which was not the case).

In that respect it is valuable to have the rapid kinetics of knock sideways which permit inactivation of a protein before severe growth defects occur (although the only partial responsiveness of MyoF clearly is not the most optimal). In contrast, the absolute loss of a gene (as is the case if diCre is used) prevents (or at least makes it extremely difficult as the timing would need to exactly hit sufficient protein reduction without killing the parasite until the end of the RSA) using this system in these experiments (again see (Mesén-Ramírez et al., 2021) where in a EXP1 diCre based knock out RSA was only possible because we complemented with a lowly, episomally expressed EXP1 copy to have parasites with only a partial phenotype to do this assay).

29) Line 261-263: the authors sate that MyoF has a function in endocytosis but at a different step compared to K13 compartment proteins. I am not sure what they mean here. Can this be clarified?

The different steps in endocytosis are explained in the introduction and we now tried to further clarify this (line 98). “So far VPS45 (Jonscher et al., 2019), Rbsn5 (Sabitzki et al., 2023), Rab5b (Sabitzki et al., 2023), the phosphoinositide-binding protein PX1 (Mukherjee et al., 2022), the host enzyme peroxiredoxin 6 (Wagner et al., 2022) and K13 and some of its compartment proteins (Eps15, AP2µ, KIC7, UBP1) (Birnbaum et al., 2020) have been reported to act at different steps in the endocytic uptake pathway of hemoglobin. While inactivation of VPS45, Rbsn5, Rab5b, PX1 or actin resulted in an accumulation of hemoglobin filled vesicles (Lazarus et al., 2008; Jonscher et al., 2019; Mukherjee et al., 2022; Sabitzki et al., 2023), indicative of a block during endosomal transport (late steps in endocytosis), no such vesicles were observed upon inactivation of K13 and its compartment proteins (Birnbaum et al., 2020), suggesting a role of these proteins during initiation of endocytosis (early steps in endocytosis).“

VPS45 has not apparent spatial connection to the K13 compartment but the fact that MyoF does - and its inactivation also results in vesicle accumulation - indicates that it is downstream of vesicle initiation, providing the first connection from the initiation phase to the transport phase. More evidence for these different steps of endocytosis has been published in a recent preprint from our lab, where we simultaneously inactivated a protein of both “endocytosis steps” (Sabitzki et al., 2023).

To clarify this in the results as requested, we changed the statement to: (line 256) “Overall, our results indicate a close association of MyoF foci with the K13 compartment and a role of MyoF in endocytosis albeit not in rings and at a step in the endocytosis pathway when hemoglobin-filled vesicles had already formed and hence is subsequent to the function of the other so far known K13 compartment proteins.”

30) Do the authors mean that it is involved in endocytosis but not in ART resistance? If so, this is a very difficult statement to make since the parasites are dying. Is there any evidence of point mutations in MyoF in the field?

We split this point to address all issues raised here. Please see response to point 29 which clarifies that this was meant in a different way and our response to point 28 which explains why the dying parasite issue is not expected to affect the RSA (please also note that we do not have evidence of actually dying parasites in the MyoF-2xFKBP-GFP-2xFKBP line, most likely the growth is slowed).

The mutation issue is interesting. In fact evidence exists that MyoF mutations may be associated with resistance (Cerqueira et al., 2017) (please note that there it is still called MyoC) but in a recent preprint from our lab we did not find any evidence for a significantly changed RSA survival in 12 tested mutations in the corresponding gene (Behrens et al., 2023).

To clarify this we added the following statement to the discussion (line 709): "Of note, mutations in myoF have previously been found to be associated with reduced ART susceptibility (Cerqueira et al., 2017), but 12 mutations tested in the laboratory strain 3D7 did not result in increased RSA survival (Behrens et al., 2023)*. *

31) Line 298: the authors state that there is no growth defect in the first cycle when rapalog is added to the KIC11 line, however based on Figure 3D, there is evidently a 25% reduction in growth compared to - rapalog at day 1 post treatment, and a 60% reduction by day 2, which is still within the 1st growth cycle. The authors should either revise their statement or provide an explanation for these findings. The authors should also explain why their Giemsa data in Fig. 3E is not in accordance with their FACS data.

We think there is a misunderstanding here, as our figure legend was not detailed enough and we apologise if this had been misleading. The growth effect is restricted to invasion or possibly the first hours of ring stage development (see point 4&5, reviewer 2), which in asynchronous cultures more rapidly takes effect as the culture also contains schizonts that immediately generate cells that re-invade but can't due to inactivation of KIC11 (due to the rapid action of the knock sideways, KIC11 is already inactivated). In contrast, in highly synchronous cultures, this effect can only be evident once the parasites reached the schizont stage (starting with rings this takes close to 2 days). We now clarify that Figure 2E (previously Figure 3D) shows growth data obtained with an asynchronous parasite culture, while in Figure 2F the growth assay is performed with tightly synchronized (4h window) parasites as stated in the Figure legend.

We now explicitly state in each Figure legend and for each growth experiment throughout the manuscript whether we used asynchronous or synchronized parasites for growth assays.

Related to this, the incorrect y-axis label of what is now Figure 2E mentioned in major comment #58 is now corrected.

32) Line 301: KIC11 could also be important very early for establishment of the ring stage for example for establishment of the PV. Also, was mislocalisation assessed in rapalog-treated parasites at 72 hours or in cycle 3?

This is a valid point and this has now been addressed. We performed an invasion/egress assay revealing similar schizont rupture rates, but significantly reduced numbers of newly formed ring stage parasites (Figure 2H, S3G), indicating an effect of KIC11 inactivation either on invasion or possibly the first hours of ring stage development. A very similar point was raised by Reviewer 2, please see reviewer 2; major comment #4. This is now also reflected in line 302, which now reads: ”… indicating an invasion defect or an effect on parasite viability in merozoites or early rings but no effect on other parasite stages (Figure 2F-H, Figure S3F-G).”

We further included an assessment of mislocalization 80 hours after the induction of knock-sideways by addition of rapalog in Figure S3E which showed mislocalization of KIC11 to the nucleus.

33) Line 311: the authors should change the sentence from 'not related to endocytosis' to 'not related to endocytosis or ART resistance'.

Done as suggested.

34) Line 323-325: Authors say that a nuclear GFP signal can be observed in early schizonts for KIC12. According to the pictures provided in Figure 4A and Figure S5A it is not very obvious. Also faint cytoplasmic GFP signal could only be background as we can see that exposure is higher for schizont pictures

We changed the sentence (line 339) to: “…nuclear signal and a faint uniform cytoplasmic GFP signal was detected in late trophozoites and early schizonts and these signals were absent in later schizonts and merozoites (Figure 3A, Figure S4A,B).” in order to emphasize that the nuclear signal disappears early during schizont development.

35) Line 326-328: The authors say that kic12 transcriptional profile indicate mRNA levels peak (no s at peak) in merozoites. Should they show live cell imaging of merozoites then? Because from the Figure 4A schizont pictures where schizonts are almost fully segmented no signal can be observed.

The observation that mRNA levels of early ring stage expressed proteins tend to increase already in mature schizonts and merozoites is well established (e.g. (Bozdech et al., 2003)). A very good example for this are exported proteins of which most show a transcription peak in schizonts but the proteins are only detected in rings see e.g. (Marti et al., 2004). Hence, our observation for KIC12 is quite typical.

We originally did not include merozoites, as in the last row of Figure 3B fully developed merozoites within a schizont with already ruptured PVM are shown and no GFP signal can be detected in these parasites. We now provide images of free merozoites in Figure S4A-B showing again no detectable GFP signal.

We thank the reviewer for pointing out the typo, "peak" has been corrected.

36) Line 347: The authors state that using the Lyn mislocaliser the nuclear pool of KIC12 is inactivated by mislocalisation to the PPM. This tends to suggest that only the nuclear pool of KIC12 is mislocalised. How is it possible that only the nuclear pool is mislocalised?

The Lyn mislocaliser is at the PPM which is continuous with the cytostomal neck where the K13 compartment likely is found. The effect of the Lyn mislocalizer on the KIC12 protein pool localizing at the K13 compartment is therefore somewhat unclear. For this reason we already had the following statement in the original submission (line 400): “Foci were still detected in the parasite periphery and it is unclear whether these remained with the K13 compartment or were also in some way affected by the Lyn-mislocaliser.” We would like to stress here that the same does not apply to the nuclear mislocaliser, which is only a trafficking signal delivering KIC12 to the nucleus and hence likely does not affect the nuclear pool of KIC12, only the K13 compartment pool (the main interest of this manuscript).

We realised that the statement towards the end of this paragraph was unnecessarily ambiguous in regards to the K13 compartment pool of KIC12 which might have caused some confusion about the function of this pool of KIC12 and therefore modified it to (line 374): "Due to the possible influence on the K13 compartment located foci of KIC12 with the Lyn mislocaliser, a clear interpretation in regard to the functional importance of the nuclear pool of KIC12 other than that it confirms the importance of this protein for asexual blood stages is not possible. In contrast, the results with the nuclear mislocaliser indicate that the K13 located pool of KIC12 is important for efficient parasite growth.". It is also important to note that this limitation does not apply to the NLS knock sideways in regard to the K13 compartment and that the endocytosis function of this pool of KIC12 seems solid which with this statement is enforced.

37) Line 368-369: Effect was also only partial for MyoF. Why didn't you measure the same metrics for MyoF?

This was now done and is provided as Figure 1J-K, S2J, confirming our previous interpretation, see also point #27 which raises the same point.

38) Line 379: you don't know if all proteins acting later in endocytosis will have an increased number of vesicles as a phenotype

This is based on our current definition as stated in the introduction. It assumes a directional vesicular transport of hemoglobin to the food vacuole where inhibition of early stages will prevent transport before HCC-filled autonomous vesicular containers have formed and entered the cell. In contrast later inhibition stops such containers from further transport, leading to their accumulation. Such an accumulation is visible after VPS45-inactivation and other proteins (Jonscher et al., 2019; Mukherjee et al., 2022; Sabitzki et al., 2023) or treatment with cytochalasin D (Lazarus et al., 2008). While it is possible that there may be smaller intermediates formed at the K13 compartment that later on unite or fuse with the compartment evident after VPS45 inactivation and these might be missed due to small size (i.e. inhibition of a step between K13 compartment and an early endosome or equivalent), this would still be upstream of the VPS45 induced containers and hence would be earlier. We therefore believe that based on the framework given in the introduction (see also (Spielmann et al., 2020)) to assume that a phenotype manifesting as reduced food vacuole bloating without formation of detectable vesicles likely signifies inhibition of the process early whereas reduced bloating but with vesicles signifies inhibition later in the process.

39) Line 413-414: The authors state that no growth defect was observed upon KS of 1365800. Is growth alone enough to say that there is no impact on endocytosis?

This is an interesting point. The endocytosis proteins we studied so far indicate that efficient impairment of endocytosis manifests as a severe growth defect. Hence, lack of a growth defect can be assumed to be an indicator for absence of an important role for endocytosis (or any other growth relevant process). Clearly there is a gradual response, such as seen in the different MyoF versions resulting in proportional growth and vesicle appearance phenotypes. Hence, a protein with a minor role might have slipped our attention but then it probably is also not a very important protein in endocytosis.

To further strengthen our assessment of PF3D7_1365800 importance for asexual blood stage development, we now also generated a cell line expressing the PPM Mislocalizer, enabling knock sideways to the PPM. This was done because this protein consistently has a focus at the nucleus that may be within the nucleus. Again this revealed no growth defect upon inactivation (Figure S7D).

40) Line 432: in this section, the authors state that KIC4 and KIC5 seem to have domains that may suggest these proteins are involved in endocytosis, based on the alpha fold data that is publicly available. Considering the authors have TGD-SLI versions of these lines (Birnbaum et al. 2020) and have already confirmed in this previous publication that they confer resistance to ART; it would make sense to look at endocytosis for these genes. This would be a relatively simple and straightforward experiment, taking no longer than two to three weeks, and would require no additional reagents or line generation. Doing these experiments would add a lot more weight to this final section. The authors later state that KIC4 and 5 are TGD lines, so not the best for endocytosis assays. It is unclear why this would be difficult to do if an adequate control is contained in the experiment (such as parental 3D7). It explains why they did not perform the MCA2 endocytosis assays further up, but in my opinion, an attempt at doing these assays is important and would significantly increase the impact of this paper. Identical as major comment #17.

As stated in the manuscript and above, we were originally hesitant to do these assays due to the fact that we can't induce inactivation which is less ideal than comparing the identical parasite population split into plus and minus and is further complicated by the likely smaller effect as the TGDs still permitted growth. However, we see the point of the reviewer and now performed these assays using 3D7 as controls and taking extra care to account for stage differences between the TGD lines and 3D7. However, there was no significant difference in the bloated food vacuole assays with these cell lines. Due to the reasons mentioned in major point 17, we are not sure this indeed means these proteins have no role in endocytosis. One possible reason why we were able to obtain these TGDs may have been because the effect on endocytosis is less than in the essential proteins (or is ring stage specific) and in a TGD an endocytosis defect may therefore not be detectable with our assays (see details and further possible explanations in response to point 17).

In an attempt to address the TGD issue, we generated knock sideways cell lines for KIC4 and KIC5. Unfortunately, the mislocalization of KIC5 to the nucleus was inefficient (see figure below). As this did not result in a growth defect (in contrast to the clear KIC5-TGD growth defect (Birnbaum et al., 2020)), this line is not suitable to study a potential role of this protein in endocytosis. Therefore, we performed the bloated food vacuole assay only with KIC4-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser parasites. However, this revealed no effect on HHC uptake, which is in line with the normal growth of KIC4-TGD parasites (Birnbaum et al., 2020) and suggests that this protein could only have a minor or redundant role in endocytosis (it is the line that shows the smallest effect in RSA). As the KIC4 and KIC5 knock sideway lines did not permit any conclusions, we did not include them into the revised manuscript but they can be found here:

[Figure KIC4 knock sideways & KIC5 knocksideways]

Figure legend: (A) Live-cell microscopy of knock sideways (+ rapalog) and control (without rapalog) KIC4-2xFKBP-GFP-2xFKBPendo+ 1xNLS mislocaliser parasites 4 and 20 hours after the induction of knock-sideways by addition of rapalog. Scale bar, 5 µm. Relative growth of asynchronous KIC4-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser plus rapalog compared with control parasites over five days. Three independent experiments were performed. Growth of knock sideways (+ rapalog) compared to control (without rapalog) KIC4-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser (blue) or KIC5-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser (red) parasites over five days. Mean relative parasitemia ± SD is shown. (B) Live-cell microscopy of knock sideways (+ rapalog) and control (without rapalog) KIC5-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser parasites 4 and 20 hours after the induction of knock-sideways by addition of rapalog. Scale bar, 5 µm. Growth of asynchronous KIC5-2xFKBP-GFP-2xFKBPendo+ 1xNLSmislocaliser plus rapalog compared with control parasites over five days. Four independent experiments were performed. __(C) __Bloated food vacuole assay with KIC4-2xFKBP-GFP-2xFKBPendo+1xNLSmislocaliser parasites 8 hours after inactivation of KIC4 (+rapalog). Cells were categorized as with ‘bloated FV’ or ‘non-bloated FV’ and percentage of cells with bloated FV is displayed; n = 3 independent experiments with each n=19-30 (mean 21.4) parasites analysed per condition. Representative DIC are displayed. Area of the FV, area of the parasite and area of FV divided by area of the corresponding parasites were determined. Mean of each independent experiment indicated by coloured symbols, individual datapoints by grey dots. Data presented according to SuperPlot guidelines (Lord et al., 2020); Error bars represent mean ± SD. P-value determined by paired t-test. Area of FV of individual cells plotted versus the area of the corresponding parasite. Line represents linear regression with error indicated by dashed line.

41) Line 490-493: the authors state that the K13 compartment proteins fall in two groups, some that are involved in ART resistance AND endocytosis, and some that have different functions. However, in this manuscript the authors have demonstrated 3 flavours that K13 compartment proteins can come in: • Some that confer ART resistance and are involved in HCCU (MCA2) • Some that are involved in HCCU but not ART resistance (MyoF & KIC12) • Some that are involved in neither (KIC11) The authors should therefore revise this statement.

We agree that this was not well phrased. To account for the fact that not all endocytosis proteins confer increased RSA survival to the parasites when inactivated we changed this statement (line 604): "This analysis suggests that proteins detected at the K13 compartment can be classified into at least two groups of which one comprises proteins involved in endocytosis or in vitro ART resistance whereas the other group might have different functions yet to be discovered.“

Generally, we believe that endocytosis is the overarching criterion and we therefore would like to keep the definitions of the main groups (endocytosis or not). As indicated by the title, the focus of the manuscript is on the K13 compartment for which so far endocytosis is the only experimentally associated function. That this group contains proteins that do not confer reduced ART susceptibility when conditionally inactivated (KIC12 and MyoF) is explained by their stage-specificity, making this a subgroup of the overarching endocytosis group.

We realise that with the endocytosis data on the KIC4, KIC5 and MCA2 TGD there is now also a subgroup we were unable to demonstrate an endocytosis effect in trophozoites although they show changes in RSA survival. However, as indicated above, we would be hesitant to fully exclude some role of these proteins in endocytosis in rings. Particularly as a comparably small reduction in endocytosis protein activity or abundance is sufficient to increase RSA survival (Behrens et al., 2023). A principal classification of "endocytosis or ART resistance" or "neither endocytosis nor ART resistance" still accounts for this and therefore seems to us to be the most useful, particularly also in light of our domain identification that then can be linked with one or the other group.

42) Line 508: the authors state that they expanded the repertoire of K13 compartments, when in fact they functionally analysed them - they did not do another BioID to identify more candidates.

We respectfully disagree with the reviewer in this point, we did expand the repertoire of known K13 compartment proteins. Only independently experimentally validated proteins from proximity biotinylation experiments can be considered part of the K13 compartment (or any other cellular site or complex). Without validation of the location, the identified proteins can only be considered candidates. This is highlighted in this manuscript by the finding that several proteins of the list did not localize at the K13 compartment.

43) Line 570-572: has anyone ever tested whether CytoD or JAS treatment in rings, is sufficient to mediate ART resistance? Something similar to what was done in PMID 21709259 with protease inhibitors. If not this would be a pretty interesting experiment for the authors to do that could shed more light on the MyoF data. It would take maybe 2 weeks to do and not require the generation of any new lines. This would clarify whether other Myosins other than MyoF are involved in endocytosis, as is suggested by previous publications (PMID: 17944961).

We now included this experiment. In agreement with a lacking need of MyoF in rings and no effect on RSA survival, there was no increased survival of the parasites in RSA (neither on 3D7 nor on K13 C580Y parasites) after cytD treatment (new part in Figure 1M). We thank the reviewer for pointing out that this experiment might also inform on whether other myosins influence endocytosis in ring stages. We added (line 250): “Similarly, also incubation with the actin destabilising agent Cytochalasin D (Casella et al., 1981), had no effect on RSA survival in 3D7 or K13C580Y (Birnbaum et al., 2020) parasites, indicating an actin/myosin independent endocytosis pathway in ring stage parasites (Figure 1M) and speaking against other myosins taking over the MyoF endocytosis function in rings.”

44) Line 608: inhibitors targeting the metacaspase domain of MCA2 may inadvertently inactivate other essential parts of the protein. They authors should acknowledge this possibility in the text.

The inhibitors used in the cited studies (Kumari et al., 2018) are validated metacaspase inhibitors, such as Z-FA-FMK (Lopez-Hernandez et al., 2003). Activity against the other parts of PfMCA2 - which apart from the MCA domain shows no homology to other proteins - is therefore unlikely.

45) Line 624-625: the authors state that MyoF is 'lowly expressed in rings' - indeed this is the case in their MyoF-2xFKBP-GFP-2xFKBP line which the authors established has defects due to the tag, but it appears from their MyoF-3xHA tagged line that it is expressed in rings. The authors should therefore revise their statement, and be careful of making claims based on their defective line and using fluorescence imaging as their only metric. If they do want to make the statement that it is not there in rings, they should also do a western blot, which is much more sensitive since it amplifies the signal compared to an image of one parasite.

This comment is related to major point #24. We also would like to stress that while the MyoF-GFP line already shows a phenotype, the impression of defectiveness based on its location is due to a mix up (see major point #23).

We now provide a comprehensive time course of the MyoF-GFP signal (Figure 1C, S2A) showing that there is no detectable MyoF-GFP signal until the transition from ring to trophozoite stage. As this is all under the endogenous promoter, we do not think the partial functional inactivation of the tagging is the reason for the absence of the signal. If anything, we would have expected adding a stably folded structure such as GFP to increase the stability of the protein. The main reason for the discrepancy of MyoF signal in rings between the GFP-tagged line (of note there is also no detectable MyoF-GFP signal in MyoF-2xFKBP-GFP ring stage parasites (Figure S2B)) and the HA-tagged line likely is that IFA is much more sensitive than live GFP detection (similar to the high sensitivity the reviewer mentions in regards to WB). This discrepancy therefore is likely due to the fact that the lowly expressed MyoF only become apparent with the HA-tagged line due to the IFA. We therefore believe that MyoF is 'lowly expressed in rings' is an appropriate description of our results obtained with three different cell lines (MyoF-2xFKBP-GFP-2xFKBP, MyoF-2xFKBP-GFP and MyoF-3xHA). We hope this is sufficiently well reflected in the manuscript where we write ‘a low level of expression of MyoF in ring stage parasites.’ not that it is ‘not there in rings’ (line 174).

46) Line 635: arguably this is the 3rd variety and not the 2nd (the authors already mentioned 2 types - ones that are involved in HCCU AND ART and those involved in HCCU only). See comment for line 490-493 above.

See response for major comment #41, we now consistently used "or" instead of "and". See line 490-493 how this was resolved for what previously was line 635.

47) Line 785: Bloated food vacuole assay/E64 hemoglobin uptake assay method specify that a concentration of 33mM E64protease inhibitor was used. However, in reference 44, cited in the manuscript, a concentration of 33µM E64 was used. Please confirmed if this is just a typo or if 1000x E64 concentration was used which renders the experiment invalid.

We thank the reviewer for pointing this out, we corrected this typo and will look out for symbol font conversion errors for the resubmission.

48) Line 788: it is unclear from this section what is considered a bloated food vacuole - is there an area above which the FV is considered bloated? Do the authors do these measurements manually or use an addon in FIJI/ImageJ? What is the cutoff for if a FV is bloated? Please clarify. Additionally, for the representative images + rapalog for Figures 2H and 4H, it would be useful to see where the authors delineate the FV (add a white circle showing what is actually measured).

The bloated FV assay is well established (Jonscher et al., 2019; Birnbaum et al., 2020; Sabitzki et al., 2023). Although the bloating of the FV is a human judgment call, it is actually quite obvious: bloating appears as an easily spotted bulging of the FV in DIC. As also minor bloating is scored as 'bloated', it is a very conservative assay. Using an-add on to measure this is not straight forward. It is unclear how this bulging effect of the FV in DIC could be spotted by a software and due to the obviousness to human operators, potentially lengthy and complicated efforts to design appropriate machine learning options were not undertaken. The situation faced by the scorer of the assay is evident from Figure S4F-G which contains close to 50 "on rapalog" cells and close to 50 control cells, giving representative cells from all replicas of bloated FV assays with KIC12. Please note that these images shows the most complicated situation as far as bloated assays go, because the phenotype is not 100% (see Figure 3F) compared to e.g. KIC7 inactivation which leads to lack of bloating in almost all cells (see (Birnbaum et al., 2020) Figure 3E) but nevertheless the difference is still obvious. We are aware that in such situations (less than absolute inhibition) this assay scoring of "yes" or "no" is a surrogate for the actual level of inhibition and may be more subjective. This is why in this case we also did the FV size measurements (which are less dependent on human judgment) to further support this and give a better quantifiable measure. Of note, the bloated food vacuole judgments are done "blinded", i.e. the examiner does not know which sample they are looking at.

In response to this reviewer's point we now also added the FV size refinement of the assay for MyoF inactivation which is one of the cases where inhibition of bloating is not in 100% of the cells (see major comment #27). Please also note here the advantage of the rapidly acting knock sideways technique for these assays which shows the sum of effect 8 h after initiating inactivation and for which we carefully control size of the cells which shows that there is no significant growth reduction over the assay time, excluding secondary effects due to a generally reduced viability. Compared to slower acting systems suggested to have been used instead (see introductory part and significance of this review), the rapid speed of knock sideways reduces the risk of potential pleiotropic or compensatory effects due to the time needed for proteins to be depleted if the gene or mRNA is targeted instead.

The suggestion to include a ‘white circle’ (raised also as minor comment#27) is useful as an aid to see the food vacuole. However, in contrast to the Figures in (Birnbaum et al., 2020) (where we did add such a circle), we here included the DHE staining images in the figure, labelling the parasite cytosol which readily shows the FV (the FV corresponds to the region where there is no DHE staining). As this shows the position of the FV we would prefer to not obscure the DIC images with additional features to permit the reader to see the difference between bloated or non-bloated food vacuoles and keeping the image as natural as possible.

49) Line 863-864: this sentence seems to be out of place.

We thank the reviewer for pointing this out, the details of nucleus staining were moved to the correct part.

50) Line 875: the authors state that there is a light blue wedge, when the circle consists of grey and black wedges. Please revise this.

This has been corrected.

51) Line 1059-1061: it is unclear whether the individual growth curves are different clones or whether they are just the same experiment repeated? If it is the latter, then why are they not combined, as is traditionally done?

These are the individual replicates of the growth curves shown in Figure 1G of the same cell lines done on a different occasion. We always try to show as much of the primary data as possible and believe that showing individual data points from the different experiments is better than only the combined values which obscure the actual course of each experiment.

52) Line 919-924: the authors mention a blue and red line, but there is only a black line in figure 3D. Moreover, the experiment of using the LYN mislocaliser was only done for KIC12 according to the manuscript. Additionally, the y axis of the figure states relative growth day 4[%] compared to rapalog, but then on the x axis there are several days. In the text it says there is no growth defect until the second cycle, but from this graph it appears the growth defect is evident as early as 1 day post rapalog treatment. Can the authors please clarify and correct the issues pointed out.

We thank the reviewer for pointing this out, this was due to a copy & paste error in the figure legend that was now amended. We also fixed the incorrect axis label. For the last part (growth defect) please see detailed answer to Major comment#31 raising the same concern for KIC11 (in synchronous parasites the defect only takes effect once the cells reached the relevant stage whereas in asynchronous cultures there are always cells in the relevant stage that due to the rapid effect of the knock sideways already have a growth phenotype).

53) Figure 1 panel B & C: the label of the figure where the signal from MCA2Y1344STOP-GFP is shown with the DAPI signal overlayed is deceptive since it suggests that this is the signal of full length MCA2. Please change the label of this panel from MAC2/DAPI to MCA2Y1344STOP/DAPI. The same is true for Panel C for the image labeled MCA2/K13 - please change this to MCA2Y1344STOP/K13.

Done as requested.

54) Figure 2B: what stages are these parasites? Please state this in the figure. Based on the MyoF pattern, it looks like rings in the upper panel and trophs in the bottom pannel. Why were schizonts not shown?

Both are trophozoites (early trophozoite in top panel and late trophozoite in bottom panel). This is now labelled in what now is figure 1B. As stated above, schizont stages are less relevant for the topic of this manuscript and in order to prevent the manuscript from getting more disjointed and keeping it more focussed on the main topic, we decided to not include a schizont in the manuscript. Nevertheless, we included an example image below.

[Figure MyoF_p40px schizont]

55) Figure 2D&F: it is not very meaningful when growth assays are shown as a final bar after 4 days of growth. It is much more useful and informative to see a growth curve instead (as is shown in the supplementary), since it shows if the defect is apparent in the first growth cycle or later. With the way the data is currently shown, this is not apparent. I would advise the authors to switch the graph in 2F out of a combined graph of all the biological replicates growth curves for S3D - showing error bars.

While we in principle fully agree with the reviewer in showing the course of the full experiment (which is available in Figure S2E), the key here is to show the overall difference. Hence, we would like to keep this comparison of the overall effect on growth in what now is Figure 1E and G. It is part of the argument to the doubts this reviewer raises to the function of MyoF (mainly in the overall assessment and the significance statement) to show that the phenotype is actually very consistent (partial inactivation through tagging or further inactivation using knock sideways increases endocytosis phenotypes, correlating with parasite viability).

Please also note, that the growth curves upon knock sideways shown in Figure 1G, S2E are performed with asynchronous parasite cultures, which doesn’t allow us to draw direct conclusions about growth cycle effects.

Nevertheless, we now also included the suggested combined data representation in Figure S2E.

56) Figure 3: why were the calculation of FV area, parasite area and FV/parasite area only done for KIC12 and not done for MyoF? It would be interesting to see if any of these values are different for MyoF - whether the parasites are smaller in area and therefore FV smaller. Please present them Figure 2. Images should be already available and would not require further experiments to be done, only the analysis.

This now has been done (confirming our results) and is included as Figure 1J-K, S2J. This point was also raised as major comment #37, please also see detailed answer there.

57) Figure 3B: why is there no spatial association assessment for KIC11 and K13 as was done for the MCA2 and MyoF? The authors should show a pie chart showing the degree of association here as was done for the other proteins.

This is now included in Figure 2C.

58) Figure 3D: The y axis of the figure states relative growth day 4[%] compared to rapalog, but then on the x axis the experiment takes place over several days. Is this a typo in the y axis? Additionally, the authors state in line 287-290 that the growth defect upon addition of rapalog is only seen in the second cycle, but from this graph it appears the growth defect is already evident 1 day post rapalog addition. The figure legend also does not make sense for this figure since it mentions a blue and a red line, when there is only a black line present. The legend also mentions the LYN mislocaliser which was used for KIC12 not KIC 11 (see above).

We apologise for the inadequate legend and colour issues, this was amended. This point was also raised in major comment #31 and #52, please find detailed answer there.

59) Figure 3E: the colour for Control and Rapalog 4 hpi are very similar and very hard to discern. Please choose an alternative colour or add a pattern to one of the samples. The y axis is also missing a label. Is this supposed to be parasitemia (%)?

We thank the reviewer for pointing this out, the missing label is now included and the colour has been adapted to make them better distinguishable.

60) Figure 4A: the ring shown in this figure does not appear to be a ring (it is far too large and appears to have multiple nuclei?). Do the authors have any other representative images to show instead?

This is in fact a ring, but we realize that we accidentally included an incorrect size bar in the ring image of Figure 4A (now Figure 3A) (size bar for 63x objective instead of the correct one for the 100x objective), we apologise for this oversight. We don’t think this parasite has multiple nuclei, instead the Hoechst signal shows the often elongated nucleus seen in rings that can appear as two foci in Giemsa stained smears which leads to the typical diagnostic feature of P. falciparum rings in diagnostics. In order to exclude any doubts about the nuclear localization of KIC12 in rings, we here attached a panel with more examples of KIC12-2xFKBP-GFP-2xFKBP ring stage parasites.

[Figure KIC12]

61) Figure 4B: why is there no spatial association assessment for KIC12 and K13 as was done for the MCA2 and MyoF? The authors should show a pie chart showing the degree of association here as was done for the other proteins. This should be done for the different life cycle stages considering the changing localisation of KIC12.

This is now provided in Figure S4A. As suggested by the reviewer, we independently quantified the association for ring stage, early trophozoite and late trophozoites stage. As there is no KI12 signal in schizonts, we did not include a quantification for this stage.

62) Figures 4C&E: it is extremely important to show the DNA stain in both these samples considering that a portion of KIC12 is in the nucleus! Please add the DAPI signal for these figures (as for all other figures!).

Please see major comment #64 for a detailed answer why we did not include DNA staining in the imaging used to assess mislocalization upon knock-sideways.

63) Figure 4E: this figure should be presented before 4D (considering the line being presented in 4E is used in an experiment in 4D). The authors should switch the order of these two.

We see the point the reviewer is raising here, Figure 4D (now Figure 3D) also contains the data with the Lyn mislocaliser while we first talk about the NLS mislocaliser. This permits a better comparison between the two mislocaliser lines. However, first explaining the Lyn-mislocaliser and then going back to the NLS would make it rather complicated for the reader to follow the storyline and therefore we would like to keep the order as it is. We realise that this means the reader has to go back one figure part for seeing the Lyn growth data, but believe this is worth the benefit that the data is there compared to the NLS result.

64) It is unclear why in many of the fluorescence images the authors do not show the DAPI signal - particularly when colocalising with K13 and when doing the knock sideways experiments. Please add these images to the figures - I would assume they have already been taken, so would simply involved adding the images to the panel.

We did not include DNA staining (DAPI or Hoechst) for any of the images used to assess the efficacy of mislocalization, as we would prefer to keep the parasites as representative of a viable parasites in culture as possible. Hence they were imaged without DNA stain (these stains are toxic). We would like to point out that a DNA stain is not necessary, as the mislocaliser already marks the nucleus (in the case of the NLS mislocaliser), actually even somewhat more accurately, as it fills the entire nuclear space rather than only the DNA which is marked by DAPI or Hoechst.

For LYN this admittedly is not the case, there the mislocaliser marks the plasma membrane. However, we think the proper control for efficient mislocalisation is the comparison between the GFP-tagged protein of interest and the mCherry mislocaliser to show mislocalisation, as previously done in our lab (e.g. (Birnbaum et al., 2017; Jonscher et al., 2019; Birnbaum et al., 2020)).

Due to their toxicity, we also avoided nuclear staining in some other parts of the manuscript when we were of the opinion that a nucleus signal was not necessary.

65) Throughout the manuscript, there is no western blot confirming the correct size of their modified proteins. This should be provided.

We did perform Western blot analysis for both MCA2 cell lines. MCA2 is the only gene-product for which we generated a disruption for this work, and together with the severe truncation from previous work, we provided a Western blot-based confirmation of the correct size.

The MCA2 disruptions are at least partially dispensable for in vitro parasite growth, hence if degradation occurred, this might not have been noticed. In that case we considered it relevant to show that the truncations were of the expected size. The other proteins in the main figures are essential for growth. Hence, if the tagging approach would lead to unexpected changes in protein integrity (which we assume is what was intended by this concern to be assessed with a Western blot), the parasites expressing the tagged MyoF, KIC11 and KIC12 would - due to their importance for asexual blood stage development - not have been obtained. Hence, we can assume the integrity of the tagged protein is very unlikely to have been affected in a functionally relevant way.

66) None of the figures are appropriate for individuals with colour blindness, limiting their accessibility to the paper. Please change the colour schemes for all fluorescent images using magenta/green or an alternative colour combination appropriate for colourblind individuals.

We thank the reviewer for this comment. This has now been amended, individual channels of fluorescence microscopy images are now shown in greyscale, while the overlay was changed to green/magenta.

Minor Comments

1) line 29: remove 'are'.

Done.

2) Line 29: the text says "HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins are among the few proteins so far functionally linked to this process." The sentence should be: 'HCCU is critical for parasite survival but is poorly understood, with the K13 compartment proteins among the few proteins so far functionally linked to this process."

Done.

3) line 44: remove 'the'

Done.

4) Line 48: consider mentioning here that malaria is caused by the parasite Plasmodium - otherwise the first mention of parasite in line 52 is confusing for the non-specialist reader.

Done.

5) Line 49: estimated malaria-related death and case numbers are from the 2021 WHO World malaria report. You cite the 2020 WHO World malaria report.

We now cite the newest WHO report.

6) Line 53: please insert the word 'have' between now and also.

Done.

7) Line 54: please change 'was linked' to is linked

Done

8) Line 72: I would specify that free heme is toxic to the parasite. Especially as you mention that hemozoin is nontoxic.

Sentence would be "where digestion results in the generation of free heme, toxic to the parasite, which is further converted into nontoxic hemozoin"

Done.

9) Line 90: authors should either say "in previous works" or "in a previous work"

The text has been altered to say: “ in a previous work”.

10) Line 91: "We designated these proteins as K13 interaction candidates (KICs)"

Done.

11) Line 95: please change 'rate' to number

Done.

12) Line 109: Please include a coma before (ii).

Done.

13) Line 112: as shown by Rudlaff et al in the paper you are citing, PPP8 is actually associated with the basal complex. You can say that "(ii) were either linked or had been shown to localise to the inner membrane complex (IMC) or the basal complex (PF3D7...).

Done.

14) Line 114: Protein PF3D7_1141300 is called APR1 in the manuscript but ARP1 in Supplementary Table 1. Please correct.

Done.

15) Line 131: please define SNP - this is the first use of the acronym.

Done.

16) Line 133-134: South-East Asia instead of "South Asia"

Done.

17) Line 135: please explain what TGD is - it is referred to over and over again in the manuscript without ever being explained.

We apologise for this oversight. We now explain what is meant with TGD at the suggested point of the manuscript.

18) Line 145: change 'Western blot' to western blot - only Southern blot is capitalised since it is named after an individual, while the other techniques are not.

To the best of our knowledge this issue has not been resolved, some Journals capitalize the “W” (e.g. Science), while others don’t (e.g. Nature). We would prefer to continue to capitalize the “W”, as this is consistent with the original publication from (Burnette, 1981), but if there are strong objections, we would be happy to change this____.

19) Line 152: add "the" between 'and spatial'

Done.

20) Line 158: please define SLI as selected linked integration, since it is the first use of the acronym.

Done.

21) Line 178: introduce a coma after protein. Sentence should be "Proliferation assays with the MCAY1344STOP-GFPendo parasites which express a larger portion of this protein, yet still lacking the MCA domain (Figure 1), indicated no growth ...

Done.

22) Line 195: the authors could mention that MyoF was previously called MyoC in the Birnbaum 2020 paper. I wanted to check back in the Birnbaum 2020 paper and could not find MyoF

Good point, this was done.

23) Line 200: "Expression and localisation of the fusion protein was analysed by fluorescent microscopy". Why expression was not analysed also by western Blot same as for MCA2?

Please see major comment #64 for a detailed answer.

24) Line 204: I could not find any mention of MyoF (Pf3D7_1329100) in reference 65. Please remove reference 65 if not correct. Also reference 66 looks at Plasmodium chabaudii transcriptomes so I would specify that "This expression pattern is in agreement with the transcriptional profile of its Plasmodium chabaudii orthologue"

Reference 65 (Wichers et al., 2019) provides an RNAseq transcriptome dataset for asexual blood stage development of 3D7 (originating from the same source as the 3D7 used in this study). While Ref 66 (Subudhi et al., 2020) indeed contain transcriptomic data from P. chabaudi, the authors also provide a nice 2h window RNAseq transcriptome dataset for asexual blood stage development of Plasmodium falciparum. Both datasets are therefore suitable as reference for the statement about myoF transcription pattern. Both datasets are also easily accessible and show the pattern in a graph in PlasmoDB.

25) Line 208: Please indicate a reference for P40 being a marker of the food vacuole

Done.

26) Line 220-224: The authors should consider changing to " Taken together these results show that MyoF is in foci that are mainly close to K13 and, at times, overlapping, indicating that MyoF is found in a regular close spatial association with the K13 compartment."

The suggested wording introduces "mainly" for "frequently" and likely was in part motivated by the discrepancy in location between cell lines that we hope we now could clarify to be only minor (see major point #23). We therefore think the original wording appropriately summarises the findings (line 178): “*Taken together these results show that MyoF is in foci that are frequently close or overlapping with K13, indicating that MyoF is found in a regular close spatial association with the K13 compartment and at times overlaps with that compartment.” *

27) Line 255: In Figure 2H, and subsequent figures showing bloated FV assay, I would delineate the food vacuole with dashed line as in Birnbaum et al. 2020 to help the reader understanding where the food vacuole is.

In contrast to the Figures in Birnbaum et al. 2020, we here included the DHE staining (parasite cytosol) in images of bloated FV assays which visualizes the FV. We therefore decided to avoid any further marking, to keep the image as unprocessed as possible (see also major point 48).

28) Line 265-266: Here the title says that KIC11 is a K13 compartment associated protein, but the title of Figure 3 says KIC11 is a K13 compartment protein. I noticed that you make the difference between K13 compartment protein et K13 compartment associated protein for MyoF for example which is not clearly associated with the K13 compartment. Which one is it for KIC11?

The interpretation of the reviewer is correct, we indeed graded this subconsciously based on level of overlap. Based on the newly added quantification shown in Figure 2C, we describe KIC11 now as K13 compartment protein.

29) Line 309-310: indicate a reference for your statement "which is in contrast to previously characterised essential K13 compartment proteins".

Done, we now included Birnbaum et al. 2020 as reference for this.

30) Line 377: Figure 4I, please correct 1st panel Y axis legend

Done.

31) Line 404: replace "dispensability" with dispensable

Done.

32) Line 416: can the authors provide any speculation as to why they observed these proteins as hits in the BioID experiments?

As some of these proteins were less well or less consistently enriched, they could be background of the experiment. Alternatively, some could be proteins that only transiently interact with the K13 compartment.

33) Line 451: Where the "97% of proteins containing these domains also contain an Adaptin_N domain and function in vesicle adaptor complexes as subunit a" come from. Do you have a reference?

The statement now includes references and reads (with small changes to original submission): "More than 97% of proteins containing these domains also contain an Adaptin_N (IPR002553) domain (Blum et al., 2021) and in this combination typically function in vesicle adaptor complexes as subunit α (Hirst and Robinson, 1998; Traub et al., 1999) (Figure 5D) but no such domain was detectable in KIC5."

34) Line 465-467: the same could be said for KIC4 as it also has a VHS domain.

The critical issue is the combination of domains and their position within the protein. While KIC4 also contains a VHS domain, the VHS domain in KIC4 is N-terminal, not in a central position and it is also not the first structural domain to be identified in KIC4. The similarity to adaptin domains was already described ((Birnbaum et al., 2020) and annotated in PlasmoDB) and these domains are also involved in vesicle formation and trafficking. These aspects of the statement can therefore not be extended to KIC4. With regards to VHS domains being involved in vesicle trafficking, this is already stated in line 538: «KIC4 contained an N-terminal VHS domain (IPR002014), followed by a GAT domain (IPR004152) and an Ig-like clathrin adaptor α/β/γ adaptin appendage domain (IPR008152) (Figure 5A-C, Figure S8). This is an arrangement typical for GGAs (Golgi-localised gamma ear-containing Arf-binding proteins) which are vesicle adaptors first found to function at the trans-Golgi (Dell’Angelica et al., 2000; Hirst et al., 2000).»

35) Line 477-479: Can be rephrased to "However, we found this protein as being likely dispensable for intra-erythrocytic parasite development and no colocalisation with K13 could be demonstrated, suggesting a limited role for PF3D7_1365800 in endocytosis. Or something like that. Makes it clearer.

We rephrased this sentence and it now reads (line 592): “However, we found this protein as being likely dispensable for intra-erythrocytic parasite development and no colocalisation with K13 was observed, suggesting PF3D7_1365800 is not needed for endocytosis“.

36) Line 535: Have AP-2a or AP-2b been shown to be at the K13 compartment?

AP2m is at the K13 compartment (Birnbaum et al., 2020). Adaptor complexes are heterotetramers and their subunits do not typically function on their own and this is conserved across evolutionarily distant organisms. In agreement that this is also the case in P. falciparum, Henrici et al. (Henrici et al., 2020a) showed that both, AP-2a and AP-2b, were present in an AP2µ Co-IP, indicating that the AP2 complex consist of the ‘classical’ subunits in P. falciparum. Therefore, the presence of all subunits at the K13 compartment is very likely, although this has only been experimentally confirmed for AP2µ. Of note, for Toxoplasma gondii the presence of AP-2a and AP-2b at the micropore has been experimentally confirmed (Wan et al., 2023; Koreny et al., 2023) and interaction suggested by presence in the same IP as DRPC (Heredero-Bermejo et al., 2019).

37) Line 569: reference 43 is wrong

We thanks the reviewer for pointing this out – we removed Ref 43.

38) Line 746: typo "ot" instead of or.

Changed.

39) Line 801: method for Domain Identification using AlphaFold specify that RMSDs of under 5Å over more than 60 amino acids are listed in the results. However, there is a typo in Figure 5B for KIC5 where it says "RMSD 4.0 Å over 8 aa". Please correct.

Done. In addition, we have now applied a more stringent cut-off of 4Å over more than 60 amino acids to ensure a higher reliability of our hits. This decision was based on results from our preprint (Behrens and Spielmann, 2023). Because of this the phosphatase domain in KIC12 is no longer included in this manuscript and accordingly the following sentence has been deleted. “In KIC12 we identified a potential purple acid phosphatase (PAP) domain. However, with the high RMSD of 4.9 Å, the domain might also be a divergent similar fold, such as a C2 domain, which targets proteins to membranes.”

40) Line 856: In Figure 1E, please use the same Y axis legend as in Figure 2D "relative growth at day 4 [%] compared with 3D7"

Done.

41) Figure S1: Some PCR gels check for integration are presented as 5', 3' and ori whereas other gels are presented as ori, 5' and 3'. This is confusing.

We agree that ideally the order of sample loading should be consistent and we apologise for this. The explanation for this is that these gels were run by different people at different times before we were able to better standardize the loading scheme. However, in the interest of not unnecessarily using resources for something that has a similar meaning, we would prefer not to repeat these PCRs and re-run them only for consistency reasons (as the conclusion is not affected by the different loading schemes).

42) Figure S1: Why was the expression of only MCA2 was verified by Western blot? What about the other proteins?

See response to major comment 56.

43) Line 493: Considering KIC11 was not involved in HCCU or ART resistance it might be worth mentioning in this section that it is of note that there are no domains detected that would be involved in endocytosis.

We agree that this is the case, however it is also the case for all other proteins that either are not involved in endocytosis and/or lowered susceptibility to ART. We therefore now added a summary statement addressing this in line 602: “In contrast, the K13 compartment proteins where no role in ART resistance (based on RSA) or endocytosis was detected, KIC1, KIC2, KIC6, KIC8, KIC9 and KIC11, do not contain such domains (Figure 5E).” We did not add this at the suggested part of the manuscript as at that point the domain search results are not yet introduced and doing this each time for all the individual proteins would disconnect the flow of the manuscript.

44) Line 503-506: is it wise to generate more drugs that target a pathway that is already highly susceptible to mutations? The authors should add a statement explaining how this might be avoided.

The only protein for which mutations do not have a large fitness cost is K13 (see also our preprint on fitness cost of ubp1 mutation (Behrens et al., 2023) and even with K13 the level of resistance seems to be limited by amino acid deprivation when endocytosis is reduced (Mesén-Ramírez et al., 2021). We therefore do not think that this pathway is particularly prone for mutations. Further, the number of commercial drugs targeting the "endproduct" of endocytosis (hemoglobin digestion and detoxification of heme) highlight it as the most prominent vulnerability for drug-based intervention if we go by number of commercially available drugs acting on things associated with a single process.

45) Throughout, scale bars are stated in the figure legends at the end of the legend. This is a slightly confusing format. The authors should consider stating the scale bar for each sub-legend where a fluorescence image is taken.

Done.

** Referees cross-commenting**

After reading reviewer 2 and 3's comments, I think there are significant overlaps in the key points raised in terms of questions about fusion proteins and their potential partial mis-localisation, better descripton of results and target selection. Overall I think we agree that the work has potential, but in its current form does not represent a major advance. It would be immensely helpful if the manuscript would be carefully edited for a better flow and linear description of results.

We now rearranged the manuscript for better flow but would like to highlight that the many requests for smaller experimental issues (and "better description of results") worked somewhat in the opposite way of a more linear description. We hope the rearranged version acceptably balances these two issues. The issues raised in regards to target selection and potential partial mis-localisation are addressed in our responses mainly to this reviewer. Please also see comments on systems used at the end of the rebuttal.

Reviewer #1 (Significance (Required)):

The authors set out to test whether other proteins that are in the vicinity of K13 are involved in mediating ART resistance and endocytosis. This is an interesting question. However, other than MCA2 which was already known to be involved in mediating ART resistance (and was not tested for its involvement in endocytosis), none of their candidate proteins seem to be involved in mediating both these functions. The authors show that the other proteins tested appear important for parasite growth, with KIC12 and MyoF involved in mediating endocytosis. While these findings are novel, the KS approach used by the authors casts some doubt over the findings, and would mean that these findings would have to be re-tested with a more reliable approach, such as the GlmS system or generating a conditional knockout using the DiCre system. Despite not advancing our understanding of ART resistance, or identifying further players involved in this process, this manuscripts provides two candidates that are involved in mediating endocytosis and a further candidate that appears to be important for parasite growth. Further work on these proteins will be required to understand their exact roles. As stated above, there is currently limited interest for these results (limited to researchers working on endocytosis in apicomplexan parasites and possibly the wider endocytosis field from an evolutionary perspective), however with further work, this could increase the impact and interest of this work substantially.

The authors do not describe any novel methods/approaches within this work.

In the significance statement the reviewer indicates that other systems would have been more reliable for the work here. This is addressed in our response above and in a detailed considerations on the properties of conditional inactivation systems at the end of the rebuttal. The systems used in this work were not only chosen because they permit rapid targeting of many different proteins, but because they have merits that are beneficial for our assays. In fact many of the functional assays in this manuscript are difficult or impossible to carry with the suggested conditional inactivation systems (please note that we have extensive experience with the systems considered preferable:

• DiCre (Birnbaum et al., 2017; Mesén-Ramírez et al., 2019; Mesén-Ramírez et al., 2021; Wichers et al., 2022; Kimmel et al., 2023)

• glmS (Wichers et al., 2021c; Wichers et al., 2021a; Wichers et al., 2022; Wichers-Misterek et al., 2023)).

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

In a previous publication the Spielmann lab identified the molecular mechanism of ART resistance in P. falciparum by connecting reduced levels of the protein K13 to decreased endocytosis (uptake of hemoglobin from the RBC cytosol), which results in reduced ART susceptibility. Using quantitative BioID the authors further identified proteins belonging to a K13 compartment, highlighting an unusual endocytosis mechanism.

In the present manuscript the authors follow up on this work and closely examine ten more proteins of the K13/Eps15-related "proxiome". They successfully link MCA2 to ART resistance in vitro, while the proteins MyoF and KIC12 are involved in endocytosis but do not confer in vitro ART resistance when impaired. They further characterize one candidate (KIC11) that partially colocalizes with K13 in trophozoites but to a lesser degree in schizonts. Growth assays suggest an important function for KIC11 in late stages of the intraerythrocytic developmental cycle. Five analyzed proteins however do not colocalize with the K13 compartment, while a sixth was refractory to endogenous tagging.

Using AlphaFold predictions of the KIC protein structures the author identify domains in most constituents of the K13 compartment, highlighting vesicle trafficking-related features that were not identified on primary sequence level before.

The combination of functional data together with structure predictions leads them to propose a refinement of the K13 compartment as being divided into proteins participating in endocytosis and proteins that have an unknown function.

We thank the reviewer for the assessment of the manuscript and the constructive comments.

Major comments:

1) -Table 1 is missing

We apologise for this mistake; Table 1 is now included.

2) -Lines 117-123: Given the total list of uncharacterized candidates encompasses 13 proteins, can the author gives the reason why only the top 10 and not all 13 were characterized in this study?

A similar point has been raised by Reviewer 1 in major comment #12, please see our response there for an explanation why we chose which targets.

3) -Line 174: 20% of observed MCA2 foci show no overlap with K13 and 21% only partially overlap, can the author confirm that the observed MCA2 foci in schizonts are the ones that co-localize with K13. (Addition of a schizont stage image in Fig 1C would be sufficient).

We now extended Figure 4C with images of MCA2-Y1344STOP-GFP+mCherryK13 parasites covering the schizont and merozoite stage, showing that the majority of the MCA2 foci in schizonts are also mCherry-K13 positive.

4) -The localization and observed phenotype of KIC11 is interesting but unfortunately the authors do not explore it further. Does KIC11 localize with markers of e.g. the secretory organelles (micronemes or rhoptries) in schizonts and could therefore be involved in RBC invasion?

While we intended to focus mainly on the endocytosis aspect of these proteins, we see the reviewer's point and now generated new cell lines enabling assessment of spatial association of KIC11 with markers for rhoptry (ARO), micronemes (AMA1), and inner membrane complex (IMC1c). This revealed that the KIC11-GFP signal in schizonts does not overlap with apical organelle markers and the signal does not resemble a typical apical localization. In addition, we assessed all three organelle markers after inactivating KIC11 by knock sideways which showed that KIC11 inactivation has no apparent effect on the appearance of these markers, suggesting no major alterations in schizont morphology in respect to apical markers. These results are now presented as Figure S3A and in line 304 of the results.

5) Can the author distinguish if KIC11 is involved in RBC invasion or in establishment of the ring-stage parasite?

In order to look into this, we performed egress/invasion assays, quantifying schizont and ring stage parasites in tightly synchronized parasites at two different time points (pre-egress: 38-42 hpi & post-egress: 46-50 hpi). This revealed a significant decrease in newly formed ring stage parasite per ruptured schizont in parasites with inactivated KIC11, while the egress efficacy remained unaffected. This indicated an invasion or very early ring stage development defect (new Figure 2H, Figure S3G). To further determine at which point exactly the phenotype occurs (ie during invasion or early after invasion) would require extensive experimentation that goes beyond the scope of this study (e.g. invasion assays using video microscopy with a representative number of parasites or sophisticated flow based quantification assays). We hope by excluding egress and gross changes of apical organelles as well as no indication for similar number of early rings (indicating it is invasion or a very early ring-establishment phenotype) will sufficiently narrow down the phenotype for labs interested in invasion to more definitely answer this question.

Minor comments:

1) Table S1: Please add the criterion for the order of proteins (abundance in "proxiome"?) in the table as a separate column. I would also suggest adding a new column that highlights the 10 proteins investigated in this study as I found the color-coding slightly confusing.

Done as suggested: we now include the “average log2 Ratio normalized Kelch13” values from the four DiQ-BioID experiments performed with K13 in (Birnbaum et al., 2020), as well as the suggested column to highlight the investigated proteins. Please also see reviewer 1 major point # 12 for additional information on the selection criteria and how this was added to the manuscript.

2) -154-155: There is a discrepancy between the text and Fig1C regarding the % of partial overlapping and non-overlapping foci.

We thank the reviewer for pointing this out, this was corrected.

3) -The y-axis label is missing in Fig 3E

Done.

4) -Fig 4I left graph, the superscript 2 is missing in μm2

We thank the reviewer for pointing this out, this is now changed.

5) -Did the author colocalize KIC11 in schizonts with other proteins found in the K13 compartment group of proteins not involved in endocytosis/ART resistance? This may help to further subgroup these proteins.

This is an interesting point but would actually be technically challenging to do. For this we would need to generate a KIC11endo parasite line for each of these KICs and then do co-localisation in schizonts. However, the outcome of this likely would not be very clear. The reason for this is as follows. There are foci of KIC11 that do overlap with K13 in schizonts. One can expect that these foci show KIC11 at the K13 compartment and that the other KICs would overlap with KIC11 in these K13 foci in schizonts. Hence, we would also need to see K13 to find the non-K13 compartment KIC11 foci and see if these contained the KIC of interest. This is technically challenging because it would mean we would need a third fluorescent protein which is not that trivial to do. Due to the difficulty to do this and the large amount of work involved and the already considerable amount of data in this manuscript, we believe this will be better suited for a different study.

6) -As a general comment: to make the beautiful IFAs more accessible to a broader readership, I would encourage the authors to switch the color-coding to green/magenta/blue or an equivalent color system or add grayscale images.

This was done as suggested, all fluorescence images are now provided as greyscale images and the overlays are shown in magenta/green.

Reviewer #2 (Significance (Required)):

Characterizing the molecular components involved in Plasmodium endocytosis will not only reveal interesting biology in these highly adapted parasites, but will more importantly lead to a better understanding and potentially open new avenues for intervention of ART resistance. The here presented manuscript is a carefully executed follow-up on previous work done in Dr. Spielmann's lab focusing on the K13 compartment. The authors use established assays to characterize novel components and reveal three new players in endocytosis with one mediating ART resistance in vitro. The proposition that parts of the K13 compartment have a function other than endocytosis is interesting, but will have to await more data from future studies. Taken together this manuscript adds significantly to our understanding of endocytosis in P. falciparum.

This work is of interest for cell and molecular biologists working on Apicomplexa, but especially for the Plasmodium community.

We thank the reviewer for this positive assessment.

I am a cell and molecular biologist working on Toxoplasma gondii

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary: The authors characterized 4 proteins from P. falciparum via cellular (co-)localization, endocytosis, parasite growth, and artemisinin resistance assays. These proteins have been identified as candidates for Kelch13 compartment and a possible role in endocytosis in their previously work with quantitative BioID for potential proximity to K13 and Eps15 (Birnbaum et al. 2020). In the current work, additional 6 proteins were not confirmed as being associated to the K13 compartment. This experimental work was complemented by an in-silico analysis of protein domains based on AlphaFold algorithm. For this protein structure evaluation all proteins were chosen, which were experimentally confirmed to be linked to the K13 compartment in the current publication and previous work. With the work 3 novel proteins linked to artemisinin resistance or endocytosis could be functionally described (KIC12, MCA2, and MyoF) and a number of hypotheses were generated.

We thank the reviewer for the assessment of the manuscript and the constructive comments.

Major comments:

The quality of the presented work is solid, the experimental design is adequate, and methods are presented clearly. The publication contains a lot of results both presented in text and in the figures and it is not always straight forward for the reader to follow the descriptions due to many details presented and a lack of context for some of these experiments.

We thank the reviewer for this overall positive assessment.

We now reordered the results section in an attempt to increase the flow of the manuscript. We also made changes to improve the context for the results. Given the further (very valid) requests for data on schizonts and invasion, there was an increased danger for a less linear manuscript that we hope to have acceptably managed with the re-arrange.

Specific suggestions for consideration by the authors to improve the manuscript. Abstract: 1) R 31: Mention how the 4 proteins were identified as candidates, you need to refer to previous work to clarify this

To clarify this the sentence was changed to (line 31): "Here we further defined the composition of the K13 compartment by analysing more hits from a previous BioID, showing that MyoF and MCA2 as well as Kelch13 interaction candidate (KIC) 11 and 12 are found at this site."

2) R38: "Second group of proteins" is confusing - different from the 4 mentioned above? Significance to endocytosis unclear. Please unify terminology in the manuscript, see also comment below on proxiome.

We changed the wording to clarify the group issue in the abstract as follows line 34: "Functional analyses, tests for ART susceptibility as well as comparisons of structural similarities using AlphaFold2 predictions of these and previously identified proteins showed that canonical vesicle trafficking and endocytosis domains were frequent in proteins involved in resistance or endocytosis (or both), comprising one group of K13 compartment proteins, While this strengthened the link of the K13 compartment to endocytosis, many proteins of this group showed unusual domain combinations and large parasite-specific regions, indicating a high level of taxon-specific adaptation of this process. Another group of K13 compartment proteins did not influence endocytosis or ART susceptibility and lacked detectable vesicle trafficking domains. We here identified the first protein of this group that is important for asexual blood stage development and showed that it likely is involved in invasion.”

3) Abstract can only be understood after reading the full publication

We attempted to amend this by expanding the abstract, particularly the changes highlighted in the previous two points.

Results: 4) Table 1 is missing from the submitted materials

We apologise for this mistake. Table 1 is now included.

5) Consider to shorten and stratify the result section to focus on the significant data

We rearranged the results in an attempt to streamline this section and are now starting with MyoF in the revised manuscript. However, as highlighted by the requests from reviewer 1, many details need to be available to support our conclusions. For instance the fact that GFP-tagging partially inactivated MyoF asked for further data to support our conclusion (HA-tagged version, showing that the location of the GFP-tagged version was consistent with the HA-tagged version, showing to what extent the different constructs affected growth and correlated with number of vesicles and bloating, see new figure 1M) or that KIC12 has two locations. Overall, we are therefore hesitant to remove data or description from the result part.

6) Unclear how the localization and functionalization assays might be impaired by the fusion proteins Significance of ART resistance assay is not clear, in presence of strong growth effects due to inactivation or truncation of genes/proteins

As indicated also in the example given in the previous point (this reviewer #5), the use of different cell lines (GFP-tagged live cells and small epitope tag in IFA) for targets with an indication for an effect of the tagging confirm that the location we assigned is reasonable. In the case of MyoF, the HA-tagged line, the partial inactivation due to GFP and the further inactivation in the GFP-tagged line by knock sideways show plausible increase of phenotypes (vesicle accumulation and bloated FV assays). Thereby the GFP-tagged line can be seen as a partial inactivation line that further supports our conclusions and overall this paints a consistent picture of the function of this protein in endocytosis (see new Figure 1M better illustrating this). Please note that the difference in location shown by this line compared to the HA-tagged proteins is only small (see also reviewer 1 major point 23ff). See also general discussion on tags at the end of this rebuttal.

Significance of ART resistance assay: The ‘ART resistance assay’ is done comparing +/- ART (DHA) in identical parasites (originating from the same culture and the same condition). Hence, any growth effects are cancelled out and effects in reducing ART susceptibility would - if at all - be underestimated (see more detailed response to point 28, reviewer 1 and controls in Birnbaum et al., 2020 where we tested an unrelated essential protein, unrelated chemical insult and rapalog on 3D7 and did not detect any effect on RSA survival).

MCA 7) Stratify results, order by significance of findings, it appears to be described in chronological order, improve readability/flow, eg ART resistance if mentioned in r138, but only reported in r183ff

We attempted to stratify, but then the reason for generating the partial MCA2 disruption parasite line becomes very arbitrary and would leave the reader wondering why we at all truncated the protein at two thirds of the protein. Hence, we do not see a way around this chronological reporting. However, this part is now not at the start of the experimental results section anymore, possibly making it overall a bit more palatable.

MyoF 8) R195 to 197 - consider moving to discussion as it is distracting here

This was shortened and additional information (asked for by reviewer 1, major point 22) to clarify that MyoF was previously called MyoC, was added (line 147): “The presence of MyosinF (MyoF; PF3D7_1329100 previously also MyoC), in the K13 proxiome could indicate an involvement of actin/myosin in endocytosis in malaria parasites. "

9) Term proxiome is introduced above, but not used in result section - suggest to unify language, eg r195 uses "K13 compartment DiQ-BioIDs" instead, which is not very convenient for the reader

We carefully reviewed this and made this more consistent.

10) What is the enrichment factor? Please provide for this and the following proteins, eg in Table 1

The enrichment factor is log2 enrichment over control and this is now provided in table S1 (see also detailed answer for Reviewer 1 major point 12).

11) R225 to 243 - overall significance of the growth experiments with mislocaliser is not clear, consider removing from manuscript or explain relevance more clearly

See also point 28, reviewer 1: This experiment is actually quite important. It shows that if we conditionally inactivate the GFP-tagged MyoF, the growth is further reduced, as stated in line 208. It might have been confusing that the mislocalisation is only partial, but this is equivalent to a partial knock down and hence is useful. This becomes even more relevant with the specific assays following in the next paragraph: while the tagging of MyoF already resulted in vesicles, conditional inactivation with KS generated even more vesicles, showing that the same phenotype was rapidly increased when MyoF was further inactivated by a different means and this also correlated with growth. Hence, this is actually a very consistent phenotype that despite some shortcomings of the tools available to analyse this protein (due to the partial inactivation by the GFP tag) in our eyes looks very convincing. We now added a graph showing the correlation of growth and phenotypes to illustrate this (Figure 1L).

We also tried to make this clearer by changing line 200 to: “Hence, conditional inactivation of MyoF further reduced growth despite the fact that the tag on MyoF already led to a substantial growth defect, indicating an important role for MyoF during asexual blood stage development.” And line 208 to:“ This was even more pronounced upon conditional inactivation of MyoF by KS (Figure 1H), suggesting this is due to a reduced function of MyoF.”

12) KIC11/KIC12 Enrichment factor?

The enrichment (’average log2 Ratio normalized Kelch13 from Birnbaum et al. 2020’) is 1.65 for KIC11 and 1.32 for KIC12, which is now also explicitly shown in column D of Table S1.

** Referees cross-commenting**

I would like to applaud reviewer #1 for a great, very thorough review and lots of detailed suggestions. I agree with the conclusions mentioned in the significance evaluation from reviewer #1 and #2: the work presented does not contain novel methods and the scope is rather narrow with the current results. (I am working on clinical studies with novel antimalarial agents)

Reviewer #3 (Significance (Required)):

On the one hand side, the authors have wrapped up some of the remaining protein candidates of the K13 compartment and could verify 4 of 10 proteins. The work is of interest for the scientific community working on endocytosis and malaria drug resistance mechanisms. Overall, the conclusions and findings from the previous work, Birnbaum et al. 2020, could be confirmed and extended mainly using the methods previously described. On the other hand, the authors made use of progress in protein structure predictions and identified domains linking the K13 compartment proteins to putative functions. The overlaid protein folds of the newly identified domains in figure 5 look convincing, but I can't comment on the technical details or cut-off used for this in-silico analysis.

Extended general remarks on the systems used for this work:

Mainly reviewer 1 suggest (in the general comments and the significance statement) that other systems would have been better suited to use for this work, namely glmS and diCre and also has concerns about the large tag which is seconded by a comment of reviewer 3. In light of this we here provide some extended considerations on the properties for conditional systems and tagging in regards to the goals of this work.

We would like to point out that we do have experience with the systems considered better-suited by the reviewer (one of the first authors has extensively used glmS (Wichers et al., 2021c; Wichers et al., 2021a; Wichers et al., 2022; Wichers-Misterek et al., 2023) and our lab was one of the first to adopt the diCre system in P. falciparum parasites and we regularly us it (Birnbaum et al., 2017; Mesén-Ramírez et al., 2019; Kimmel et al., 2023)). Clearly, these methods have a lot of strengths but there are a number of issues to be considered for the assays we use in this work (see the next section on conditional inactivation systems). In a nutshell, we believe diCre would give a more reliable readout of the absolute level of "essentiality" (i.e. importance for growth) but is unsuitable or at least difficult to use for the assays that reveal the function of our interest in this work. GlmS basically combines the drawbacks of diCre and knock sideways and hence for most targets is not expected to give a better readout of level of "essentiality" but is similarly difficult to use for our specific assays. The fact that both of these systems are possible to use without adding a tag to the target may be an advantage but without tag one loses some very important features that can be critical to understand the outcome with a given system (see considerations on the tag further below).

Conditional inactivation systems:

1. __ speed of inactivation:__ glms acts on mRNA and diCre on the gene level, which makes them slower than techniques acting directly on the protein such as DD or KS. With diCre, mRNA and protein is still left, even if the gene is very rapidly excised. For instance for Kelch13 it takes 3-4 days after excising the gene until protein levels have waned enough that this manifests in a reduced growth (Birnbaum et al., 2017). While in some instances diCre permits same cycle analyses if the protein has a very rapid turn-over (e.g. Rab5a, (Birnbaum et al., 2017)), control in a few hours is still difficult. For vesicle accumulation and bloated food vacuole assays, which are done over comparably short time frames and with specific stages, it is rather challenging to hit the correct time of induction to have all the cells at the correct stage with suitably (and uniformly, ie all cells) sufficiently reduced target protein levels during the assay time. Slow acting systems are also more prone to secondary effects. The more immediate the inactivation, the closer it is to the core of the affected function. With vesicle trafficking processes this is particularly relevant as all vesicle trafficking in a cell is interconnected and there are always recycling pathways that maintain the membrane and protein homeostasis of individual compartments. Particularly for endocytosis there seem to be compensatory capacities at least in other organisms (see e.g. (Chen and Schmid, 2020)). One reason why knock sideways was developed is that it permitted to avoid compensatory changes when vesicle adaptors are inactivated (Robinson et al., 2010).

The comparably short time frame for malaria parasites to go through different stages during blood stage development also is an issue relevant for inactivation speed. The advantage of speed and the danger of obscured phenotypes is highlighted by our work on VPS45 which showed that in trophozoites this protein is involved in the transport of hemoglobin to the FV whereas in late stages it also has a role in secretory processes. Both of these functions we were able to specifically assess in the same growth cycle using KS to rapidly inactivate the protein (Bisio et al., 2020) but with a slower system would have been more complicated to dissect.

Speed of effect with glmS: unless the KS does not work well, glmS is slower acting than KS (it does not target the already synthesised protein which can remain in the cell) and also often suffers from only partial inactivation, hence the benefit of using it here is unclear. The option to have an untagged protein is a plus, however it also is a minus, as assessing efficiency (particularly in live cells e.g. for bloated assays etc a fluorescent tag is the only direct option to assess inactivation of target) is critical to ensure the phenotype manifests at the stage of interest.

lethality/absolute phenotypic effects are detrimental to some assays to study the functions we are interested in for this work: no RSA can be conducted, if the gene is lost and the parasites die. Again, with diCre, one could attempt to hit the point when the parasites have lost sufficient amounts of the target protein when they are placed under ART but then the parasites need to continue growing for ~3 days, which is not possible if the cKO is lethal except for very slowly turning over proteins. However, in that latter case, the parasites likely still had full functionality of the target protein at the beginning of the RSA, when the drug pulse happens and there would be no effect. Knock sideways solves these problems by permitting knock sideways inactivation only under ART (or with a few hours pre-incubation depending on the inactivation speed) to not yet affect growth in a severe manner but inhibiting the process the protein is involved in. It may be possible to use glmS for RSAs, but the slow speed would complicate it (it would not permit control of target protein levels in a matter of a few hours to inactivate the target protein and then re-install it).

None-absolute inactivation is also a strength for some functional assays. While we really like using diCre, in the case of EXP1 it made it necessary to complement the exp1 cKO parasites with low levels of EXP1 to be able to do functional assays without killing the parasites (Mesén-Ramírez et al., 2019; Mesén-Ramírez et al., 2021). While the lethality issue does not apply to glmS (like knock sideways, it also can be tuned), it is unclear what would be gained over knock sideways. Knockdown levels with glmS vary from gene to gene and cannot be predicted, it is in most cases considerably slower than KS, it requires glucosamine which becomes toxic at higher concentrations and might introduce off target effects and tracking protein levels during the assay would equally need GFP tagging.

Integration of properties of conditional systems

Given the above discussed properties, several factors have to be considered to be able to use a system for a given assay. Stage-specific transcription is one example. For diCre a protein not expressed in e.g. rings permits to remove the gene and the protein is never made in that parasite development cycle. We exploited this for instance for two proteins only expressed from the trophozoite stage onwards (Kimmel et al., 2023). However, if lethal (absolute effect problem), this also means one can also only see the phenotype on onset of expression of the target (e.g. if in mitosis, the first nuclear division in case the protein is absolutely essential for the process). This is just one example of such issues. Expression timing, turnover of the protein and homogeneity of stage-specific loss of protein will all influence how clearly the phenotype can be determined. All this will decide the exact time of loss/inactivation of the target protein to levels generating a phenotype and ideally therefore can be monitored during an assay (see considerations on tagging).

For these reasons vesicle accumulation or bloated food vacuole assays are difficult with slow systems as ideally the target should rapidly be inactivated at the trophozoite stage and the result monitored before the cells have moved to the schizont stage. For this a well responding knock sideways is ideal as the protein can be rapidly taken away (sometimes within seconds) to visualise the immediate, direct effect in the cell.

As shown for KIC11, there is also no disadvantage of using KS for proteins with other assays or proteins that result in different phenotypes. It permits stage-specific same cycle inactivation without having to worry about the turnover of mRNA and protein (Fig. 2F,G). Thus, besides the advantages of knock sideways for endocytosis related assays and RSAs, we also see no disadvantage of using knock sideways for the functional study of KIC11 which has a role other than endocytosis. KS also permits to specifically target the K13 pool of KIC12, something impossible or very difficult to do with other systems. Hence, we are of the opinion that the system for inactivation was adequate for most of the proteins analysed in this manuscript.

Large tag: we agree that GFP-tagging can be a disadvantage but in our opinion its benefits often outweigh the drawbacks because it permits easy and immediate (on individual cell level, if need be) monitoring of the presence/location of the target protein (e.g. after KS, but given the discrepancy of the timing between gene excision and protein loss, it might be even more important for techniques such as diCre). No fixing/permeabilisation (prone to artifacts, prevents immediate view of cells) to detect a target with specific antibodies or via a small tag is needed with GFP. Similarly, the use of Western blots to do this is time consuming and impractical if monitoring of left-over protein in the course of an assay such as a bloated food vacuole assay is needed.

In many cases, adding GFP has no negative effect. In addition, if the bulky folded structure of GFP is tolerated, it usually also tolerates the 2 to 4 12kDa FKBP domains in our standard tag. We also typically add a linker. This approach has worked for a large number of different proteins, including many essential ones for which we would not otherwise have obtained the integration cell lines (Birnbaum et al., 2017; Jonscher et al., 2019; Hoeijmakers et al., 2019; Birnbaum et al., 2020; Kimmel et al., 2023; Sabitzki et al., 2023). Hence, whenever a cell line is obtained with it, this tag in most cases is not a disadvantage. Admittedly an exception in this is MyoF and to some extent maybe MCA2 (we would like to stress that in the case of MCA2 the reason for not being able to obtain the full length tagged cell line is unclear: the protein can be severely truncated to less than 3% of its amino acid sequence and a GFP-tag is tolerated on the version with 2/3s of the protein left, which gives no good reason why the full length was not obtained; a potential reason could be a dominant negative effect). However, we obtained the full length with a small tag detected by IFA for both, MyoF and MCA2 and the location of these agreed well with the GFP tagged versions, indicating that the GFP-tagged versions are useful to show the location of these proteins in live cells.

There are also tricks to attempt monitoring the effect of e.g. diCre without tagging the target. For instance, if a fluorescent protein is connected to excision without actually being fused to the target (ie excision of the gene leads to its expression of e.g. GFP), which would avoid adding a tag to the target itself. However, the problem with this is that expression of GFP does only show excision, but mRNA producing the target protein and left over target protein may still be there in the cell. All in all, the GFP-tag on the target, while with some drawbacks, is still our preferred method to control to monitor the target protein in the cell (in principle permitting quantification of ablation efficiency on the individual cell level).

Conclusion on these considerations for this manuscript

Based on these considerations we do not see the immediate benefit of changing the system for the conclusions drawn from this study and are unsure if they are indeed better suited for this work as suggested. While a more exact readout of "essentiality" might be possible with the diCre system we are of the opinion this is less important than learning the function of a protein which - as outlined above - we believe to be considerably more difficult with diCre and even more so with glmS considering our target functions. The same applies to target specific cellular pools of a protein as done here for KIC12. Clearly MyoF is one example where the employed systems shows limitations, but with the new Figure part showing consistency in phenotype with degree of inactivation (importantly with two different forms of inactivation) and the clarification that the location of the GFP-tagged and HA-tagged versions are actually quite similar in location, we do not think employing an extra system is warranted for the conclusions of this work. Admittedly, the apparent lack of need in ring stags might give an opening to attack MyoF using diCre (by excision before its major expression peak), but depending on lethality this might preclude extended analyses (possibly vesicle assays, for sure not RSAs).

In the end the question is, if our approach provides the function of target analysed in this work and based on the data in our manuscript and the arguments in the rebuttal, we are reasonably confident that this is the case. It is not very likely the other mentioned techniques would result in a different conclusion on the function of the here studied proteins. In fact, we expect other commonly used techniques to be less suitable for the key assays in this work.

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Author Response

Reviewer #3 (Public Review):

This manuscript proposes to tackle a very interesting and methodologically challenging topic: the mechanistic underpinnings of neural specialization in the infant brain. The authors presented 4- to 7-month-old infants with social and non-social stimuli while their neural, hemodynamic, and metabolic activity was monitored, and they report a complex pattern of relationships between neural and metabolic or hemodynamic responses during social processing on the one hand, and during non-social processing on the other hand.

The approach described in this manuscript is very interesting and the combined use of EEG and bNIRS data appears very promising. However, there is some confusion between the initial aims of the study, and the analyses performed, which jeopardizes the clarity and the impact of this manuscript. Besides, the predictions of the authors are often underspecified which complexifies the interpretation of the results.

Based on its abstract, the goal of this work is to "combine simultaneous measures of coordinated neural activity metabolic rate and oxygenated blood supply to measure emerging specialization in the infant brain". The introduction nicely elaborates on the "interactive specialization theory" and the potential role of the interplay between brain energy consumption and neural activity in the emergence of functionally specialized brain regions during development. The authors present a novel multimodal approach, with potentially important implications for the study of brain specialization as a function of experience or maturation. Yet the experimental procedure presented in this manuscript only assesses specialized brain activity in response to social processing in 4- to 7-month-old infants, using multimodal neuroimaging.

Indeed, the authors presented 4- to 7-month-old infants with social and non-social stimuli while their neural, hemodynamic, and metabolic activity was monitored. The authors report significant differences between the two conditions in terms of neural activity in the delta, alpha, beta, and gamma bands; as well as in the pattern of hemodynamic to metabolic coupling. Using a GLM approach, the authors report on fNIRS channels and EEG sensors showing significant relationships between the evoked neural activity in the beta and gamma frequency bands, and each of the bNIRS signals (HbO, HbR & CCO), in the social and in the non-social conditions. The authors identify a particular fNIRS channel overlaying posterior STS, showing a positive relationship between Pz EEG beta activity and HbO, as well as CCO, together with a negative relationship between that same neural activity and HbR, in the social condition. This pattern of activity was not observed in the non-social condition.

Overall, these results indicate differential neural responses to social and non-social stimuli, coupled metabolic and hemodynamic activity in response to social as well as nonsocial stimuli.

These results additionally indicate coordinated metabolic, hemodynamic, and neural responses in brain regions selective for social processing, but it does not allow us to conclude that this coordinated activity is actually related to the functional specialization process (e.g. last sentence of the abstract).

We would like to thank the reviewer for their detailed comments. Based on their suggestions, we have made several changes to the manuscript. This study was the first to combine EEG and broadband NIRS and therefore served as a proof of principle study. At the onset of this work, there were many elements to develop such as the technical aspect of simultaneous bNIRS – EEG measurements as well as the methodology to combine the signals from both techniques with such different time resolutions. Therefore, we focused on one age group of infants rather than performing a study involving multiple age groups. The 4-to-7-month-old age group has been studied extensively using fNIRS, particularly to look at social brain development using similar stimuli as those used in the present study. Previous studies have demonstrated that social selectivity can be detected at 4 – 8 months of age (Grossmann et al., 2010; Lloyd-Fox et al., 2012, 2013, 2017). As this was a proof of principle study, we wanted to ensure that we were able to replicate results from previous studies with this new methodology. We therefore used one age group of 4-to-7-months. This has also been added to the introduction of the manuscript to provide clearer reasoning for using this age group.

The reviewer is correct that the current study does not provide direct evidence of developmental change in functional specialisation or the hypothesised interactive process through which functional specialisation may occur. Rather, we are measuring the status of functional specialisation (the idea that different areas in the brain are specialised for different functions) at the age we study, by testing whether the signals we observe are selective to social but not non-social stimuli. We have reframed the abstract and introduction of the manuscript to ensure this is clear, and we additionally now focus more on the methodology developed to answer such questions. Future studies can leverage our methodology to study different age groups to establish how the relationships between neural and vascular/metabolic signals changes over developmental time, which may provide greater insight into the specialisation process.

Grossmann, T., Oberecker, R., Koch, S. P., & Friederici, A. D. (2010). The Developmental Origins of Voice Processing in the Human Brain. Neuron, 65(6), 852–858. https://doi.org/https://doi.org/10.1016/j.neuron.2010.03.001

Lloyd-Fox, S., Begus, K., Halliday, D., Pirazzoli, L., Blasi, A., Papademetriou, M., Darboe, M. K., Prentice, A. M., Johnson, M. H., Moore, S. E., & Elwell, C. E. (2017). Cortical specialisation to social stimuli from the first days to the second year of life: A rural Gambian cohort. Developmental Cognitive Neuroscience, 25, 92–104. https://doi.org/10.1016/j.dcn.2016.11.005

Lloyd-Fox, S., Blasi, A., Elwell, C. E., Charman, T., Murphy, D., & Johnson, M. H. (2013). Reduced neural sensitivity to social stimuli in infants at risk for autism. Proceedings of the Royal Society B: Biological Sciences, 280(1758), 20123026. https://doi.org/10.1098/rspb.2012.3026

Lloyd-Fox, S., Blasi, A., Mercure, E., Elwell, C. E., & Johnson, M. H. (2012). The emergence of cerebral specialization for the human voice over the first months of life. Social Neuroscience, 7(3), 317–330. https://doi.org/10.1080/17470919.2011.614696

Another weakness of this manuscript relates to the unclear or underspecified motivations behind some of the performed analyses. For example, the authors contrast brain responses to social vs. baseline, non-social vs. baseline, and social vs. non-social. For clarity in the manuscript, the authors should specify the motivation behind each of these contrasts and their predictions.

We thank the reviewer for their suggestion. We have added the predictions for each of the analyses in the introduction section, lines 436 – 527. We have removed the “social minus non-social” comparison for the EEG topographical maps from Figure 2 as there was no value added by including this comparison.

Another example is in the analysis of the hemodynamic and metabolic coupling analysis, here the authors analyze only the social vs. baseline and non-social vs. baseline contrast, and they do not analyze the social vs non-social contrast. It would be useful for the reader to understand why only these two contrasts are performed and not the social vs. non-social, and what are the predictions of the authors.

We have now added this into the manuscript and the results can be seen in Figure 3c. We have clarified our predictions both at the end of the introduction (lines 436 - 527) and at the beginning of the discussion (lines 685 – 755).

The following has been added to the introduction:

For EEG, we expected an increase in neural activity in response to the social condition and a decrease in neural activity in response to the non-social condition. Based on previous work, this was expected to be strongest in the theta frequency band [3]. Moreover, for the combined bNIRS-EEG analyses, we hypothesised differentiated haemodynamic/metabolic coupling with neural activity for the social and non-social stimulus conditions. We performed two types of statistical tests: a) individual comparisons of the social and non-social conditions and b) comparison of the social condition versus the non-social condition. The individual condition tests were performed to show the scale and spatial location/sensitivity of the coupling between haemodynamics/metabolism and neural activity for each condition. Meanwhile, the social versus non-social comparison was performed to show where there was a significant difference in the coupling between the two conditions. With comparison (a) we aimed to identify regions involved in the processing of social and non-social stimuli by identifying the regions where the coupling was significant. With comparison (b) we aimed to identify regions where coupling was significantly different between conditions. We predicted that for the individual comparison of the social condition, we would observe positive associations between bNIRS and EEG measures, i.e. coordinated increases in haemodynamics/metabolism and neural oscillatory activity in the beta and gamma frequency bands (based on previous combined EEG – fMRI studies [16], [18]–[21], [23], [30]) which would be localised to core social brain regions. We hypothesised that for the non-social condition, over the same brain regions, positive associations would be observed between bNIRS and EEG measures, but they would be coordinated decreases in haemodynamics/metabolism and oscillatory activity. We also expected coordinated increases in haemodynamics/metabolism and oscillatory activity localised to the parietal brain region. These predictions are based on our previous work [29] where we demonstrated that stronger coupling between haemodynamics and metabolism was observed in the temporo-parietal regions for the social condition and in parietal region for the non-social condition which is known to play an important role in object processing [31], [32]. For the social versus the non-social contrast, we predicted that haemodynamic activity and metabolism would be coupled with neuronal oscillatory activity more strongly for the social stimuli in comparison to the non-social stimuli, with significant differences being observed in the temporo-parietal regions.

The following has been added to the discussion:

As a proof of principle, we examined the relationship between these measures to identify regional selectivity to social versus non-social stimuli. To first demonstrate the scale and spatial sensitivity of the coupling between haemodynamic/metabolic activity and neuronal oscillatory activity, comparisons were performed individually for the social and non-social conditions. For this, we predicted coordinated increases in haemodynamics/metabolism and neural activity in the beta and gamma frequency band. We predicted that for the social condition this would be localised to the core social brain regions (temporo-parietal region) while for the non-social condition, we expected the coupling to be localised to parietal regions, known to be involved in object processing [31], [32]. We additionally expected coordinated decreases in haemodynamic/metabolic activity and neural activity over the temporo-parietal region for the non-social condition, in accordance with our previous work [29]. Next, to demonstrate differential coupling for social and non-social stimuli, we performed a comparison of the social condition versus the non-social condition. For this, we hypothesised that in the beta and gamma frequency bands, there would be stronger coupling between haemodynamics/metabolism and neural activity for the social condition over the temporo-parietal region.

Finally, the core result of this work derives from the final GLM analysis which relates EEG activity to hemodynamic or metabolic responses. This analysis implies the inspection of interactions between 3 neuroimaging modalities, with 4 EEG measures, 2 hemodynamic measures, and 1 metabolic measure, which represents a very rich and relatively complex analytic approach. Unfortunately, the predictions are not clearly specified, which makes results interpretation difficult.

We appreciate that the methods are complex, and the hypotheses should be stated more clearly. The hypotheses have now been explicitly stated both at the end of the introduction (lines 436 - 527) and at the beginning of the discussion (lines 685 – 755).

Based on the results (L160-162) and discussion (L233-235) sections, it appears that the authors aim at identifying brain regions showing a precise pattern of activity, with a positive relationship between EEG activity and HbO/CCO responses together with a concurrent negative relationship between EEG and HbR responses in response to social events, but not in response to non-social events. Importantly, the social vs. non-social contrast seems crucial to assess the selectivity of the response. Yet, the authors analyze the 3 chromophores separately, and they do not contrast the two conditions (figure 3). As a result, the authors are limited to reporting a descriptive pattern of relationships between EEG and HbO/HbR/CCO activations for the social condition. And another one for the non-social condition. Overall, the authors conclude that channel 14, overlaying the right TPJ, shows the expected pattern of activity, specifically in response to social stimuli. Yet, this statement is only supported by visual inspection/comparison of the results between the social vs baseline and non-social vs baseline conditions. The authors do not assess analytically the differential patterns of activations between the two conditions. Instead, a GLM including all 3 chromophores and contrasting the two experimental conditions would allow us to directly test the predicted pattern of activity, and the selectivity of the activity for social stimuli.

As per the reviewer’s comment, we have now included the comparison of the social and non-social conditions, shown in Figure 3c. The results from this comparison showed that haemodynamics and metabolic activity at channels 11 and 14 (located spatially close to one another) had a significantly greater association to EEG electrode “Pz” for the social condition, in comparison to the non-social condition for the beta and gamma bands. These results support/indicate the selectivity of the response to the social condition, analytically.

We have kept the results showing the individual comparison of the social and non-social conditions. The individual condition tests were performed to show the scale and spatial location/sensitivity of the coupling between haemodynamics/metabolism and neural activity for each condition. Meanwhile, the social versus non-social comparison was performed to show where there was a significant difference in the coupling between the two conditions. With comparison (a) we aimed to identify regions involved in the processing of social and non-social stimuli by identifying the regions where the coupling was significant. With comparison (b) we aimed to identify regions where coupling was significantly different between conditions. The following has been added on line 533 – 541 to explain the reasoning behind the comparisons performed.

We performed two types of statistical tests: a) individual comparisons of the social and non-social conditions and b) comparison of the social condition versus the non-social condition. The individual condition tests were performed to show the scale and spatial location/sensitivity of the coupling between haemodynamics/metabolism and neural activity for each condition. Meanwhile, the social versus non-social comparison was performed to show where there was a significant difference in the coupling between the two conditions. With comparison (a) we aimed to identify regions involved in the processing of social and non-social stimuli by identifying the regions where the coupling was significant. With comparison (b) we aimed to identify regions where coupling was significantly different between conditions.

As our interest was in looking at the selectivity of the response and not comparing the chromophores, we did not perform a comparison between chromophores.

Author Response

The following is the authors’ response to the current reviews.

We will make some minor changes to address the issues in the revised manuscript during preparation of the Version of Record.

1) Acknowledge the previous discovery that COUPTFII expression is confined to the ventral hippocampus in early human fetal forebrain (doi: 10.1093/cercor/bhx185).

We agree. We will incorporate the previous discovery that COUPTFII expression is confined to the ventral hippocampus in early human fetal forebrain (doi: 10.1093/cercor/bhx185) in the discussion section of "COUP-TFII governs the distinct characteristics of the ventral hippocampus".

2) Give some consideration to this observation from my original review "Abnormalities in the trisynaptic circuit. No studies of actual synapses, either physiological or morphological, were carried out. I wonder to what extent these immunohistochemical studies just further reflect the abnormalities in hippocampal morphology presented earlier in the manuscript without specifically telling us about synaptic circuits? Although the immunohistochemical preparations are beautiful, they are inadequate on their own in telling us much about what sort of synaptic circuitry exists in the transgenic animals".

Our data in Figure 4 show clearly that at the neural circuit level, compared with the corresponding control, the trisynaptic circuit is abnormal in all three models; therefore, in the discussion section of "COUP-TF genes are imperative for the formation of the trisynaptic circuit", we will add the following sentence, "We would like to investigate what sort of synaptic circuitry is compromised either physiologically or morphologically in the trisynaptic circuit of individual animal model in detail in the future studies.

In addition, we will correct a reference related to the COUP-TFII gene and congenital heart defects.

The reference of "High, F. A., Bhayani, P., Wilson, J. M., Bult, C. J., Donahoe, P. K., & Longoni, M. (2016). De novo frameshift mutation in COUP-TFII (NR2F2) in human congenital diaphragmatic hernia. Am J Med Genet A, 170(9), 2457-2461. doi:10.1002/ajmg.a.37830" was replaced with "Al Turki, S., Manickaraj, A. K., Mercer, C. L., Gerety, S. S., Hitz, M. P., Lindsay, S., . . . Hurles, M. E. (2014). Rare variants in NR2F2 cause congenital heart defects in humans. Am J Hum Genet, 94(4), 574-585. doi:10.1016/j.ajhg.2014.03.007".

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The following is the authors’ response to the original reviews.

Reviewer #1(Recommendations For The Authors):

1) Better presentation of the western blot results

We agree with the reviewer. Based on the suggestion, new information about the western blot results has been added in the revised Figure 1Ap. We added a dash to each western blot image to indicate the target band of COUP-TFI (46 KDa), COUP-TFII (45 KDa), and GAPDH (37 KDa), respectively. There were two bands in the blot of COUP-TFII, with the upper band corresponding to mouse IgG at 50 KDa, and the bottom band corresponding to COUP-TFII protein at 45 KDa. Therefore, only the lower bands of COUP-TFII are used for the quantitative analysis. The expression of COUP-TFII in the ventral hippocampus is clearly higher than that in the dorsal hippocampus.

2) Full presentation of the Immunohistochemistry and qPCR results for at E11.5 and E14.5 in double knockdown mice.

Thanks for the suggestion. Based on the suggestion, we added immunofluorescent data in the double knockout mice at E11.5 in the Figure 5Ba-h. Meanwhile, given that it takes time to prepare animal samples at E14.5 for RT-qPCR assays, we performed immunofluorescent assays at both E13.5 and E14.5 to make sure that the changes of Lhx5 and Lhx2 expression in the hippocampal regions between the control and mutant mice were consistent. As shown in the new Figure 5B, consistent with the downregulated expression of Lhx5 transcripts in the double mutant, the expression of the Lhx5 protein was reduced in the CH in the double mutants at E11.5; moreover, the numbers of Lhx5-positive Cajal-Retzius cells decreased in the double mutant embryos at E11.5, E13.5 and E14.5 (Figure 5Ba-d, a’-d’, a’’-d’’, i-l, i’-l’, q-t, q’-t’). Consistent with RT-qPCR data, the expression of Lhx2 was comparable between the control and double-mutant mice at E11.5 (Figure 5Be-h, e’-h’). Interestingly, the expression of the Lhx2 protein was increased in the hippocampal primordium in the COUP-TF double-mutant mice at E13.5 and E14.5 (Figure 5Bm-p, m’-p’, u-x, u’-x’). Please find the altered descriptions in the Page 15, lines 347-351, 353-358 and Page 21, lines 500-503 in the revised manuscript.

3) Minor corrections. Lines 159-162, prospected not quite the right word. I would suggest "an ectopic CA-like region was observed medially in the temporal hippocampus in the COUP1TFII mutant, where the prospective posterior part of the medial amygdaloid nucleus was situated, (MeP), indicated by the star (Figure 1Ba-f). The presence of the ectopic CA-like region in the ventral but not dorsal hippocampus of the mutant was further confirmed by the presence of the prospective MeP and amygdalohippocampal area (AHi) in sagittal sections, as indicated by the star. See also line 251. Line437/438 I would suggest "... most important breakthroughs in understanding the role of the hippocampus in memory."

Thanks for the suggestion. We made the changes based on the suggestion. Please find the amendments in Page 8, lines 178-181; Page 12, lines 270, 276; Page 14, line 318; Page 19, lines 451; Page 20, lines 461-462 in the revised manuscript.

Reviewer #2 (Recommendations For The Authors):

1) It is also important to point out that the immunofluorescence data in Figure 5B is contrary to what is known for Lhx5 (it's not expressed in the neocortical and hippocampal vz) and Lhx2 (it's not expressed in the choroid plexus). Authors should explain how their conclusions could align more clearly, and consider the possibility that their results are due to a possible artifact of image setting issues or worse, antibody specificity issues.

Very good point. Based on the comments and suggestions, we first tested another Lhx5 antibody, R&D, Cat # AF6290, in the immunofluorescence assays. Indeed, there was something wrong with the previous Lhx5 antibody, Millipore, Cat # AB5762. With the new Lhx5 antibody, consistent with the reported in situ data, the expression of Lhx5 was detected specifically in the CH at E11.5, and in the Cajal-Retzius cells in the marginal zone of the telencephalon. The same Lhx2 antibody, Santa Cruz, Cat # sc-19344, which has been used successfully in one of our previous studies (Tang et al., Development, 2012) (PMID: 22492355), was used in the present study. We believe that the observations at the MP and DP of the samples are really associated with the expression of Lhx2 protein. We performed new immunofluorescence assays with the new Lhx5 antibody and confirmed with the Lhx2 antibody. As shown in new Figure 5B, consistent with the downregulated expression of Lhx5 transcripts in the double mutant, the expression of the Lhx5 protein was reduced in the CH in the double mutants at E11.5; moreover, the numbers of Lhx5-positive Cajal-Retzius cells decreased in the double mutant embryos at E11.5, E13.5 and E14.5 (Figure 5Ba-d, a’-d’, a’’-d’’, i-l, i’-l’, q-t, q’-t’). Consistent with RT-qPCR data, the expression of Lhx2 was comparable between the control and double-mutant mice at E11.5 (Figure 5Be-h, e’-h’). Interestingly, the expression of the Lhx2 protein was increased in the hippocampal primordium in the COUP-TF double-mutant mice at E13.5 and E14.5 (Figure 5Bm-p, m’-p’, u-x, u’-x’). Please find the changed descriptions in Page 15, lines 347-351, 353-358 and Page 21, lines 500-503 in the revised manuscript.

The reference:

Tang, K., Rubenstein, J. L., Tsai, S. Y., & Tsai, M. J. (2012). COUP-TFII controls amygdala patterning by regulating neuropilin expression. Development, 139(9), 1630-1639. doi:10.1242/dev.075564

2) The expression domain of RxCre remains poorly explained, and the early expression of COUPTFI and II (E10.5-E12.5) could be considered major weaknesses of the paper.

Thanks for the suggestion. The generation of RXCre was reported by Swindell et al., Genesis, 2006 (PMID: 16850473). Given that the activation of the LacZ expression serves as an indicator for the deletion of the COUP-TFII gene (Tang et al., Development, 2012) (PMID: 22492355), we performed the immunofluorescent data with antibodies against COUP-TFII and LacZ on the sagittal sections of RXCre/+; COUP-TFIIF/+ heterozygous mutant and RXCre/+; COUP-TFIIF/F homozygous mice at E11.5. As shown in the new Figure 1—figure supplement 1Da-f, COUP-TFII was readily detected at the hippocampal primordium of the heterozygous mutant embryo at E11.5 (Figure 1—figure supplement 1Da, c, g); in contrast, the expression of COUP-TFII significantly decreased in the homozygous mutant (Figure 1—figure supplement 1Dd, f, j). In addition, compared with the heterozygous mutant embryo, the LacZ signals increased distinctly in the hippocampal primordium of the homozygous mutant embryo at E11.5 (Figure 1—figure supplement 1Db-c, e-f, h, k), suggesting that RXCre recombinase can efficiently excise the COUP-TFII gene in the hippocampal primordium as early as E11.5. Please find the corresponding changes in Page 7, lines 149-159 and Page 8, lines 160-164 in the revised manuscript.

Meanwhile, we also added the early expression of COUP-TFI and -TFII at E10.5 and E11.5 in new Figure 1—figure supplement 1Aa-d. At embryonic days 10.5 (E10.5), COUP-TFI was detected in the dorsal pallium (DP) laterally and COUP-TFII was expressed in the MP and CH medially (Figure 1—figure supplement 1Aa, b). At E11.5, the expression of COUP-TFII remained in the hippocampal primordium, including MP and CH (Figure 1—figure supplement 1Ac, d). Please find the corresponding changes in Page 6, lines 129-132 and Page 9, lines 202-203 in the revised manuscript.

The references:

Swindell, E. C., Bailey, T. J., Loosli, F., Liu, C., Amaya-Manzanares, F., Mahon, K. A., . . . Jamrich, M. (2006). Rx-Cre, a tool for inactivation of gene expression in the developing retina. Genesis, 44(8), 361-363. doi:10.1002/dvg.20225

Tang, K., Rubenstein, J. L., Tsai, S. Y., & Tsai, M. J. (2012). COUP-TFII controls amygdala patterning by regulating neuropilin expression. Development, 139(9), 1630-1639. doi:10.1242/dev.075564

Reviewer #3 (Recommendations For The Authors):

1) Regarding the RxCre line, I was also confused about its spatiotemporal expression, as this line is not a commonly used Cre line and no detailed description is provided in the manuscript. Searching this line shows a previous paper by the authors (PMID: 22492355) in which they tested the RxCre recombinase activity. At E12.5, RxCre induced high LacZ expression in the ventral telencephalon but much less in the dorsal telencephalon. But they did not check later stage. Therefore, it's hard to explain the defective dorsal hippocampus in RxCre, CFI CKO. They should check later stage.

The generation of RXCre was reported by Swindell et al., Genesis, 2006 (PMID: 16850473), which reveals high Cre recombinase activity of RXCre in the eye and ventral telencephalon. Given that the activation of the LacZ expression serves as an indicator for the deletion of COUP-TFII gene, Tang et al., Development, 2012 (PMID: 22492355), we performed the immunofluorescent data with antibodies against COUP-TFII and LacZ on the sagittal sections of RXCre/+; COUP-TFIIF/+ heterozygous mutant and RXCre/+; COUP-TFIIF/F homozygous mice at E11.5. As shown in new Figure 1—figure supplement 1D, compared with the heterozygous mutant embryo, the expression of COUP-TFII was significantly decreased in the homozygous mutant; in addition, the LacZ signals evidently increased in the hippocampal primordium of the homozygous mutant embryo at E11.5, suggesting that RXCre recombinase can efficiently excise the target gene in the hippocampal primordium as early as E11.5. The expression of COUP-TFI is barely detectable in the early developing hippocampal primordium including MP at E10.5, E11.5 and E12.5. The expression of COUP-TFI is high in the MP of the control (Figure 1Cj, l); in contrast, the COUP-TFI expression is barely detectable in the MP of the homozygous double mutant at E14.5, indicating that RXCre can efficiently delete the COUP-TFI gene in the hippocampal primordium at E14.5. The loss of the COUP-TFI gene in the MP as early as E14.5 by RXCre initiates the defective dorsal hippocampus in RXCre/+; COUP-TFIF/F knockout mice.

2) Authors should check and review extensively for improvements to the use of English.

We carefully checked and made changes throughout the manuscript accordingly. For example, “imperative” was used 6 times in the previous manuscript, lines 20, 255, 486, 499, 522, 553; “imperative” was used only once in Page 22, line 522 in the revised manuscript.

3) Please correct the manuscript; 1-month-old mice are not adult mice.

Thanks for the suggestion. Based on the suggestion, we have corrected related words and sentences in the manuscript. Please find the amendments in the revised manuscript (Page 7, line 146; Page 9, lines 203-204; Page 10, line 213; Page 13, lines 299-300; Page 17, line 406; Page 20, line 476).

4) Additional ref should be added at line 93 on page 5.

Based on the suggestion, we added some new references (Bertacchi et al., EMBO J, 2020) (PMID: 32572460); (Del Pino et al., Cereb Cortex, 2020) (PMID: 32484994); (J. Feng et al., Sci Adv, 2021) (PMID: 34215582) at line 96 on page 5.

The references:

Bertacchi, M., Romano, A. L., Loubat, A., Tran Mau-Them, F., Willems, M., Faivre, L., . . . Studer, M. (2020). NR2F1 regulates regional progenitor dynamics in the mouse neocortex and cortical gyrification in BBSOAS patients. Embo j, 39(13), e104163. doi:10.15252/embj.2019104163

Del Pino, I., Tocco, C., Magrinelli, E., Marcantoni, A., Ferraguto, C., Tomagra, G., . . . Studer, M. (2020). COUP-TFI/Nr2f1 Orchestrates Intrinsic Neuronal Activity during Development of the Somatosensory Cortex. Cereb Cortex, 30(11), 5667-5685. doi:10.1093/cercor/bhaa137

Feng, J., Hsu, W. H., Patterson, D., Tseng, C. S., Hsing, H. W., Zhuang, Z. H., . . . Chou, S. J. (2021). COUP-TFI specifies the medial entorhinal cortex identity and induces differential cell adhesion to determine the integrity of its boundary with neocortex. Sci Adv, 7(27). doi:10.1126/sciadv.abf6808

5) I am confused why the authors analyzed 1-month-old mice in some instances but 3-month-old mice in others.

The RXCre/+; COUP-TFIF/F; COUP-TFIIF/F double mutant mice barely survived beyond postnatal 3 weeks. To make our findings consistent and comparable, we mainly prepared figures with observations on about 1-month-old mice in the RXCre related single or/and double gene mutant mouse models. In the study of the Emx1Cre related COUP-TFI mouse model, due to behavioral tests such as the Morris water maze test, experiments were performed with the adult experimental animal about postnatal 3 months. In order to be consistent with the stage of the mice for the behavioral tests, we only displayed morphological data with observations on the control and Emx1Cre/+; COUP-TFIF/F mutant mice at about postnatal 3-month.

Author Response:

We would like to thank the eLife reviewers for the considerable time and effort they have invested to review these manuscripts. We have also benefited from a previous round of review of the manuscript describing the proposed burial features, which underwent two rounds of revisions in a high-impact journal over a period of approximately 8 months during 2022 and early 2023. Both sets of reviews have reflected mixed responses to the evidence we have presented, with one reviewer recommending acceptance with minor editorial revisions, two recommending acceptance with minor revisions and the fourth recommending rejection based upon similar arguments to those reflected by some of the reviewers in this current round of reviews in eLife. Ultimately the managing editor of this first journal took the decision that the review process could not be completed in a timely manner and rejected the manuscript although the submission here reflected our consideration of these reviewers suggestions.

We have chosen in this initial response to the eLife reviews to include some references to the previous anonymous reviews in order to illustrate differences of opinion and differences in revision suggestions within the review process. Our goal is to offer maximal insight into our decision-making process and to acknowledge the considerable time and effort put into the assessment of these manuscripts by reviewers (for eLife and in the case of the earlier review process). We hope that this approach will assist the readers, and reviewers, of our manuscripts in understanding why we are proceeding with certain decisions during the revision process.

This is a new process for us and the reviewers, and one way in which it significantly differs from more traditional review is that both the reviews and our reply will be public well in advance of our revisions to the manuscript. Indeed, considering the scope of the reviews, some of those revisions may take considerable time, although many can be accomplished fairly easily. Thus, we are not in a position to say that we have solved every issue raised by the reviewers. Instead, we will examine what appear to be the key critical issues raised regarding the data and the analyses and how we propose to address these as we revise the papers. We will also address several philosophical and ethical issues raised by the reviews and our proposal for dealing with these. More specific editorial and citational recommendations will be dealt with on a case-by-case basis, and we do not address these point-by-point in this reply. Please note, this response to the reviewers is not the revision of the manuscript and is only the initial opinion of the corresponding authors with some guidance from the larger group of authors of all three papers. Our final submitted revision will reflect the input of all authors included on those submissions.

We took the decision to submit three separate papers consciously. The two different categories of evidence, burials and engravings, involve different kinds of analysis and different (although overlapping) teams of researchers, and we recognized that each deserved their own presentation and assessment. Meanwhile, together they inform the context of H. naledi in a way that requires some synthetic discussion, in which both kinds of evidence are relevant, leading to a third paper. But the mutual relevance of these different kinds of evidence and their review by a common set of reviewers naturally raises cross-cutting issues, and the reviewers have cross-referenced the three articles. This has sometimes led to suggestions about one manuscript based on the contents of another. Considering the situation, we accepted the recommendation that it would be clearer to consider all three articles in a single reply. Thus, while each of the three papers will proceed separately during the revision process, it will be necessary to highlight across all three papers occasionally in our responses.

Scientific Issues:

In reading the reviews, we feel there are 9 critical points/assertions raised by one or more of the reviewers that present a problem for, or challenge to, our hypothesis that the observed evidence (bone accumulations and engravings) described in the Dinaledi subsystem are of intentional naledigenic origin. These are:

1. The evidence presented does not demonstrate a clear interruption of the floor sediments, thus failing to demonstrate excavated holes.

2. The sediments infilling the holes where the skeletal remains are found have not been demonstrated to originate from the disruption of the floor sediments and thus could be part of a natural geological process (e.g. water movement, slumping) or carnivore accumulations.

3. Previous geological interpretations by our research group have given alternative geological explanations for formation of the bony accumulations that contradict the present evidence presented here and result in alternative origins hypotheses.

4. Burial cannot be effectively assessed without complete excavation of the features and site.

5. The skeletal remains as presented do not conform clearly to typical body arrangement/positions associated with human (Homo sapiens) burials.

6. There is no evidence of grave goods or lithic scatters that are typically associated with human burials.

7. Humans may have been involved with the creation of either the Homo naledi bone accumulations, the engravings, or both.

8. Without a date of the engravings, the null hypothesis should be the engravings were created by Homo sapiens.

9. The null hypothesis for explanation of the skeletal remains in this situation should be “natural accumulation”.

Our analysis of the Dinaledi Feature 1 leads us to accept that the laminated orange-red mudstone (LORM) sedimentary layer is interrupted, indicating a non-natural intervention, and that the hole created by the interruption was then filled by both a fleshed body (and perhaps parts of other bodies) which were then covered by sediment that originated from the hole that was dug. We recognize that the four eLife reviewers are not convinced that our presentation is sufficient to establish this. Interestingly, this was not the universal opinion of earlier reviewers of the initial manuscript several of whom felt we had adequately supported this hypothesis. The lack of clarity in this current version of the burial manuscript is our responsibility. In the upcoming revision of this paper to be submitted, we will take the reviewers’ critiques to heart and add additional figures that illustrate better the disruption of the LORM and clarify the sedimentological data showing the material covering the skeletal remains in the hole are the disrupted sediments excavated from the same hole. We are proposing to isolate this most critical evidence for burial into a separate section in the revised submission based on the reviewers’ comments. The fact that the LORM layer is disrupted, a fleshed body was placed in the hole created by this disruption, and the body (and perhaps parts of other bodies) was/were then covered by the same sediments from the hole is the central feature of our hypothesis that the bone accumulations observed reflect a burial and not a natural process.

The possibility of fluvial transport or involvement in the subsystem is a topic that we have addressed extensively in past work, and it is clear from these reviews that we must enhance our current manuscript to discuss this issue at greater length. Our previous work (Dirks et al. 2015; Dirks et al. 2017) emphasized that fluvial transport of whole bodies into the subsystem was precluded by several lines of sedimentological evidence. We excavated a rich accumulation of skeletal remains, including articulated limbs and other elements in subvertical orientations inconsistent with slow sedimentary infill, which were difficult to explain without positing either a large and dense pile of bodies and/or sediment movement. We encountered fractured chunks of laminated orange-red mudstone (LORM) in random orientations within our excavation area, within and among skeletal remains, which directly refuted that the remains were inundated with water at the time of burial, and this limited the possibility of fluvial transport. Water flow sufficient to displace bodies or complete skeletal evidence would also transport large and course sediment, which is absent from the subsystem, and would sort the commingled skeletal material that we found by size, which we do not observe. But our excavation only covered less than a square meter at very limited depth, and this was the limit to our knowledge of subsurface sediment. We thus were left with uncertainty that led us to suggest the possibility of sediment slumping or movement into subsurface drains, although these were not observed near our excavation. Our current work expands our knowledge of the subsurface and presents an alternative explanation for the disposition of skeletal remains from our earlier excavation. But we acknowledge that this new explanation is vulnerable to our own previous published proposals, and we must do a better job of explaining how the new information addresses our previous suggestions. By not clearly creating a section where we explained how these previous hypotheses were now nullified by new evidence, we clearly confused the reviewers with our own previous work. We will revise the manuscript by enhancing the review of the significant geological evidence demonstrating that there is no significant fluvial action in the system and making it clear how the burial hypothesis provides a clearer explanation for the situation of skeletal remains from our previous excavation work.

One of the central issues raised by reviewers has been a perceived need to excavate these features completely, totally exhuming all skeletal remains from them. Reviewers have written that it is necessary to identify every skeletal element that is present and account for any missing elements. On this point, we have both ethical and scientific differences from these reviewers. We express our ethical concerns first. Many of the best-preserved possible burials ever discovered by archaeologists were subjected to total excavation and exhumation. Cases like La Chapelle-aux-Saints, La Ferrassie, and Skhūl were fully excavated at a time when data recording and excavation methods did not include the range of spatial and geomorphological approaches that later became routine. The judgment of early investigators that these situations were intentional burials was challenged by later workers, and the kind of information that might enable better tests had been irrevocably lost (Gargett 1999; Dibble et al. 2015; Rendu et al. 2014).

Later, improved excavation standards have not sufficed to remove uncertainty or debate about possible burials. For example, it was long presumed that well-preserved remains of young children were by themselves diagnostic of intentional burial, such as those from Dederiyeh, Border Cave, or Roc de Marsal. Such cases were also fully excavated, with adequate documentation of the positioning of skeletal remains and their surrounding stratigraphic situation, but such cases were later challenged on several bases and the complete exhumation of material has confused or precluded testing of new hypotheses (e.g. Gargett 1999). The case of Roc de Marsal is one in which data from the initial excavation combined with data from the initial excavation combined with re-excavation and geoarchaeological analysis led to a naturalistic interpretation of the skeletal material (Sandgathe et al. 2011; Goldberg et al. 2017). But even in this case, the researchers erred in their interpretation of the skeleton’s situation due to a lack of identification of parts of the infant’s skeleton (Gómez-Olivencia and García-Martinez 2019). That is to say, it is not only the burial hypothesis but other hypotheses that suffer from complete excavation. Researchers concerned with preserving all possible information have sometimes taken extraordinary measures to remove and study possible burials at high-resolution in the laboratory. Such was the case of the Shanidar IV burial removed from the site and transported in plaster jacket by Solecki, which led to the disruption and loss of internal stratigraphic information (Pomeroy et al. 2020). Arguably, the current state of the art is full excavation with partial preparation, such as that undertaken at Panga ya Saidi (Martinón-Torres et al. 2021). But again, any future attempt to reinterpret or test the hypothesis of burial must rely on the adequacy of documentation as the original context has been removed.

In our decision to leave material in place as much as possible, we are expanding upon standard practice to leave witness sections and unexcavated areas for future research. The situation is novel, representing possible burials by a nonhuman species, and that makes it doubly important in our opinion to be conservative in not fully exhuming the skeletal material from its context. We anticipate that many other researchers, including future investigators, will suggest additional methods to further test the hypothesis of burial, something that would be impossible if we had excavated the features in their entirety prior to publishing a description of our work. We believe strongly that our ethical responsibility is to publish the work and the most likely interpretation while leaving as much evidence in place as possible to enable further testing and replication. We welcome the suggestions of additional methods/analyses to test the H. naledi burial hypothesis.

This being said, we also observe that total exhumation would not resolve the concerns raised by the reviewers. The recommendation of total exhumation is in pursuit of a full account of all skeletal material present and its preservation and spatial situation, in order to demonstrate that they conform to body positions comparable to human burials. As has been highlighted in forensic casework, the excavation of an inhumation feature does not necessarily provide an accurate spatial or anatomical manifest of the stratigraphical relationships between the body, encapsulating matrix, and any cut present due to preservational, taphonomic and operational factors (Dirkmaat and Cabo, 2016; Hunter, 2014). In particular, in cases where skeletal elements are highly fragmented, friable, or degraded (such as through bioerosion) then complete excavation—even under controlled laboratory conditions—may destroy bone and severely limit skeletal identification (Henderson, 1997; Hochrein, 2002; Owsley and Compton, 1997), particularly in elements where the ratio of trabecular to cortical bone is high (Darwent and Lyman, 2002; Lyman, 1994). As such, non-invasive methods of 3D and 4D modelling (preservation in situ) are often considered preferable to complete necropsy or excavation (preservation by record) where appropriate (Bolliger and Thali, 2009; Dell’Unto and Landeschi, 2022; Randolph-Quinney et al., 2018; Silver, 2016). 

The test of burial is not primarily positional, but taphonomic and geological. The position and number of bones can elaborate on process-driven questions of decay and destruction in the burial environment, or post-mortem modification, but are not singularly indicative of whether the remains were intentionally buried – the post-mortem narrative of all the processes affecting the cadaveric island is required (Knüsel and Robb, 2016). In previous cases, researchers have disputed or accepted the hypothesis of intentional hominin burial based upon assumptions about how modern humans or Neandertals would have positioned bodies, with the idea that some positions reflect ritual intent while others do not. But applying such assumptions is unjustifiable, particularly for a species like H. naledi, whose culture may have differed fundamentally from our own. Our work acknowledges that the present evidence does not enable a full reconstruction of the burial positions, but it does show that fleshed remains were encased in sediment prior to decomposition of soft tissue, and that subsequent spatial changes can be most parsimoniously explained by natural decomposition within sedimentary matrix contained within a burial feature (after Green, 2022; Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022). If the argument is that extraordinary claims require extraordinary evidence, we feel that the evidence documents excavation and interment (and will do so more clearly in the revision) and the fact of the remains do not match a “typical” human burial in body positioning is not in itself evidence that these are not H. naledi burials.

We feel that the reviewers (in keeping with many palaeoanthropologists) have a clear idea of what they “think” a burial should look like in an idealised sense, but this platonic ideal of burial form is not matched by the extensive literature in archaeothanatology, funerary archaeology and forensic science which indicates enormous variability in the activity, morphology and post-mortem system experienced by the human body in cases of interment and body disposal (e.g. Aspöck, 2008; Boulestin and Duday, 2005 and 2006; Connelly et al., 2005; Channing and Randolph-Quinney, 2006; Cherryson, 2008; Donnelly et al., 1995; Finley, 2000; Hunter, 2014; Parker Pearson, 1999; Randolph-Quinney, 2013). Decades of experience in the identification, recovery and interpretation of clandestine, deviant, and non-formal burials indicates the platonic ideal is rare, and in many contexts, the exception (Cherryson, 2008; Parker Pearson, 1999). This variability is particularly relevant to morphological traits in burial context, such as the informal nature of the grave cut in plan and section, shallow burial depth, and initial disposition of body (placement) during the early post-mortem period. These might run counter to the expectations of reviewers or others referencing the fossil hominin record, but are well accepted within the communities of researchers investigating Holocene archaeological sites and forensic contexts.

It is encouraging to see reviewers beginning to incorporate the extensive (often experimentally derived) literature from archaeothanatology and forensic taphonomy in their deliberations, and we will be taking these comments on board going forward. In particular, we acknowledge reviewers’ comments and the need to construct a more detailed post-mortem narrative, accounting for joint disarticulation (labile versus persistent joints etc), displacement, and final disposition of elements within the burial space. As such we will incorporate the hierarchy of decomposition (rank order disarticulation), associations between regions of anatomical association, areas of disassociation, and the voids produced during decomposition (after Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022) into our narrative. In doing so we acknowledge the tensions between the inductive archaeolothanatological narrative-driven approach (e.g. Duday, 2005 & 2009) versus robust decomposition data derived from human forensic taphonomic experimentation recently articulated by Schotsmans and colleagues (2022) - noting that we will highlight comparative data based on forensic experimental casework and actualistic modelling over inductive intuitive approaches which come with significant evidential shortcomings (Bristow et al. 2011).

Finally, from a taphonomic perspective it is worth pointing out to reviewers that we have already addressed the issue of lack of taphonomic evidence for carnivore involvement in the formation of the Dinaledi assemblage (Dirks, et al., 2016). Absence of any carnivore-induced bone surface modifications, patterns of skeletal part representation, and a total absence of any carnivore remains found within the Dinaledi chamber (following Kuhn and colleagues, 2010) lead us to reject carnivores as possible vectors of body accumulation within the Dinaledi Chamber and Hill Antechamber.

Reviewers suggest that without a date derived from geochronological methods, the engravings cannot be associated with H. naledi, and that it is possible (or probable) that the engravings were done in the recent past by H. sapiens. This suggestion neglects the context of the site. We have previously documented the structure and extremely limited accessibility of the Dinaledi subsystem. This subsystem was not recorded on maps of the documented Rising Star Cave system prior to our work and its discovery by our teams. Furthermore, there is no evidence of prehistoric human activity in the areas of the cave related to possible subterranean entrances There is no evidence that humans in the past typically ventured into such extreme spaces like those of Rising Star. It is clear from the presence of the remains of many individuals that H. naledi ventured into these spaces again and again. It is likely that H. naledi moved through these spaces more easily than humans do based on their physique. We show that the engravings overlay each other suggesting multiple engraving events.  These engravings took time and effort and the only evidence for use of the Dinaledi subsystem by any hominin is by H. naledi. The context leads to the null hypothesis that H. naledi made the marks. In our revision, we will elaborate on this argument to clarify the evidence for our stance on this hypothesis. Several reviewers took issue with the title of the engraving paper as we did not insert a qualifier in front of the suggested date range for the engravings. We deliberately left out qualifying language so that the title took the form of a testable hypothesis rather than a weak assertation. Should future work find the engravings were not produced within this time range, then we will restate this hypothesis.

Finally, with regards to the engravings we have chosen to report them because they exist. Not reporting the presence of engraved marks on the walls of a cave above hypothesized burials would be tantamount to leaving relevant evidence out of the description of an archeological context. We recognize and state in our manuscript that these markings require substantial further study, including attempts at geochronological dating. But the current evidence is clearly relevant to the archaeological context of the subsystem. We take a similar stance with reporting the presence of the tool shaped artefact near the hand of the H. naledi skeleton in the Hill Antechamber. It is evident that this object requires further study, as we stated in our manuscript, but again omitting it from our study would be leaving out relevant evidence.

Some have suggested that the null hypothesis should be that all of these observed circumstances are of natural origin. Our team took this approach in our early investigation of the Dinaledi subsystem (Dirks et al. 2015). We adopted the null hypothesis that the geological processes involved in the accumulation of H. naledi skeletal remains were “natural” (e.g., non-naledigenic involvement), and we were able to reject many alternative explanations for the assemblage, including carnivore accumulation, “death trap” accumulation, and fluvial transport of bodies or bones (Dirks et al. 2015). This led us to the hypothesis that H. naledi were involved in bringing the bodies into the spaces where they were found. But we did not hypothesize their involvement in the formation of the deposit itself beyond bringing the bodies to the location.

This approach seems conservative. It followed the traditional view that small-brained hominins do not engage in cultural practices. But we recognize in hindsight that this null hypothesis approach did harm to our analyses. It impeded us from recognizing within our initial excavations of the puzzle box area and other excavations between 2014 – 2017 that we might be encountering remains that were intrusive in the sedimentary floor of the chamber. If we had approached the accumulation of a large number of hominins from the perspective of the null hypothesis being that the situation was likely cultural, we perhaps would have collected evidence in a slightly different manner. We certainly note that if the Dinaledi system had been full of the remains of modern humans, there would have been little doubt that the null hypothesis would have been that this was a cultural space and not a “natural space”.  We therefore respectfully disagree with the reviewers who continue to support the idea that we should approach hominin excavations with the null hypothesis that they will be natural (specifically non-cultural) in origins. If excavations continue with this mindset we believe that potential cultural evidence is almost certain to be lost.

There has been a gradient across paleoanthropological excavations, archaeological work, and forensic investigation, with increasing precision of context. The reality is that the recording precision and frame of approach is typically different in most paleontological excavations than in those related to contemporary human remains. If anything comes from the present discussion of whether the Dinaledi system is a burial site for H. naledi or not, we hope that by taking seriously the possibility of deep cultural dynamics of hominins, we will encourage other teams to meet the highest standards of excavation in order to preserve potential cultural evidence. Given H. naledi’s cranial capacity we suggest that even very early hominin skeletal assemblages should be re-examined, if there is sufficient evidence or records available.  These would include examples such as the A.L. 333 Au. afarensis site (the so called First Family site in Hadar Ethiopia), the Dikika infant skeleton, WT 15000 (Turkana Boy) and even A.L. 288 (Lucy) as such unusual taphonomic situations where skeletons are preserved cannot be simply explained away as “natural” in origin, based solely on the cranial capacity and assumed lack of cognitive and cultural complexity of the hominins as emphasized by us in Fuentes et al. (2023). We are not the first to observe that some very early hominin situations may represent early mortuary activity (Pettitt 2013), but we would advocate a step further. We suggest it may be damaging to take “natural accumulation” as the standard null hypothesis for hominin paleoanthropology, and that it is more conservative in practice to engage remains with the null hypothesis of possible cultural formation.

We are deeply grateful for the time and effort all of the 8 reviewers (across three reviews) have taken with this work.  We also acknowledge the anonymous reviewers from previous submissions who’s opinions and comments will have made the final iterations of these manuscripts better for their efforts. As this process is rather public and includes commentary outside of the eLife forum, we ask that the efforts of all 37 authors and 8 reviewers involved be respected and that the discourse remain professional in all venues as we study this fascinating and quite complex occurrence. We appreciate also the efforts of members of the public who have engaged with this relatively new process where preprints are posted prior to the reviews allowing comments and interactions from colleagues and the public who are normally not part of the internal peer review process.  We believe these interactions will make for better final papers. We feel we have met the standards of demonstrating burials in H. naledi and that the engraving are most likely associated with H. naledi. However, given the reviews we see many areas where our clarity and context, and analyses, were less strong than they can be. With the clarifications and additions taken on board through these review processes the final papers will be stronger and clearer. We, recognize that this is an ongoing process of scientific investigation and further work will allow continued, and possibly better, evaluation of these hypothesis and others.

Lee R Berger, Agustín Fuentes, John Hawks, Tebogo Makhubela

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Author Response:

We would like to thank the eLife reviewers for the considerable time and effort they have invested to review these manuscripts. We have also benefited from a previous round of review of the manuscript describing the proposed burial features, which underwent two rounds of revisions in a high-impact journal over a period of approximately 8 months during 2022 and early 2023. Both sets of reviews have reflected mixed responses to the evidence we have presented, with one reviewer recommending acceptance with minor editorial revisions, two recommending acceptance with minor revisions and the fourth recommending rejection based upon similar arguments to those reflected by some of the reviewers in this current round of reviews in eLife. Ultimately the managing editor of this first journal took the decision that the review process could not be completed in a timely manner and rejected the manuscript although the submission here reflected our consideration of these reviewers suggestions.

We have chosen in this initial response to the eLife reviews to include some references to the previous anonymous reviews in order to illustrate differences of opinion and differences in revision suggestions within the review process. Our goal is to offer maximal insight into our decision-making process and to acknowledge the considerable time and effort put into the assessment of these manuscripts by reviewers (for eLife and in the case of the earlier review process). We hope that this approach will assist the readers, and reviewers, of our manuscripts in understanding why we are proceeding with certain decisions during the revision process.

This is a new process for us and the reviewers, and one way in which it significantly differs from more traditional review is that both the reviews and our reply will be public well in advance of our revisions to the manuscript. Indeed, considering the scope of the reviews, some of those revisions may take considerable time, although many can be accomplished fairly easily. Thus, we are not in a position to say that we have solved every issue raised by the reviewers. Instead, we will examine what appear to be the key critical issues raised regarding the data and the analyses and how we propose to address these as we revise the papers. We will also address several philosophical and ethical issues raised by the reviews and our proposal for dealing with these. More specific editorial and citational recommendations will be dealt with on a case-by-case basis, and we do not address these point-by-point in this reply. Please note, this response to the reviewers is not the revision of the manuscript and is only the initial opinion of the corresponding authors with some guidance from the larger group of authors of all three papers. Our final submitted revision will reflect the input of all authors included on those submissions.

We took the decision to submit three separate papers consciously. The two different categories of evidence, burials and engravings, involve different kinds of analysis and different (although overlapping) teams of researchers, and we recognized that each deserved their own presentation and assessment. Meanwhile, together they inform the context of H. naledi in a way that requires some synthetic discussion, in which both kinds of evidence are relevant, leading to a third paper. But the mutual relevance of these different kinds of evidence and their review by a common set of reviewers naturally raises cross-cutting issues, and the reviewers have cross-referenced the three articles. This has sometimes led to suggestions about one manuscript based on the contents of another. Considering the situation, we accepted the recommendation that it would be clearer to consider all three articles in a single reply. Thus, while each of the three papers will proceed separately during the revision process, it will be necessary to highlight across all three papers occasionally in our responses.

Scientific Issues:

In reading the reviews, we feel there are 9 critical points/assertions raised by one or more of the reviewers that present a problem for, or challenge to, our hypothesis that the observed evidence (bone accumulations and engravings) described in the Dinaledi subsystem are of intentional naledigenic origin. These are:

1. The evidence presented does not demonstrate a clear interruption of the floor sediments, thus failing to demonstrate excavated holes.

2. The sediments infilling the holes where the skeletal remains are found have not been demonstrated to originate from the disruption of the floor sediments and thus could be part of a natural geological process (e.g. water movement, slumping) or carnivore accumulations.

3. Previous geological interpretations by our research group have given alternative geological explanations for formation of the bony accumulations that contradict the present evidence presented here and result in alternative origins hypotheses.

4. Burial cannot be effectively assessed without complete excavation of the features and site.

5. The skeletal remains as presented do not conform clearly to typical body arrangement/positions associated with human (Homo sapiens) burials.

6. There is no evidence of grave goods or lithic scatters that are typically associated with human burials.

7. Humans may have been involved with the creation of either the Homo naledi bone accumulations, the engravings, or both.

8. Without a date of the engravings, the null hypothesis should be the engravings were created by Homo sapiens.

9. The null hypothesis for explanation of the skeletal remains in this situation should be “natural accumulation”.

Our analysis of the Dinaledi Feature 1 leads us to accept that the laminated orange-red mudstone (LORM) sedimentary layer is interrupted, indicating a non-natural intervention, and that the hole created by the interruption was then filled by both a fleshed body (and perhaps parts of other bodies) which were then covered by sediment that originated from the hole that was dug. We recognize that the four eLife reviewers are not convinced that our presentation is sufficient to establish this. Interestingly, this was not the universal opinion of earlier reviewers of the initial manuscript several of whom felt we had adequately supported this hypothesis. The lack of clarity in this current version of the burial manuscript is our responsibility. In the upcoming revision of this paper to be submitted, we will take the reviewers’ critiques to heart and add additional figures that illustrate better the disruption of the LORM and clarify the sedimentological data showing the material covering the skeletal remains in the hole are the disrupted sediments excavated from the same hole. We are proposing to isolate this most critical evidence for burial into a separate section in the revised submission based on the reviewers’ comments. The fact that the LORM layer is disrupted, a fleshed body was placed in the hole created by this disruption, and the body (and perhaps parts of other bodies) was/were then covered by the same sediments from the hole is the central feature of our hypothesis that the bone accumulations observed reflect a burial and not a natural process.

The possibility of fluvial transport or involvement in the subsystem is a topic that we have addressed extensively in past work, and it is clear from these reviews that we must enhance our current manuscript to discuss this issue at greater length. Our previous work (Dirks et al. 2015; Dirks et al. 2017) emphasized that fluvial transport of whole bodies into the subsystem was precluded by several lines of sedimentological evidence. We excavated a rich accumulation of skeletal remains, including articulated limbs and other elements in subvertical orientations inconsistent with slow sedimentary infill, which were difficult to explain without positing either a large and dense pile of bodies and/or sediment movement. We encountered fractured chunks of laminated orange-red mudstone (LORM) in random orientations within our excavation area, within and among skeletal remains, which directly refuted that the remains were inundated with water at the time of burial, and this limited the possibility of fluvial transport. Water flow sufficient to displace bodies or complete skeletal evidence would also transport large and course sediment, which is absent from the subsystem, and would sort the commingled skeletal material that we found by size, which we do not observe. But our excavation only covered less than a square meter at very limited depth, and this was the limit to our knowledge of subsurface sediment. We thus were left with uncertainty that led us to suggest the possibility of sediment slumping or movement into subsurface drains, although these were not observed near our excavation. Our current work expands our knowledge of the subsurface and presents an alternative explanation for the disposition of skeletal remains from our earlier excavation. But we acknowledge that this new explanation is vulnerable to our own previous published proposals, and we must do a better job of explaining how the new information addresses our previous suggestions. By not clearly creating a section where we explained how these previous hypotheses were now nullified by new evidence, we clearly confused the reviewers with our own previous work. We will revise the manuscript by enhancing the review of the significant geological evidence demonstrating that there is no significant fluvial action in the system and making it clear how the burial hypothesis provides a clearer explanation for the situation of skeletal remains from our previous excavation work.

One of the central issues raised by reviewers has been a perceived need to excavate these features completely, totally exhuming all skeletal remains from them. Reviewers have written that it is necessary to identify every skeletal element that is present and account for any missing elements. On this point, we have both ethical and scientific differences from these reviewers. We express our ethical concerns first. Many of the best-preserved possible burials ever discovered by archaeologists were subjected to total excavation and exhumation. Cases like La Chapelle-aux-Saints, La Ferrassie, and Skhūl were fully excavated at a time when data recording and excavation methods did not include the range of spatial and geomorphological approaches that later became routine. The judgment of early investigators that these situations were intentional burials was challenged by later workers, and the kind of information that might enable better tests had been irrevocably lost (Gargett 1999; Dibble et al. 2015; Rendu et al. 2014).

Later, improved excavation standards have not sufficed to remove uncertainty or debate about possible burials. For example, it was long presumed that well-preserved remains of young children were by themselves diagnostic of intentional burial, such as those from Dederiyeh, Border Cave, or Roc de Marsal. Such cases were also fully excavated, with adequate documentation of the positioning of skeletal remains and their surrounding stratigraphic situation, but such cases were later challenged on several bases and the complete exhumation of material has confused or precluded testing of new hypotheses (e.g. Gargett 1999). The case of Roc de Marsal is one in which data from the initial excavation combined with data from the initial excavation combined with re-excavation and geoarchaeological analysis led to a naturalistic interpretation of the skeletal material (Sandgathe et al. 2011; Goldberg et al. 2017). But even in this case, the researchers erred in their interpretation of the skeleton’s situation due to a lack of identification of parts of the infant’s skeleton (Gómez-Olivencia and García-Martinez 2019). That is to say, it is not only the burial hypothesis but other hypotheses that suffer from complete excavation. Researchers concerned with preserving all possible information have sometimes taken extraordinary measures to remove and study possible burials at high-resolution in the laboratory. Such was the case of the Shanidar IV burial removed from the site and transported in plaster jacket by Solecki, which led to the disruption and loss of internal stratigraphic information (Pomeroy et al. 2020). Arguably, the current state of the art is full excavation with partial preparation, such as that undertaken at Panga ya Saidi (Martinón-Torres et al. 2021). But again, any future attempt to reinterpret or test the hypothesis of burial must rely on the adequacy of documentation as the original context has been removed.

In our decision to leave material in place as much as possible, we are expanding upon standard practice to leave witness sections and unexcavated areas for future research. The situation is novel, representing possible burials by a nonhuman species, and that makes it doubly important in our opinion to be conservative in not fully exhuming the skeletal material from its context. We anticipate that many other researchers, including future investigators, will suggest additional methods to further test the hypothesis of burial, something that would be impossible if we had excavated the features in their entirety prior to publishing a description of our work. We believe strongly that our ethical responsibility is to publish the work and the most likely interpretation while leaving as much evidence in place as possible to enable further testing and replication. We welcome the suggestions of additional methods/analyses to test the H. naledi burial hypothesis.

This being said, we also observe that total exhumation would not resolve the concerns raised by the reviewers. The recommendation of total exhumation is in pursuit of a full account of all skeletal material present and its preservation and spatial situation, in order to demonstrate that they conform to body positions comparable to human burials. As has been highlighted in forensic casework, the excavation of an inhumation feature does not necessarily provide an accurate spatial or anatomical manifest of the stratigraphical relationships between the body, encapsulating matrix, and any cut present due to preservational, taphonomic and operational factors (Dirkmaat and Cabo, 2016; Hunter, 2014). In particular, in cases where skeletal elements are highly fragmented, friable, or degraded (such as through bioerosion) then complete excavation—even under controlled laboratory conditions—may destroy bone and severely limit skeletal identification (Henderson, 1997; Hochrein, 2002; Owsley and Compton, 1997), particularly in elements where the ratio of trabecular to cortical bone is high (Darwent and Lyman, 2002; Lyman, 1994). As such, non-invasive methods of 3D and 4D modelling (preservation in situ) are often considered preferable to complete necropsy or excavation (preservation by record) where appropriate (Bolliger and Thali, 2009; Dell’Unto and Landeschi, 2022; Randolph-Quinney et al., 2018; Silver, 2016). 

The test of burial is not primarily positional, but taphonomic and geological. The position and number of bones can elaborate on process-driven questions of decay and destruction in the burial environment, or post-mortem modification, but are not singularly indicative of whether the remains were intentionally buried – the post-mortem narrative of all the processes affecting the cadaveric island is required (Knüsel and Robb, 2016). In previous cases, researchers have disputed or accepted the hypothesis of intentional hominin burial based upon assumptions about how modern humans or Neandertals would have positioned bodies, with the idea that some positions reflect ritual intent while others do not. But applying such assumptions is unjustifiable, particularly for a species like H. naledi, whose culture may have differed fundamentally from our own. Our work acknowledges that the present evidence does not enable a full reconstruction of the burial positions, but it does show that fleshed remains were encased in sediment prior to decomposition of soft tissue, and that subsequent spatial changes can be most parsimoniously explained by natural decomposition within sedimentary matrix contained within a burial feature (after Green, 2022; Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022). If the argument is that extraordinary claims require extraordinary evidence, we feel that the evidence documents excavation and interment (and will do so more clearly in the revision) and the fact of the remains do not match a “typical” human burial in body positioning is not in itself evidence that these are not H. naledi burials.

We feel that the reviewers (in keeping with many palaeoanthropologists) have a clear idea of what they “think” a burial should look like in an idealised sense, but this platonic ideal of burial form is not matched by the extensive literature in archaeothanatology, funerary archaeology and forensic science which indicates enormous variability in the activity, morphology and post-mortem system experienced by the human body in cases of interment and body disposal (e.g. Aspöck, 2008; Boulestin and Duday, 2005 and 2006; Connelly et al., 2005; Channing and Randolph-Quinney, 2006; Cherryson, 2008; Donnelly et al., 1995; Finley, 2000; Hunter, 2014; Parker Pearson, 1999; Randolph-Quinney, 2013). Decades of experience in the identification, recovery and interpretation of clandestine, deviant, and non-formal burials indicates the platonic ideal is rare, and in many contexts, the exception (Cherryson, 2008; Parker Pearson, 1999). This variability is particularly relevant to morphological traits in burial context, such as the informal nature of the grave cut in plan and section, shallow burial depth, and initial disposition of body (placement) during the early post-mortem period. These might run counter to the expectations of reviewers or others referencing the fossil hominin record, but are well accepted within the communities of researchers investigating Holocene archaeological sites and forensic contexts.

It is encouraging to see reviewers beginning to incorporate the extensive (often experimentally derived) literature from archaeothanatology and forensic taphonomy in their deliberations, and we will be taking these comments on board going forward. In particular, we acknowledge reviewers’ comments and the need to construct a more detailed post-mortem narrative, accounting for joint disarticulation (labile versus persistent joints etc), displacement, and final disposition of elements within the burial space. As such we will incorporate the hierarchy of decomposition (rank order disarticulation), associations between regions of anatomical association, areas of disassociation, and the voids produced during decomposition (after Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022) into our narrative. In doing so we acknowledge the tensions between the inductive archaeolothanatological narrative-driven approach (e.g. Duday, 2005 & 2009) versus robust decomposition data derived from human forensic taphonomic experimentation recently articulated by Schotsmans and colleagues (2022) - noting that we will highlight comparative data based on forensic experimental casework and actualistic modelling over inductive intuitive approaches which come with significant evidential shortcomings (Bristow et al. 2011).

Finally, from a taphonomic perspective it is worth pointing out to reviewers that we have already addressed the issue of lack of taphonomic evidence for carnivore involvement in the formation of the Dinaledi assemblage (Dirks, et al., 2016). Absence of any carnivore-induced bone surface modifications, patterns of skeletal part representation, and a total absence of any carnivore remains found within the Dinaledi chamber (following Kuhn and colleagues, 2010) lead us to reject carnivores as possible vectors of body accumulation within the Dinaledi Chamber and Hill Antechamber.

Reviewers suggest that without a date derived from geochronological methods, the engravings cannot be associated with H. naledi, and that it is possible (or probable) that the engravings were done in the recent past by H. sapiens. This suggestion neglects the context of the site. We have previously documented the structure and extremely limited accessibility of the Dinaledi subsystem. This subsystem was not recorded on maps of the documented Rising Star Cave system prior to our work and its discovery by our teams. Furthermore, there is no evidence of prehistoric human activity in the areas of the cave related to possible subterranean entrances There is no evidence that humans in the past typically ventured into such extreme spaces like those of Rising Star. It is clear from the presence of the remains of many individuals that H. naledi ventured into these spaces again and again. It is likely that H. naledi moved through these spaces more easily than humans do based on their physique. We show that the engravings overlay each other suggesting multiple engraving events.  These engravings took time and effort and the only evidence for use of the Dinaledi subsystem by any hominin is by H. naledi. The context leads to the null hypothesis that H. naledi made the marks. In our revision, we will elaborate on this argument to clarify the evidence for our stance on this hypothesis. Several reviewers took issue with the title of the engraving paper as we did not insert a qualifier in front of the suggested date range for the engravings. We deliberately left out qualifying language so that the title took the form of a testable hypothesis rather than a weak assertation. Should future work find the engravings were not produced within this time range, then we will restate this hypothesis.

Finally, with regards to the engravings we have chosen to report them because they exist. Not reporting the presence of engraved marks on the walls of a cave above hypothesized burials would be tantamount to leaving relevant evidence out of the description of an archeological context. We recognize and state in our manuscript that these markings require substantial further study, including attempts at geochronological dating. But the current evidence is clearly relevant to the archaeological context of the subsystem. We take a similar stance with reporting the presence of the tool shaped artefact near the hand of the H. naledi skeleton in the Hill Antechamber. It is evident that this object requires further study, as we stated in our manuscript, but again omitting it from our study would be leaving out relevant evidence.

Some have suggested that the null hypothesis should be that all of these observed circumstances are of natural origin. Our team took this approach in our early investigation of the Dinaledi subsystem (Dirks et al. 2015). We adopted the null hypothesis that the geological processes involved in the accumulation of H. naledi skeletal remains were “natural” (e.g., non-naledigenic involvement), and we were able to reject many alternative explanations for the assemblage, including carnivore accumulation, “death trap” accumulation, and fluvial transport of bodies or bones (Dirks et al. 2015). This led us to the hypothesis that H. naledi were involved in bringing the bodies into the spaces where they were found. But we did not hypothesize their involvement in the formation of the deposit itself beyond bringing the bodies to the location.

This approach seems conservative. It followed the traditional view that small-brained hominins do not engage in cultural practices. But we recognize in hindsight that this null hypothesis approach did harm to our analyses. It impeded us from recognizing within our initial excavations of the puzzle box area and other excavations between 2014 – 2017 that we might be encountering remains that were intrusive in the sedimentary floor of the chamber. If we had approached the accumulation of a large number of hominins from the perspective of the null hypothesis being that the situation was likely cultural, we perhaps would have collected evidence in a slightly different manner. We certainly note that if the Dinaledi system had been full of the remains of modern humans, there would have been little doubt that the null hypothesis would have been that this was a cultural space and not a “natural space”.  We therefore respectfully disagree with the reviewers who continue to support the idea that we should approach hominin excavations with the null hypothesis that they will be natural (specifically non-cultural) in origins. If excavations continue with this mindset we believe that potential cultural evidence is almost certain to be lost.

There has been a gradient across paleoanthropological excavations, archaeological work, and forensic investigation, with increasing precision of context. The reality is that the recording precision and frame of approach is typically different in most paleontological excavations than in those related to contemporary human remains. If anything comes from the present discussion of whether the Dinaledi system is a burial site for H. naledi or not, we hope that by taking seriously the possibility of deep cultural dynamics of hominins, we will encourage other teams to meet the highest standards of excavation in order to preserve potential cultural evidence. Given H. naledi’s cranial capacity we suggest that even very early hominin skeletal assemblages should be re-examined, if there is sufficient evidence or records available.  These would include examples such as the A.L. 333 Au. afarensis site (the so called First Family site in Hadar Ethiopia), the Dikika infant skeleton, WT 15000 (Turkana Boy) and even A.L. 288 (Lucy) as such unusual taphonomic situations where skeletons are preserved cannot be simply explained away as “natural” in origin, based solely on the cranial capacity and assumed lack of cognitive and cultural complexity of the hominins as emphasized by us in Fuentes et al. (2023). We are not the first to observe that some very early hominin situations may represent early mortuary activity (Pettitt 2013), but we would advocate a step further. We suggest it may be damaging to take “natural accumulation” as the standard null hypothesis for hominin paleoanthropology, and that it is more conservative in practice to engage remains with the null hypothesis of possible cultural formation.

We are deeply grateful for the time and effort all of the 8 reviewers (across three reviews) have taken with this work.  We also acknowledge the anonymous reviewers from previous submissions who’s opinions and comments will have made the final iterations of these manuscripts better for their efforts. As this process is rather public and includes commentary outside of the eLife forum, we ask that the efforts of all 37 authors and 8 reviewers involved be respected and that the discourse remain professional in all venues as we study this fascinating and quite complex occurrence. We appreciate also the efforts of members of the public who have engaged with this relatively new process where preprints are posted prior to the reviews allowing comments and interactions from colleagues and the public who are normally not part of the internal peer review process.  We believe these interactions will make for better final papers. We feel we have met the standards of demonstrating burials in H. naledi and that the engraving are most likely associated with H. naledi. However, given the reviews we see many areas where our clarity and context, and analyses, were less strong than they can be. With the clarifications and additions taken on board through these review processes the final papers will be stronger and clearer. We, recognize that this is an ongoing process of scientific investigation and further work will allow continued, and possibly better, evaluation of these hypothesis and others.

Lee R Berger, Agustín Fuentes, John Hawks, Tebogo Makhubela

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Author Response:

We would like to thank the eLife reviewers for the considerable time and effort they have invested to review these manuscripts. We have also benefited from a previous round of review of the manuscript describing the proposed burial features, which underwent two rounds of revisions in a high-impact journal over a period of approximately 8 months during 2022 and early 2023. Both sets of reviews have reflected mixed responses to the evidence we have presented, with one reviewer recommending acceptance with minor editorial revisions, two recommending acceptance with minor revisions and the fourth recommending rejection based upon similar arguments to those reflected by some of the reviewers in this current round of reviews in eLife. Ultimately the managing editor of this first journal took the decision that the review process could not be completed in a timely manner and rejected the manuscript although the submission here reflected our consideration of these reviewers suggestions.

We have chosen in this initial response to the eLife reviews to include some references to the previous anonymous reviews in order to illustrate differences of opinion and differences in revision suggestions within the review process. Our goal is to offer maximal insight into our decision-making process and to acknowledge the considerable time and effort put into the assessment of these manuscripts by reviewers (for eLife and in the case of the earlier review process). We hope that this approach will assist the readers, and reviewers, of our manuscripts in understanding why we are proceeding with certain decisions during the revision process.

This is a new process for us and the reviewers, and one way in which it significantly differs from more traditional review is that both the reviews and our reply will be public well in advance of our revisions to the manuscript. Indeed, considering the scope of the reviews, some of those revisions may take considerable time, although many can be accomplished fairly easily. Thus, we are not in a position to say that we have solved every issue raised by the reviewers. Instead, we will examine what appear to be the key critical issues raised regarding the data and the analyses and how we propose to address these as we revise the papers. We will also address several philosophical and ethical issues raised by the reviews and our proposal for dealing with these. More specific editorial and citational recommendations will be dealt with on a case-by-case basis, and we do not address these point-by-point in this reply. Please note, this response to the reviewers is not the revision of the manuscript and is only the initial opinion of the corresponding authors with some guidance from the larger group of authors of all three papers. Our final submitted revision will reflect the input of all authors included on those submissions.

We took the decision to submit three separate papers consciously. The two different categories of evidence, burials and engravings, involve different kinds of analysis and different (although overlapping) teams of researchers, and we recognized that each deserved their own presentation and assessment. Meanwhile, together they inform the context of H. naledi in a way that requires some synthetic discussion, in which both kinds of evidence are relevant, leading to a third paper. But the mutual relevance of these different kinds of evidence and their review by a common set of reviewers naturally raises cross-cutting issues, and the reviewers have cross-referenced the three articles. This has sometimes led to suggestions about one manuscript based on the contents of another. Considering the situation, we accepted the recommendation that it would be clearer to consider all three articles in a single reply. Thus, while each of the three papers will proceed separately during the revision process, it will be necessary to highlight across all three papers occasionally in our responses.

Scientific Issues:

In reading the reviews, we feel there are 9 critical points/assertions raised by one or more of the reviewers that present a problem for, or challenge to, our hypothesis that the observed evidence (bone accumulations and engravings) described in the Dinaledi subsystem are of intentional naledigenic origin. These are:

1. The evidence presented does not demonstrate a clear interruption of the floor sediments, thus failing to demonstrate excavated holes.

2. The sediments infilling the holes where the skeletal remains are found have not been demonstrated to originate from the disruption of the floor sediments and thus could be part of a natural geological process (e.g. water movement, slumping) or carnivore accumulations.

3. Previous geological interpretations by our research group have given alternative geological explanations for formation of the bony accumulations that contradict the present evidence presented here and result in alternative origins hypotheses.

4. Burial cannot be effectively assessed without complete excavation of the features and site.

5. The skeletal remains as presented do not conform clearly to typical body arrangement/positions associated with human (Homo sapiens) burials.

6. There is no evidence of grave goods or lithic scatters that are typically associated with human burials.

7. Humans may have been involved with the creation of either the Homo naledi bone accumulations, the engravings, or both.

8. Without a date of the engravings, the null hypothesis should be the engravings were created by Homo sapiens.

9. The null hypothesis for explanation of the skeletal remains in this situation should be “natural accumulation”.

Our analysis of the Dinaledi Feature 1 leads us to accept that the laminated orange-red mudstone (LORM) sedimentary layer is interrupted, indicating a non-natural intervention, and that the hole created by the interruption was then filled by both a fleshed body (and perhaps parts of other bodies) which were then covered by sediment that originated from the hole that was dug. We recognize that the four eLife reviewers are not convinced that our presentation is sufficient to establish this. Interestingly, this was not the universal opinion of earlier reviewers of the initial manuscript several of whom felt we had adequately supported this hypothesis. The lack of clarity in this current version of the burial manuscript is our responsibility. In the upcoming revision of this paper to be submitted, we will take the reviewers’ critiques to heart and add additional figures that illustrate better the disruption of the LORM and clarify the sedimentological data showing the material covering the skeletal remains in the hole are the disrupted sediments excavated from the same hole. We are proposing to isolate this most critical evidence for burial into a separate section in the revised submission based on the reviewers’ comments. The fact that the LORM layer is disrupted, a fleshed body was placed in the hole created by this disruption, and the body (and perhaps parts of other bodies) was/were then covered by the same sediments from the hole is the central feature of our hypothesis that the bone accumulations observed reflect a burial and not a natural process.

The possibility of fluvial transport or involvement in the subsystem is a topic that we have addressed extensively in past work, and it is clear from these reviews that we must enhance our current manuscript to discuss this issue at greater length. Our previous work (Dirks et al. 2015; Dirks et al. 2017) emphasized that fluvial transport of whole bodies into the subsystem was precluded by several lines of sedimentological evidence. We excavated a rich accumulation of skeletal remains, including articulated limbs and other elements in subvertical orientations inconsistent with slow sedimentary infill, which were difficult to explain without positing either a large and dense pile of bodies and/or sediment movement. We encountered fractured chunks of laminated orange-red mudstone (LORM) in random orientations within our excavation area, within and among skeletal remains, which directly refuted that the remains were inundated with water at the time of burial, and this limited the possibility of fluvial transport. Water flow sufficient to displace bodies or complete skeletal evidence would also transport large and course sediment, which is absent from the subsystem, and would sort the commingled skeletal material that we found by size, which we do not observe. But our excavation only covered less than a square meter at very limited depth, and this was the limit to our knowledge of subsurface sediment. We thus were left with uncertainty that led us to suggest the possibility of sediment slumping or movement into subsurface drains, although these were not observed near our excavation. Our current work expands our knowledge of the subsurface and presents an alternative explanation for the disposition of skeletal remains from our earlier excavation. But we acknowledge that this new explanation is vulnerable to our own previous published proposals, and we must do a better job of explaining how the new information addresses our previous suggestions. By not clearly creating a section where we explained how these previous hypotheses were now nullified by new evidence, we clearly confused the reviewers with our own previous work. We will revise the manuscript by enhancing the review of the significant geological evidence demonstrating that there is no significant fluvial action in the system and making it clear how the burial hypothesis provides a clearer explanation for the situation of skeletal remains from our previous excavation work.

One of the central issues raised by reviewers has been a perceived need to excavate these features completely, totally exhuming all skeletal remains from them. Reviewers have written that it is necessary to identify every skeletal element that is present and account for any missing elements. On this point, we have both ethical and scientific differences from these reviewers. We express our ethical concerns first. Many of the best-preserved possible burials ever discovered by archaeologists were subjected to total excavation and exhumation. Cases like La Chapelle-aux-Saints, La Ferrassie, and Skhūl were fully excavated at a time when data recording and excavation methods did not include the range of spatial and geomorphological approaches that later became routine. The judgment of early investigators that these situations were intentional burials was challenged by later workers, and the kind of information that might enable better tests had been irrevocably lost (Gargett 1999; Dibble et al. 2015; Rendu et al. 2014).

Later, improved excavation standards have not sufficed to remove uncertainty or debate about possible burials. For example, it was long presumed that well-preserved remains of young children were by themselves diagnostic of intentional burial, such as those from Dederiyeh, Border Cave, or Roc de Marsal. Such cases were also fully excavated, with adequate documentation of the positioning of skeletal remains and their surrounding stratigraphic situation, but such cases were later challenged on several bases and the complete exhumation of material has confused or precluded testing of new hypotheses (e.g. Gargett 1999). The case of Roc de Marsal is one in which data from the initial excavation combined with data from the initial excavation combined with re-excavation and geoarchaeological analysis led to a naturalistic interpretation of the skeletal material (Sandgathe et al. 2011; Goldberg et al. 2017). But even in this case, the researchers erred in their interpretation of the skeleton’s situation due to a lack of identification of parts of the infant’s skeleton (Gómez-Olivencia and García-Martinez 2019). That is to say, it is not only the burial hypothesis but other hypotheses that suffer from complete excavation. Researchers concerned with preserving all possible information have sometimes taken extraordinary measures to remove and study possible burials at high-resolution in the laboratory. Such was the case of the Shanidar IV burial removed from the site and transported in plaster jacket by Solecki, which led to the disruption and loss of internal stratigraphic information (Pomeroy et al. 2020). Arguably, the current state of the art is full excavation with partial preparation, such as that undertaken at Panga ya Saidi (Martinón-Torres et al. 2021). But again, any future attempt to reinterpret or test the hypothesis of burial must rely on the adequacy of documentation as the original context has been removed.

In our decision to leave material in place as much as possible, we are expanding upon standard practice to leave witness sections and unexcavated areas for future research. The situation is novel, representing possible burials by a nonhuman species, and that makes it doubly important in our opinion to be conservative in not fully exhuming the skeletal material from its context. We anticipate that many other researchers, including future investigators, will suggest additional methods to further test the hypothesis of burial, something that would be impossible if we had excavated the features in their entirety prior to publishing a description of our work. We believe strongly that our ethical responsibility is to publish the work and the most likely interpretation while leaving as much evidence in place as possible to enable further testing and replication. We welcome the suggestions of additional methods/analyses to test the H. naledi burial hypothesis.

This being said, we also observe that total exhumation would not resolve the concerns raised by the reviewers. The recommendation of total exhumation is in pursuit of a full account of all skeletal material present and its preservation and spatial situation, in order to demonstrate that they conform to body positions comparable to human burials. As has been highlighted in forensic casework, the excavation of an inhumation feature does not necessarily provide an accurate spatial or anatomical manifest of the stratigraphical relationships between the body, encapsulating matrix, and any cut present due to preservational, taphonomic and operational factors (Dirkmaat and Cabo, 2016; Hunter, 2014). In particular, in cases where skeletal elements are highly fragmented, friable, or degraded (such as through bioerosion) then complete excavation—even under controlled laboratory conditions—may destroy bone and severely limit skeletal identification (Henderson, 1997; Hochrein, 2002; Owsley and Compton, 1997), particularly in elements where the ratio of trabecular to cortical bone is high (Darwent and Lyman, 2002; Lyman, 1994). As such, non-invasive methods of 3D and 4D modelling (preservation in situ) are often considered preferable to complete necropsy or excavation (preservation by record) where appropriate (Bolliger and Thali, 2009; Dell’Unto and Landeschi, 2022; Randolph-Quinney et al., 2018; Silver, 2016). 

The test of burial is not primarily positional, but taphonomic and geological. The position and number of bones can elaborate on process-driven questions of decay and destruction in the burial environment, or post-mortem modification, but are not singularly indicative of whether the remains were intentionally buried – the post-mortem narrative of all the processes affecting the cadaveric island is required (Knüsel and Robb, 2016). In previous cases, researchers have disputed or accepted the hypothesis of intentional hominin burial based upon assumptions about how modern humans or Neandertals would have positioned bodies, with the idea that some positions reflect ritual intent while others do not. But applying such assumptions is unjustifiable, particularly for a species like H. naledi, whose culture may have differed fundamentally from our own. Our work acknowledges that the present evidence does not enable a full reconstruction of the burial positions, but it does show that fleshed remains were encased in sediment prior to decomposition of soft tissue, and that subsequent spatial changes can be most parsimoniously explained by natural decomposition within sedimentary matrix contained within a burial feature (after Green, 2022; Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022). If the argument is that extraordinary claims require extraordinary evidence, we feel that the evidence documents excavation and interment (and will do so more clearly in the revision) and the fact of the remains do not match a “typical” human burial in body positioning is not in itself evidence that these are not H. naledi burials.

We feel that the reviewers (in keeping with many palaeoanthropologists) have a clear idea of what they “think” a burial should look like in an idealised sense, but this platonic ideal of burial form is not matched by the extensive literature in archaeothanatology, funerary archaeology and forensic science which indicates enormous variability in the activity, morphology and post-mortem system experienced by the human body in cases of interment and body disposal (e.g. Aspöck, 2008; Boulestin and Duday, 2005 and 2006; Connelly et al., 2005; Channing and Randolph-Quinney, 2006; Cherryson, 2008; Donnelly et al., 1995; Finley, 2000; Hunter, 2014; Parker Pearson, 1999; Randolph-Quinney, 2013). Decades of experience in the identification, recovery and interpretation of clandestine, deviant, and non-formal burials indicates the platonic ideal is rare, and in many contexts, the exception (Cherryson, 2008; Parker Pearson, 1999). This variability is particularly relevant to morphological traits in burial context, such as the informal nature of the grave cut in plan and section, shallow burial depth, and initial disposition of body (placement) during the early post-mortem period. These might run counter to the expectations of reviewers or others referencing the fossil hominin record, but are well accepted within the communities of researchers investigating Holocene archaeological sites and forensic contexts.

It is encouraging to see reviewers beginning to incorporate the extensive (often experimentally derived) literature from archaeothanatology and forensic taphonomy in their deliberations, and we will be taking these comments on board going forward. In particular, we acknowledge reviewers’ comments and the need to construct a more detailed post-mortem narrative, accounting for joint disarticulation (labile versus persistent joints etc), displacement, and final disposition of elements within the burial space. As such we will incorporate the hierarchy of decomposition (rank order disarticulation), associations between regions of anatomical association, areas of disassociation, and the voids produced during decomposition (after Mickleburgh and Wescott, 2018; Mickleburgh et al., 2022) into our narrative. In doing so we acknowledge the tensions between the inductive archaeolothanatological narrative-driven approach (e.g. Duday, 2005 & 2009) versus robust decomposition data derived from human forensic taphonomic experimentation recently articulated by Schotsmans and colleagues (2022) - noting that we will highlight comparative data based on forensic experimental casework and actualistic modelling over inductive intuitive approaches which come with significant evidential shortcomings (Bristow et al. 2011).

Finally, from a taphonomic perspective it is worth pointing out to reviewers that we have already addressed the issue of lack of taphonomic evidence for carnivore involvement in the formation of the Dinaledi assemblage (Dirks, et al., 2016). Absence of any carnivore-induced bone surface modifications, patterns of skeletal part representation, and a total absence of any carnivore remains found within the Dinaledi chamber (following Kuhn and colleagues, 2010) lead us to reject carnivores as possible vectors of body accumulation within the Dinaledi Chamber and Hill Antechamber.

Reviewers suggest that without a date derived from geochronological methods, the engravings cannot be associated with H. naledi, and that it is possible (or probable) that the engravings were done in the recent past by H. sapiens. This suggestion neglects the context of the site. We have previously documented the structure and extremely limited accessibility of the Dinaledi subsystem. This subsystem was not recorded on maps of the documented Rising Star Cave system prior to our work and its discovery by our teams. Furthermore, there is no evidence of prehistoric human activity in the areas of the cave related to possible subterranean entrances There is no evidence that humans in the past typically ventured into such extreme spaces like those of Rising Star. It is clear from the presence of the remains of many individuals that H. naledi ventured into these spaces again and again. It is likely that H. naledi moved through these spaces more easily than humans do based on their physique. We show that the engravings overlay each other suggesting multiple engraving events.  These engravings took time and effort and the only evidence for use of the Dinaledi subsystem by any hominin is by H. naledi. The context leads to the null hypothesis that H. naledi made the marks. In our revision, we will elaborate on this argument to clarify the evidence for our stance on this hypothesis. Several reviewers took issue with the title of the engraving paper as we did not insert a qualifier in front of the suggested date range for the engravings. We deliberately left out qualifying language so that the title took the form of a testable hypothesis rather than a weak assertation. Should future work find the engravings were not produced within this time range, then we will restate this hypothesis.

Finally, with regards to the engravings we have chosen to report them because they exist. Not reporting the presence of engraved marks on the walls of a cave above hypothesized burials would be tantamount to leaving relevant evidence out of the description of an archeological context. We recognize and state in our manuscript that these markings require substantial further study, including attempts at geochronological dating. But the current evidence is clearly relevant to the archaeological context of the subsystem. We take a similar stance with reporting the presence of the tool shaped artefact near the hand of the H. naledi skeleton in the Hill Antechamber. It is evident that this object requires further study, as we stated in our manuscript, but again omitting it from our study would be leaving out relevant evidence.

Some have suggested that the null hypothesis should be that all of these observed circumstances are of natural origin. Our team took this approach in our early investigation of the Dinaledi subsystem (Dirks et al. 2015). We adopted the null hypothesis that the geological processes involved in the accumulation of H. naledi skeletal remains were “natural” (e.g., non-naledigenic involvement), and we were able to reject many alternative explanations for the assemblage, including carnivore accumulation, “death trap” accumulation, and fluvial transport of bodies or bones (Dirks et al. 2015). This led us to the hypothesis that H. naledi were involved in bringing the bodies into the spaces where they were found. But we did not hypothesize their involvement in the formation of the deposit itself beyond bringing the bodies to the location.

This approach seems conservative. It followed the traditional view that small-brained hominins do not engage in cultural practices. But we recognize in hindsight that this null hypothesis approach did harm to our analyses. It impeded us from recognizing within our initial excavations of the puzzle box area and other excavations between 2014 – 2017 that we might be encountering remains that were intrusive in the sedimentary floor of the chamber. If we had approached the accumulation of a large number of hominins from the perspective of the null hypothesis being that the situation was likely cultural, we perhaps would have collected evidence in a slightly different manner. We certainly note that if the Dinaledi system had been full of the remains of modern humans, there would have been little doubt that the null hypothesis would have been that this was a cultural space and not a “natural space”.  We therefore respectfully disagree with the reviewers who continue to support the idea that we should approach hominin excavations with the null hypothesis that they will be natural (specifically non-cultural) in origins. If excavations continue with this mindset we believe that potential cultural evidence is almost certain to be lost.

There has been a gradient across paleoanthropological excavations, archaeological work, and forensic investigation, with increasing precision of context. The reality is that the recording precision and frame of approach is typically different in most paleontological excavations than in those related to contemporary human remains. If anything comes from the present discussion of whether the Dinaledi system is a burial site for H. naledi or not, we hope that by taking seriously the possibility of deep cultural dynamics of hominins, we will encourage other teams to meet the highest standards of excavation in order to preserve potential cultural evidence. Given H. naledi’s cranial capacity we suggest that even very early hominin skeletal assemblages should be re-examined, if there is sufficient evidence or records available.  These would include examples such as the A.L. 333 Au. afarensis site (the so called First Family site in Hadar Ethiopia), the Dikika infant skeleton, WT 15000 (Turkana Boy) and even A.L. 288 (Lucy) as such unusual taphonomic situations where skeletons are preserved cannot be simply explained away as “natural” in origin, based solely on the cranial capacity and assumed lack of cognitive and cultural complexity of the hominins as emphasized by us in Fuentes et al. (2023). We are not the first to observe that some very early hominin situations may represent early mortuary activity (Pettitt 2013), but we would advocate a step further. We suggest it may be damaging to take “natural accumulation” as the standard null hypothesis for hominin paleoanthropology, and that it is more conservative in practice to engage remains with the null hypothesis of possible cultural formation.

We are deeply grateful for the time and effort all of the 8 reviewers (across three reviews) have taken with this work.  We also acknowledge the anonymous reviewers from previous submissions who’s opinions and comments will have made the final iterations of these manuscripts better for their efforts. As this process is rather public and includes commentary outside of the eLife forum, we ask that the efforts of all 37 authors and 8 reviewers involved be respected and that the discourse remain professional in all venues as we study this fascinating and quite complex occurrence. We appreciate also the efforts of members of the public who have engaged with this relatively new process where preprints are posted prior to the reviews allowing comments and interactions from colleagues and the public who are normally not part of the internal peer review process.  We believe these interactions will make for better final papers. We feel we have met the standards of demonstrating burials in H. naledi and that the engraving are most likely associated with H. naledi. However, given the reviews we see many areas where our clarity and context, and analyses, were less strong than they can be. With the clarifications and additions taken on board through these review processes the final papers will be stronger and clearer. We, recognize that this is an ongoing process of scientific investigation and further work will allow continued, and possibly better, evaluation of these hypothesis and others.

Lee R Berger, Agustín Fuentes, John Hawks, Tebogo Makhubela

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Need to be added: Blanton, M. L., Berenson, S. B., & Norwood, K. S. (2001).Exploring a pedagogy for the supervision of prospective mathematics teachers. Journal of Mathematics Teacher Education 4, 177 - 204. https://doi.org/10.1023/A:1011411221421

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Author Response

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

(1) What's the rationale of trypsinizing the tissue prior to mitochondrial isolation? This is not standard for subsequent proteomics analysis. This step will inevitably cause protein loss, especially for the post mitochondrial fractions (PMF). Treating samples with 0.01ug/uL trypsin for 37oC 30 min is sufficient to partially digest a substantial portion of the proteome. If samples from different subjects were not of the same weight, then this partial digestion step may introduce artificial variability as variable proportions of proteins from different subjects would be lost during this step. In addition, the mitochondrial protein enrichment in the mito fraction, despite statistically significant, does not look striking (Figure 1E, ~30% mitochondrial proteins in the mito fraction). As a comparison, Williams et al., MCP 2018 seem to have obtained high mitochondrial protein content in the mito fraction without trpsinizing the frozen quadriceps using a similar SWATH-MS-based approach.

Trypsinisation of the tissue prior to mitochondrial isolation is based on previous work and a Nature Protocol (1, 2) which isolated mitochondria for skeletal muscle. The rationale is that it aids in mechanical homogenisation from highly fibrous tissues such as quadriceps muscle by digesting extracellular matrix proteins. The trypsin/protein ratio used to aid in this process is at least 400 times lower than the amount of trypsin used for formal proteomic tryptic digestion. Three pieces of evidence suggest this step has negligible effect on downstream proteomic analysis. First, because the trypsinisation buffer is detergent free, trypsin will only affect extracellular or exposed membrane proteins. Filtering our PMF dataset for proteins with ‘extracellular matrix’ gene ontology identifies at least 90 unique extracellular matrix proteins indicating good retention of proteins susceptible to partial digestion. Second, the trypsin dose used is 50 times lower than the concentration used for passaging cultured cells, which retain viability after trypsinisation. Third, and contrary to the point raised by the reviewer, we observe less missingness in PMF samples compared to mitochondrial samples. We thank the reviewer for bringing the Williams et al. 2018 MCP paper to our attention. We note that mitochondrial enrichment between the two papers is comparable (~2- fold). To improve clarity line 408 now reads: “Whole quadriceps muscle samples were prepared as previously described with modification (99, 100). First, tissue was snap frozen with liquid nitrogen…” and line 95 reads: “Mitochondrial proteins were defined based on their presence in MitoCarta 3.0 (24) and consistent with previous work (25) were approximately two-fold enriched in the mitochondrial fraction relative to the PMF (Fig 1E).”

(2) The authors mentioned that the proteomics data were Log2 transformed and median- normalized. Would it be possible to provide a bit more details on this? Were the subjects randomized?

Samples were randomised prior to sample processing and mass spectrometry analysis. Because of possible variation in total protein content, it is critical to normalise protein intensities between samples. Median normalisation adjusts the samples so that they have the same median, thereby accounting for technical variation. Log2 normalisation helps to achieve normal distributions, critical for many downstream statistical tests. Line 471 now reads: “…to achieve normal distributions and account for technical variation in total protein.”

(3) In Figure 1D, what were the numbers of mice the authors used for the CV comparisons in each group? Were they of similar age and sex? Were the differences in CV values statistically significant?

The mitochondrial and PMF proteomes originated from the same quadriceps sample from the same mouse, and thus the age and sex are the same across both proteomes. After quality control, we had mitochondrial proteomes for 194 mice and PMF proteomes for 215 mice. The overall CV in the mitochondrial fraction was significantly greater than in the PMF, however whether the source of this variation is biological, or the result of mitochondrial isolation is unclear and as such we have avoided making a statement within the body of the manuscript. We have now more clearly described the nature of the samples in the revised manuscript and added sample sizes to figure 1F.

(4) The authors stated in lines 155-157 that proteins negatively associated with the Matsuda index were further filtered by presence of their cis-pQTLs. Perhaps more explanations would be needed to justify this filtering criterion? Having a cis-pQTL would mean the protein abundance variation is explained by the variation in its coding gene, this however conceptually would not be relevant to its association with the Matsuda index. With the data that the authors have in hand, would it not be natural to align the Matsuda index QTL with the pQTLs (cis and trans if available), and/or to perform mediation analysis to examine causal relationships with statistical significance?

The rationale for filtering by cis-pQTL was not to study the genetics of either Matsuda or associated proteins but rather to identify proteins that were more likely to be causally associated with Matsuda Index as opposed to adaptively associated. To clarify this line 165 now reads: “Filtering based on cis-pQTL presence was based on the rationale that if genetic variation can explain protein abundance differences between mice, then we can be confident that phenotype (Matsuda Index) is not driving the observed differences and therefore the protein-phenotype associations are likely causal. Importantly, this assumption can only be made for cis-acting pQTLs.” Previous work by Matthew et al. (see https://qtlviewer.jax.org/) has demonstrated that cis-pQTL have markedly higher LOD scores than trans-pQTLs, and our own unpublished work suggests that trans-pQTLs do not reproduce well between datasets. The reviewer rightfully suggests aligning protein QTL with those for Matsuda. This is our long-term goal but to identify genome wide significant peaks associated with altered Matsuda will require many more mice than studied here.

(5) It seems a bit odd that the first half of the paper focused extensively on the authors' discoveries in the mitochondrial proteome, and how proteins involved in mitochondrial processes (such as complex I) were associated with Matsuda Index, but the final fingerprint list of insulin resistance, which contained 76 proteins, only had 7 mitochondrial proteins. Was this because many mitochondrial proteins were filtered out due to no cis-pQTL presenting?

There are three reasons our fingerprint is lacking mitochondrial proteins: 1) there are more non-mitochondrial than mitochondrial proteins in the muscle proteome; 2) we focussed on negatively associated proteins, and as demonstrated in figure 2c, the mitochondrial proteome is enriched for positively associated proteins; 3) as implied by the reviewer, we filtered for pQTL presence, further reducing the number of mitochondrial proteins in our fingerprint. To improve clarity, line 170 now reads: “Low mitochondrial representation in the fingerprint is the result of selecting negatively associating proteins, and as seen (Figure 2C) previously, the mitochondrial proteome is enriched for positive contributors to insulin resistance.”

(6) The authors found that thiostrepton-induced insulin resistance reversal effects were not through insulin signalling. It activated glycolysis but the mechanism of action was not clear. What are the proteins in the fingerprint list that led to identification of thiostrepton on CMAP?

Is thiostrepton able to bind or change the expression of these proteins? Since thiostrepton was identified by searching the insulin resistance fingerprint protein list against CMAP, it would be rational to think that it exerts the biological effects by directly or indirectly acting on these protein targets.

This is indeed the implication of our data. Because of the timescales involved it is unlikely that thiostrepton is changing fingerprint protein levels but could be binding to and inhibiting them. Searching the CMAP thiostrepton signature reveals ARHGDIB and NAGK as the fingerprint proteins with the most positive and negative fold-changes respectively perhaps suggesting they play a role in thiostrepton’s mechanism of action. Experiments are underway to test this hypothesis however these are beyond the scope of the current paper.

Reviewer #2 (Public Review):

Line 105: The observation that variance in respiratory proteins is stable while lipid pathways is variable is quite interesting. Is this due to lower overall levels of lipid metabolism enzymes (ex. do these differ substantially from similar pathways ranked from high-low abundance?).

The relationship between coefficient of variation (CV) and relative abundance of proteins is important to consider. To address this, we have now also performed GSEA on proteins ranked from high to low relative abundance. These comparisons have been added to supplementary figure 1 and line 110 now reads: “As a control experiment, we also performed enrichment analysis on proteins ranked by LFQ relative abundance. High CV pathways (enriched for high CV proteins) tended to be lower in relative abundance (enriched for low relative abundance proteins) (Supplementary Fig 1a, b). However, many high variability pathways, lipid metabolism for example, were not enriched in either direction based on relative abundance suggesting differences in relative abundance do not fully explain pathway variability differences.”

Line 154: the 664 associations are impressive and potentially informative. It would be valuable to know which of these co-map to the same locus - either to distinguish linkage in a 2mb window or identify any cis-proteins which directly exert effects in trans-

To assess this, we have analysed pQTL position relative to gene position to generate a ‘hotspot’ plot. We have also generated a histogram of this pQTL density (in a 2 Mbp window) and added these figures to figure 3. We did not detect any obvious pQTL hotspots, and the distribution of pQTLs across the genome appears fairly uniform. Line 159 now reads: “These were distributed across the genome and were predominately cis acting (Figure 3A)...”

Line 194: Cross-platform validation of the CMAP fingerprint results is an admirable set of validations. It might be good to know general parameters like how many compounds were shared/unique for each platform. Also the concordance between ranking scores for significant and shared compounds.

The Connectivity Map (CMap) query included 5163 compounds, the Prestwick library included 1120, and the overlap was 420. We have added these comparisons to supplementary figure 2. Supplementary figure 2 now also contains a comparison of CMap scores between overlapping compounds (found in CMap and the Prestwick library) against all significant compounds identified by CMap (supplementary figure 2b). Interestingly, compounds present in both platforms scored higher on average, suggesting the Prestwick library captures a significant proportion of highly scoring CMap candidates. Line 206 now reads: “In total, 420 compounds were found across both platforms, and these consensus compounds captured a significant proportion of highly scoring CMap compounds (Supplementary Figure 2A, B).”

Line 319: Another consideration in the molecular fingerprint is how unique these are for muscle. While studies evaluating gene expression have shown that many cis-eQTLs are shared across tissues, to my knowledge, this hasn't been performed systematically for pQTLs. Therefore, consider adding a point to the discussion pointing out that some of the proteins might be conserved pQTLs whereas others which would be more relevant here present unique druggable targets in muscle.

To examine tissue specificity, we determined whether our skeletal muscle fingerprint proteins were detected and contained a pQTL in two metabolically important tissues, liver and adipose. Despite detecting almost all the fingerprint proteins in both adipose and liver tissue, they were depleted for pQTL compared to skeletal muscle. These data have now been added to figure 3c. Line 172 now reads: “To assess the tissue specificity of our fingerprint we searched for the same proteins in metabolically important adipose and liver tissues. Despite detecting 94% and 82% of muscle fingerprint proteins across each tissue respectively, both adipose and liver were depleted for pQTL presence (Figure 3C) suggesting that regulation of our fingerprint protein abundance is specific to skeletal muscle.”

Line 332: These are fascinating observations. 1, that in general insulin signaling and ampk were not themselves shown as top-ranked enrichments with matsuda and that this was sufficient to alter glucose metabolism without changes in these pathways. While further characterization of this signaling mechanism is beyond the scope of this study, it would be good to speculate as to additional signaling pathways that are relevant beyond ROS (ex. CNYP2 and others)

We have now added further discussion to the manuscript to address this point., Line 347 now reads: “Aside from glycolysis, other pathways may be involved in enhancing insulin sensitivity. For example, the negatively associated protein ARHGDIA (Figure 2F) is a potent negative regulator of insulin sensitivity, and our fingerprint of insulin resistance contained its homologue ARHGDIB. Both ARHGDIA and ARHGDIB have been reported to inhibit the insulin action regulator RAC1 thus lowering GLUT4 translocation and glucose uptake. Further investigations may uncover a role for thiostrepton in modulating the RAC1 signalling pathway via ARHGDIB.”

Line: 314: Remove the statement: "While this approach is less powerful than QTL co- localisation for identifying causal drivers,", as I don't believe that this has been demonstrated. Clearly, the authors provide a sufficient framework to pinpoint causality and produce an actionable set of proteins.

We have edited line 314, which now reads: “Moreover, our approach has the major advantage that it requires far fewer mice to obtain meaningful outcomes (222 mice in this study) compared to that required for genetic mapping of complex traits like Matsuda Index.”

Line 346: I would highlight one more appeal of the approach adopted by the authors. Given that these compound libraries were prioritized from patterns of diverse genetics, these observations are inherently more-likely to operate robustly across target backgrounds.

This point is further supported by our thiostrepton results in both C57BL6/j and BXH9 mice. Line 317 now reads: “Furthermore, because we have used genetically diverse datasets (DOz mice and multiple cell lines in Connectivity Map) our findings are likely robust across diverse target backgrounds.”

Line 434: I might have missed but can't seem to find where the muscle data are available to researchers. Given the importance and novelty of these studies, it will be important to provide some way to access the proteomic data.

These data are now available via the ProteomeXchange Consortium. Line 465 now reads: “The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (104) partner repository with the dataset identifier PXD042277.”

1. Frezza C, Cipolat S, Scorrano L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured filroblasts. Nat Protoc. 2007;2(2):287-95.

2. Acin-Perez R, Benador IY, Petcherski A, Veliova M, Benavides GA, Lagarrigue S, et al. A novel approach to measure mitochondrial respiration in frozen biological samples. The EMBO Journal. 2020;39(13):e104073.

3. Chick JM, Munger SC, Simecek P, Huttlin EL, Choi K, Gatti DM, et al. Defining the consequences of genetic variation on a proteome-wide scale. Nature. 2016;534(7608):500- 5.

4. Gatti DM, Svenson KL, Shabalin A, Wu L-Y, Valdar W, Simecek P, et al. Quantitative Trait Locus Mapping Methods for Diversity Outbred Mice. G3 Genes|Genomes|Genetics. 2014;4(9):1623-33.

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Reply to the reviewers

We thank the reviewers for their insights and comments on this manuscript. Specific responses to reviewer concerns are detailed below. We made a couple of significant changes based on the feedback. First, we performed more experiments to increase biologic replicates and then quantified image data for multiple figures. The new quantitative information added to Figure 3 fully supports our original conclusions about changes to the ONH in Hes-TKO mutants. The quantification of Atoh7, Otx2, Rbpms and Crx expressing cells among the different genotypes revealed interesting differences in Notch intracellular gene requirements for both RGC and cone development. The most startling outcome is that changes in both cell types correlate with significant changes in Otx2, but not Atoh7. This singular finding suggests interesting future work is needed, well beyond the scope of this paper about the molecular mechanisms underlying these cell fates. Second, our data presentation was reorganized with new information added to Fig 1 that clarifies the relationships between Hes1, Hes5, Foxg1 and Pax2; old Figs 6 & 7 about neurogenesis were merged; and some data moved to new Suppl Figs 2 and 5. The numbering for multiple figures changed and a new summary model (now Fig 8) is provided. In addition, the manuscript was completely rewritten to improve clarity. We hope this revised manuscript is acceptable for publication.

Reviewer #1 Summary:

In this study, the authors employed an impressive set of mouse mutant or Cre lines to investigate the complexity of Notch signaling across different stages of retinal development. These comprehensive analyses led to two main findings: 1. Sustained hes1 in the OHS/OS is Notch-independent; 2. Rbpj and Hes1 exhibited opposing roles in cone photoreceptor development. Although the study is potentially interesting, the current manuscript needs the essential research background and quantification, a lack of which significantly reduced the clarity of the manuscript and the credibility of the major conclusions. Also, how the authors organized the results is quite confusing, making the manuscript very difficult to follow.

Response: We agree with all reviewers concerning incomplete quantification of the data. We directly addressed this shortcoming in revised Figs 3 and 6 (the latter combines old Figs 6 +7). To do this, we repeated some IHC experiments to add more replicates and reorganized all of the neurogenesis phenotypic data figures. Our quantifications uncovered several surprising outcomes that clarify our model. For these reasons, the manuscript was exhaustively rewritten. We merged E13 neurogenesis data into revised Figure 6 and moved the most relevant E16 analyses to new supplemental data Fig 5. All changes made should make the paper easier to understand for retinal development, neurogenesis, and Notch pathway aficionados, in addition to readers lacking such expertise.

Major comments: 1. The authors needed to make the quantification for many analyses to strengthen the conclusions, such as Fig. 1F, 1G, and etc.

Response: We quantified optic nerve head (ONL) immunohistochemistry data in the revised Fig 3. We also quantified neurogenesis markers Atoh7, Otx2, Rbpms (RGCs), and Crx at E13 in revised Fig 6 (former Figs 6 and 7). Older stages were moved to a new Suppl Fig 5.

Respectfully, Hes5 mRNA expression in old Fig 1F and 1G shows that Hes5, like other retinal progenitor cell (RPC) markers, expanded in Rax-Cre deletion but not Chx10-Cre deletion conditions. This is analogous to Pax6 and Rax expansion in Rax-Cre;Hes1 CKO eyes and Pax2 mutants (doi: 10.1523/JNEUROSCI.2327-19.2020) (1). In revised Fig 1, we now show analogous expansion of Hes5 mRNA in Pax2 mutant retinas (compare Figs 1F-1I). Because Hes5 RNA in situ hybridization experiments are nonquantitative, we do not discuss the possibility of Hes5 mRNA level changes in labeled cells.

The authors reported many exciting results. However, further mechanistic insights are largely missing. They may focus on one of these exciting findings and give some mechanistic insights. For example, hes1 suppresses hes5 expression as the ONH boundary forms; hes1 expression in the ONH is Notch independent; differential influences of Rbpj and Hes1 on cone development. It is better for the authors to select one of these exciting findings and provide a deeper mechanistic study.

Response: This revision brings fresh focus to Notch regulation of RGC and photoreceptor development, particularly differential influences for Rbpj versus Hes1. We also better support our interpretation of image data in Fig 1. We include new data about the spatial relationships between Hes5-GFP/Pax2 and Hes5-GFP/Foxg1. In summary, we find that as Pax2 becomes restricted to the nasal optic cup prior to the onset of RGC genesis, it becomes mutually exclusive with Hes5-GFP, at the same time that Hes5-GFP+ cells coexpress Hes1. This is consistent with Hes1 indirectly regulating Hes5-GFP as a marker of neurogenic RPCs at the forming ONH. Furthermore, it emphasizes the importance of genetically teasing apart the separate and potentially compensatory roles for Hes1 versus Hes5 undertaken here. These relationships remain poorly resolved during vertebrate CNS development.

Some analyses lack an explanation of the rationale. For example, "To understand if the loss of multiple Hes genes is more catastrophic than Hes1 alone..."(PAGE 7). Please explain its significance.

Response: We assume the reviewer is referring to the first sentence of the last paragraph on this page. We analyzed Hes triple mutant mice (TKO) to understand if removing multiple Hes genes reveals redundant functions. This is an open question, given that Hes1 is expressed in the ONH/OS, which is normally devoid of Hes5 by the time retinal neurogenesis begins. These questions have only been explored in a handful of tissues throughout the body. Also see response to point 2 above. In general, we have expanded the rationale for all of the experiments throughout the revised manuscript.

Significance: In general, many results are quite interesting. However, the significance of these findings is largely hampered in the following aspects: 1. The authors were unable to provide the sufficient research contexts that are essential for understanding many results.2. Many conclusions were solely based on descriptive images but lacked statistical quantification, which significantly weakened many conclusions. 3. Many interesting findings are quite descriptive, and some mechanistic understandings of one of these exciting findings will be beneficial to improve the focus and significance of the study. Current format of the manuscript fits more specialized audience.

Response: During in vivo development, we wished to understand which particular Notch pathway genes can interact in a Notch-dependent versus a Notch-independent manner. Genetic (phenotypic) studies produce extremely rigorous datasets, in our opinion. This revision now extensively quantifies key findings. Here we dissected the "receipt" of a Notch signal by identically testing the functional requirements of particular pathway members. For Mastermind (Maml), there are 3 paralogues, double mutants for Maml1 and Maml3 are early lethal, and no floxed alleles exist, so it was logical to employ the ROSA-dnMaml mouse strain, particularly since it has been discussed throughout the Notch literature as "analogous" to removing either a Notch receptor or Rbpj. Our finding that the dnMAML allele does not function like a Rbpj null in the retina is important for researchers in the broad Notch field to consider when designing and interpreting experiments.

Reviewer #2: Hes genes are effectors of the Notch signaling pathway but can also act down-stream of other signaling cascades. In this manuscript the authors attempt to address the complexity of Hes effectors during optic cup development and retinal neurogenesis. To do so, they compared optic cup patterning and retinal neurogenesis in seven germline or conditional mutant mouse embryos generated with two spatio-temporally distinct Cre drivers. These lines allowed for the analysis of the consequences of perturbing the Notch ternary complex and multiple Hes genes alone or in combination. The authors show that the optic disc/nerve head is regulated by Notch independent Hes1 function. They also confirm that perturbation of Notch signaling interferes with cell proliferation enhancing the production of differentiated ganglion cells, whereas photoreceptor genesis requires both Rbpj and Hes1 with Notch dependent and independent mechanisms. This is a rather complex study that dissects further the role of the Notch pathway and Hes proteins during eye development, a topic that has been addressed in many previous studies but perhaps not with the details that the authors have used here. In this respect, this study adds to current literature but will likely be of interest to retina aficionados. The manuscript reads well and the figures are of very good quality. However, many of the statements are based on qualitative rather than on quantitative analysis. This should be, at least in some cases, remediated, despite the effort that this may require given the number of mouse lines used in the study.

Response: As described in the response to Reviewer 1, we agree and present considerably more quantification data. We extensively reorganized and rewrote this manuscript to emphasize that Hes1 in the ONH/OS is fully Notch-independent and highlight branchpoints in Notch-dependent signaling, for Rbpj versus Hes,1 during early retinal neurogenesis. It is too simplistic that the ternary complex (Rbpj-NICD-Maml) simply activates Hes1 (and/or multiple Hes genes) to regulate downstream signaling targets. This paradigm has been portrayed in the literature numerous times for many processes throughout vertebrate development, homeostasis or relative to particular diseases. By focusing on one tissue and a narrow window of development, our phenotypic studies delved more deeply to show the greater complexity and molecular cross-talk that we think underlie the modulation of signaling levels with in vivo context. Thus, our results are of broad interest and impact to the greater Notch field.

1. The title is somewhat misleading. The authors have explored mostly the role of Hes1, 3 and5. Although these are Notch effectors, there is already evidence that they participate in other pathways This is confirmed by the data present here. I would suggest to eliminate Notch from the title and use instead "Hes" to better reflect the findings. Furthermore, it is unclear why there is a reference to "mutations" or what are the Notch branchpoints to which the authors refer at the beginning of the discussion.

Response: We appreciate the reviewer’s viewpoint but disagree this paper is mostly about Hes genes, as there is a critical direct, comparable evaluation with Rbpj and dn-Maml. Direct comparison of 7 genotypes highlights where each pathway member exhibits idiosyncratic phenotypes. We are striving for a clear, simple title about a very complex topic, involving the in vivo genetic dissection of a signaling pathway. We modified the title to: "Notch pathway mutations do not equivalently perturb mouse embryonic retinal development "

1. "Although the Pax6-Pax2 boundary is intact in Rax-Cre;RbpjCKO/CKO eyes, ONH shape was attenuated compared to controls (Fig 3I)". This statement is arguable as the difference seems subtle. Perhaps some kind of quantification would help.

Response: We quantified Pax2+ cells (ONH domain) using the adjacent proximal terminus of the retinal pigmented epithelium (RPE) to indicate a transition from ONH to optic stalk (OS). We also quantified the number of Pax2+Pax6+ double positive cells where the 2 domains abut (boundary cells). Some higher magnification examples are now provided in Fig 3H';3K';3N'. Grossly, the imaging data support that the Pax2+ ONH is expanded in Chx10-Cre;TKO eyes, while boundary cells are most affected in Rax-Cre;HesTKO eyes, due to an expansion of retinal tissue. This is supported by our quantitative data (Fig 3O,3P). We observed even in controls that Pax2-expressing cells show some numerical variability. We attributed this to the position of the section through the ONH, which is a 3-dimsenional ring (torus). Therefore, we quantified additional wild-type controls and mutant samples in the new Fig 3O,3P graphs, improving statistical power, and allowing us to detect quantitative differences.

Page 12 first paragraph. "....but all other genotypes were unaffected". This statement is unclear. All lines in which the Rax-Cre has been used seem to have an increased number of apoptotic cells. This should be better explained

Response: Respectfully, only one genotype, Rax-Cre;Rbpj mutants contain a statistically significant increase in apoptotic cells (Fig 5P). This is demonstrated by one-way ANOVA analyses that included all pairwise comparisons. To ensure that the quantification was not misleading due to changes in tissue morphology, data in Figs 5, 6, and 7 were normalized to optic cup area. The area was traced in FIJI, creating a polygon whose area was determined in square microns. For every section image, the marker+ cells were divided by the square micron area of the retina (excluding the opening for the optic nerve). Such a method is critical for comparison across this allelic series, given the morphologic changes, differences in cell clustering where rosettes form, and reduced proliferation whenever Notch signaling is lost or reduced.

Page 12, end of second paragraph: "E13.5 Chx10-Cre;HesTKO eyes had a milder RGC phenotype (Figs 6G, 6N, 6U), but all other mutants were unaffected (Figs 6E, 6F, 6L, 6M, 6S, 6T). This statement is also rather subjective. The phenotype of Chx10-Cre;HesTKO is quite strong and the other mutants seem to have a phenotype. Some quantifications here will help.

Response: We agree and provide quantification for both Atoh7 and Rbpms positive cells in the revised Figure 6. This is now in the same figure with quantification of Otx2+, Otx2+Atoh7+ and Crx+ cells. The reviewer is correct that both ROSA-dnMaml and both HesTKO mutants have a statistically significant increase in RGCs. Surprisingly, neither of the Rbpj CKO mutants have this outcome (Fig 6Y).

1. Page 13, toward the bottom..."...but noted that Chx10-Cre RbpjCKO/CKO eyes were not different from controls (Figs 7E, 7AA)". Again, this statement is questionable as staining for both CRX and Rbpms seem reduced as compared to controls as quantifications in 7AA seems also to indicate (about half?). Did the authors calculate whether there is a statistical difference between controls and Chx10-Cre RbpjCKO/CKO ?

Response: Rbpms+ RGCs and Crx+ photoreceptor precursors were colabeled and quantified on sections for all genotypes. All counts were normalized to area as described above. Upon quantification and ANOVA with pairwise comparisons, there was no statistical difference in Crx+ or Rbpms+ cells between control and Chx10-Cre;Rbpj mutants (new Fig 6Y and Z).

In Fig 7CC the authors should make the effort of including at least one additional sample, 2 biological replicates seem insufficient to draw a conclusion.

Response: The Rax-Cre;Hes1CKO/+ X Hes1CKO/CKO matings stopped producing litters in late 2022. While this manuscript was out for review, we obtained younger mice, from which new control and Rax-Cre; Hes1 mutant littermates were collected, stained, imaged and quantified. Upon adding samples, we found that the outcome was unchanged, but the data better support the lack of a statistical difference in rods between genotypes at E17. These data were moved to revised Suppl Fig 5.

Significance: This is a rather complex study that dissects further the role of the Notch pathway and Hes proteins during eye development, a topic that has been addressed in many previous studies but perhaps not with the details that the authors have used here. In this respect, this study adds to current literature but will likely be of interest to retina aficionados. The manuscript reads well and the figures are of very good quality. However, many of the statements are based on qualitative rather than on quantitative analysis. This should be, at least in some cases, remediated, despite the effort that this may require given the number of mouse lines used in the study.

Response: To increase the impact of our manuscript, we quantified all markers except Tubb3, since its localization in cell bodies and axons make it impossible to assign to individual cells. We feel that this additional quantification strongly improves the quality of our findings and allowed us to make well-supported and novel conclusions. While we certainly believe that the retinal development community will find this paper of interest, it will also be of value to the broader Notch pathway scientific community. In this manuscript, we simultaneously compared phenotypes for Notch pathway genes in signal receiving cells. We could find essentially no studies like this for the mouse CNS and only a few from the Kopan lab about the kidney and immune system. Interestingly, one of us (NLB) is a coauthor on a recent paper about Notch signaling in the cortex, in which ROSA-dnMaml behaves analogously to Notch1CKO or RbpjCKO. This emphasizes that findings in one organ may not recapitulate the "rules" for this pathway for other cell types or tissues (doi: 10.1242/dev.201408)(2). Deeper understanding of how the Notch pathway in the retina functions, analogously or differently, is important. We feel our revised study advances when and where there are "branchpoints" in canonical signaling that may be overlooked in other developing tissues and organs.

Reviewer #3: I have reviewed a manuscript submitted by Bosze et al., which is entitled "Not all Notch pathway mutations are equal in the embryonic mouse retina". The authors focused on Notch signaling pathway. Notch signaling is deeply conserved across vertebrate and invertebrate animal species: in general, two transmembrane proteins, Delta and Notch, interact as a ligand and a receptor, respectively, which induces proteolytic cleavage of Notch receptors to generate Notch intracellular domain (NICD). NICD is translocated into nucleus, then forms the transcription factor complex including Rbpj (also referred to as CBF1) and Mastermind-like (Maml), and activates the transcription of Hes family transcription factors. Three Hes proteins, Hes1, 3, and 5, are important for nervous system development. In the vertebrate developing retina, these Hes proteins inhibit neurogenesis to maintain a pool of neural progenitor cells. In addition to their primary role in neurogenesis, the authors recently reported that Hes1 promotes cone photoreceptor differentiation. In the later stages of development, Hes proteins also promote Müller glial differentiation. In addition, Hes1 is highly expressed in the boundary between the neural retina and optic stalk and required for this boundary maintenance. To understand precise regulation of Notch component-mediated signaling network for retinal neurogenesis and cell differentiation, the authors compared retinal phenotypes in the knockdown of three Notch pathway components, that is (1) Hes1/3/5 cTKO, (2) Rbpj KO, and (3) dominant-negative Maml (dnMaml) overexpression, under the control of two Cre derivers; Rax-Cre and Chx10-Cre. First, the authors found that Hes1 expression in the boundary between optic stalk and neural retina is lost in Rax-Cre; Hes1/3/5 cTKO, but still retained in Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression, suggesting that Delta-Notch interaction is not required for Hes1 expression in the boundary between optic stalk and neural retina. Furthermore, Hes1 expressing boundary region expands distally at the expense of the neural retina in Chx10-Cre; Hes1/3/5 cTKO. Maintenance of ccd2 expression in this expanded boundary area suggests that Hes1 normally maintains a proliferative state in the optic stalk, which may allow these cells to differentiate into astrocyte in later stages. Second, in addition to precocious RGC differentiation in all the Notch component KO, the authors found that, as compared with wild-type, cone and rod photoreceptor genesis is highly enhanced in Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression and mildly enhanced in Chx10-Cre; dnMaml overexpression. On the other hand, in Rax-Cre; Hes1/3/5 cTKO, cone and rod photoreceptor genesis is not enhanced but similar to wild-type level. Since the authors previously reported that cone genesis is reduced in Rax-Cre; Hes1 cKO and Chx10-Cre; Hes1 cKO, so Rax-Cre; Hes1/3/5 cTKO may rescue decrease in cone genesis in single Hes1 cKO. The authors raise the possibility that elevated Hes5 expression in single Hes1 cKO may suppress cone photoreceptor genesis. The authors also found that amacrine cell genesis is significantly suppressed in Rax-Cre; Rbpj KO but not changed in Rax-Cre; dnMaml overexpression and Rax-Cre; Hes1/3/5 cTKO, suggesting that Rbpj is specifically required for amacrine cell genesis. From these observations, the authors propose that there are at least two branchpoints for photoreceptor and amacrine cell genesis in Notch component-mediated signaling network. Their findings are very interesting and provide some new insight on how Notch signaling components are integrated into other signaling pathways and promote to generate diverse but well-balanced retinal cell-types during retinal neurogenesis and cell differentiation, in addition to conventional classic view of Notch signaling pathway. However, one weak point is that, although the authors figured out what kinds of phenotypic difference appear in the KO retinas between these Notch components, the research result is descriptive and less analytical. Most of their conclusions may be supported by their previous works or others; it is still hypothetical. So, it is important to show more analytical data to support their interpretation and more clearly show what is new conceptual advance for Notch signaling pathways.

For example, sustained Hes1 expression in the boundary region between optic stalk and neural retina may be reminiscent to brain isthmus situation. I would like to request the authors to show more direct evidence that Hes1 regulation in optic stalk/retina boundary is independent of Delta-Notch interaction. One possible experiment is whether DAPT treatment phenocopies Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression (Hes1 in optic stalk boundary is normal?).

Response: Usage of the gamma secretase inhibitor DAPT is an interesting experiment as it can phenocopy the loss of Notch signaling in developing tissues. However, the reviewer's proposed DAPT experiment is problematic for two major reasons. First, DAPT blocks the gamma secretase complex, which has more than 90 protein targets in the cell membrane (3). Therefore, DAPT may not be informative for Hes1 regulation given the myriad of expected off-target effects. Second, it would be difficult to treat embryos at the relevant stages with DAPT. Injections into pregnant mice are lethal and we cannot localize drug to the relevant area during in vivo development. Our direct phenotypic comparisons with two Cre drivers strongly indicate that Hes1 is independent of canonical Notch signaling in the developing optic stalk.

We include an extra related data figure (Reviewer Fig 1) showing anti-Hes1 immunolabeling of E13.5 Rax-Cre;Notch1CKO/CKO (n=2) and E13.5 Rax-Cre;Notch2CKO/CKO eyes (n=3). The Notch1 mutant lost oscillating Hes1 expression in retinal progenitors, but the uniform Hes1 ONH domain remains. Interestingly, the Notch2 mutant had essentially no effect on Hes1 (oscillating or sustained), or Hes5 mRNA expression. A Notch2 RNA in situ hybridization demonstrates that Notch2 mRNA was lost in the E13 optic cup and RPE (Rax-Cre expressing tissues). These data emphasize: A) the Notch1-specific dependency of oscillating Hes1 expression in retinal progenitors is absent from the ONH; B) although coexpressed in the same tissue, Notch receptors have unequal activities.

Does Rax-Cre; Rbpj KO; Hes1-cKO phenocopy Rax-Cre; Hes1-cKO (or Rax-Cre; Hes1/3/5 cTKO)?

Response: This is a good question! The first author tried very hard to produce Rax-Cre; Rbpj CKO;Hes1 CKO double mutant embryos. However, these progeny could not be recovered from E10-E13 embryos, despite collecting more than 10 litters. Thus, it is likely that this genotype is lethal before eye formation.

Could the authors identify an enhancer element that drives Hes1 transcription in optic stalk/retina boundary, which should be not overlapped with that of NICD/ Rbpj binding motif? Such additional evidence will make their conclusion more convincing.

Response: Another interesting question. We have been working for >3 years on Hes1 cis regulatory enhancers, but the pandemic greatly delayed progress. The proximal Hes1 600bp upstream region is a generic enhancer that contains Hes1 binding sites for repressing its own expression (4) and has a pair of Rbpj consensus sites for Notch ternary complex activation of Hes1 expression (5,6). Nearby is a binding site occupied by Gli2 in the E16 mouse retina (7). Recently, it was shown that Ikzf4 binds slightly farther away (8). The upstream 1.8 kb region (including the 600bp just described) can drive destabilized GFP or dsRed reporters in early postnatal retinal explants (9). However, this sequence was used to make and analyze a classic Hes1-GFP transgenic reporter mouse, in which GFP was not expressed in the early embryonic mouse optic vesicle or cup (10). Therefore, any early eye-specific enhancer(s) are located farther upstream, in an intron, or downstream (or combination thereof). Public domain epigenetic and chromatin accessibility datasets support this idea. Identifying the gene regulatory logic for Hes1 expression in the eye will be an exciting future story, well beyond this manuscript. We are excited to use live imaging of enhancer reporters to discern oscillating versus sustained activity patterns during early ocular development.

Regarding the conclusion on new branchpoints on photoreceptor and amacrine cell genesis, a model shown in Figure 9 is still hypothetical. Figure 9B indicate a model in which the increase of Otx2+ cells and Crx+ cells in Rax-Cre; Rbpj KO is mediated by Hes1, which is presumed to be activated in Notch-independent signaling. However, Hes1 expression in the neural retina is markedly reduced in Rax-Cre; Rbpj KO (Fig. 2I), which does not fit in with the model.

Response: We removed Fig 9B and now present new models about the Notch-dependent versus -independent roles for both Rbpj and Hes1. The new summary is Fig 8.

So, I would like to request the authors to examine whether the increase of Otx2+ cells and Crx+ cells in Rax-Cre; Rbpj KO, (or Rax-Cre; dnMaml overexpression and Chx10-Cre; dnMaml overexpression) is inhibited by Hes1 KO.

Response: If we understand this correctly, it would mean generating double mutants, some of which we determined are not viable (see the response above, and Suppl Table 2). Given there is only a partial knockdown of Hes1 or Hes5 in either dnMaml mutant we do not believe repeating this in the Hes1 CKO genetic background to be informative and it would take 3 generations to perform.

Second, the authors concluded that both cone and rod genesis are enhanced in Rax-Cre; Rbpj KO by showing the data on Crx/Nr2e3 labeling in Rax-Cre; Hes1 cKO in Fig. 7BB. However, as the authors mentioned in the manuscript, Hes5 expression is elevated in Rax-Cre; Hes1 cKO (Fig. 1G). So, since Rax-Cre; Hes1 cKO has residual Hes activity in the retina, Fig. 7BB should be replaced with labeling of Crx/Nr2e3 in Rax-Cre; Hes1/3/5 cTKO.

Response: Unfortunately, Rax-Cre;HesTKO embryos do not live past E13 (Suppl Table 2). Thus, we cannot evaluate rods, whose genesis starts around E13.5. Revised Fig 1G shows the Hes5 domain is shifted with the expansion of retinal tissue in E13.5 Hes1 single mutants, but importantly, also analogously shifted in Pax2 mutants (Fig 1H). We do not conclude that mRNA levels are "elevated" since mRNA in situ hybridization is not a quantitative technique. Our initial examination of rods in E17 Rax-Cre;Hes1 CKO mutants tested the idea of a fate shift from cones to rods. However, deeper quantification (Suppl Fig 5) do not support such a fate change.

Furthermore, possibly, it is best to examine labeling of the retinas of Rax-Cre; Rbpj KO with rod and cone-specific markers and confirm that the number of both rods and cones is significantly increased. Third, as for defects in amacrine cells genesis in Rax-Cre; Rbpj KO, I would like to request the authors to show the data on Crx10-Cre; Rbpj KO. Although Rbpj KO is mosaic in Crx10-Cre; Rbpj KO, we can distinct Rbpj KO cells by GFP expression (Fig. S2C, C', C'). So, the authors can confirm that amacrine cell genesis is inhibited in a cell-autonomous manner in Crx10-Cre; Rbpj KO retinas but not in Crx10-Cre; dnMaml overexpression. Addition of such data will make the authors' conclusion is more convincing.

Response: Suppl Table 1 lists multiple references (two from the NLB lab) that demonstrated both a rod and cone increase in Rbpj loss-of-function conditions. Chx10;Rbpj CKO animals were evaluated by Zheng et al., who showed an amacrine loss phenotype in these mutants (11). This is equivalent to what we see in our Rax-Cre;Rbpj CKO data, but without the complications of Chx10 mosaic Cre expression upon Rbpj deletion.

Other comments: 1) Title of this manuscript is "Not all Notch pathway mutations are equal in the embryonic mouse retina". However, this title is quite obscure in what is research advancement of their findings. I suggest the authors to include more concrete and conclusive sentence in the title, for example "Hes and Rbpj differentially promotes retina/optic stalk boundary maintenance and photoreceptor genesis, in parallel with neurogenic inhibition by Notch signaling pathway".

Response: We appreciate the reviewer's perspective. We are striving for a relatively simple title about a very complex topic, involving the in vivo genetic dissection of a signaling pathway. We modified the title to "Notch pathway mutations do not equivalently perturb mouse embryonic retinal development ".

2) The "Results" section is a bit difficult to follow logics without detailed knowledge on roles of Notch signaling in mouse retinal development. I suggest the authors to improve a writing style of "Results" section for readers without such detailed knowledge on mouse Notch mutant phenotypes to follow logical flow more easily. There are many additional descriptions on research background before start to mention results. Such introductory sentences should be moved to the "Introduction" section, by which logical flow in the Results section should be simpler. In addition, the authors should show a concrete question at the beginning of each result subsection. Furthermore, the authors sometimes jump over from one result subsection and suddenly move to cite another figure panel in a far ahead subsection whose data has not been explained. Such a back-and-forth citation of figure data generally makes it difficult to follow logical flow.

Response: We now present a considerable amount of new quantified data, reorganized multiple figures, and extensively rewrote the paper. We significantly revised the summary figure to improve clarity. In addition, Suppl Table 1 provides a wealth of background information to orient the reader on this topic. We feel that this extensive revision has greatly improved the quality, logical flow, and readability of the manuscript.

3) In addition, figure configuration is not well organized. Each figure compared some particular marker expression in wild-type, Rax-Cre; HesTKO, Rax-Cre; Rbpj cKO, Rax-Cre; dn-Maml-GFP, Chx10-Cre; HesTKO, Chx10-Cre; Rbpj cKO, Chx10-Cre; dn-Maml-GFP. For example, Fig. 2 shows Hes1 for inhibition of neurogenesis, Fig. 3 shows Vsx2; Mitf and Pax2; Pax6 for retinal pigmented epithelium and optic stalk, Fig. 6 shows Atoh7, Rbpms, and Tubb3 for retinal ganglion cells. Fig. 7 shows Crx, Otx2, and Thrb2 for photoreceptor differentiation. Fig. 8 shows Prdm1, and Ptf1a for photoreceptors and amacrine cells. Although this figure configuration is convenient to show phenotypic difference between different genetic mutations, it is difficult to know how each differentiation steps are spatially and temporally coordinated during development. At least, I recommend the authors to show one summary figure, which shows spatio-temporal expression profile of retinal markers in wild-type mouse retinas.

Response: We recognize this point and completely reorganized and combined Figs 6 and 7 to improve clarity. New Figure 6 presents E13 quantification for Atoh7, Otx2, Atoh7/Otx2, Rbpms and Crx expressing retinal populations. E16-E17 data were condensed and moved to a new Suppl Fig 5.

4a) Page 7, line 7-10 "With earlier deletion using Rax-Cre, hes5 mRNA abnormally extended into the optic stalk": I wonder how the authors define the optic stalk. It is likely that optic stalk area (Pax2+, Vax1+ area) is shifted to more proximal (depart from the optic cup and move toward the brain), and neural retina is expanded accordingly (Fig. 4B, 4F), resulting in expansion of hes5 expression. Thus, it may be better to mention that optic stalk/neural retina boundary is abnormally shifted toward the brain.

Response: The retina, including the optic nerve head, ends where the adjacent RPE terminates. This is conspicuous morphologically in our sections. We also defined this by colabeling for Pax2 and Pax6, which is now quantified in revised Fig 3. To clarify this further, we added the words " in all panels the brain is to the right" in the Fig 4 legend.

4b) Page 8, line 14-15, "ONH/OS cells still express it (Hes1), demonstrating that sustained Hes1 is independent of Notch": I presume that Cre-Rax drives Cre in neural retina as well as optic stalk and pigmented epithelium. However, it is likely that Rbpj is not expressed in optic stalk/neural retina boundary area in wild type (Fig. S2A). No expression of Rbpj in optic stalk/neural retina boundary may support that Hes1 expression in this boundary area is Notch-independent. However, Rbpj expression is retained in some vitreal cells near optic nerve head in Rax-Cre; Rbpj-CKO retinas (Fig. S2B). What are these Rbpj+ cells? I would like to request the authors to confirm that Rbpj expression is completely absent in both neural retina and optic stalk in Rax-Cre; Rbpj-CKO mice. Otherwise, this conclusion is still not fully supported.

Response: We show the Rax-Cre lineage in Suppl Fig 2 via the Ai9 (tomato) reporter. The results are striking, with all of the optic cup derivatives (retina, RPE, ONH, optic stalk, and presumptive ciliary tissue and iris) being tomato positive, while the well-described population of vascular cells in the hyaloid space lack tomato expression. Furthermore, our figure shows that Rbpj expression is only absent from the optic cup derivates, rather than the vascular structures in the vitreous. Vascular cells also depend on the Notch pathway and express Rbpj. Based on considerable evidence from the literature and our lineage experiments, the population of cells the reviewer highlights represents the hyaloid vasculature and associated cell types. It does not represent any population that derives from neuroectoderm.

4c) Page 9, line 16-18, "Foxg1 had spread into the nasal optic stalk": Is Foxg1 expanded nasal area really "OS" rather than expanded retina? I suggest the authors to confirm molecular markers Pax2 expression is overlapped with Foxg1. Otherwise, it is difficult to conclude that foxg1 is expanded into the optic stalk territory, because foxg1 is normally a marker of retina. Indeed, Fig. 3K shows pax2 expression is shifted into more inside towards the brain, suggesting that neural retina is expanded. Please explain the situation.

Response: Foxg1 (BF-1) mRNA and protein are found in the nasal retina and are expressed in other brain tissues. Multiple studies show Foxg1 in the nasal side of the E10 optic cup/retina/optic stalk and developing hypothalamus (See extra data figure Reviewer Fig 2; top row figure is data from Smith et al., 2017 (12) with Foxg1 mRNA in purple. Also see our new manuscript panel Fig 1C. We include here for reviewers (extra data Reviewer Fig 2 showing E13 ocular cryosections colabeled for Foxg1 and Pax2, highlighting their relationship in the retina, optic stalk and adjacent forming hypothalamus. On page 9 the text now reads "At E13.5 Rax-Cre;HesTKO eyes, the Foxg1 nasal retinal domain was contiguous with the nasal optic stalk (Suppl Fig 4D). This is reminiscent of younger stages (Fig 1C), since normally at E13.5, Foxg1 in the nasal optic cup/retina is separated from expression in the ONH/OS (Suppl Fig 4A). Based on the expansion of Pax6, Vsx2 and Hes5 RPC domains into the optic stalk, we conclude that the change in Foxg1 similarly reflects an extension of retinal tissue."

4d) Page 10, line 4-5, In Rax-Cre; Hes1/3/5 cTKO eye, this tissue (RPE) extended into the optic stalk": This description seems to be incorrect. A part of Pax2 area, which is adjacent to the neural retina, contacts with RPE in wild type (Fig. 3AH), so most of RPE covers the neural retina even in Fig. 3DK.

Response: We disagree with the reviewer’s interpretation. Fig 3D shows Mitf labeling of RPE nuclei. Figure 3K shows the adjacent section labeled with Pax2 and Pax6 (labels both retina and RPE). As the retina extended "towards the brain", the RPE analogously extends and surrounds the retinal domain. We also added higher magnification data panels 3H, 3K and 3N, showing merged and single channels.

4e) Page 10, line 22-23, "For Chk10-Cre; Hes1/3/5 cTKO, there was a unique presence of ectopic Pax2 within the retinal territories": I wonder if this description is correct. I suspect that proliferative Pax2+ cells expand into regressing territory of Hes KO retinal cells, which undergo precocious neurogenesis and lose proliferative activity, in Chk10-Cre; HesTKO. In this case, it is possible that the Pax2/Pax6 interface may be maintained. Please show red and green channel panels for Fig. 3N to confirm that there is ectopic pax2 and pax6 double positive cells.

Response: New quantification in revised Fig 3 (see panels O,P) fully supports our original conclusion. Only Chx10-Cre;HesTKO mutants have a statistically significant increase in Pax2+ cells. There are not more Pax2+Pax6+ double labeled cells. Only this particular genotype has an increase in Pax2+ single labeled cells.

5a) Page 11, line 20-25. There seems to be inconsistency between result description and image data of Fig. 5A-G, and histogram Fig. 5O. Authors mentioned that a modest loss of pH3+ cell fraction in Chx10-Cre; Hes1/3/5 cTKO but not in Rax-Cre; Hes1/3/5 cTKO. However, Fig. 5D indicates severe reduction of pH3+ cell fraction in Rax-Cre; Hes1/3/5/ cTKO, which is similar to reduction of pH3+ cell fraction in Rex-Cre; Rbpj (Fig. 5B), but histogram data is different (Fig. 5O). Furthermore, pH3+ cell fraction is severely reduced in Chx10-Cre; ROSA(dn-Maml-GFP) (Fig. 5F) and modestly reduced in Chx10-Cre; Hes1/3/5 cTKO (Fig. 5G). However, pH3+ cell fraction seems to be normal in Chx10-Cre; Rbpj (Fig. 5E). These Chx10-Cre image data do not match the histogram of Fig. 5O. Please check their situation.

Response: Images in old Figs 5-8 were normalized using area measurements, see methods and above comments (note: old Figs 6&7 were combined into new Fig 6). One-way ANOVA with pairwise comparisons for each mutant genotype compared to control were calculated using Prism. All genotypes except two have a statistically significant loss of M phase cells and we discuss possibilities for this outcome (Fig 5O). A normalization method for the sampled area is an essential component of these studies since morphologic differences are apparent for particular genotypes. The quantitative data are consistent with our original conclusions.

5b) Fig. 5H-N, P: I wonder if the stage E13 is appropriate to evaluate cell death and survival because optic cup already becomes smaller in Rax-Cre; Rbpj, Hes1/3/5 cTKO, or ROSA(dn-MAML-GFP) than in wild-type control. I suggest the authors examine more earlier stage.

Response: While an earlier effect is possible, we only observed size differences in a subset of the genotypes. Thus, E13 serves as a critical timepoint to examine early developmental phenotypes across the totality of our mutant conditions. It is also first age when the ONH is fully formed.

5c) Page 12, line 19-20, "all other mutants (Chx10-Cre; Rbpj, and Chx10-Cre; ROSA(dn-MAML-GFP) were unaffected (Fig. 6EF, LM, ST)": It is likely that atoh7 expressing cells are mildly decreased and neuronal marker, Tubb3 and Rbpms-expressing cells are increased in Chx10-Cre; Rbpj, and Chx10-Cre; ROSA(dn-MAML-GFP). I requested the authors to evaluate the fraction of these markers in retinal area statistically in all the cases.

Response: As described above, we quantified Atoh7 and Rbpms nuclear expression by immunohistochemistry. We do not believe that Tubb3+ cells can be reliably quantified. Nonetheless, it is useful to qualitatively show the extent of excess neuron formation. Importantly, we observed that it is not the Atoh7 status that matters for RGC formation, rather it is the Otx2 expression status. This is in good agreement with single cell-RNA transcriptomics data from Wu et al 2021 showing that Atoh7 mRNA in all early transitional RPCs remains fairly constant and its loss does not block the formation of early RGC cell states (13). By contrast Otx2 fluctuates but remains expressed in transitional RPCs that progress to photoreceptor lineages.

6a) Page 7, line 19 "Ectopic blood vessels protruded from the ONH (Fig. 1K, 1L)": It is difficult to see blood vessel structures in these panels (Fig. 1I-L). Please show some molecular marker of blood vessels to confirm how blood vessel is organized in Hes1/3/5 cTKO.

Response: These vascular structures are highly conspicuous by morphology in the H&E insets. Nonetheless, we used adjacent P21 sections to immunolabel for Endomuscin (14) and Tubb3 antibodies. This colabeling confirms the morphology and position of ectopic blood vessels in the abnormal tissue masses in Chx10-Cre;HesTKO mutant eyes. Ectopic tissue contains only rare Tubb3+ cells or cell processes suggesting it is overwhelmingly nonneural. All P21 data were moved to a new Suppl Fig 2. A full detailing of vascular phenotypes is beyond the scope of this manuscript and, interestingly, would be potentially attributable to non-autonomous effects of perturbing the Hes genes in the adjacent retina.

6b) Fig. 5: Increase of pH3 fraction indicates several possibilities, for example (1) increased fraction of mitotic cells due to precocious neurogenesis, (2) increased fraction of mitotic cells due to activated cell proliferation of retinal progenitor cells, (3) increased cell-cycle arrest in M phase due to some stress response of progenitor cells. So, I suggest the authors to examine (1) BrdU percentage of retinal section area, (2) the percentage of pH3+ cells in PCNA+ retinal cells.

Response: The data listed in Suppl Table 1 presents a unified picture that disrupting Notch signaling reduced proliferation. This paradigm extends to other model organisms (e.g., Drosophila, chick, frog, zebrafish and even to nonneural tissues). We included the phospho-histone H3 staining so readers would see how the six mutants evaluated in this study align with this paradigm, providing confidence for the novel findings in other figures. A full evaluation of cell cycle kinetics is interesting, but beyond the scope and focus of this manuscript.

6c) Fig. 5: It is better that cell death fraction will be evaluated by TUNEL and labeling with anti-activated caspase 3 antibody.

Response: We disagree. The DNA repair enzyme PARP is inactivated upon cleavage by activated caspase 3. There are currently ~3,600 citations that use it as a marker of apoptosis. PARP also has a separate and very specific role in maintaining the integrity of sperm DNA. This antibody works on all metazoans and is amenable to many tissue preparations and fixatives, making it easy to use, robust and quantifiable.

7a) Please show red channel (Hes1) image in Fig1BC.

Response: This was added to Revised Fig 1 (Fig 1A).

7b) Fig. 1DH should be shown in neighbor. Fig. 1H should be assigned as Fig. 1E.

Response: The new Fig 1 layout addresses this point.

7c) Fig. S2D, F, H, J: Please show GFP green channel as well. Otherwise, it is difficult to see non-overlapping expression in optic stalk area.

Response: In the revision, this is Suppl Fig 3. Chx-10-Cre is not expressed by ONH-OS cells (1). The green and fuchsia overlap (coexpression) in RPCs is white, we feel this is fairly clear. If needed, all readers can turn on and off the green channel in the final PDF version of this figure to compare GFP with Hes1 expression for those panels.

7d) Fig. 9B: It is better to show Rax-Cre: Hes1/3/5 TKO rather than Rax-Cre: Hes1 cKO. 7e) Fig. 9B: Lettering "Rbpj mutant" should be revised as "Rax-Cre: Rbpj KO".

Response: Fig 9B was removed so these terms are now irrelevant. Our models are presented in new Fig 8.

Significance: The senior author of this manuscript, Dr. Nadean Brown, is an expert scientist who has investigate the role of Notch signaling pathway in vertebrate ocular tissue, including the neural retina and lens. In general, Notch signaling pathway consists of signaling stream from the interaction of Delta and Notch, Notch receptor activation by proteolytic cleavage, translocation of Notch intracellular domain (NICD) into nucleus, formation of transcription factor complex consisting of NICD/Rbpj/Maml, to the transcriptional activation of Notch target genes, Hes family transcription factors. Finally, Hes suppresses neurogenic program and maintain a pool of neural progenitor cells. Therefore, Notch is a key factor to regulate the balance between neurogenesis and progenitor proliferation. In this manuscript, the authors investigated retinal phenotypes in the knockout mice of different Notch signaling components, including Rbpj, Maml, and Hes. They found that functions of these three factors are not always equal in retinal cell differentiation; rather, they specifically regulate a particular step of retinal development. The authors propose the possibility that each of Notch signaling components may be modified by other signaling pathways and achieve some new roles beyond the conventional frame of classic Notch signaling pathway. In this point, this work has a potential to provide a new conceptual advance in the field of developmental and cell biology.

We fully agree this work is a significant advance for the fields of developmental and cell biology. Our findings provide new information and stimulate fresh ideas for anyone working on signal transduction and signal integration.

References cited:

1. Bosze et al., 2020 Journal of Neuroscience Vol 40:1501-13; Bosze et al. 2021 Dev Biol Vol 472:18-29.
2. Han et al., 2023 Development Vol 150 dev201408.
3. Kopan and Ilagan, 2004 Nat Rev Cell Biol. Vol 5:499-504
4. Hirata et al., 2002 Science Vol 298:840-3
5. Friedmann and Kovall, 2010 Protein Sci. Vol 19:34-46
6. Ong et al., 2006 JBC Voll24:5106-19
7. Wall et al., 2009 J Cell Biol. Vo 184: 101-12.
8. Javed et al., 2023 Development Vol 150:dev200436
9. Matuda and Cepko 2007 PNAS Vol 104: 1027-1032
10. Ohtsuka et al., 2006 Mol. Cell Neurosci. Vol 31:109-22
11. Zheng et al., 2009 Molecular Brain Vol 2:38
12. Smith et al., 2017 Journal of Neuroscience Vol 37:7975-93.
13. Wu et al., 2021 Nature Communications Vol 12:1465: doi 10.1038/s41467-021-21704-4
14. Saint-Geniez et al., 2009 IOVS Vol 50: 311-21.

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Referee #3

Evidence, reproducibility and clarity

I have reviewed a manuscript submitted by Bosze et al., which is entitled "Not all Notch pathway mutations are equal in the embryonic mouse retina". The authors focused on Notch signaling pathway. Notch signaling is deeply conserved across vertebrate and invertebrate animal species: in general, two transmembrane proteins, Delta and Notch, interact as a ligand and a receptor, respectively, which induces proteolytic cleavage of Notch receptors to generate Notch intracellular domain (NICD). NICD is translocated into nucleus, then forms the transcription factor complex including Rbpj (also referred to as CBF1) and Mastermind-like (Maml), and activates the transcription of Hes family transcription factors. Three Hes proteins, Hes1, 3, and 5, are important for nervous system development. In the vertebrate developing retina, these Hes proteins inhibit neurogenesis to maintain a pool of neural progenitor cells. In addition to their primary role in neurogenesis, the authors recently reported that Hes1 promotes cone photoreceptor differentiation. In the later stages of development, Hes proteins also promote Müller glial differentiation. In addition, Hes1 is highly expressed in the boundary between the neural retina and optic stalk and required for this boundary maintenance.

To understand precise regulation of Notch component-mediated signaling network for retinal neurogenesis and cell differentiation, the authors compared retinal phenotypes in the knockdown of three Notch pathway components, that is (1) Hes1/3/5 cTKO, (2) Rbpj KO, and (3) dominant-negative Maml (dnMaml) overexpression, under the control of two Cre derivers; Rax-Cre and Chx10-Cre.

First, the authors found that Hes1 expression in the boundary between optic stalk and neural retina is lost in Rax-Cre; Hes1/3/5 cTKO, but still retained in Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression, suggesting that Delta-Notch interaction is not required for Hes1 expression in the boundary between optic stalk and neural retina. Furthermore, Hes1 expressing boundary region expands distally at the expense of the neural retina in Chx10-Cre; Hes1/3/5 cTKO. Maintenance of ccd2 expression in this expanded boundary area suggests that Hes1 normally maintains a proliferative state in the optic stalk, which may allow these cells to differentiate into astrocyte in later stages.

Second, in addition to precocious RGC differentiation in all the Notch component KO, the authors found that, as compared with wild-type, cone and rod photoreceptor genesis is highly enhanced in Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression and mildly enhanced in Chx10-Cre; dnMaml overexpression. On the other hand, in Rax-Cre; Hes1/3/5 cTKO, cone and rod photoreceptor genesis is not enhanced but similar to wild-type level. Since the authors previously reported that cone genesis is reduced in Rax-Cre; Hes1 cKO and Chx10-Cre; Hes1 cKO, so Rax-Cre; Hes1/3/5 cTKO may rescue decrease in cone genesis in single Hes1 cKO. The authors raise the possibility that elevated Hes5 expression in single Hes1 cKO may suppress cone photoreceptor genesis. The authors also found that amacrine cell genesis is significantly suppressed in Rax-Cre; Rbpj KO but not changed in Rax-Cre; dnMaml overexpression and Rax-Cre; Hes1/3/5 cTKO, suggesting that Rbpj is specifically required for amacrine cell genesis. From these observations, the authors propose that there are at least two branchpoints for photoreceptor and amacrine cell genesis in Notch component-mediated signaling network.

Their findings are very interesting and provide some new insight on how Notch signaling components are integrated into other signaling pathways and promote to generate diverse but well-balanced retinal cell-types during retinal neurogenesis and cell differentiation, in addition to conventional classic view of Notch signaling pathway. However, one weak point is that, although the authors figured out what kinds of phenotypic difference appear in the KO retinas between these Notch components, the research result is descriptive and less analytical. Most of their conclusions may be supported by their previous works or others; it is still hypothetical. So, it is important to show more analytical data to support their interpretation and more clearly show what is new conceptual advance for Notch signaling pathways.

For example, sustained Hes1 expression in the boundary region between optic stalk and neural retina may be reminiscent to brain isthmus situation. I would like to request the authors to show more direct evidence that Hes1 regulation in optic stalk/retina boundary is independent of Delta-Notch interaction. One possible experiment is whether DAPT treatment phenocopies Rax-Cre; Rbpj KO and Rax-Cre; dnMaml overexpression (Hes1 in optic stalk boundary is normal?). Does Rax-Cre; Rbpj KO; Hes1-cKO phenocopy Rax-Cre; Hes1-cKO (or Rax-Cre; Hes1/3/5 cTKO)? Could the authors identify an enhancer element that drives Hes1 transcription in optic stalk/retina boundary, which should be not overlapped with that of NICD/ Rbpj binding motif? Such additional evidence will make their conclusion more convincing.

Regarding the conclusion on new branchpoints on photoreceptor and amacrine cell genesis, a model shown in Figure 9 is still hypothetical. Figure 9B indicate a model in which the increase of Otx2+ cells and Crx+ cells in Rax-Cre; Rbpj KO is mediated by Hes1, which is presumed to be activated in Notch-independent signaling. However, Hes1 expression in the neural retina is markedly reduced in Rax-Cre; Rbpj KO (Fig. 2I), which does not fit in with the model. So, I would like to request the authors to examine whether the increase of Otx2+ cells and Crx+ cells in Rax-Cre; Rbpj KO, (or Rax-Cre; dnMaml overexpression and Chx10-Cre; dnMaml overexpression) is inhibited by Hes1 KO. Second, the authors concluded that both cone and rod genesis are enhanced in Rax-Cre; Rbpj KO by showing the data on Crx/Nr2e3 labeling in Rax-Cre; Hes1 cKO in Fig. 7BB. However, as the authors mentioned in the manuscript, Hes5 expression is elevated in Rax-Cre; Hes1 cKO (Fig. 1G). So, since Rax-Cre; Hes1 cKO has residual Hes activity in the retina, Fig. 7BB should be replaced with labeling of Crx/Nr2e3 in Rax-Cre; Hes1/3/5 cTKO. Furthermore, possibly, it is best to examine labeling of the retinas of Rax-Cre; Rbpj KO with rod and cone-specific markers and confirm that the number of both rods and cones is significantly increased. Third, as for defects in amacrine cells genesis in Rax-Cre; Rbpj KO, I would like to request the authors to show the data on Crx10-Cre; Rbpj KO. Although Rbpj KO is mosaic in Crx10-Cre; Rbpj KO, we can distinct Rbpj KO cells by GFP expression (Fig. S2C, C', C'). So, the authors can confirm that amacrine cell genesis is inhibited in a cell-autonomous manner in Crx10-Cre; Rbpj KO retinas but not in Crx10-Cre; dnMaml overexpression. Addition of such data will make the authors' conclusion is more convincing.

Other comments are shown below.

1. Title of this manuscript is "Not all Notch pathway mutations are equal in the embryonic mouse retina". However, this title is quite obscure in what is research advancement of their findings. I suggest the authors to include more concrete and conclusive sentence in the title, for example "Hes and Rbpj differentially promotes retina/optic stalk boundary maintenance and photoreceptor genesis, in parallel with neurogenic inhibition by Notch signaling pathway".
2. The "Results" section is a bit difficult to follow logics without detailed knowledge on roles of Notch signaling in mouse retinal development. I suggest the authors to improve a writing style of "Results" section for readers without such detailed knowledge on mouse Notch mutant phenotypes to follow logical flow more easily. There are many additional descriptions on research background before start to mention results. Such introductory sentences should be moved to the "Introduction" section, by which logical flow in the Results section should be simpler. In addition, the authors should show a concrete question at the beginning of each result subsection. Furthermore, the authors sometimes jump over from one result subsection and suddenly move to cite another figure panel in a far ahead subsection whose data has not been explained. Such a back-and-forth citation of figure data generally makes it difficult to follow logical flow.
3. In addition, figure configuration is not well organized. Each figure compared some particular marker expression in wild-type, Rax-Cre; HesTKO, Rax-Cre; Rbpj cKO, Rax-Cre; dn-Maml-GFP, Chx10-Cre; HesTKO, Chx10-Cre; Rbpj cKO, Chx10-Cre; dn-Maml-GFP. For example, Fig. 2 shows Hes1 for inhibition of neurogenesis, Fig. 3 shows Vsx2; Mitf and Pax2; Pax6 for retinal pigmented epithelium and optic stalk, Fig. 6 shows Atoh7, Rbpms, and Tubb3 for retinal ganglion cells. Fig. 7 shows Crx, Otx2, and Thrb2 for photoreceptor differentiation. Fig. 8 shows Prdm1, and Ptf1a for photoreceptors and amacrine cells. Although this figure configuration is convenient to show phenotypic difference between different genetic mutations, it is difficult to know how each differentiation steps are spatially and temporally coordinated during development. At least, I recommend the authors to show one summary figure, which shows spatio-temporal expression profile of retinal markers in wild-type mouse retinas.
4. There are several logically incorrect sentences or inconsistent sentences in the results section. Please respond my comment below.
   • a) Page 7, line 7-10 "With earlier deletion using Rax-Cre, hes5 mRNA abnormally extended into the optic stalk": I wonder how the authors define the optic stalk. It is likely that optic stalk area (Pax2+, Vax1+ area) is shifted to more proximal (depart from the optic cup and move toward the brain), and neural retina is expanded accordingly (Fig. 4B, 4F), resulting in expansion of hes5 expression. Thus, it may be better to mention that optic stalk/neural retina boundary is abnormally shifted toward the brain.
   • b) Page 8, line 14-15, "ONH/OS cells still express it (Hes1), demonstrating that sustained Hes1 is independent of Notch": I presume that Cre-Rax drives Cre in neural retina as well as optic stalk and pigmented epithelium. However, it is likely that Rbpj is not expressed in optic stalk/neural retina boundary area in wild type (Fig. S2A). No expression of Rbpj in optic stalk/neural retina boundary may support that Hes1 expression in this boundary area is Notch-independent. However, Rbpj expression is retained in some vitrial cells near optic nerve head in Rax-Cre; Rbpj-CKO retinas (Fig. S2B). What are these Rbpj+ cells? I would like to request the authors to confirm that Rbpj expression is completely absent in both neural retina and optic stalk in Rax-Cre; Rbpj-CKO mice. Otherwise, this conclusion is still not fully supported.
   • c) Page 9, line 16-18, "Foxg1 had spread into the nasal optic stalk": Is Foxg1 expanded nasal area really "OS" rather than expanded retina? I suggest the authors to confirm molecular markers Pax2 expression is overlapped with Foxg1. Otherwise, it is difficult to conclude that foxg1 is expanded into the optic stalk territory, because foxg1 is normally a marker of retina. Indeed, Fig. 3K shows pax2 expression is shifted into more inside towards the brain, suggesting that neural retina is expanded. Please explain the situation.
   • d) Page 10, line 4-5, In Rax-Cre; Hes1/3/5 cTKO eye, this tissue (RPE) extended into the optic stalk": This description seems to be incorrect. A part of Pax2 area, which is adjacent to the neural retina, contacts with RPE in wild type (Fig. 3AH), so most of RPE covers the neural retina even in Fig. 3DK.
   • e) Page 10, line 22-23, "For Chk10-Cre; Hes1/3/5 cTKO, there was a unique presence of ectopic Pax2 within the retinal territories": I wonder if this description is correct. I suspect that proliferative Pax2+ cells expand into regressing territory of Hes KO retinal cells, which undergo precocious neurogenesis and lose proliferative activity, in Chk10-Cre; HesTKO. In this case, it is possible that the Pax2/Pax6 interface may be maintained. Please show red and green channel panels for Fig. 3N to confirm that there is ectopic pax2 and pax6 double positive cells.
5. There seems to be some mismatch descriptions between image data and histogram (or text in the result section). Please respond my comments below.
   • a) Page 11, line 20-25. There seems to be inconsistency between result description and image data of Fig. 5A-G, and histogram Fig. 5O. Authors mentioned that a modest loss of pH3+ cell fraction in Chx10-Cre; Hes1/3/5 cTKO but not in Rax-Cre; Hes1/3/5 cTKO. However, Fig. 5D indicates severe reduction of pH3+ cell fraction in Rax-Cre; Hes1/3/5/ cTKO, which is similar to reduction of pH3+ cell fraction in Rex-Cre; Rbpj (Fig. 5B), but histogram data is different (Fig. 5O). Furthermore, pH3+ cell fraction is severely reduced in Chx10-Cre; ROSA(dn-Maml-GFP) (Fig. 5F) and modestly reduced in Chx10-Cre; Hes1/3/5 cTKO (Fig. 5G). However, pH3+ cell fraction seems to be normal in Chx10-Cre; Rbpj (Fig. 5E). These Chx10-Cre image data do not match the histogram of Fig. 5O. Please check their situation.
   • b) Fig. 5H-N, P: I wonder if the stage E13 is appropriate to evaluate cell death and survival because optic cup already becomes smaller in Rax-Cre; Rbpj, Hes1/3/5 cTKO, or ROSA(dn-MAML-GFP) than in wild-type control. I suggest the authors examine more earlier stage.
   • c) Page 12, line 19-20, "all other mutants (Chx10-Cre; Rbpj, and Chx10-Cre; ROSA(dn-MAML-GFP) were unaffected (Fig. 6EF, LM, ST)": It is likely that atoh7 expressing cells are mildly decreased and neuronal marker, Tubb3 and Rbpms-expressing cells are increased in Chx10-Cre; Rbpj, and Chx10-Cre; ROSA(dn-MAML-GFP). I requested the authors to evaluate the fraction of these markers in retinal area statistically in all the cases.
6. Some experiments are necessary to improve their design. Please respond my comments below.
   • a) Page 7, line 19 "Ectopic blood vessels protruded from the ONH (Fig. 1K, 1L)": It is difficult to see blood vessel structures in these panels (Fig. 1I-L). Please show some molecular marker of blood vessels to confirm how blood vessel is organized in Hes1/3/5 cTKO.
   • b) Fig. 5: Increase of pH3 fraction indicates several possibilities, for example (1) increased fraction of mitotic cells due to precocious neurogenesis, (2) increased fraction of mitotic cells due to activated cell proliferation of retinal progenitor cells, (3) increased cell-cycle arrest in M phase due to some stress response of progenitor cells. So, I suggest the authors to examine (1) BrdU percentage of retinal section area, (2) the percentage of pH3+ cells in PCNA+ retinal cells.
   • c) Fig. 5: It is better that cell death fraction will be evaluated by TUNEL and labeling with anti-activated caspase 3 antibody.
7. Panel configuration of Figures should be revised as below.
   • a) Please show red channel (Hes1) image in Fig1BC.
   • b) Fig. 1DH should be shown in neighbor. Fig. 1H should be assigned as Fig. 1E.
   • c) Fig. S2D, F, H, J: Please show GFP green channel as well. Otherwise, it is difficult to see non-overlapping expression in optic stalk area.
   • d) Fig. 9B: It is better to show Rax-Cre: Hes1/3/5 TKO rather than Rax-Cre: Hes1 cKO.
   • e) Fig. 9B: Lettering "Rbpj mutant" should be revised as "Rax-Cre: Rbpj KO".

Significance

The senior author of this manuscript, Dr. Nadean Brown, is an expert scientist who has investigate the role of Notch signaling pathway in vertebrate ocular tissue, including the neural retina and lens. In general, Notch signaling pathway consists of signaling stream from the interaction of Delta and Notch, Notch receptor activation by proteolytic cleavage, translocation of Notch intracellular domain (NICD) into nucleus, formation of transcription factor complex consisting of NICD/Rbpj/Maml, to the transcriptional activation of Notch target genes, Hes family transcription factors. Finally, Hes suppresses neurogenic program and maintain a pool of neural progenitor cells. Therefore, Notch is a key factor to regulate the balance between neurogenesis and progenitor proliferation. In this manuscript, the authors investigated retinal phenotypes in the knockout mice of different Notch signaling components, including Rbpj, Maml, and Hes. They found that functions of these three factors are not always equal in retinal cell differentiation; rather, they specifically regulate a particular step of retinal development. The authors propose the possibility that each of Notch signaling components may be modified by other signaling pathways and achieve some new roles beyond the conventional frame of classic Notch signaling pathway. In this point, this work has a potential to provide a new conceptual advance in the field of developmental and cell biology.

Author Response

Reviewer #1 (Public Review):

The objective of this investigation was to determine whether experimental pain could induce alterations in cortical inhibitory/facilitatory activity observed in TMS-evoked potentials (TEPs). Previous TMS investigations of pain perception had focused on motor evoked potentials (MEPs), which reflect a combination of cortical, spinal, and peripheral activity, as well as restricting the focus to M1. The main strength of this investigation is the combined use of TMS and EEG in the context of experimental pain. More specifically, Experiment 1 investigated whether acute pain altered cortical excitability, reflected in the modulation of TEPs. The main outcome of this study is that relative to non-painful warm stimuli, painful thermal stimuli led to an increase on the amplitude of the TEP N45, with a larger increase associated with higher pain ratings. Because it has been argued that a significant portion of TEPs could reflect auditory potentials elicited by the sound (click) of the TMS, Experiment 2 constituted a control study that aimed to disentangle the cortical response related to TMS and auditory activity. Finally, Experiment 3 aimed to disentangle the cortical response to TMS and reafferent feedback from muscular activity elicited by suprathreshold TMS applied over M1. The fact that the authors accompanied their main experiment with two control experiments strengthens the conclusion that the N45 TEP peak could be implicated in the perception of painful stimuli.

Perhaps, the addition of a highly salient but non-painful stimulus (i.e. from another modality) would have further ruled out that the effects on the N45 are not predominantly related to intensity/saliency of the stimulus rather than to pain per se.

We thank the reviewer for their comment on the possibility of whether stimulus salience influences the N45 as opposed to pain per se. However, we note that in Experiment 1, despite the same level of stimulus salience/intensity for all participants (46 degrees), individual differences in pain ratings were associated with the change in the N45 amplitude, suggesting that the results cannot be explained by stimulus intensity/salience.

Reviewer #2 (Public Review):

The authors have used transcranial magnetic stimulation (TMS) and motor evoked potentials (MEPs) and TMS-electroencephalography (EEG) evoked potentials (TEPs) to determine how experimental heat pain could induce alterations in these metrics.In Experiment 1 (n = 29), multiple sustained thermal stimuli were administered over the forearm, with the first, second, and third block of stimuli consisting of warm but non-painful (pre-pain block), painful heat (pain block) and warm but non-painful (post-pain block) temperatures respectively. Painful stimuli led to an increase in the amplitude of the fronto-central N45, with a larger increase associated with higher pain ratings. Experiments 2 and 3 studied the correlation between the increase in the N45 in pain and the effects of a sham stimulation protocol/higher stimulation intensity. They found that the centro-frontal N45 TEP was decreased in acute pain.

The study comes from a very strong group in the pain fields with long experience in psychophysics, experimental pain, neuromodulation, and EEG in pain. They are among the first to report on changes in cortical excitability as measured by TMS-EEG over M1.

While their results are in line with reductions seen in motor-evoked responses during pain and effort was made to address possible confounding factors (study 2 and 3), there are some points that need attention. In my view the most important are:

1) The method used to calculate the rest motor threshold, which is likely to have overestimated its true value : calculating highly abnormal RMT may lead to suprathreshold stimulations in all instances (Experiment 3) and may lead to somatosensory "contamination" due to re-afferent loops in both "supra" and "infra" (aka. less supra) conditions.

The method used to assess motor threshold was the TMS motor threshold Assessment Tool (Awiszus et al., 2003). This was developed as a quicker alternative for calculating motor threshold compared to the traditional Rossini-Rothwell method which involves determining the lowest intensity that evokes 5/10 MEPs of at least 50 microvolts. The method has been shown to achieve the same accuracy of determining motor threshold as the traditional Rossini-Rothwell method, but with fewer pulses (Qi et al., 2011; Silbert et al., 2013). Therefore, the high RMTs in our study cannot be explained by the threshold assessment method. Instead, they are likely explained by aspects of the experimental setup that increased the distance between the TMS coil and the scalp, including the layer of foam placed over the coil, the EEG cap and the fact that the electrodes we used had a relatively thick profile.

Awiszus, F. (2003). TMS and threshold hunting. In Supplements to Clinical neurophysiology (Vol. 56, pp. 13-23). Elsevier.

Qi, F., Wu, A. D., & Schweighofer, N. (2011). Fast estimation of transcranial magnetic stimulation motor threshold. Brain stimulation, 4(1), 50-57.

Silbert, B. I., Patterson, H. I., Pevcic, D. D., Windnagel, K. A., & Thickbroom, G. W. (2013). A comparison of relative-frequency and threshold-hunting methods to determine stimulus intensity in transcranial magnetic stimulation. Clinical Neurophysiology, 124(4), 708-712.

2) The low number of pulses used for TEPs (close to ⅓ of the usual and recommended)

We agree that increasing the number of pulses can increase the signal to noise ratio. During piloting, participants were unable to tolerate the painful stimulus for long periods of time and we were required to minimize the number of pulses per condition.

We note that there is no set advised number of trials in TMS-EEG research. According to the recommendations paper, the number of trials should be based on the outcome measure e.g., TEP peaks vs. frequency domain measures vs. other measures and based on previous studies investigating test-retest reliability (Hernandez-Pavon et al., 2023). The choice of 66 pulses per condition was based on the study by Kerwin et al., (2018) showing that optimal concordance between TEP peaks can be found with 60-100 TMS pulses delivered in the same run (as in the present study). The concordance was particularly higher for the N40 peak at prefrontal electrodes, which was the key peak and electrode cluster in our study.

Further supporting the reliability of the TEP data in our experiment, we note that the scalp topographies of the TEPs for active TMS at various timepoints (Figures 5, 7 and 9) were similar across all three experiments, especially at 45 ms post-TMS (frontal negative activity, parietal-occipital positive activity).

In addition to this, the interclass correlation coefficient (Two-way fixed, single measure) for the N45 to active suprathreshold TMS across timepoints for each experiment was 0.90 for Experiment 1 (across pre-pain, pain, post-pain time points), 0.74 for Experiment 2 (across pre-pain and pain conditions), and 0.95 for Experiment 3 (across pre-pain conditions). This suggests that even with the fluctuations in the N45 induced by pain, the N45 for each participant was stable across time, further supporting the reliability of our data. These ICCs will be reported in the next revision.

Hernandez-Pavon, J. C., Veniero, D., Bergmann, T. O., Belardinelli, P., Bortoletto, M., Casarotto, S., ... & Ilmoniemi, R. J. (2023). TMS combined with EEG: Recommendations and open issues for data collection and analysis. Brain Stimulatio, 16(3), 567-593

Kerwin, L. J., Keller, C. J., Wu, W., Narayan, M., & Etkin, A. (2018). Test-retest reliability of transcranial magnetic stimulation EEG evoked potentials. Brain stimulation, 11(3), 536-544.

Lack of measures to mask auditory noise.

In TMS-EEG research, various masking methods have been proposed to suppress the somatosensory and auditory artefacts resulting from TMS pulses, such as white noise played through headphones to mask the click sound (Ilmoniemi and Kičić, 2010), and a thin layer of foam placed between the TMS coil and EEG cap to minimize the scalp sensation (Massimini et al., 2005). However, recent studies have shown that even when these methods are used, sensory contamination of TEPs is still present, as shown by studies that show commonalities in the signal between active and sensory sham conditions that mimic the auditory/somatosensory aspects of real TMS (Biabani et al., 2019; Conde et al., 2019; Rocchi et al., 2021). This has led many authors (Biabani et al., 2019; Conde et al., 2019) to recommend the use of sham conditions to control for sensory contamination. To separate the direct cortical response to TMS from sensory evoked activity, Experiment 2 (n = 10) included a sham TMS condition that mimicked the auditory/somatosensory aspects of active TMS to determine whether any alterations in the TEP peaks in response to pain were due to changes in sensory evoked activity associated with TMS, as opposed to changes in cortical excitability. Therefore, the lack of auditory masking does not impact the main conclusions of the paper.

Ilmoniemi, R. J., & Kičić, D. (2010). Methodology for combined TMS and EEG. Brain topography, 22, 233-248.

Massimini, M., Ferrarelli, F., Huber, R., Esser, S. K., Singh, H., & Tononi, G. (2005). Breakdown of cortical effective connectivity during sleep. Science, 309(5744), 2228-2232.

Biabani, M., Fornito, A., Mutanen, T. P., Morrow, J., & Rogasch, N. C. (2019). Characterizing and minimizing the contribution of sensory inputs to TMS-evoked potentials. Brain stimulation, 12(6), 1537-1552.

Conde, V., Tomasevic, L., Akopian, I., Stanek, K., Saturnino, G. B., Thielscher, A., ... & Siebner, H. R. (2019). The non-transcranial TMS-evoked potential is an inherent source of ambiguity in TMS-EEG studies. Neuroimage, 185, 300-312.

Rocchi, L., Di Santo, A., Brown, K., Ibáñez, J., Casula, E., Rawji, V., ... & Rothwell, J. (2021). Disentangling EEG responses to TMS due to cortical and peripheral activations. Brain stimulation, 14(1), 4-18.

3) A supra-stimulus heat stimulus not based on individual HPT, that oscillates during the experiment and that lead to large variations in pain intensity across participants is unfortunate.

The choice of whether to calibrate or fix stimulus intensity is a contentious question in experimental pain research. A recent discussion by Adamczyk et al., (2022) explores the pros and cons of each approach and recommends situations where one method may be preferred over the other. That paper suggests that the choice of the methodology is related to the research question – when the main outcome of the research is objective (neurophysiological measures) and researchers are interested in the variability in pain ratings, the fixed approach is preferrable. Given we explored the relationship between MEP/N45 modulation by pain and pain intensity, this question is better explored by using the same stimulus intensity for all participants, as opposed to calibrating the intensity to achieve a similar of pain across participants.

Adamczyk, W. M., Szikszay, T. M., Nahman-Averbuch, H., Skalski, J., Nastaj, J., Gouverneur, P., & Luedtke, K. (2022). To calibrate or not to calibrate? A methodological dilemma in experimental pain research. The Journal of Pain, 23(11), 1823-1832.

So is the lack of report on measures taken to correct for a fortuitous significance (multiple comparison correction) in such a huge number of serial paired tests.

Note that we used a Bayesian approach for all analyses as opposed to traditional frequentist approach. In contrast to the frequentist approach, the Bayesian approach does not require corrections for multiple comparisons (Gelman et al., 2000) given that they provide a ratio representing the strength of evidence for the null vs. alternative hypotheses as opposed to accepting or rejecting the null hypothesis based on p-values. As such, throughout the paper, we frame our interpretations and conclusions based on the strength of evidence (e.g. anecdotal/weak, moderate, strong, very strong) as opposed to referring to the significance of the effects.

Gelman A, Tuerlinckx F. (2000). Type S error rates for classical and Bayesian single and multiple comparison procedures. Computational statistics, 15(3):373-90.

Reviewer #3 (Public Review):

The present study aims to investigate whether pain influences cortical excitability. To this end, heat pain stimuli are applied to healthy human participants. Simultaneously, TMS pulses are applied to M1 and TMS-evoked potentials (TEPs) and pain ratings are assessed after each TMS pulse. TEPs are used as measures of cortical excitability. The results show that TEP amplitudes at 45 msec (N45) after TMS pulses are higher during painful stimulation than during non-painful warm stimulation. Control experiments indicate that auditory, somatosensory, or proprioceptive effects cannot explain this effect. Considering that the N45 might reflect GABAergic activity, the results suggest that pain changes GABAergic activity. The authors conclude that TEP indices of GABAergic transmission might be useful as biomarkers of pain sensitivity.

Pain-induced cortical excitability changes is an interesting, timely, and potentially clinically relevant topic. The paradigm and the analysis are sound, the results are mostly convincing, and the interpretation is adequate. The following clarifications and revisions might help to improve the manuscript further.

1) Non-painful control condition. In this condition, stimuli are applied at warmth detection threshold. At this intensity, by definition, some stimuli are not perceived as different from the baseline. Thus, this condition might not be perfectly suited to control for the effects of painful vs. non-painful stimulation. This potential confound should be critically discussed.

In Experiment 3, we also collected warmth ratings to confirm whether the pre-pain stimuli were perceived as different from baseline. We did not include this data initially in the first submission, but will do so in the supplemental material in our next revision. This data showed warmth ratings were close to 2/10 on average. This confirms that the non-painful control condition produced some level of non-painful sensation.

2) MEP differences between conditions. The results do not show differences in MEP amplitudes between conditions (BF 1.015). The analysis nevertheless relates MEP differences between conditions to pain ratings. It would be more appropriate to state that in this study, pain did not affect MEP and to remove the correlation analysis and its interpretation from the manuscript.

The interindividual relationship between changes in MEP amplitude and individual pain rating is statistically independent from the overall group level effect of pain on MEP amplitude. Therefore, conclusions for the individual and group level effects can be made independently.

It is also important to note that in the pain literature, there is now increasing emphasis placed on investigating the individual level relationship between changes in cortical excitability and pain as opposed to the group level effect (Seminowicz et al., 2019; Summers et al., 2019). As such, it is important to make these results readily available for the scientific community.

Summers, S. J., Chipchase, L. S., Hirata, R., Graven-Nielsen, T., Cavaleri, R., & Schabrun, S. M. (2019). Motor adaptation varies between individuals in the transition to sustained pain. Pain, 160(9), 2115-2125.

Seminowicz, D. A., Thapa, T., & Schabrun, S. M. (2019). Corticomotor depression is associated with higher pain severity in the transition to sustained pain: a longitudinal exploratory study of individual differences. The Journal of Pain, 20(12), 1498-1506.

3) Confounds by pain ratings. The ISI between TMS pulses is 4 sec and includes verbal pain ratings. Considering this relatively short ISI, would it be possible that verbal pain ratings confound the TEP? Moreover, could the pain ratings confound TEP differences between conditions, e.g., by providing earlier ratings when the stimulus is painful? This should be carefully considered, and the authors might perform control analyses.

It is unlikely that the verbal ratings contaminated the TEP response as the subsequent TMS pulse was not delivered until the verbal rating was complete and given that each participant was cued by the experimenter to provide the pain rating after each pulse (rather than the participant giving the rating at any time). As such, it would not be possible for participants to provide earlier ratings to more painful stimuli. We will make this part of the protocol clearer in the next revision of the manuscript.

4) Confounds by time effects. Non-painful and painful conditions were performed in a fixed order. Potential confounds by time effects should be carefully considered.

Previous research suggests that pain alters neural excitability even after pain has subsided. In a recent meta-analysis (Chowdhury et al., 2022) we found effect sizes of 0.55-0.9 for MEP reductions 0-30 minutes after pain had resolved. As such, we avoided intermixing pain and warm blocks given subsequent warm blocks would not serve as a valid baseline, as each subsequent warm block would have residual effects from the previous pain blocks.

At the same time, given there was no conclusive evidence for a difference in N45 amplitude between pre-pain and post-pain conditions of Experiment 1 (Supplementary Figure 1), it is unlikely that the effect of pain was an artefact of time i.e., the explanation that successive thermal stimuli applied to the skin results an increase in the N45, regardless of whether they are painful or not. We will make this point in our next revision.

Chowdhury, N. S., Chang, W. J., Millard, S. K., Skippen, P., Bilska, K., Seminowicz, D. A., & Schabrun, S. M. (2022). The Effect of Acute and Sustained Pain on Corticomotor Excitability: A Systematic Review and Meta-Analysis of Group and Individual Level Data. The Journal of Pain, 23(10), 1680-1696.

5) Data availability. The authors should state how they make the data openly available.

We will upload the MEP, TEP and pain data on the Open science framework at the time of the next revision.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

1. General Statements [optional]

Reply to general assessment of referee #2:

1. General assessments: The current study adds some to these observations…some of these observations are incremental…biological significance is limited. While this reviewer does not suggest additional experimentation, this manuscript would be suitable as a resource paper.

Reply: It appears we were not clear enough in explaining the novel aspects of our study.

The starting points are two published studies from our lab demonstrating a global increase of ISGF3 association with ISG promoters in IFNγ-treated cells and a remarkable similarity of IFN-γ and type I IFN-induced early transcriptome changes. These findings challenge the notion in the field (as mentioned by the referee) that IFNγ specificity is produced by the predominant deployment of STAT1 homodimers. We thus tested the hypothesis that the specificity of the IFNγ-induced transcriptome is generated over time, rather than during the early response, and relies on secondary responses to transcription factors such as IRF1. In contrast, IRF1 plays no or only a small role in the type I IFN response that utilises ISGF3 and/or unknown secondary factors in the delayed response. We tested this hypothesis with PRO-seq technology to rule out confounding effects of mRNA processing over a 48h period. The data are clear in showing that many genes associated with the antibacterial or anti parasite profile of activated macrophages are indeed much more abundant in late-stage rather than briefly IFNγ-treated macrophages and these delayed changes are to a large extent dependent on IRF1. Our findings are based on the best available technologies, a combination of nascent transcript analysis with genetics and protein interaction studies. In addition, our findings rule out alternative models of sustained or secondary ISG transcription, such as the employment of alternative ISGF3 complexes (such as STAT2-IRF9) or of ISGF3 complexes formed with unphosphorylated STAT1 and STAT2. We provide evidence for higher order waves of transcription caused by unknow transcription factors that are produced by transcriptional activation of ISGF3 or IRF1 target genes and identify candidates among the AP1 and Ets transcription factor families. We agree that some of the data are confirmatory rather than novel (i.e. some of the genes we describe were known from previous literature to be IRF1 targets), but it is the systems approach of our study, and particularly the delineation of conditions under which the largely neglected delayed response diverts the IFNβ and IFNγ-induced transcriptomes, that generates a comprehensive and conclusive view of IFNγ acting predominantly as a macrophage activating factor, and IFNβ being an essential antiviral cytokine. We do think this main outcome is immunologically meaningful and not incremental. For this reason, we would prefer to publish the paper as a relevant contribution to innate immunology rather than a resource. Emphasizing our point, a paper appeared in ‘Cell’ while our study was under review, showing that human IRF1 mutations cause mendelian susceptibility to mycobacterial disease (MSMD), a term coined by JL Casanova and colleagues for immunological defects that reduce the ability of macrophages to cope with intracellular bacteria (new ref. 65). This important study emphasizes the main conclusions of our study about the relevance of IRF1 for macrophage activation. We discuss this paper on p. 14 lines 9-14.

Revision: We tried to better explain the scientific motivation for this study and the significance of the results (p. 4, lines, lines 12-25).

Revision plan: n. a.

2. Description of the planned revisions

Referee #3; major comment 1:

In Fig. 1d is difficult to interpret and misleading for many reasons. First, the cluster numbering is disconnected from the cluster order; why not numbering them based on the hierarchical clustering and writing the cluster number besides the cluster itself? Second, having a 2-color gradient is misleading; negative values shouldn't be in the same color tone than the positive values. Third, the authors did not provide adequate rationale behind using only the top 1,000 most expressed gene? Why not using all the differentially expressed genes in at least one of the condition to provide a comprehensive analysis? Could this potentially lead to bias in the data, and is there any information lost by not using the - lower - expressed genes fraction? Fourth, it is not clear what the color scale is representing and how the data was transformed. Was a mean centering of the expression values of the log2FC applied to the RNA-seq data to facilitate clustering? Mean centering and z-scoring is a common technique used to adjust expression data, but it can potentially exaggerate differences between samples. More information about the data and analysis should be provided, as it is difficult to determine whether this was a valid approach or not.

Reply:

• To create the heatmap, we used the pheatmap package from R and the cutree_rows option to separate 11 clusters with strikingly different patterns of gene expression based on visual exploration. The numbering was autogenerated by the program.
• The data is now shown in red-blue.
• We restricted our list to only 1000 genes from each comparison as we aimed to analyze the prominent patterns of gene expression across timepoints. Considering all differentially expressed genes based on a padj value would also include genes expressed at very low levels as evident from the low baseMean values obtained from DESeq2. Hence, we applied a selection of 1000 genes which effectively represented the major patterns of gene expression across timepoints.
• Variance stabilized transformation was applied on read counts obtained from PRO-seq using the DESeq2 package. The transformed reads were z-score normalized and used for performing hierarchical clustering by the “Ward.D2” method using the pheatmap package in R. A total of 3126 genes were used for this analysis. 11 distinct clusters were defined using cutree_rows option. The color scale represents z-score normalized counts. The genes represented in the heatmap were selected based on the following criteria: each timepoint of interferon treatment was compared to the homeostatic condition (untreated sample) in wildtype BMDMs. The differentially expressed genes from each comparison were selected based on the filtering criteria: absolute log2FoldChange >=1 and adjusted p value <0.01 by Wald test. Following the differential analysis, the first 1000 differentially expressed genes in each treatment condition (ordered based on adjusted p values) were selected for both IFN types and combined and selected for creating a list which consisted of 3126 unique genes. The scale in the heatmap represents z-scores of variance-stabilized reads, calculated across all genotype and treatment conditions, separately for each IFN type.

Revision plan: We will label the clusters with the cluster number next to it in addition to the color codes.

Referee #3; major comment 3:

The large standard deviation bars in the claim that ChIP data confirmed the binding of ISGF3 components to the promoter of Mx2 cast doubt on the validity of the results and conclusions. The authors should consider additional experiments or complementary analyses to validate their findings. Or alternative, to adjust their claims accordingly.

Reply: To demonstrate sufficient quality of the data the ratio of Stat1/ Stat2 was calculated for early (1.5hrs) and late (48h) separately. The unpaired two-tailed t test comparing this ratio between 1.5 hrs and 48hs, shows that they are not significantly different. This indicates that all ISGF3 components are associated with ISG during both early and delayed responses, i. e., that STAT2/IRF9 complexes are unlikely to contribute to delayed ISG control. However, we agree with the referee that the standard deviations of the kinetic ChIP experiment are high and that it would be good to generate additional data.

Revision plan: We will perform additional ChIP experiments to improve the statistical power of the results in fig. S2c.

Referee #3, major comment 6:

The authors interpret their ATAC-seq and ChIP-seq results based on a 2kb window to the TSS of genes, not considering relatively close enhancers or longer range cis-regulatory interactions in their interpretation. For example, they mention on p.7 "Contrasting the strong binding of IRF9 and IRF1 to the Mx2 (cluster 2) and Gbp2 (cluster 9) promoters, respectively, we saw no evidence for direct binding to Lrp11 (cluster 3) and Ptgs2 (cluster 10)", but on Fig 3d they show only the proximal regions. No scale bars are shown either. Moreover, exploring the same published IRF1 ChIP-seq dataset, there is a clear IRF1 binding site at the promoter of Ptgs2, while the authors report none.

Reply:

• According to the literature (e. g. refs. 11, 27), most IFN-induced accessibility changes occur in the vicinity of the TSS of ISG. This is further strengthened by the data shown in this manuscript. In addition, most functionally validated GAS and ISRE sequences are in the DNA interval chosen for our analysis. While distal ISG enhancers have been reported (e. g. DOI: 10.26508/lsa.202201823), an analysis beyond the placement of most control regions increases the risk of wrong assignments between ISG and their regulatory elements, hence the causality between transcription factor binding and accessibility changes.
• We extended the regions for the analysis of the Lrp11 and Ptgs2 regulatory regions and found no evidence for the binding of ISGF3 or IRF1. We find no evidence for a clear peak in the Ptgs2 promoter. There is a peak called by the Macs2 algorithm, but visual inspection of the track (bigwig file) shows it consists of a minor increase in reads above background that does not suggest a bona fide IRF1 binding site (see below). This view is supported by our inability to find an IRF binding site in the vicinity of the peak.

IRF1 binding indicated by bigWig browser tracks and corresponding peakfiles detected at the locus. We identified the peakfile from Langlais et al., 2016 and identified peaks using MACS2, however using mm10 genome as the analysis in the original paper was done with mm9 genome. The peak identified here appears to be an artefact of the MACS2 program as there is no evident enrichment at the gene promoter region upon inspection of the bigWig files.

Revision plan: Scales will be added to the browser tracks as requested.

Referee #3, major comment 7:

Lack of statistical analysis on chromatin accessibility claims: The authors claim that ATAC-seq data in BMDMs stimulated with IFNβ or IFNγ for a short (1.5 hours) or long (48 hours) period reveals a striking similarity between transcription and the general trends of chromatin accessibility at regions up to 1000 bp upstream of the TSS (Fig. 2a), suggesting continuous chromatin remodeling during the transcriptional response. However, I would like to know if this conclusion is well-supported by the correlation between the chromatin accessibility from ATAC-seq data from only one sample and the PRO-seq data.

Reply: See revision plan.

Revision plan: We will analyze single experiments whether they support the conclusions derived from the z-score of the triplicate samples.

Referee #3, major comment 8:

The need for additional experiments to verify claims such as the dependence of Ifi44 on IRF1 for gaining ATAC signal, as stated in the claim, "Expression required IRF1 for both, but accessibility of the Ifi44 regulatory region depended upon IRF1 whereas that of Gbp2 acquired an open structure independently of IRF1 (Fig. 5c).

Reply: We think the lack of clarity might be related to the size of figures 5a and 5b and the density of the dots in some areas of the plot. We agree it is very difficult to assign our gene labels unambiguously to a single dot.

Fig. 5a combines ATACseq data in wt and IRF1 knockout cells with the expression data from the Pro-seq experiment, Fig. 5b is the same set-up, but IRF9-deficient macrophages are analyzed.

Blue dots show ATACseq signals induced by IFN treatment. Violet dots represent genes that require IRF1 (Fig. 5a) or IRF9 (Fig. 5b) for transcriptional induction. Yellow dots mark genes such as IFI44 requiring IRF1 (Fig. 5a) or IRF9 (Fig. 5b) for both expression and the accessibility change in the promoter region. Fig. 5c visualizes representative examples of genes whose accessibility is coupled to the transcription factor dependence of the transcriptional induction (IFI44), or not (Gbp2). Thus Fig. 5c must be interpreted based on the dot color code in fig. 5a and we admit this has been difficult with the figure in its present form.

Revision plan: We will improve the clarity of figs 5a and 5b in several ways:

• We will label the panels to better indicate the intersected data sets.
• We will increase the size of the panels and figure legends and make sure that the correspondence between gene names and dots are unambiguous.
• We will include trend lines of the Ifi44 and Gbp2 genes to visualize their induction and IRF1 dependence.

Referee #3, major comment 13 (see also section 3):

The authors have not adequately addressed the methodological limitations in their discussion, which extends beyond the aforementioned comments. It is suggested they include a comprehensive discussion of the claims made pertaining to the necessity of IRF1 for accessibility and the potential biases in the interactomes, along with their associated consequences.

Reply: The contribution of IRF1 to the accessibility of ISG promoters emerges from the data in figures 5a, whose clarity will be improved (see reply to point 8). We do not interpret the impact of IRF1 beyond the data, in fact we state a relatively minor effect of IRF1 in the control of promoter accessibility (p. 10, lines 20-22) and we have added a reference in agreement with an impact of IRF1 on basal expression of antiviral genes (ref. 39, as suggested by the referee).

We have added discussion on potential limitations of the TurboID approach (p. 11, lines 22-24 and p. 15, lines 3-11).

Revision plan: Improvement of fig 5a (see ref. #3, point 8).

Referee #3, minor comment 2

Fig 1e. The color scales on the GO enrichment graphs are misleading since they use the same blue-to-red gradient for adj p-values ranging from 10-25 to 10-49 and 0.008 to 0.016, which could be considered non significant.

Reply: We agree that this is confusing. It results from automated assignments of the color gradients by the software.

Revision plan: We will investigate possibilities to change color codes for different ranges of p values.

Referee #3, minor comment 4

The incomplete schema in Figure 1a, which only focuses on PRO-seq and does not include the ATAC-seq element.

Reply: We will add a new figure to visualize the set-up of the ATAC seq experiments and their intersection with the Pro-seq data.

Revision plan: We will add a new figure in accordance with the referee’s request.

Referee #3, minor comment 6

The clearer labeling of Figure 5a and 5b.

Reply: Please refer to our reply to major point 8.

Referee #3, minor comment 10

Fig S1b, S3b. The PRO-seq was generated in triplicates, hence these graphs should include the Log2FC for the individual data points.

Reply: The Log2FC from DESeq2 were calculated from the triplicates, the software does not compute Log2FC from individual replicates.

Revision plan: We mention the p-values for the Log2FC to show the degree of consistency (figure legends). We will provide a table with log2FC and corresponding padj values of the genes represented at each timepoint (table_showing_padj_values_and_log2fc).

Referee #3, minor comment 12

In the genomic snapshot shown, only bars or fading triangles are shown in place of the gene body. The authors should provide an accurate gene structure; i.e., exons and introns.

Reply: We will try to include the exon-intron structure wherever the size of the figure allows this.

Revision: n. a.

Revision plan: If figure size permits, we will add the exon-intron structure of the genes in browser tracks as requested.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Referee #1, major comment 1

Figure 2. Difficult to interpret data as it is presented. Consider quantifying figure 2C in order to make "changes in Pol II pausing were more pronounced during IFNb signaling" statement more apparent.

Reply: We presented the pausing data in two different graphic representations (figures 2c and S2) to make the understanding of the information content easier. In hindsight we may have generated more confusion than clarity.

Revision: We removed the original figure 2c and replaced it with original figure S2. This representation is quite intuitive as the graphs represent a direct quantitative logarithmic display whether and how much the relative amount of paused polymerase changes when comparing IFN-treated and untreated cells. The calculation of these ratios is now explained better in the legend to figure 2.

Referee #1, major comment 2

How are you distinguishing autocrine signaling in the BMDMs driven by IFN treatment from late transcripts (for example, at 48 hours are differential genes due to autocrine cytokine signaling or are they truly late transcripts)?

Reply: We do not exclude autocrine effects. In case of ISG, the most likely autocrine factor would be secreted interferon. According to our Proseq data, the differentially expressed genes do not include any interferon genes. That being said, it is possible that the transcription factors from the AP1 family we hypothesize as drivers of secondary or tertiary waves of transcription are activated by non-IFN cytokines secreted from IFN-treated cells (see also reply to comment 3).

Revision: We now mention that enhanced IFN production is not sustaining ISG responses (p.5 lines 18/20). We mention the possibility that secreted factors may drive secondary or tertiary waves of ISG transcription (p. 8, lines 21/23).

Referee #1, major comment 3

Figure 3D. Authors choose Gbp2 (as positive control for IFNg driven gene), but don't show that Gbp2 is a IFNb independent gene. Consider using IRF1 KO BMDMs in this data as well.

Reply: This is a misunderstanding. Gbp2 is not shown as an IFNγ-specific gene (it’s induction by both IFN types has been shown previously and emerges from our Pro-seq analysis, see also response to minor issue no. 2). It represents the cluster of genes that are sustained specifically after IFNγ treatment in an IRF1-dependent manner. The purpose of fig. 3D is to show that not all ISGF3/IRF9-dependent genes have promoter binding sites for ISGF3 and not all IRF1-dependent genes have binding sites for IRF1. This suggests indirect effects of both transcription factors in sustaining IFN-induced transcription (in line with the referee’s comment 1).

Previous figure S3e (now S2f) confirms binding of IRF1 to the GBP2 promoter by ChIP with kinetics correlating to its transcriptional effect. This experiment is normalized with an IgG control. IRF1 knockout cells did not produce a ChIP signal with IRF1 antibody, as expected (data not shown).

Revision: We better explain the rationale behind the experiments shown in figure 3D (text on p8, lines 12-16). In addition, we show the trend line of Gbp2 expression in WT vs IRF1KO as well as that of additional genes showing delayed/sustained responses in the new Figure S3.

Referee #1, minor comment 2

Define known IFNg and IFNb driven genes when they are introduced in figure 2 rather than in discussion.

Reply: Following the referee’s suggestion we provide the examples of IFNβ and IFNγ-controlled genes and the characteristics of their regulation in the context of our description of the results displayed by fig. 2 (p.6 lines 15-21). This includes Gbp2 (see major issue no. 3).

Revision: The text on p. 6 lines 15-21 has been modified in accordance with the request.

Referee #1, minor comment 4

Unclear whether IRF1 expression in figure 3A is from whole cell lysate or nuclear fraction.

Reply: We indicate in the figure legend that whole cell lysates were used.

Revision: We added a sentence with the relevant information in the legend of figure 3.

Referee #1, minor comment 5

Authors suggest IFNb treatment induces less IRF1 at later time points, however loading control also seems slightly lower than other considerations. Is it possible that IFNb treated cells are dying at later time points, given that type I IFN signaling can be pro-apoptotic.

Reply: The graph below the blot represents quantified IRF1 signals, normalized to the loading control. It shows that the differences are not generated by unequal loading of the blotted gel. We and others have shown that IFNβ may indeed enhance macrophage death, however only when the cells are simultaneously infected with an intracellular pathogen (e.g. new ref. 25). These studies also show that treatment with IFNβ alone over periods used in the present study does not affect macrophage viability.<br /> Revision: We added a sentence about the viability of IFN-treated macrophages (p. 4, lines 31-32).

Revision plan: n. a.

Referee #2, major comment 3

The sequencing and BioID data are not submitted to public databases.

Reply: An accession number has been added.

Revision: The accession number was added on p.29, line 25.

Referee #3, major comment 1 (see also revision plan, section 2):

Revision: The rationale for using the top 1.000 genes is explained (p.5, lines 7-9). The description of the pro-seq read count processing has been extended in accordance with our reply to the referee in the legend of figure 1d and in the methods section (p. 33, lines following line 10.)

Referee #3, major comment 2

Fig 2c. The authors claim that RNA Pol II pausing is a major factor in controlling the dynamics of ISG transcription. However, they did not provide sufficient explanation of the results, and in all fairness there is not much variation between the clusters to sustain the claim that this is a major factor in ISG transcriptional control.

Reply: We agree with the referee that we cannot posit RNA pol II pausing as a major factor for the differences of transcriptional control of ISG in individual clusters. We have made sure to remove any statements suggesting this possibility. We also try to better integrate our findings with RNA pol II pausing into the existing literature.

Revision: We added relevant literature on p. 6 lines 28-30 and p. 7, lines 4-6.

Referee #3, major comment 4

On p.5, the authors mention "Representative browser tracks from the Gbp2 and Slfn1 genes further validate this observation" but they are simply referring to genome browser snapshot, i.e., specific genomic examples, extracting from the same single dataset. Without using an independent dataset, this can not "further validate" the initial findings.

Reply: We agree the wording is incorrect.

Revision: We changed the paragraph describing this experiment (p. 6, lines 15-21).

Referee #3, major comment 5

IRF1 was successfully pulled down with STAT1 bait but not in the reciprocal experiment. The author should discuss this point as it is important for the conclusions. Could it potentially indicate issues with the technique used, and if this could introduce any bias into the results. The statement, "In contrast, interactors of the IRF1 bait did not include STAT1. This discrepancy could result from steric constraints of the tagged proteins due to the limitation of the 10nm distance reached by the biotin ligase," does not seem to be sufficient to explain this discrepancy.

Reply: STAT1 was present in the IRF1 pull-down and the interaction increased significantly after IFN treatment but after normalization to the NLS control it did not conform to our criterium of a 95% confidence interval for the FDR. To be consistent we did not include it in the list of IRF1 interactors. We have observed on several occasions that the significance of proximity is not reciprocal, even for well- documented physical interactions. A prime example for this is the interaction between STAT1 and IRF9 in IFN-treated cells which is recorded in the STAT1 pull-down, but not that with IRF9 (ref. 10). Apart from steric reasons the lack of reciprocity may result from different signal/noise ratios in pull downs with different baits.

Revision: We mention that IRF1 was a STAT1 interactor below the statistical cut-off (p. 11, lines 26-28) as well as the possibility of different signal/noise ratios in the IRF1 and STAT1 pull-downs on p.11, lines 22-24.

Referee #3, major comment 9

In the figure legends, there is missing information about the number of times experiments were replicated, suggesting that some were done a single time. Moreover, some graphs are missing statistical analysis, e.g., in Fig S3cS3e, S3f, the ChIP-qPCR experiments were done on biological triplicates, there is no mention of statistical test performed, it is not mentioned what the error bars represents (SD, SEM, etc.) and the variance is large, but the authors still interpret these results as significant enrichment of the transcription factors to the Mx2 promoter.

Reply: Where missing the relevant information has been added to figure legends. In brief, all experiments represent at least three biological replicates. The only exception is the western blot shown in figure S3a, (no S2a) which represents two independent replicates. Here, the clarity of the difference of IRF1 expression and the fact that the only purpose is to show that Raw264.7 macrophages behave like bone marrow-derived macrophages in fig. 3a justifies the omission of another replicate (please see also answer to point 3).

Revision: The relevant information has been added to figure legends where necessary (figs. 1, a, 3a, 6a-f, S1, S4, S5).

Referee #3, major comment 10

Another example are the RNA Pol II pausing index ratios, which show minor variations and not are supported by statistics to support a possible significance. Proper description, replication and statistical analyses of the results are critical.

Reply: We agree.

Revision: Statistics underlying the RNA Pol II pausing data are included in supplementary data 2.

Referee #3, major comment 11

The authors used CRISPR-Cas9 genome editing to generate knockout cell lines. However, they did not verify the knockouts at the protein level. Further experiments could confirm that the targeted proteins are not expressed in the knockout cell lines.

Reply: We included a western blot showing the lack of IRF1 and STAT1 expression in the respective cell lines.

Revision: New figure S6.

Referee #3, major comment 12

On p.9, it is mentioned "IRF1 affects chromatin structure ...". Here chromatin structure is related to minor changes in chromatin accessibility, this can not be qualified as changes in chromatin structure.

Reply: ‘structure’ has been changed in accordance with the request.

Revision: ‚structure‘ has been replaced with ‘accessibility’. (p. 10, lines 19 and 21).

Referee #3, major comment 13 (see also section 2, revision plan, major comment 8)

The authors have not adequately addressed the methodological limitations in their discussion, which extends beyond the aforementioned comments. It is suggested they include a comprehensive discussion of the claims made pertaining to the necessity of IRF1 for accessibility and the potential biases in the interactomes, along with their associated consequences.

Reply: The contribution of IRF1 to the accessibility of ISG promoters emerges from the data in figures 5a, whose clarity will be improved (see reply to point 8). We do not interpret the impact of IRF1 beyond the data, in fact we state a relatively minor effect of IRF1 in the control of promoter accessibility (p. 10, lines 20-22) and we have added a reference in agreement with an impact of IRF1 on basal expression of antiviral genes (ref. 39, as suggested by the referee).

We have added discussion on potential limitations of the TurboID approach (p. 11, lines 22-24 and p. 15, lines 3-11).

Revision: Change of the discussion section (p. 11, lines 22-24 and p. 15, lines 3-11).

Revision plan: Improvement of fig 5a (see ref. #3, point 8).

Referee #3, major comment 15

The work should be discussed in the context of the demonstrated physiopathological evidence of the IRF1 and IRF9 functions. IRF9 (Hernandez et al., JEM 2018) and more recently IRF1 (Rosain et al Cell, 2023) were identified as causing non overlapping phenotypes in human patients carrying loss-of-function mutations for these genes. The authors must interpret their results in this context.

Reply: We thank the referee for reminding us about the importance of these papers for our work.

Revision: The papers have been mentioned and discussed (p. 13 lines 19-28 and p.14, lines 9-14).

Referee #3, minor comment 3

The inconsistency in the title referring to IFNb as Type 1 but using IFNg instead of Type 2 nomenclature, perhaps consistency is best.

Reply: We agree about the importance of consistency but find ourselves in yet another quandary. While the use of ‘type I IFN’ is clearly indicated and widely used as a collective name for this group of cytokines, the use of ‘type II IFN’ for IFNγ is rare because it is the only member of this type. Hence, we decided for sticking with convention at the expense of a bit of consistency. We agree about the title, though, and have changed type I IFN to IFNβ.

Revision: We adapted the title in agreement with the referee’s comment.

Referee #3, minor comment 5

Figure 6d includes a color scale of -1 to +3, but it is unclear what these values represent and how they were calculated per interactor. The figure legend should be revised to clarify this information.

Reply: We agree. The relevant information has been added to the figure legend.

Revision: We added information (log2FC with regard to the NLS control) to the legend of fig. 6d.

Referee #3, minor comment 9

Fig 1e, S1c. Graphs having circles of varying sizes in function of a value are named "bubble plots" and not "dot plots".

Reply: Thank you for pointing this out, we corrected our mistake.

Revision: We changed dot plot to bubble plot in legend to figure S1c.

Referee #3, minor comment 11

Fig S3c legend. It is mentioned "Graph represents RT-qPCR of genomic Mx2". RT-qPCR usually stands for reverse transcription quantitative PCR, hence we suggest to change to "ChIP-qPCR" or qPCR. Confusingly, in the literature the term "RT-PCR" is used for real-time PCR and "qPCR" for quantitative PCR. Also, the authors should be specific about the "genomic" region targeted; the graphs mention "promoter", hence it would be appropriate to use the same designation in the legend.

Reply: We agree and thank the referee for correction of the terminology.

Revision: We changed RT-PCR to qPCR throughout the manuscript. Moreover, we specifically refer to ‘promoter region’ as the amplified DNA.

Referee #3, minor comment 12

Fig S3e. The y-axis names are missing.

Reply: Thanks for spotting this.

Revision: The y axis in the figure received its proper label.

Referee #3, minor comment 14

Raw cells are sometimes spelled as "Raw" and other times as "RAW". Please choose one for consistency.

Revision: This inconsistency has been corrected

Referee #3, minor comment 15

In p.10 l.20, the figure number is missing.

Revision: We corrected this mistake.

4. Description of analyses that authors prefer not to carry out

Referee #1, minor comment 1

Simplify figure 4B- consider focusing on most differentially expressed genes between clusters

Reply: The purpose of fig. 4B is to provide a visual overview of the kinetics of eRNA transcription in response to both IFN types and of the effects of IRF9 and IRF1 knockouts. This information needs to be given to demonstrate the similarities and differences between the control of eRNA and the corresponding ISG transcripts in the different regulatory clusters (as shown in figs. 1d and 2a).

Simplifying the figure would mean to separate it according to time point, IFN type treatment or knock-out effect. We think this would require to mentally reassemble the figure to understand the interrelationships between these parameters. To our opinion the visual display of the data interrelationship in fig. 4B facilitates the impropriation of the information content.

Revision: n. a. - we hope our reasoning has become sufficiently clear.

Revision plan: n. a.

Referee #1, minor comment 3

Clarify which cell types (IRF1 KO vs IRF9 KO) are used in figure 5 A/B.

Reply: The cell type (bone marrow-derived macrophages) is mentioned in the first sentence of the figure legend. Since all experiments except the Bio-ID experiment were performed with this cell type we decided not to label each figure.

Revision: n. a.

Revision plan: n. a.

Referee #2, major comment 2 and referee #3, major comment 14

Ref #2: Biological significance is limited as this study is largely descriptive and they do not test the hits obtained from BioID.

Ref #3: Although the TurboID experiments identify known STAT1 and IRF1 interactors, the proposed new interactors are numerous, and none are validated through independent co-IP experiments. Moreover, the results are very noisy, with little differences between untreated BMDMs (where IRF1 is barely expressed) and IFN-treated conditions.

Reply: The big advantage of BioID or TurboID is the ability to score proximity and very transient interactions. Validating BioID hits with technologies such as coIP is not particularly useful as the two technologies will obviously produce different interactomes. In fact, we show in this manuscript that IRF1 and STAT1 show proximity, but they do not form a stable complex under co-IP conditions. This leaves genetic approaches (LOF or GOF) as alternatives. However, apart from the workload (> 100 genes would have to be knocked out or their products overexpressed), most of our hits are expected to produce very broad effects in such experiments, hard to interpret regarding ISGF3 and IRF1 activities.

In view of this situation, we publish exclusively the high confidence nuclear interactors identified in our screen: biological replicates were performed in triplicate, a stringent internal control (TurboID-NLS) was used, and a stringent statistical cut-off for high-confidence interactors (95% FDR between groups) was applied. We further account for the experimental situation by limiting interpretation of the data to confirmed molecular events. For example, STAT1 dimers and the ISGF3 complex are required for histone acetylation in response to IFN, and ISGF3 is known to contribute to the exchange of the H2AZ histone variant (refs 11, 14, 71, 72). Our data show that IRF1 contributes to promoter accessibility changes and this is in line with its proximity to a remodelling complex. Thus, the BioID data indeed validate previous findings. However, in agreement with the referee’s comment, some of the data remain descriptive (such as the intriguing proximity of both STAT1 and IRF1 to nuclear products of ISG). To determine the importance of this molecular proximity is a major undertaking and beyond the scope of this study.

Revision: We added discussion to state the difficulty of validating TurboID-based interactions and the limitations of the TurboID experiments (p.15 lines 3-11).

Referee #3, minor comment 1

In most graphs the expression values or log2FC are shown separately for IFNb and IFNg, however in the heatmaps (Fig 1d, S1d) the IFNb and IFNg results are intercalated keeping them side-by-side for each time point, which makes them more difficult to interpret.

Reply: We are in a quandary about the design of the figure. On the one hand our goal is to visualize gene clusters with distinct behaviors for each IFN type. For this purpose, it would be advantageous to separate the IFN types. On the other hand, we aim at showing similarities and differences between genes induced by each IFN type, for this purpose it is better to maintain the current sample order. While understanding the referee’s point, we prefer to keep the figure as it is, because the suggested change will not increase its overall clarity.

Revision: n. a.

Revision plan: n. a.

Referee #3, minor comment 7

The statement that "IFN-I are the more important mediators of antiviral immunity" is not entirely accurate and may be an oversimplification, as there are certainly articles which suggest a larger role for type ll IFN elements than type l (ref: Yamane D et al., 2019 Nature microbiology). While yes, IFN-I plays a critical role in the innate immune response to viral infections, IFNγ also has antiviral activity and is involved in the adaptive immune response to viral infections, and in some instances to a larger extent than IFN l.

Reply: The Yamane et al study (now mentioned on p 10, lines 22-25 and referenced) agrees with our findings because it shows that IRF1 contributes to the basal expression of an ISRE-driven ISG subset. Our statement about the predominant role of type I IFN versus IFNγ refers to genetic data in both humans (mainly Casanova’s work including effects of autoantibodies against type I IFN, see also the paper about human STAT2 deficiency in the June 15th issue of the JCI, https://doi.org/10.1172/JCI168321) and mice (hundreds of papers) showing that disruption of type I IFN synthesis or response causes profound effects of antiviral immunity (i.e. resulting susceptibilities are first and foremost to viral pathogens) whereas susceptibilities as a consequence of disrupting the IFNγ pathway are first and foremost to intracellular nonviral pathogens such a mycobacteria. In fact, the term mendelian susceptibility to mycobacterial disease (MSMD) was coined by Casanova and colleagues to describe a variety of human mutations that include those of the IFNγ, but not the type I IFN pathway.

Maybe more importantly, the Rosain et al. paper mentioned by the referee which appeared in ‘Cell’ while our study was under review, shows that human IRF1 mutations also fall into the MSMD category (new ref. 65). In contrast, the authors did not observe diminished antiviral immunity. This emphasizes the main conclusions of our study about the relevance of IRF1 for macrophage activation. We discuss this paper on p 14. lines 9-14.

Obviously, this does not exclude a role of type I IFN in nonviral infection or of IFNγ in viral infection, in fact much of our own work has been dedicated to a role of type I IFN in infections with L. monocytogenes. Nevertheless, we think that in a generic statement about the difference between type I IFN and IFNγ it is correct to label the former as predominantly antiviral and the latter predominantly as a macrophage activating factor against nonviral, intracellular pathogens.

Revision: We added discussion of Rosain et al. (ref. 65) on p 14. lines 9-14.

Referee #3, minor comment 8

The authors claim that a significant portion of ISG promoters is associated with ISGF3 upon IFNγ receptor engagement and that the transcriptomes of macrophages treated briefly with IFNβ or IFNγ exhibit remarkable similarity and sensitivity to Irf9 deletion. However, I am uncertain about the extent of consensus on this claim.

Reply: The data were surprising but supported by ChIP-seq and RNA-seq in wt and IRF9 ko macrophages (ref 10). Data in a follow-up study (ref. 11) and in this manuscript support our original conclusion by demonstrating the impact of the IRF9 ko on IFNγ responses. Importantly, we don’t claim this is true in all cell types, it may well depend on STAT/IRF9 expression levels and tonic IFN signaling.

Revision: n. a.

Revision plan: n. a.

Reviewer #3 (Public Review):

Summary:<br /> The present article attempts to answer both the ultimate question of why different stinging behaviours have evolved in Cnidiarians with different ecological niches and shed light on the proximate question of which electro-physiological mechanisms underlie these distinct behaviours.

Account of major methods and results:<br /> In the first part of the paper, the authors try to answer the ultimate question of why distinct dependencies of the sting response on internal starvation levels have evolved. The premise of the article that Exaiptasia's nematocyte discharge is independent of the presence of prey (Artemia nauplii) as compared to Nematostella's significant dependence of the discharge on the presence of actual prey, is shown be a robust phenomenon justified by the data in Figure 1.

The hypothesis that defensive vs. predatory stinging leads to different nematocyte discharge behaviours is analysed in mathematical models based on the suitable framework of optimal control/decision theory. By assuming functional relations between the:<br /> 1) cost of a full nematocyte discharge and the starvation level.<br /> 2) probability of successful predation/avoidance on the discharge level.<br /> 3) desirability/reward of the reached nutritional state.

Based on these assumptions of environmental and internal influences, the optimal choice of attack intensity is calculated using Bellman's equation for this problem. The model predictions are validated using counted nematocytes on a coverslip. The scaling of normalised nematocyte discharge numbers with scaled starvation time is qualitatively comparable to what is predicted from the models. The abundance of nematocytes in the tentacles was, on the other hand, independent of the starvation state of the animals.

Next, the authors turn to investigate the proximate cause of the differential stinging behaviour. The authors have previously reported convincing evidence that a strongly inactivating Cav2.1 channel ortholog (nCav) is used by Nematostella to prevent stinging in the absence of prey (Weir et al. 2020). This inactivation is released by hyperpolarising sensory inputs signalling the presence of prey. In this article, it is clearly shown by blocking respective currents that Exaiptasia, too, relies on extracellular Ca2+ influx to initiate stinging. Patch clamp data of the involved currents is provided in support. However, the authors find that in addition to the nCav with a low-inactivation threshold, Exaiptasia has a splice variant with a higher inactivation threshold expressed (Figure 3D).

The authors hypothesise that it is this high-threshold nCav channel population that amplifies any voltage depolarisation to release a sting irrespective of the presence of prey signals. They found that the β subunit that is responsible for Nematostella's unusually low inactivation threshold exists in Exaiptasia as two alternative splice isoforms. These N-terminus variants also showed the greatest variation in a phylogenetic comparison (Figure 5), rendering it a candidate target for mutations causing variation in stinging responses.

Appraisal of methodology in support of the conclusions:<br /> The authors base their inference on a normative model that yields quantitative predictions which is an exciting and challenging approach. The authors take care in stating the model assumptions as well as showing that the data indeed does not contradict their model predictions. The interesting comparative nature of the modelling part of the study is complicated by slightly different cost assumptions for the two scenarios. Hence, Figure 2 needs to be carefully digested by readers.

It would be even more prudent to analyse the same set of cost-of-discharge vs. starvation scenarios for both species. Specifically, for Nematostella the complete cost-of-discharge vs starvation-state curves as for Exaiptasia (Figure 2E, example 2-4) could be used. It is likely that the differential effect size of Nematostella and Exaiptasia behaviour is the strongest if only the flat cost-of-discharge vs starvation is used (Figure 2A) for Nematostella. But as a worst-case comparison the other curves, where the cost to the animal scales with starvation would be a good comparison. This could help the reader to understand when the different prediction of Nematostella's behaviour breaks down. In addition, this minor change could shed light on broader topics like common trade-offs in pursuit predation.

The qualitatively similar scaling of the model-derived relation between starvation and sting intensity with the counted nematocytes for different feeding pauses is evidence that feeding has indeed been optimised for the two distinct ecological niches.<br /> To prove that Exaiptasia uses a similar Ca2+ channel ortholog as well as a different splice variant, the authors employed both clean electrophysiological characterisaiton (Figure 3) as well as transcriptomics data (Figure 4S1).

To strengthen the authors' hypothesis that variation in the N-termini leads to changes in Ca2+ channel inactivation and hence altered stinging, the response sequence variability of 6 Cnidaria was analysed.

Additional context:<br /> Although, the present article focuses on nematocytes alone, currently, there has been a refocus in neurobiology on the nervous systems of more basal metazoans, which received much attention already in the works of Romanes (1885). In part, this is driven by the goal to understand the early evolution of nervous systems. Cnidarians and Ctenophors are exciting model organisms in this venture. This will hopefully be accompanied by more comparative studies like the present one. Some of the recent literature also uses computational models to understand mechanisms of motor behaviour using full-body simulations (Pallasdies et al. 2019; Wang et al. 2023), which can be thought of as complementary to the normative modelling provided by the authors.

Comparative studies of recent Cnidarians, such as the present article, can shed light on speculative ideas on the origin of nervous systems (Jékely, Keijzer, and Godfrey-Smith 2015). During a time (the Ediacarium/Cambrium transition) that has seen the genesis of complex trophic foodwebs with preditor-prey interaction, symbioses, but also an increase of body sizes and shapes, multiple ultimate causes can be envisioned that drove the increase in behavioural complexity. The authors show that not all of it needs to be implemented in dedicated nerve cells.

References:

Jékely, Gáspár, Fred Keijzer, and Peter Godfrey-Smith. 2015. "An Option Space for Early Neural Evolution." Philosophical Transactions of the Royal Society B: Biological Sciences 370 (December): 20150181. https://doi.org/10.1098/rstb.2015.0181.

Pallasdies, Fabian, Sven Goedeke, Wilhelm Braun, and Raoul-Martin Memmesheimer. 2019. "From Single Neurons to Behavior in the Jellyfish Aurelia Aurita." eLife 8 (December). https://doi.org/10.7554/elife.50084.

Romanes, G. J. 1885. Jelly-Fish, Star-Fish and Sea-Urchins: Being a Research on Primitive Nervous Systems. Appleton.

Wang, Hengji, Joshua Swore, Shashank Sharma, John R. Szymanski, Rafael Yuste, Thomas L. Daniel, Michael Regnier, Martha M. Bosma, and Adrienne L. Fairhall. 2023. "A Complete Biomechanical Model of hydra Contractile Behaviors, from Neural Drive to Muscle to Movement." Proceedings of the National Academy of Sciences 120 (March). https://doi.org/10.1073/pnas.2210439120.

Weir, Keiko, Christophe Dupre, Lena van Giesen, Amy S-Y Lee, and Nicholas W Bellono. 2020. "A Molecular Filter for the Cnidarian Stinging Response." eLife 9 (May). https://doi.org/10.7554/elife.57578.

From Title 7-AGRICULTURE CHAPTER 54-TRANSPORTATION, SALE, AND HANDLING OF CERTAIN ANIMALS

§2131. Congressional statement of policy The Congress finds that animals and activities which are regulated under this chapter are either in interstate or foreign commerce or substantially affect such commerce or the free flow thereof, and that regulation of animals and activities as provided in this chapter is necessary to prevent and eliminate burdens upon such commerce and to effectively regulate such commerce, in order-

(1) to insure that animals intended for use in research facilities or for exhibition purposes or for use as pets are provided humane care and treatment;

(2) to assure the humane treatment of animals during transportation in commerce; and

(3) to protect the owners of animals from the theft of their animals by preventing the sale or use of animals which have been stolen.

The Congress further finds that it is essential to regulate, as provided in this chapter, the transportation, purchase, sale, housing, care, handling, and treatment of animals by carriers or by persons or organizations engaged in using them for research or experimental purposes or for exhibition purposes or holding them for sale as pets or for any such purpose or use.

( Pub. L. 89–544, §1(b), formerly §1, Aug. 24, 1966, 80 Stat. 350 ; Pub. L. 91–579, §2, Dec. 24, 1970, 84 Stat. 1560 ; renumbered and amended Pub. L. 94–279, §2, Apr. 22, 1976, 90 Stat. 417 .)

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Referee #3

Evidence, reproducibility and clarity

Chakraborty et al describe the biochemical and structural characterization of Spiroplasma FtsZ and report that the protein has unusual properties compared to other FtsZ. Sedimentation and GTPase measurements showed that whereas the wild-type protein has a high critical concentration and low GTPase activity, a mutant predicted to facilitate FtsZ cleft opening (F224M) exhibited lower critical concentration and higher GTPase activity. In addition, the crystal structures of both wild-type and F224M SmFtsZ revealed a unique domain-swapped dimer configuration in which one of the monomers in each dimer exhibited an R/T hybrid or intermediate conformation, with the NTD in the T state and the CTD in the R state. The T state of FtsZ has only been observed before when the protein crystallizes as filaments. Thus, the crystal structure of SmFtsZ - which is not assembled in filaments - was interpreted as capturing a conformational state that could explain the kinetic polarity of FtsZ (preferential addition of subunits to the CTD-exposed end of FtsZ filament).

This is a good quality manuscript overall, but which could still be improved by the suggestions below. In terms of significance, it provides new data to support current models for FtsZ assembly mechanism but no major new insights. The findings are interesting for a more specialized audience.

Major points

1. The peculiar biochemical properties of SmFtsZ (high CC, low GTPase) are well documented and interesting but deserve further critical assessment to rule out artifacts. The EM in Fig 1B suggests abundant aggregated protein (not monomers), in addition to filament bundles, which suggests that SmFtsZ is not stable under the experimental conditions used. There are reports that some FtsZ will lose nucleotide during purification and become partially unfolded and unstable (doi.org/10.1111/febs.15235). Figure S1E suggests that the same may be happening here, as the amount of GDP released by SmFtsZ seems to be lower than expected if all the protein had nucleotide. Perhaps the authors should repeat their experiments with SmFtsZ purified in the presence of GDP, which should stabilize the protein, to confirm that the biochemical properties of the protein stay the same.
2. Another unexpected observation is that the SmFtsZ bundles are quite short despite the low GTPase activity of the protein, whereas mutant F224M forms much longer bundles and is a stronger GTPase. In general, filament length correlates inversely with GTPase activity, if measurements are being made at steady state. However, no kinetic (light scattering or fluorescence) experiments seem to have been done to ensure measurements were done in steady state. The authors do try to explain the odd behavior of SmFtsZ but the idea that the increase in GTPase reflects a faster kinetics of nucleation and elongation is not necessarily true. GTP turnover is usually limited by the kinetics of filament disassembly not by assembly. However, it is possible that in reactions with a mutant that is much better at nucleation there will be many more filaments than with a poorly nucleating protein and, thus, more filament ends for subunit turnover. A complicator to these experiments is that they were carried out in high magnesium and at pH 6.5 which favor bundling, and bundling affects subunit and GTP turnover in ways that are hard to account for. Ideally, experiments aimed at properly determining the kinetic properties of FtsZ should be carried out under conditions that avoid bundling (pH 7.4-7.7, 2-5 mM Mg2+) and include proper kinetic measurements, such as light scattering. Thus, before any hard conclusions can be drawn about the properties of SmFtsZ, the authors may wish to revisit some of their biochemical experiments in light of the caveats pointed out here.
3. A central part of the paper is the description of the intermediate R/T conformation but that was a bit confusing and perhaps could be improved. The first thing would be to more clearly define what are the structural changes of the NTD in the T conformation. From other publications, it seems that the NTD undergoes little alteration upon switching to the T conformation, the main one being the flipping of the guanine of the bound nucleotide. But if the NTD structure remains essentially the same, what causes the flipping of the guanine? My impression was that guanine flipping was caused by the downward movement of H7 but if H7 and its attached elements (H6, S6) are moving, why is this not manifested as a significant structural change in the NTD in the T state? Moreover, from Figs. 3C and 5A we conclude that the relative position of H7 in the R/T structures is the same as in R structures. If H7 has not changed in the R/T structure, can you call this a T structure? Also, if there is no H7 movement, what caused the change in guanine angle?
4. The observation that the intermediate conformation was detected in a swapped-dimer is always a matter of some concern, as domain swapping imposes additional constraints on the conformational freedom of a protein and generates structures that are often different from their non-swapped counterparts. This seems to be the case for other FtsZ domain-swapped structures, which were outliers in the extensive comparisons made by Wagstaff et al (doi.org/10.1128/mBio.00254-17) and also stand out in the analysis in Fig. 3BC. Perhaps the authors should discuss more thoroughly why this structure must reflect a natural conformation of FtsZ.
5. Still regarding the structural basis of kinetic polarity, it would be desirable to present a more complete view of the debate in the field about this issue. For example, Ruiz et al, (doi.org/10.1371/journal.pbio.3001497) recently provided structural arguments for the NTD being the face used for monomer addition without detecting the same intermediate form reported in this manuscript. How do their data and arguments differ from your findings? More generally, isn´t the fact that the NTD does not change substantially as FtsZ transitions from R to T already an argument for the NTD being the surface used for monomer addition?
6. l. 74 "led us to propose a structural basis for the kinetic polarity of FtsZ, where transition of the NTD to the T-state conformation driven by GTP binding is sufficient to add a GTP-bound monomer to the bottom interface of the FtsZ filament." This statement suggests that GTP is necessary for the intermediate conformation but this is not supported by the data, as the GDP bound 7YSZ structure also has one monomer in the intermediate conformation. As far as I can tell, there is no structural evidence to suggest that the nucleotide gamma phosphate plays any role in the R-T transition. Even the role of the gamma phosphate in organizing the T3 loop in an assembly-conducive conformation seems to still be a controversial matter in the field. According to Matsui 2014 (doi.org/10.1074/jbc.M113.514901) "based on the results of the present study as well as on the structures deposited previously by other groups (PDB codes 2RHL, 2RHO, 2Q1X, and 2Q1Y) (43, 44), nucleotide exchange appears not to directly induce a structural change in the monomer, including the T3 loop."
7. The experiments with the reciprocal cleft mutation in E. coli are not very informative as it is difficult to correlate the division defect in vivo with specific kinetic defects of the mutant FtsZ. The authors should have at least done a basic characterization of the E. coli mutant in vitro to demonstrate that it is altered in its CC alone. In fact, the dominant negative effect of the mutation in vivo is not something one expects from a poorly nucleating protein, which, if anything, should have a hard time poisoning the endogenous protein. The effect on ring compaction also suggests that the mutation must affect the protein in a broader way, perhaps including filament geometry. I would suggest that this part of the manuscript could be excluded without any loss for the SmFtsZ conclusions.

Minor points

1. l. 14 "CTD of the nucleotide-bound monomer cannot bind to the NTD-exposed end of the filament unless relative rotation of the domains leads to cleft opening." This is not accurate. There is no steric impediment to this reaction. Monomers in R conformation should be able to add to the NTD end of the filament as well, even if this is slower than the opposite reaction. The absence of growth from the NTD end is because the rate of addition/conformational change is slower than the rate of GTP hydrolysis.
2. The comparison between the 7YOP (B) structure and the S. aureus 3WGN structure to show the effect of the gamma phosphate on T3 loop structure should be presented in a single figure, instead of being split between Fig. 4 and Fig. S2, and preferably using similar poses of the two structures. In the current state, it is quite hard to visualize the similarities mentioned by the authors.
3. In contrast to what´s in the main text (l. 130), the chain with continuous density in Figure 2 is assigned as B, not A. Please clarify which is correct.
4. l. 271 pBAD is the plasmid name, not the promoter. The promoter is PBAD(subscript).

Significance

This is a good quality manuscript overall, but which could still be improved by the suggestions below. In terms of significance, it provides new data to support current models for FtsZ assembly mechanism but no major new insights. The findings are interesting for a more specialized audience.

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Reply to the reviewers

__Reviewer 1____: __

1-Localization of ESYT1 and SYNJ2BP

The claim of a localization at ER-mitochondria contacts relies on two type of assays. Light microscopy and subcellular fractionation. Concerning microscopy, while the staining pattern is obviously colocalizing with the ER (a control of specificity of staining using KO cells would nevertheless be desirable)

the idea that ESYT1 foci "partially colocalized with mitochondria" is either trivial or unfounded

Every cellular structure is "partially colocalized with mitochondria" simply by chance at the resolution of light microscopy

If the meaning of the experiment is to show that ESYT1 'specifically' colocalizes with mitochondria, then this isn't shown by the data

There is no quantification that the level of colocalization is more than expected by chance

nor that it is higher than that of any other ER protein

Moreover, the author's model implies that ESYT1 partial colocalization with mitochondria is, at least partially, due to its interaction with SYNJ2BP. This is not tested.

• To analyze and measure MERCs parameters and functions, we used a set of validated methods described in the following specialized review articles (Eisenberg-Bord, Shai et al. 2016, Scorrano, De Matteis et al. 2019).
• To support and confirm the localization of ESYT1-SYNJ2BP complex at MERCs, we performed supplementary BioID analysis using ER target BirA*, OMM targeted BirA* and ER-mitochondria tether BirA* (Table S1, Figure S1 and Figure 1 A and B). These results confirmed the specificity of the interaction of the 2 partners. ESYT1 is not identified as a prey in OMM BioID and SYNJ2BP is not identified in ER BioID, on the other hand both partners are identified in the ER-mitochondria tether BioID.
• To improve our description of the partial localization of ESYT1 at mitochondria, we performed a quantitative analysis using confocal microscopy on control human fibroblasts stably overexpressing SEC61B-mCherry as an ER marker which were labelled with ESYT1 and TOMM40 for mitochondria. We measured the % of ESYT1 signal colocalizing with mitochondria and the % of mitochondria positive for ESYT1 (Figure 1E).

• To demonstrate than ESYT1 partial colocalization with mitochondria is, at least partially, due to its interaction with SYNJ2BP, we performed a quantitative analysis using confocal microscopy. Human control fibroblasts, KO SYNJ2BP fibroblasts and SYNJ2BP overexpressing fibroblasts were labelled with ESYT1, TOMM40 for mitochondria and CANX for ER. We measured the % of ESYT1 signal colocalizing with mitochondria in each condition (Figure 3C). Membranes (MAM) can be purified and are enriched for proteins that localize at ER-mitochondria contacts. This idea originated in the early 90's and since then, myriad of papers has been using MAM purification, and whole MAM proteomes have been determined. Yet the evidence that MAM-enriched proteins represent bona fide ER-mitochondria-contact-enriched proteins (as can nowadays be determined by microscopy techniques) remain scarce. Here, anyway, ESYT1 fractionation pattern is identical to that of PDI, a marker of general ER, with no indication of specific MAM accumulation.

• To highlight the enrichment of ESYT1 in the MAM fraction, we quantified the ESYT1 signal in each fraction. Those results show a similar fractionation pattern than the MAM resident protein SIGMAR1 (Figure 1F). For SYNJ2BP, it is different as it is more enriched in the MAM than the general mitochondrial marker PRDX3. However, PRDX3 is a matrix protein, making it a poor comparison point, since SYNJ2BP is an OMM protein.

• To confirm the partial enrichment of SYNJ2BP in the MAM fraction compared to another outer mitochondrial membrane protein, we added the signal of the well characterized OMM protein CARD19 (Rios, Zhou et al. 2022). Again, the model implies that ESYT1 and SYNJ2BP accumulation in the MAM should be dependent on each other. This is not tested.

• As describe above, we demonstrated in Figure 3C than the accumulation of ESYT1 at mitochondria is, at least partially, dependent on the quantity of SYNJ2BP.

• We moreover showed a reciprocal effect in Figure 3E. A quantitative analysis using confocal microscopy demonstrated that the effect of SYNJ2BP overexpression on MERCs formation is partially dependent of the presence of ESYT1. 2-ESYT1-SYNJ2BP interaction.

The starting point of the paper is a BioID signal for SYNJ2BP when BioID is fused to ESYT1. One confirmation of the interaction comes in figure 4, using blue native gel electrophoresis and assessing comigration. Because BioID is promiscuous and comigration can be spurious, better evidence is needed to make this claim. This is exemplified by the fact that, although SYNJ2BP is found in a complex comigrating with RRBP1, according to the BN gel, this slow migrating complex isn't disturbed by RRBP1 knockdown, but is somewhat disturbed by ESYT1 knockdown. More than a change in abundance, a change in migration velocity when either protein is absent would be evidence that these comigrating bands represent the same complex.

• We showed in Figure 4C that the presence of SYNJ2BP in a complex of a similar molecular weight that ESYT1 (410KDa) is totally dependent of the presence of ESYT1, suggesting an interaction of the 2 proteins.

• To confirm this interaction, in figure 4A we analyzed on BN cells overexpressing SYNJ2BP together with a 3xFlag tagged version of ESYT1. As a result of the addition of the Flag tag, the complex positive for ESYT1 shifted to a higher molecular weight. The complex positive for SYNJ2BP shifted to a similar the molecular weight, demonstrating the interaction and dependence of the 2 partners. ESYT1-SYNJ2BP interaction needs to be tested by coimmunoprecipitation of endogenous proteins, yeast-2-hybrid, in vitro reconstitution or any other confirmatory methods.

• To confirm the interaction of the 2 partners, we performed co-immunoprecipitation of the ESYT1-3xFlag protein that we showed in Figure 1H to form complexes similar to the endogenous protein. SYNJ2BP is found as the strongest prey, followed by ESYT2 and SEC22B two described interactors of ESYT1, confirming the quality of the analysis (Table S2) (Giordano, Saheki et al. 2013, Gallo, Danglot et al. 2020). 3-Tethering by ESYT1- SYNJ2BP.

This is assessed by light and electron microscopy. Absence of ESYT1 decreases several metrics for ER-mitochondria contacts (whether absence of SYNJ2BP has the same effect isn't tested).

• Using PLA (proximity ligation assay) we demonstrated that the loss of SYNJ2BP leads to a decrease in MERCs (Figure 7 H and I), confirming previous studies (Ilacqua, Anastasia et al. 2022, Pourshafie, Masati et al. 2022). This interesting phenomenon could be due to many things, including but not limited to the possibility that "ESYT1 tethers ER to mitochondria".

This statement and the respective subheading title are therefore clearly overreaching and should be either supported by evidence or removed.

Indeed, absence of ESYT1 ER-PM tethering and lipid exchange could have knock-on effects on ER-mito contacts, therefore strong statements aren't supported.

Moreover, the effect on ER-mitochondria contact metrics could be due to changes in ER-mitochondria contact indeed but may also reflect changes in ER and/or mitochondria abundance and/or distribution, which favour or disfavour their encounter. Abundance and distribution of both organelles are not controlled for.

• The mitochondrial phenotypes caused by the loss of ESYT1 are all rescued by the introduction of an artificial mitochondrial-ER tether, demonstrating that they are due to loss of the tethering function of ESYT1. Finally, the authors repeat a finding that SYNJ2BP overexpression induces artificial ER-mitochondria tethering. Again, according to the model, this should be, at least in part, due to interaction with ESYT1. Whether ESYT1 is required for this tethering enhancement isn't tested.

• As described above, we demonstrated in Figure 3C that the accumulation of ESYT1 at mitochondria is, at least partially, dependent on the quantity of SYNJ2BP.

• We moreover showed a reciprocal effect in Figure 3F. A quantitative analysis using confocal microscopy demonstrated that the effect of SYNJ2BP overexpression on MERC formation is partially dependent of the presence of ESYT1. 4-Phenotypes of ESYT1/SYNJ2BP KD or KO.

The study goes in details to show that downregulation of either protein yields physiological phenotypes consistent with decreased ER-mitochondria tethering. These phenotypes include calcium import into mitochondria and mitochondrial lipid composition.

Figure 5 shows that histamine-evoked ER-calcium release cause an increase in mitochondrial calcium, and this increase is reduced in absence of ESYT1, without detectable change in the abundance of the main known players of this calcium import. This is rescued by an artificial ER-mitochondria tether. However, Figure 5D shows that the increase in calcium concentration in the cytosol upon histamine-evoked ER calcium release is equally impaired by ESYT1 deletion, contrary to expectation. Indeed, if the impairment of mitochondrial calcium import was due to improper ER-mitochondria tethering in ESYT1 mutant cells, one would expect more calcium to leak into the cytosol, not less.

The remaining explanation is that ESYT1 knockout desensitizes the cells to histamine, by affecting GPCR signalling at the PM, something unexplored here.

In any case, a decreased calcium discharge by the ER upon histamine treatment, explains the decreased uptake by mitochondria.

The authors argue that ER calcium release is unaffected by ESYT1 KO, but crucially use thapsigargin instead of histamine to show it. Thus, the most likely interpretation of the data is that ESYT1 KO affects histamine signalling and histamine-evoked calcium release upstream of ER-mitochondria contacts.

• Silencing ESYT1 impairs SOCE efficiency in Jurkat cells (Woo, Sun et al. 2020), but not in HeLa cells (Giordano, Saheki et al. 2013, Woo, Sun et al. 2020). Analysis of the role of ESYT1 in HeLa cells prevents confounding effects due to the loss of ESYT1 at ER-PM. In this model, knock-down of ESYT1 led to a decrease of mitochondrial Ca2+ uptake from the ER upon histamine stimulation, as monitored by genetically encoded Ca2+ indicator targeted to mitochondrial matrix (Figure 5A and B). ESYT1 silencing in HeLa cells did not impact ER Ca2+ store measured by the ER-targeted R-GECO Ca2+ probe (Figure 5C and D). The expression of the artificial mitochondria-ER tether was able to rescue mitochondrial Ca2+ defects observed in ESYT1 silenced cells (Figure 5B), confirming that the observed anomalies are specifically due to MERC defects.
• In contrast loss of ESYT1 impaired SOCE efficiency in fibroblasts (Figure 6 A and B). This phenotype was fully rescued by re-expression of ESYT1-Myc but not the artificial tether. We therefore investigated the influence of ESYT1 loss on cytosolic Ca2+ concentration following ATP (Figure 6F to H) or histamine stimulation (Figure S3 D to F), both of which showed a reduced cytosolic Ca2+ concentration and uptake in ESYT1 KO cells. This phenotype was fully rescued by the re-expression of ESYT1-Myc but not the artificial tether. Measurment of cytosolic Ca2+ after tharpsigargin treatment in Ca2+-fee media, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase SERCA that blocks Ca2+ pumping into the ER, showed that ESYT1 KO does not influence the total ER Ca2+ pool (Figure 6K and L). However, ER-Ca2+ release capacity upon histamine stimulation (Figure 6I and J) is decreased in ESYT1 KO cells. This phenotype was fully rescued by the re-expression of ESYT1-Myc but not the artificial tether. Loss of ESYT1 decreased the Ca2+ uptake capacities of mitochondria after activation with histamine (Figure S3 A to C) or ATP (Figure 6 C to E). This phenotype was rescued by re-expression of ESYT1-Myc and also the engineered ER-mitochondria tether. Thus, despite the ER-Ca2+ release defect observed after ESYT1 loss, the artificial tether fully rescued the mitochondrial phenotype.

• These results highlight the distinct and dual roles of ESYT1 in Ca2+ regulation at the ER-PM and at MERCs. The data with SYNJ2BP deletion are more compatible with decreased ER-mito contacts, as no decreased in cytosolic calcium is observed. This is compatible with the previously proposed role of SYNJ2BP in ER-mitochondria tethering, but the difference with ESYT1 rather argue that both proteins affect calcium signaling by different means, meaning they act in different pathways.

• We explain the different results concerning cytosolic calcium by the fact that ESYT1 is a bi-localized protein with dual functions on cellular calcium. Implicated both in SOCE at ER-PM and in mitochondrial calcium uptake at MERCs. On the other hand, SYNJ2BP is only present at MERCs and its loss do not influence PM-ER signaling or ER-Ca2+ release. Finally, the study delves into mitochondrial lipids to "investigated the role of the SMP-domain containing protein ESYT1 in lipid transfer from ER to mitochondria". In reality, it is not ER-mitochondria lipid transport that is under scrutiny, but general lipid homeostasis, and changes in ER-PM lipids could have knock-on effects on mitochondrial lipids without the need to invoke disruptions in ER-mitochondria transfer activity.

• The fact that the artificial tether, which specifically rescue MERCs, fully rescue the lipid phenotype argue for a direct loss of MERCs tethering function when ESYT1 is missing. The changes observed are interesting but could be due to anything. Surprisingly, PCA analysis shows that the rescue of the knockout by the ESYT1 gene clusters with the rescue by the artificial tether, and not with the wildtype. This indicates that overexpressing either ESYT1 or a tether cause similar lipidomic changes. These could be due, for instance, to ER stress caused by protein overexpression, and not to a rescue.

• In order to verify if the overexpression of ESYT1 or the artificial tether induces ER stress, we performed a WB analysis to compare markers of ER stress in control fibroblasts, KO ESYT1 fibroblasts, KO ESYT1 fibroblasts overexpressing ESYT1-Myc or the tether (Figure S4C). This showed no changes in the levels of several different markers of ER stress or cell death. __Reviewer 2____: __

1) the interaction between those proteins is direct,

2) if SYNJ2BP is necessary and sufficient to localize E-Syt1 at MERC, and

3) if MERCs extension induced by SYNJ2BP is dependent on E-Syt1.

Those points are important to investigate because SYNJ2BP has already been shown to induce MERCs by interacting with the ER protein RRBP1. In addition, some experiments need to be better quantified.

Major comments: E-syt1/SYNJ2BP in MERCs formation: the authors provide several convincing lines of evidence that both proteins are in the same complex (proximity labelling, localization in the same complex in BN-PAGE, localization in MAM) but it is not clear in which extent the direct interaction between both proteins regulates ER-mitochondria tethering. 1- Pull down experiments or BiFC strategy could be performed to show the direct interaction between both proteins.

• We showed in Figure 4C that the presence of SYNJ2BP in a complex of a similar molecular weight to that ESYT1 (410KDa) is totally dependent of the presence of ESYT1, suggesting an interaction of the 2 proteins.
• To confirm this interaction, in figure 4A we analyzed on BN cells overexpressing SYNJ2BP together with a 3xFlag tagged version of ESYT1. As a result of the addition of the Flag tag, the complex positive for ESYT1 shifted to a higher molecular weight. Significantly, the complex positive for SYNJ2BP shifted to a similar the molecular weight, demonstrating the interaction and dependence of the 2 protein partners.

• To confirm the interaction of the 2 partners, we performed co-immunoprecipitation of the ESYT1-3xFlag protein (Table S2). SYNJ2BP was found as the strongest prey, followed by ESYT2 and SEC22B two described interactors of ESYT1, confirming the quality of the analysis (Giordano, Saheki et al. 2013, Gallo, Danglot et al. 2020). 2- SYNJ2BP OE has already been demonstrated to increase MERCs and this being dependent on the ER binding partners RRBP1 (10.7554/eLife.24463). Therefore, it would be of interest to perform OE of SYNJ2BP in KO Esyt1 to address the question of whether ESyt1 is also required to increase MERCs.

• A quantitative analysis using confocal microscopy demonstrated that the effect of SYNJ2BP overexpression on MERCs formation is partially dependent of the presence of ESYT1 (Figure 3F). 3- The authors show that Esyt1 punctate size increases when SYNJ2BP is OE (Fig3C), but this can be indirectly linked to the increase of MERCs in the OE line. Thus, it could be interesting to test if the number/shape of E-syt1 punctate located close to mitochondria decreases in KO SYNJ2B. This could really show the dependence of SYNJ2BP for E-syt1 function at MERCs.

• To improve our description of the partial localization of ESYT1 at mitochondria, we performed a quantitative analysis using confocal microscopy on control human fibroblasts stably overexpressing SEC61B-mCherry as an ER marker which were labelled with ESYT1 and TOMM40 for mitochondria. We measured the % of ESYT1 signal colocalizing with mitochondria and the % of mitochondria colocalizing with ESYT1 (Figure 1E).

• To demonstrate than ESYT1 partial colocalization with mitochondria is, at least partially, due to its interaction with SYNJ2BP, we performed a quantitative analysis using confocal microscopy. Human control fibroblasts, KO SYNJ2BP fibroblasts and SYNJ2BP overexpressing fibroblasts were labelled with ESYT1, TOMM40 for mitochondria and CANX for ER. We measured the % of ESYT1 signal colocalizing with mitochondria in each condition (Figure 3C). Lipid analyses: the results of MS on isolated mitochondria clearly show that mitochondrial lipid homeostasis is affected on KO-Syt1 and rescued by expression of Syt1-Myc and artificial mitochondria-ER tether. However, p.15, the authors wrote "The loss of ESYT1 resulted in a decrease of the three main mitochondrial lipid categories CL, PE and PI, which was accompanied by an increase in PC ». As the results are expressed in mol%, this interpretation can be distorted by the fact that mathematically, if the content of one lipid decreases, the content of others will increase. I would suggest to express the results in lipid quantity (nmol)/mg of mitochondria proteins instead of mol%. This will clarify the role of E-Syt1 on mitochondrial lipid homeostasis and which lipid increase and decrease.

• We changed the sentence in the text as suggested. Also it could be of high interest to have the lipid composition of the whole cells to reinforce the direct involvement of E-Syt1 in mitochondrial lipid homeostasis and verify that the disruption of mitochondrial lipid homeostasis is not linked to a general perturbation of lipid metabolism as this protein acts at different MCSs.

• This is beyond the scope of the project and we would argue that the results of such an experiment would be difficult to interpret. To better understand the impact of Esyt1 of mitochondria morphology, the author could analyze the mitochondria morphology (size, shape, cristae) on their EM images of crt, KO and OE lines. Indeed, on OE (Fig3A), the mitochondria look bigger and with a different shape compared to crt.

• As we do not observe obvious differences in mitochondrial morphology between control, KO and OE fibroblasts we do not think that quantitative analysis would add to the understanding of the effect of ESYT1 on mitochondrial function. Also, they performed a lot of BN-PAGE. Is it possible to check whether the mitochondrial respiratory chain super-complexes are affected on Esyt1 KO line compared to crt?

• We decided to remove the data on the metabolic consequences of ESYT1 loss since it was too preliminary and required deeper investigations, focusing instead on the effect of ESYT1 loss on calcium homeostasis. Quantifications: some western blots needs to be quantified (Fig 5K, 6J, S3E);

• We did not observe obvious differences in the protein levels so we think that quantitation would not add significantly to the understanding of the differences in calcium dynamics that we report. Fig1A: Can the author provide a higher magnification of the triple labeling and perform quantification about the proportion of E-Syt1 punctate located close to mitochondria?

• We added higher magnification of the same area in all channels and arrows that point to the foci of ESYT1 colocalizing with both ER and mitochondria (Figure 1D).

• To improve our description of the partial localization of ESYT1 at mitochondria, we performed a quantitative analysis using confocal microscopy on control human fibroblasts stably overexpressing SEC61B-mCherry as an ER marker which were labelled with ESYT1 and TOMM40 for mitochondria. We measured the % of ESYT1 signal colocalizing with mitochondria and the % of mitochondria colocalizing with ESYT1 (Figure 1E). Minor comments:

• Fig1E + text: according to the legend, the BN-PAGE has been performed on Heavy membrane fraction. Why the authors speak about complexes at MAM in the text of the corresponding figure? Is-it the MAM or the heavy fraction (MAM + mito + ER...)? If BN have been performed from heavy membranes, it is not a real proof that E-syt1 is in MAMs.

• Heavy membranes have been used in this experiment. The text and conclusions have been changed accordingly.

• On fig3C (panel crt): it seems like SYNJ2BP dots are not co-localizaed with mito. Is this protein targeted to another organelle beside mitochondria?

• It is not described that SYNJ2BP would be targeted to another organelle beside mitochondria. It is possible that those dots outside of mitochondria could be non-specific signals from the antibody we used.

• Fig4A: can the author provide a control of protein loading (membrane staining as example) to confirm the decrease of E-Syt1 in siSYNJ2BP?

• As we performed this experiment only once we have removed the statement suggesting a decrease in ESYT1 protein in response to the siSYNJ2BP.

• Fig5E/F: it is not clear to me why the expression of E-Syt1 in the KO is not able to complement the KO phenotype for cytosolic Ca++. Can the authors comment this?

• We performed further analysis using ATP to trigger calcium release from the ER (figure 6 F to H). In those conditions, expression of ESYT1 in the KO is able to complement the KO phenotype for cytosolic Ca2+. __Reviewer 3____: __

Main points 1. Confirming the MERC localization of ESYT1 should include some more of tethering factors as demonstrated interactors (some are mentioned above) and should not be limited to lipid homeostasis.

• As shown in Figure 1B, VAPB, PDZD8 and BCAP31 are found as preys in the ESYT1 bioID analysis. Those proteins have been described as MERC tethers, their loss leading to mitochondrial calcium defects. To support and confirm the specificity of ESYT1-SYNJ2BP complex at MERCs, we performed a supplementary BioID analysis using ER targeted BirA* and OMM targeted BirA* (Table S1, Figure S1 and Figure 1 A and B). These results confirmed the specificity of the interaction of the 2 partners. ESYT1 is not identified as a prey in OMM BioID and SYNJ2BP is not identified in ER BioID. Additional ER-mitochondria tether BirA* analyses showed that tether-BirA* identified both ESYT1 and SYNJ2BP as a prey at MERCs, confirming the localisation of this interaction. Interestingly, a large majority of the known MERCs tethers VAPB-PTPIP51, MFN2, ITPRs, BCAP31 are also found as preys in the tether-BirA* (Figure 1B), confirming the quality of these data.
• To confirm the interaction of the 2 partners, we performed co-immunoprecipitation of the ESYT1-3xFlag protein. SYNJ2BP is found as the strongest prey, followed by ESYT2 and SEC22B two described interactors of ESYT1, confirming the quality of the analysis (Table S2) (Giordano, Saheki et al. 2013, Gallo, Danglot et al. 2020).

The fact that in ESYT1 KO cells both mitochondrial calcium transfer and cytosolic calcium accumulation are accompanied by decreased ER-cepia1ER signal decay upon histamine addition suggest that the main reason for ER-mitochondria calcium transfer defects are due to impaired SOCE. Calcium-free medium and histamine are used to show that ESYT1 does not affect ER calcium content. However, if it affects SOCE, then the absence of extracellular calcium would abolish such an effect; moreover, histamine does not test for leak effects. As additional information, the authors should investigate whether ER calcium content is affected by the presence of extracellular calcium in the ko scenario using thapsigargin. The authors should inhibit SOCE to test whether this mechanism is affected in ESYT1 KO and could account for observed signal differences. Excluding SOCE is critical, since any change in calcium entry from the outside would potentially negate a role of ESYT1 in mitochondrial calcium uptake.

• Silencing ESYT1 impairs SOCE efficiency in Jurkat cells (Woo, Sun et al. 2020), but not in HeLa cells (Giordano, Saheki et al. 2013, Woo, Sun et al. 2020). Analysis of the role of ESYT1 in HeLa cells prevents confounding effects due to the loss of ESYT1 at ER-PM. In this model, knock-down of ESYT1 led to a decrease of mitochondrial Ca2+ uptake from the ER upon histamine stimulation, as monitored by genetically encoded Ca2+ indicator targeted to mitochondrial matrix (Figure 5A and B). ESYT1 silencing in HeLa cells did not impact ER Ca2+ store measured by the ER-targeted R-GECO Ca2+ probe (Figure 5C and D). The expression of the artificial mitochondria-ER tether was able to rescue mitochondrial Ca2+ defects observed in ESYT1 silenced cells (Figure 5B), confirming that the observed anomalies are specifically due to MERC defects.
• In contrast loss of ESYT1 impaired SOCE efficiency in fibroblasts (Figure 6 A and B). This phenotype was fully rescued by re-expression of ESYT1-Myc but not the artificial tether. We therefore investigated the influence of ESYT1 loss on cytosolic Ca2+ concentration following ATP (Figure 6F to H) or histamine stimulation (Figure S3 D to F), both of which showed a reduced cytosolic Ca2+ concentration and uptake in ESYT1 KO cells. This phenotype was fully rescued by the re-expression of ESYT1-Myc but not the artificial tether. Measurment of cytosolic Ca2+ after tharpsigargin treatment in Ca2+-fee media, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase SERCA that blocks Ca2+ pumping into the ER, showed that ESYT1 KO does not influence the total ER Ca2+ pool (Figure 6K and L). However, ER-Ca2+ release capacity upon histamine stimulation (Figure 6I and J) is decreased in ESYT1 KO cells. This phenotype was fully rescued by the re-expression of ESYT1-Myc but not the artificial tether. Loss of ESYT1 decreased the Ca2+ uptake capacities of mitochondria after activation with histamine (Figure S3 A to C) or ATP (Figure 6 C to E). This phenotype was rescued by re-expression of ESYT1-Myc and also the engineered ER-mitochondria tether. Thus, despite the ER-Ca2+ release defect observed after ESYT1 loss, the artificial tether fully rescued the mitochondrial phenotype.
• These results highlight the distinct and dual roles of ESYT1 in Ca2+ regulation at the ER-PM and at MERCs.

The authors claim that ER-Geco measurements show that no change of ER calcium was observed. However, they use thapsigargin treatment and then get a peak, when the signal should show a decrease due to leak. This suggests they did not use ER-Geco in Figure S3C. What was measured and what does it mean?

• We used R-GECO (not ER-GECO) which measures the cytosolic calcium.
• We measured total ER Ca2+ store using the cytosolic-targeted R-GECO Ca2+ probe upon thapsigarin treatment, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase SERCA that blocks Ca2+ pumping into the ER (Figure 5C and D) and observed no difference in our different conditions.

The findings on growth in galactose medium are intriguing but are not accompanied by respirometry to confirm mitochondria are compromised upon ESYT1 KO.

• We decided to remove the data on the metabolic consequences of ESYT1 loss since it was to preliminary and required deeper investigations, focusing instead on the effect of ESYT1 loss on calcium homeostasis

Minor points: 1. The authors mention they measure mitochondrial uptake of "exogenous" calcium by applying histamine. They should specify that these measures transferred calcium from the ER rather than uptake of calcium from the exterior (directly at the plasma membrane).

• The text was clarified as suggested.

• Expression levels of IP3Rs are not very indicative of any change of their activity. The authors should discuss how ESYT1 could affect their PTMs.

• A large numer of post translational modifications are known to regulate IP3R activity (Hamada and Mikoshiba 2020), and it is possible that the loss of ESYT1 could interfere with these modifications, but an exploration of this issue is beyond the scope of this study. The text was clarified as suggested. Eisenberg-Bord, M., N. Shai, M. Schuldiner and M. Bohnert (2016). "A Tether Is a Tether Is a Tether: Tethering at Membrane Contact Sites." Dev Cell 39(4): 395-409.

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Author Response

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

She et al studied the evolution of gene expression reaction norms when individuals colonise a new environment that exposes them to physiologically challenging conditions. Their objective was to test the "plasticity first" hypothesis, which suggest that traits that are already plastic (their value changes when facing a new environment compared to the original environment) facilitates the colonisation of novel environments, which, if true, would be predicted to result in the evolution of gene expression values that are similar in the population that colonised the new environment and evolved under these particular selection pressures. To test this prediction, they studied gene expression in cardiac and muscle tissues in individuals originating from three conditions: lowland individuals in their natural environment (ancestral state), lowland individuals exposed to hypoxia (the plastic response state), and a highland population facing hypoxia for several generations (the coloniser state). They classified gene expression patterns as maladaptive or adaptive in lowland individuals responding to short term hypoxia by classifying gene expression patterns using genes that differed between the ancestral state (lowland) and colonised state (highland). Genes expressed in the same direction in lowland individuals facing hypoxia (the plastic state) as what is found in the colonised state are defined as adaptative, while genes with the opposite expression pattern were labelled as maladaptive, using the assumption that the colonised state must represent the result of natural selection. Furthermore, genes could be classified as representing reversion plasticity when the expression pattern differed between the plasticity and colonised states and as reinforcement when they were in the same direction (for example more expressed in the plastic state and the colonised state than in the ancestral state). They found that more genes had a plastic expression pattern that was labelled as maladaptive than adaptive. Therefore, some of the genes have an expression pattern in accordance with what would be predicted based on the plasticity-first hypothesis, while others do not.

Thank you for a precise summary of our work. We appreciate the very encouraging comments recognizing the value of our work. We have addressed concerns from the reviewer in greater detail below.

Q1. As pointed out by the authors themselves, the fact that temperature was not included as a variable, which would make the experimental design much more complex, misses the opportunity to more accurately reflect the environmental conditions that the colonizer individuals face at high altitude. Also pointed out by the authors, the acclimation experiment in hypoxia lasted 4 weeks. It is possible that longer term effects would be identifiable in gene expression in the lowland individuals facing hypoxia on a longer time scale. Furthermore, a sample size of 3 or 4 individuals per group depending on the tissue for wild individuals may miss some of the natural variation present in these populations. Stating that they have a n=7 for the plastic stage and n= 14 for the ancestral and colonized stages refers to the total number of tissue samples and not the number of individuals, according to supplementary table 1.

We shared the same concerns as the reviewer. This is partly because it is quite challenging to bring wild birds into captivity to conduct the hypoxia acclimation experiments. We had to work hard to perform acclimation experiments by taking lowland sparrows in a hypoxic condition for a month. We indeed have recognized the similar set of limitations as the review pointed out and have discussed the limitations in the study, i.e., considering hypoxic condition alone, short time acclimation period, etc. Regarding sample sizes, we have collected cardiac muscle from nine individuals (three individuals for each stage) and flight muscle from 12 individuals (four individuals for each stage). We have clarified this in Supplementary Table 1.

Q2. Finally, I could not find a statement indicating that the lowland individuals placed in hypoxia (plastic stage) were from the same population as the lowland individuals for which transcriptomic data was already available, used as the "ancestral state" group (which themselves seem to come from 3 populations Qinghuangdao, Beijing, and Tianjin, according to supplementary table 2) nor if they were sampled in the same time of year (pre reproduction, during breeding, after, or if they were juveniles, proportion of males or females, etc). These two aspects could affect both gene expression (through neutral or adaptive genetic variation among lowland populations that can affect gene expression, or environmental effects other than hypoxia that differ in these populations' environments or because of their sexes or age). This could potentially also affect the FST analysis done by the authors, which they use to claim that strong selective pressure acted on the expression level of some of the genes in the colonised group.

The reviewer asked how individual tree sparrows used in the transcriptomic analyses were collected. The individuals used for the hypoxia acclimation experiment and represented the ancestral lowland population were collected from the same locality (Beijing) and at the same season (i.e., pre-breeding) of the year. They are all adults and weight approximately 18g. We have clarified this in the Supplementary Table S1 and Methods. We did not distinguish males from females (both sexes look similar) under the assumption that both sexes respond similarly to hypoxia acclimation in their cardiac and flight muscle gene expression.

The Supplementary Table 2 lists the individuals that were used for sequence analyses. These individuals were only used for sequence comparisons but not for the transcriptomic analyses. The population genetic structure analyzed in a previously published study showed that there is no clear genetic divergence within the lowland population (i.e., individuals collected from Beijing, Tianjing and Qinhuangdao) or the highland population (i.e., Gangcha and Qinghai Lake). In addition, there was no clear genetic divergence between the highland and lowland populations (Qu et al. 2020).

Q4. Impact of the work

There has been work showing that populations adapted to high altitude environments show changes in their hypoxia response that differs from the short-term acclimation response of lowland population of the same species. For example, in humans, see Erzurum et al. 2007 and Peng et al. 2017, where they show that the hypoxia response cascade, which starts with the gene HIF (Hypoxia-Inducible Factor) and includes the EPO gene, which codes for erythropoietin, which in turns activates the production of red blood cell, is LESS activated in high altitude individuals compared to the activation level in lowland individuals (which gives it its name). The present work adds to this body of knowledge showing that the short-term response to hypoxia and the long term one can affect different pathways and that acclimation/plasticity does not always predict what physiological traits will evolve in populations that colonize these environments over many generations and additional selection pressure (UV exposure, temperature, nutrient availability). Altogether, this work provides new information on the evolution of reaction norms of genes associated with the physiological response to one of the main environmental variables that affects almost all animals, oxygen availability. It also provides an interesting model system to study this type of question further in a natural population of homeotherms.

Erzurum, S. C., S. Ghosh, A. J. Janocha, W. Xu, S. Bauer, N. S. Bryan, J. Tejero et al. "Higher blood flow and circulating NO products offset high-altitude hypoxia among Tibetans." Proceedings of the National Academy of Sciences 104, no. 45 (2007): 17593-17598.

Peng, Y., C. Cui, Y. He, Ouzhuluobu, H. Zhang, D. Yang, Q. Zhang, Bianbazhuoma, L. Yang, Y. He, et al. 2017. Down-regulation of EPAS1 transcription and genetic adaptation of Tibetans to high-altitude hypoxia. Molecular biology and evolution 34:818-830.

Thank you for highlighting the potential novelty of our work in light of the big field. We found it very interesting to discuss our results (from a bird species) together with similar findings from humans. In the revised version of manuscript, we have discussed short-term acclimation response and long-term adaptive evolution to a high-elevation environment, as well as how our work provides understanding of the relative roles of short-term plasticity and long-term adaptation. We appreciate the two important work pointed out by the reviewer and we have also cited them in the revised version of manuscript.

Reviewer #2 (Public Review):

This is a well-written paper using gene expression in tree sparrow as model traits to distinguish between genetic effects that either reinforce or reverse initial plastic response to environmental changes. Tree sparrow tissues (cardiac and flight muscle) collected in lowland populations subject to hypoxia treatment were profiled for gene expression and compared with previously collected data in 1) highland birds; 2) lowland birds under normal condition to test for differences in directions of changes between initial plastic response and subsequent colonized response. The question is an important and interesting one but I have several major concerns on experimental design and interpretations.

Thank you for a precise summary of our work and constructive comments to improve this study. We have addressed your concerns in greater detail below.

Q1. The datasets consist of two sources of data. The hypoxia treated birds collected from the current study and highland and lowland birds in their respective native environment from a previous study. This creates a complete confounding between the hypoxia treatment and experimental batches that it is impossible to draw any conclusions. The sample size is relatively small. Basically correlation among tens of thousands of genes was computed based on merely 12 or 9 samples.

We appreciate the critical comments from the reviewer. The reviewer raised the concerns about the batch effect from birds collected from the previous study and this study. There is an important detail we didn’t describe in the previous version. All tissues from hypoxia acclimated birds and highland and lowland birds have been collected at the same time (i.e., Qu et al. 2020). RNA library construction and sequencing of these samples were also conducted at the same time, although only the transcriptomic data of lowland and highland tree sparrows were included in Qu et al. (2020). The data from acclimated birds have not been published before.

In the revised version of manuscript, we also compared log-transformed transcript per million (TPM) across all genes and determined the most conserved genes (i.e., coefficient of variance ≤  0.3 and average TPM ≥ 1 for each sample) for the flight and cardiac muscles, respectively (Hao et al. 2023). We compared the median expression levels of these conserved genes and found no difference among the lowland, hypoxia-exposed lowland, and highland tree sparrows (Wilcoxon signed-rank test, P<0.05). As these results suggested little batch effect on the transcriptomic data, we used TPM values to calculate gene expression level and intensity. This methodological detail has been further clarified in the Methods and we also provided a new supplementary Figure (Figure S5) to show the comparative results.

The reviewer also raised the issue of sample size. We certainly would have liked to have more individuals in the study, but this was not possible due to the logistical problem of keeping wild bird in a common garden experiment for a long time. We have acknowledged this in the manuscript. In order to mitigate this we have tested the hypothesis of plasticity following by genetic change using two different tissues (cardiac and flight muscles) and two different datasets (co-expressed gene-set and muscle-associated gene-set). As all these analyses show similar results, they indicate that the main conclusion drawn from this study is robust.

Q2. Genes are classified into two classes (reversion and reinforcement) based on arbitrarily chosen thresholds. More "reversion" genes are found and this was taken as evidence reversal is more prominent. However, a trivial explanation is that genes must be expressed within a certain range and those plastic changes simply have more space to reverse direction rather than having any biological reason to do so.

Thank you for the critical comments. There are two questions raised we should like to address them separately. The first concern centered on the issue of arbitrarily chosen thresholds. In our manuscript, we used a range of thresholds, i.e., 50%, 100%, 150% and 200% of change in the gene expression levels of the ancestral lowland tree sparrow to detect genes with reinforcement and reversion plasticity. By this design we wanted to explore the magnitudes of gene expression plasticity (i.e., Ho & Zhang 2018), and whether strength of selection (i.e., genetic variation) changes with the magnitude of gene expression plasticity (i.e., Campbell-Staton et al. 2021).

As the reviewer pointed out, we have now realized that this threshold selection is arbitrarily. We have thus implemented two other categorization schemes to test the robustness of the observation of unequal proportions of genes with reinforcement and reversion plasticity. Specifically, we used a parametric bootstrap procedure as described in Ho & Zhang (2019), which aimed to identify genes resulting from genuine differences rather than random sampling errors. Bootstrap results suggested that genes exhibiting reversing plasticity significantly outnumber those exhibiting reversing plasticity, suggesting that our inference of an excess of genes with reversion plasticity is robust to random sampling errors. We have added these analyses to the revised version of manuscript, and provided results in the Figure 2d and Figure 3d.

In addition, we adapted a bin scheme (i.e., 20%, 40% and 60% bin settings along the spectrum of the reinforcement/reversion plasticity). These analyses based on different categorization schemes revealed similar results, and suggested that our inference of an excess of genes with reversion plasticity is robust. We have provided these results in the Supplementary Figure S2 and S4.

The second issue that the reviewer raised is that the plastic changes simply have more space to reverse direction rather than having any biological reason to do so. While a causal reason why there are more genes with expression levels being reversed than those with expression levels being reinforced at the late stages is still contentious, increasingly many studies show that genes expression plasticity at the early stage may be functionally maladapted to novel environment that the species have recently colonized (i.e., lizard, Campbell-Staton et al. 2021; Escherichia coli, yeast, guppies, chickens and babblers, Ho and Zhang 2018; Ho et al. 2020; Kuo et al. 2023). Our comparisons based on the two genesets that are associated with muscle phenotypes corroborated with these previous studies and showed that initial gene expression plasticity may be nonadaptive to the novel environments (i.e., Ghalambor et al. 2015; Ho & Zhang 2018; Ho et al. 2020; Kuo et al. 2023; Campbell-Staton et al. 2021).

Q3. The correlation between plastic change and evolved divergence is an artifact due to the definitions of adaptive versus maladaptive changes. For example, the definition of adaptive changes requires that plastic change and evolved divergence are in the same direction (Figure 3a), so the positive correlation was a result of this selection (Figure 3d).

The reviewer raised an issue that the correlation between plastic change and evolved divergence is an artifact because of the definition of adaptive versus maladaptive changes, for example, Figure 3d. We agree with the reviewer that the correlation analysis is circular because the definition of adaptive and maladaptive plasticity depends on the direction of plastic change matched or opposed that of the colonized tree sparrows. We have thus removed previous Figure 3d-e and related texts from the revised version of manuscript. Meanwhile, we have changed Figure 3a to further clarify the schematic framework.

Reviewer #1 (Recommendations For The Authors):

Q1. Here are private recommendations that I think could help improve the manuscript. West-Eberhard was a pioneer back in 2003 in explicating the hypothesis of "plasticity first". I think it is important to cite their main work in the first paragraph of introduction and to use the term "plasticity-first", which is widely known among evolutionary biologists studying phenotypic plasticity, instead of "plasticity followed by genetic change", since the three papers cited in paragraph 1 call it « plasticity first ».

West-Eberhard, M.J. (2003) Developmental Plasticity and Evolution, Oxford University Press.

Thank you for suggesting West-Eberhard (2003) and we have cited this important work. We have also changed “plasticity followed by genetic change” to “plasticity first”.

Q2. Introduction. Line 5, Change for « On the one hand, if plasticity changes ... »

We have modified as suggested.

Q3. Line 52, Change for « ...same direction as adaptive evolution does ...»

We have modified as suggested.

Q4. Line 66,When presenting papers that address the plasticity and evolution of gene expression in response to environmental variables, paper by Morris et al is another example that could be useful to include (but this is only a suggestion in case the authors missed it).

Thank you for suggesting this nice work. We have cited Morris et al. (2014).

Q5. Line 94, Change for "We acclimated"

We have modified as suggested.

Q6. In Figure 3, the figure in panel A and B is labelled "normaxia", but I think that "normoxia" is usually the term used.

Thank you for spot the typo. We have modified Figure 3a and we no longer used the term “normaxia”.

Material and methods

It would be important to merge supplementary table 1 and 2 and only present the individuals that were used with their respective cardiac and muscle libraries (if they come from the same individual?). Also, the origin of the individuals used in the hypoxia experiment should be explained at the beginning of the methods section and explicated in the supplementary table. Information on sex or stage of development (juvenile? Adult? Male? female?) and time of year (in breeding stage? Pre-migration (if any), etc) would allow the reader to see that individuals from lowland differed only in their exposure to hypoxia or not, or if other variables may affect gene expression patterns. Similarly, if all individuals form the highland are males and the lowland hypoxia exposed individuals are females (or juveniles versus breeders, or different time of year, etc) this should be stated in the methods. Gene expression is labile so the reader should know if other variables influence the results presented or not.

Thank you for suggestion. We have added detailed information (i.e., age, collecting time and season) to the supplementary Table 1. We have also added this information to the Methods. Because the birds used in transcriptomic analysis (Supplementary Table 1) were different individuals from those used in the sequence analyses (Supplementary Table 2), these two tables cannot be merged.

References:

Campbell-Staton SC, Velotta JP, Winchell KM. 2021. Selection on adaptive and maladaptive genes expression plasticity during thermal adaptation to urban heat islands. Nat. Commun. 12: 6195.

Ghalambor CK, Hoke KL, Ruell EW, Fischer EK, Reznick DN, Hughes KA. 2015. Non-adaptive plasticity potentiates rapid adaptive evolution of gene expression in nature. Nature 525:372–375.

Hao et al. 2023. Divergent contributions of coding and noncoding sequences to initial high-altitude adaptation in passerine birds endemic to the Qinghai–Tibet Plateau. Mol. Ecol. Doi: 10.1111/mec.16942.

Ho WC, Zhang J. 2018. Evolutionary adaptations to new environments generally reverse plastic phenotypic changes. Nat. Commun. 9: 350.

Ho WC, Zhang J. 2019. Genetic gene expression changes during environmental adaptations tend to reverse plastic changes even after correction for statistical nonindependence. Mol. Biol. Evol. 36: 604–612.

Ho WC, Li D, Zhu Q, Zhang J. 2020. Phenotypic plasticity as a long-term memory easing readaptations to ancestral environments. Sci. Adv. 6: eaba3388.

Kuo KC, Yao CT, Liao BY, Weng MP, Dong F, Hsu YC, Hung CM. 2023. Weak gene-gene interaction facilitates the evolution of gene expression plasticity. BMC Biol. 21: 57.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity):

1) It is interesting MxDnaK1 seems to prefer cytosolic proteins while Mx-DnaK2 prefers inner membrane proteins. The domain-swapping experiments seem to suggest that the NBD is important for this difference. How NBD is important is not addressed. Is it due to ATP hydrolysis, NBD-SBD interaction, or co-chaperone interactions?

Answer: Thanks for your comments. We speculate that the co-chaperone interaction might be the key factor contributing to substrate differences. According to the working principle of Hsp70, its functional diversity is largely determined by substrate differences. Co-chaperones, such as JDPs, play a crucial role in this process as they possess the ability to bind substrates and facilitate their targeted delivery. Therefore, much of the functional diversity of the HSP70s is driven by a diverse class of JDPs 1,2. We found that NBD played important roles in cochaperone recognition of MxDnaKs. Additionally, it is generally accepted that the efficiency of ATP hydrolysis does not significantly impact the substrate recognition of Hsp70. Furthermore, if the NBD-SBD interaction is crucial, the substitution of either the NBD or SBDβ domain might result in similar cell phenotypes, as both alterations disrupt the original NBD-SBDβ interaction. We believe the DnaK proteins and their cochaperones both determine the substrate spectrums. We made corresponding modifications in the revised manuscript. (Page22; Line 488-494 in the marked-up manuscript)

2) About the interactome analysis, since apyrase was added to remove ATP, it's surprising multiple Hsp40s were found in their analysis. Hsp70-Hsp40 interaction is known to require ATP. This may suggest some of the proteins found in their interactome analysis are artifacts. The authors should perform negative controls for their interactome analysis, such as using a control antibody for their CO-IP and analyze any non-specific binding to their resin.

In addition, since JDPs were pull-down, is it possible some of the substrates identified are actually substrates for JDPs, not binding directly to DnaKs?

Answer: This is an interesting question. As you correctly noted, the interaction between Hsp70 and Hsp40 requires ATP. In our experiment, we used apyrase to remove ATP in order to promote tight binding of substrate by DnaK. This methodology was initially described by Calloni, G. et al in 20123, and the authors also identified the co-chaperone protein DnaJ, but with a concentration higher than 77% of the interactors. In our opinions, the incomplete removal of ATP could be the underlying cause of this phenomenon.

We apologize for the undetailed description in Methods. Actually, we implemented negative controls for each MxDnaK in order to eliminate the potential non-specific interactions with Protein A/G beads or antibodies. Specifically, we conducted a CO-IP experiment without the presence of antibodies to assess any non-specific binding to the Protein A/G beads. To further investigate non-specific binding to the antibodies of MxDnaK2 and MxDnaK1, we utilized the mxdnak2-deleted mutant (strain YL2216) and the MxDnaK1 swapping strain with the MxDnaK2 SBDα (strain YL2204), respectively. As the SBDα of MxDnaK1 was employed as antigen to generate antibodies, and YL2204 can’t be recognized by anti-MxDnaK1 (Figure S5). We believe these controls allowed us to evaluate and exclude the non-specific interactions in our CO-IP. We have improved our description in methods. (Page 27; Line 596-607)

While one of the main functions of JDPs is to interact with unfolded substrates and facilitate their delivery to Hsp70, there may still be substrates that do not directly bind to Hsp70. It’s thus possible that some of the substrates identified only bind to JDPs. We made corresponding modifications in the revised manuscript. (Page 14; Line 290-292)

3) For Figure S7, the pull-down assay used His6-tagged JDPs. Ni resin is known to bind Hsp70s non-specifically. It's not surprising DnaK showed up in all the pull-down lanes, especially considering how much DnaK was over-expressed. For some pull-down lanes, the amount of DnaK is much more than that of JDPs, further indicating artifact. The author should include negative controls such as JDPs without His6-tag or any irrelevant protein with His6 tag.

Answer: Thanks for your suggestion. As you and another reviewer pointed out, there were some flaws in the experimental design of the pulldown assay. These include the non-specific binding of Hsp70 proteins to nickel resin, the absence of a negative control without a tag, and the inappropriate selection of the MBP tag. Thus, we employed the nLuc assay as an alternative to the pulldown experiment to validate the interaction between DnaK and JDP (Figure S9). While our manuscript employed nLuc to confirm protein dimerization, it is worth noting that nLuc assay was originally devised for investigating protein interactions 4.

4) For the proposed dimer formation in Fig. 4C, there are multiple bands above the monomer bands. What are these forms? It seems the majority of the Cys residues that could form disulfide bonds are in the NBD of MxDnaK2 since constructs with MxDnaK2-NBD form some sort of high-MW bands above the monomer. Does MxDnaK1-NBD also contain Cys at the analogous positions? The fact that MxDnaK1 didn't show disulfide-bonded bands doesn't mean it doesn't form dimer. It depends on where the Cys residues are.

It's nice the authors did Fig. 4D. However, the authors should include a positive control to show how strong the signal is for a true interaction before interpreting their results.

Answer: Thank you very much for your comments. In at least three independent experiments, we consistently observed two unidentified bands within the molecular weight range of 70-100 kDa during the purification process of His6-MxDnaK2. These bands appeared to be intermediate in size between the monomeric and dimeric forms of His6-MxDnaK2, and disappeared upon DTT treatment. the unidentified band compositions have been confirmed by LC/MS. The upper band included MxDnaK2 (65.3 kDa) and anti-FlhDC factor of E. coli (WP_001300634.1, 27 kDa). In the lower band, we detected the presence of MxDnaK2 and the 50S ribosomal protein L28 of E. coli (WP_000091955.1, 9 kDa). Based on these findings, we conclude that these two additional bands are the result of the interaction between His6-MxDnaK2 and these two E. coli proteins. The related explanations have been added in the legend of Figure 5. (Page 42; Line 938-942)

We analyzed the presence of Cys in MxDnaK1 and MxDnaK2. The NBD region of MxDnaK2 contains two Cys, located at positions 15 and 319. MxDnaK1-NBD contain a Cys at position of 316, which is the analogous position of 319-Cys of MxDnaK2. The analogous position of 15-Cys of MxDnaK2 is a Val in MxDnaK1, which might be an important factor contributing to the inability of MxDnaK1 to form oligomers.

Thanks for your suggestion to add the positive control. We re-performed the nLuc assays including a positive control(αSyn). According to the working principle of the nLuc assay, the amount of fluorescent substrate is limited. Therefore, even for proteins that interact with each other, the fluorescence value gradually decreases and reaches a plateau, similar to the negative control. This gradual decline in fluorescence is a significant indicator of protein interaction. In Figure 4D (Figure 5D in the revision version), we only presented the results of the first 20 minutes of detection. The complete two-hour detection results have been added in the supplementary figure (Figure S14).

5) line 48: "human HSC70 and HSP70 are 85% identical, and the phenotypes of their knockout mutants are different, which is consistent with their largely nonoverlapping substrates" The authors completely ignored that the promoters of HSC70 and HSP70 are very different.

Answer: This is our carelessness. Yes, HSC70 and HSP70 exhibit distinct expression patterns, which play important roles in their functional diversity. We modified the sentence in the new version (Page 5; Line 58)

6) Line 69: "The two PRK00290 proteins, not the other Myxococcus Hsp70s, could alternatively compensate the functions of EcDnaK (DnaK of E. coli) for growth." Please add references for this statement.

Answer: Added, thanks.

7) line 191: What's the mechanism for DnaK's role in oxidative stress? Is the disulfide bond formation in Fig. 4 related? Does disulfide-bond change the activity of DnaK?

Answer: Thanks for your pertinent comments. Honestly, we have no idea about the mechanism for MxDnaK2's role in oxidative stress. In our previous studies, we determined that the deletion of mxdnaK2 resulted in a longer lag phase after H2O2 treatment. Here, our aim was to investigate the impact of region swapping on the cellular function of MxDnaK2. In other bacteria, the critical role that DnaK plays in resistance to oxidative stress stems from the pleotropic functions of this chaperone. By shortening the dwelling time that proteins spend in the unfolded state, the DnaK/DnaJ chaperone system minimizes the risk of metal-catalyzed carbonylation of the side chains of proline, lysine, arginine, and threonine residues, but none of them linked to the dimerization characteristic of DnaK 5-7.

8) Fig. S9 seems redundant.

Answer: Deleted, thanks.

9) line 263, "but the NBD exchange was almost equal to the deletion of the gene with respect to phenotypes." But, the mutant has >50% activity in Fig. 3F.

Answer: We apologize for the confusing description. The “phenotypes” here indicates “cell phenotypes”. What we really tried to say with this sentence is that the NBD swapping strain of either MxDnaK1 or MxDnaK2 presented identical cell phenotypes with the gene-deleted strain. As we have already provided a detailed description of this result earlier, now we consider this sentence to be redundant and have therefore deleted it. (Page 17; Line 355-356)

10) line 221-226: the logic is not quite clear.

Answer: We apologize for the confusing description. In M. xanthus DK1622, MxDnaK1 is essential for cell survival, and an insertion of a second copy of mxdnaK1 in the genome is required for deletion of the in-situ gene. Thus, To verify whether the NBD region is required for the essentiality of MxDnaK1, we performed the region swapping of the in situ MxDnaK1 gene in the att::mxdnaK1 mutant (a DK1622 mutant containing a second copy of mxdnaK1 at attB site), and successfully obtained the MxDnaK1 mutant swapped with the MxDnaK2 NBD region. The experiment indicated that the NBD of MxDnaK1 is essential for the cellular functions of the chaperone. We have added the information and modified the sentences in the manuscript. (Page 15; Line 308-319)

Minor concerns:

Please check spelling. There are some typos such as "HPPES" in the Methods.

Answer: Corrected. Many thanks.

My areas of expertise are protein biochemistry, genetics, and structural biology on heat shock proteins.

Reviewer #2 (Evidence, reproducibility and clarity):

Major comments:

The manuscript is very nice and interesting, although some of the authors' conclusions are perhaps not well supported by their data. For example:

1) In the pulldown experiments the lack of interaction between 2747-MxDnaK2, 3015-MxDnaK2 and 1145-MxDnaK1 should be shown in order to support the conclusion made in line 197-198,

Answer: This is our carelessness. As you and another reviewer pointed out, there are some flaws in the experimental design of the pulldown assay. These include the non-specific binding of Hsp70 proteins to nickel resin, the absence of a negative control without a tag, and the inappropriate selection of the MBP tag. Thus, we employed the nLuc assay as an alternative to the pulldown experiment to validate the interaction between DnaK and JDP (including 2747-MxDnaK2, 3015-MxDnaK2 and 1145-MxDnaK1 interaction) (Figure S9). While our manuscript employed nLuc to confirm protein dimerization, it is worth noting that nLuc assay was originally devised for investigating protein interactions 4.

2) The only evidence that the NBD of MxDnaK1 is essential for bacterial growth is that this mutation couldn´t be obtained in M. xanthus. However, it could be purified in E. coli. Could the authors do some experiments with the M. xanthus strain without the chromosomal MxDnaK1 and then introduce a plasmid with the mutated gene?

Answer: We apologize for the confusing description. Actually, we determined the NBD is essential not only from the mutation couldn’t be obtained. In M. xanthus DK1622, MxDnaK1 is essential for cell survival, and in-situ deletion of the gene could be obtained after an insertion of a second copy of mxdnaK1 in the genome at the attB site. To verify whether the NBD region is required for the essentiality of MxDnaK1, we performed the region swapping of the in situ MxDnaK1 gene in the att::_mxdnaK_1 mutant (a DK1622 mutant containing a second copy of _mxdnaK_1), and successfully obtained the MxDnaK1 mutant swapped with the MxDnaK2 NBD region. The experiment indicated that the NBD of MxDnaK1 is essential for the cellular functions of the chaperone. We have added the information and modified the sentences in the manuscript. (Page 15; Line 308-319)

3) All the experiments with purified proteins were done with MxDnaKs bearing His-tags. It doesn't say explicitly its position, but as they employed a pET28A it is likely that the tag is at the N-terminus, which is close to the linker region. As this tag might interfere, it should be removed for the experiments, or at least a control done with the tag removed.

Answer: We apologize for the lack of detailed description. As you pointed out, the His-tags are located at the N-terminus of DnaKs. The full lengths of MxDnaK1 and MxDnaK2 are 638 and 607 amino acids. The linker regions are located at amino acid positions 381-386 for MxDnaK1 and 387-392 for MxDnaK2. Therefore, we believe that the His-tag is not close to the linker regions. We have included the information in new manuscript. (Page 24; Line 544-546)

The purified His6-DnaK proteins were employed for holdase activity assays and in vitro dimerization assays. Several previous studies have utilized the same holdase activity assay method with His-tagged DnaK 8,9. We suggested that the His-tag did not interfere with the holdase activity of DnaK. To exclude the influence of His-tag on oligomerization, we conducted a control with the tag removed in the in vitro dimerization assay and the result show no difference (Figure S13).

4) The authors state that MxDnaK dimerized in vitro with the NBD, and to disrupt the dimer they used 100 mM DTT, which is a very high concentration. As the protein has the His-tag, it should be removed to corroborate that it is not interfering with the dimerization.

Answer: Thanks for your suggestion. As mentioned above, to exclude the influence of the His-tag on oligomerization, we conducted a control with the tag removed in the in vitro dimerization assay and the result show no difference (Figure S13).

5) Why were the pulldown experiments done with MBP-MxDnaKs? Can you show a negative control between the MBP and the JDPs to rule out this interaction? It will be more suitable to do the pulldown assays with the purified MxDnaK´s without the His-tags (and the His-tags JDP that were employed).

Answer: Thanks for your suggestion. As mentioned above, there are some flaws in the experimental design of the pulldown assay. Thus, we employed the nLuc assay as an alternative to the pulldown experiment to validate the interaction between MxDnaKs and JDPs (Figure S9).

Minor comments:

• E. coli´s DnaK is only essential in heat shock conditions and for lambda phage cycle. If MxDnaK1 is similar to this Hsp70, why the substitution of its NBD for the NBD MxDnaK2 would be lethal for bacterial growth?

Answer: Thanks for the comments. As you correctly point out, DnaK is nonessential in E. coli. But in some other bacteria, DnaK also plays an essential role in cell growth for different reasons 10-12. In our previous studies, we determined that MxDnaK1 is essential in M. xanthus DK1622, and the MxDnaK2 is nonessential. In this study, we performed region swapping and found that only the NBD of MxDnaK1 was unreplaceable. In our opinions, the result indicated that NBD play important roles in the functional diversity between MxDnaK1 and MxDnaK2.

• I think that the writing should be revised and in the supporting information the captions of the figures should include more information.

Answer: Thanks a lot for the suggestion. We revised the manuscript and added more information in the legends of supplementary figures.

Reviewer #2 (Significance):

-General assessment: This is a nice piece of work which would benefit from revision to address the comments above. The authors showed the roles and differences between two DnaK in the same organism. They track these differences to the subdomains of the MxDnaK´s and co-chaperones. It will be interesting for future works to explore more deeply the co-chaperones and their interactions.

-Advance: I think that this manuscript fills a gap regarding the role of DnaK duplicated in bacterial strains. -Audience: I would say that the audience is broad and includes scientists interested in protein folding and chaperones, as well as myxobacteria.

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2. Kampinga, H. H. & Craig, E. A. The HSP70 chaperone machinery: J proteins as drivers of functional specificity. Nat Rev Mol Cell Biol 11, 579-592, doi:10.1038/nrm2941 (2010).
3. Calloni, G. et al. DnaK functions as a central hub in the E. coli chaperone network. Cell Rep 1, 251-264, doi:10.1016/j.celrep.2011.12.007 (2012).
4. Dixon, A. S. et al. NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells. ACS Chem Biol 11, 400-408, doi:10.1021/acschembio.5b00753 (2016).
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7. Fredriksson, A., Ballesteros, M., Dukan, S. & Nystrom, T. Induction of the heat shock regulon in response to increased mistranslation requires oxidative modification of the malformed proteins. Mol Microbiol 59, 350-359, doi:10.1111/j.1365-2958.2005.04947.x (2006).
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9. Thompson, A. D., Bernard, S. M., Skiniotis, G. & Gestwicki, J. E. Visualization and functional analysis of the oligomeric states of Escherichia coli heat shock protein 70 (Hsp70/DnaK). Cell Stress Chaperones 17, 313-327, doi:10.1007/s12192-011-0307-1 (2012).
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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary: Kellner and Berlin present their research findings pertaining to the effect of GRIN2B variants that modify NMDA receptor function and pharmacology. While these mutations were published previously, the new manuscript provides a more thorough investigation into the effects that these variants pose when incorporated into heteromeric complexes with either wildtype GluN2B or GluN2A - NMDA receptors containing only a single mutated GluN2B subunits is more relevant to the disease cases because the associated patients are heterozygous for the variant. The authors achieved selective expression of receptor heteromeric complexes by utilising an established trafficking control system. The authors found that while a single variant subunit in the receptor complex is largely dominant in its effect on reducing glutamate potency of the NMDA receptor, it 's effect on receptor pharmacology varied. Unlike diheteromeric receptors containing mutated subunits, polyamine spermine potentiated GluN1/2B (but not GluN1/2A/2B) receptors that contained a single mutated GluN2B. In contrast, the neurosteroid, pregnenolone-sulfate (PS), was effective at potentiating the NMDA receptor currents (to varying degrees) regardless of the subunit composition. The potentiation of NMDA receptor currents by PS was also observed in neurons overexpressing the variants.

The techniques used in this study were appropriate to address the objectives and the overall effects are large, and generally convincing. I like the way the results are presented, although have a few (easily addressable) comments.

We thank the reviewer for the positive remarks on our manuscript.

Major comments:

#1 When incrementally adding drugs (e.g. traces in figures 5 and 6), it doesn't always appear like the response has plateaued before changing the solutions/drugs. Therefore, I am curious to what extent the effects observed are underestimated.

The reviewer is correct to note that some responses do not necessarily reach a plateau, despite our efforts reach steady-state (as shown in most figures, e.g., Figs. 1-4, 6b, etc.), in particular when applying pregnenolone-sulfate (PS) (Fig. 5a, all traces in middle and bottom rows). However, in several instances, this was unobtainable due the very slow effect of the neurosteroid (its mode of action is from within the membrane) and the very large size of the cell (~1 mm). For these reasons, these experiments mandated excessively long exposures (~minutes) of oocytes to glutamate and PS (see scale bar- 20 secs) to try to reach steady-state, however this also caused deterioration to some cells (which did not return to baseline- and were therefore discarded). Thus, we eventually converged on settings whereby we did not expose oocytes to more than 4 minutes of the drug. Nevertheless, to try to estimate the extent of the underestimation (if any), we fitted the currents (standard mono-exponential fit, as previously reported1–3 (Suppl. 5a). We found that our application times of PS were, on average, three time the response’s time constants (tau) (Suppl. 5b), and we found a very weak relationship (R2 = 0.09) between the response to PS and time of its application (Suppl. 5c). These are now explicitly mentioned in the text (line #203), and in the legend of Suppl. 5. These thereby suggest that the reaction reached approximately 95% (1 - 1/e^3) of the steady-state value, and we are therefore confident that we have very small, if any, underestimation the extent of PS potentiation.

2 Also, in relation to figure 6, to what extent does agonist application cause desensitization here? Looking at traces in Figure 6b it appears that there is some desensitization and it isn’t clear to what extent this persists during the solution changes.

Agonist desensitization of NMDARs-currents is a well-known phenomenon, but it is very well established that it is not always observed in cells, including neurons (e.g., 4–7). In general, we did not observe very frequent desensitization’s (we provide a larger variety of traces of desensitizing and non-desensitizing currents (Fig. 6b Suppl. 7e and Suppl. 8a). Nevertheless, we explicitly note that in neurons, currents that didn’t reach steady-state after application of 100 mM NMDA were excluded from analysis (Methods - Patch clamping of cultured neurons, line #474), and in most cases desensitization was minor (or absent) following application of 100 mM NMDA and 100 mM PS (Fig. 6b).

3 Could the authors conduct/show the controls where NMDA alone (for 50-60s), or NMDA followed by PE-S (without ifenprodil).

These recordings are now shown in Fig. 6b and Suppl. 8a, (as opposed to Suppl. 7e).

#4 Finally, figure 5 shows the effect of the neurosteroid (and ifenprodil) on NMDA-evoked currents in neurons overexpressing the GluN2B variants in neurons. However, there currents probably reflect a mixture of extrasynaptic and synaptic receptors. To what extent are synaptic NMDA receptors affected by the variants?

To show the extent of the effect of the variants over synaptic receptors, we recorded miniature NMDA-dependent EPSCs; mEPSCNMDA), as described in our previous report8. We find that the varinats completely eliminate the appearance of mEPSCs (Suppl. 7a, b). Change in minis’ frequency is not the result of a presynaptic change or a change in synapse number9, as we have shown that AMPAR-mEPSC frequency was unaffected by the variants (i.e., synapse number and probability of presynaptic release are unchanged by the variants).

  To further address this, we also explored the relative synaptic vs. extrasynaptic distribution of the variants by using the established MK-801-protocol (to block all synaptic receptors during spontaneous activity, leaving extrasynaptic receptors unblocked)10,11. In neurons overexpressing the GluN2B-*wt* subunit, we obtained an extrasynaptic fraction of 38%, highly consistent with previous reports12,13. Overexpression of the variants, however, yielded a significantly and higher fraction (~50%) of the remaining current, supposedly suggesting more variant receptors at extrasynaptic loci (__Suppl. 8b, c__). However, due to the experimental settings we have chosen, the results from this experiment represent quite the inverse when involving extreme LoF variants. Firstly, 100 mM NMDA does not saturate variant receptors (whether pure, mixed di- or tri-heteromers, see __Table 1__). Secondly, normal neurotransmission does not open synaptic receptors containing mutant GluN2B-subunits, attested by the complete absence of mEPSCs (see __Suppl. 7a, b and __8,9). Thus, during the 10 minutes exposure to MK-801, only (mostly) purely *wt* receptors are blocked by spontaneous synaptic activity, and thus the second bout of 100 mM NMDA solely exposes the remaining *wt*-receptors. An increase in the number reflects more *wt*-receptors at the extrasynapse than the synapse. Thus, the observed increase in the fraction of extrasynaptic receptors in neurons overexpressing the variants, implies that the number of *wt*-receptors is necessarily decreased from the synapse and increases at the extrasynapse. We deem this to ensue due to the incorporation of the variants at the synapse. This increase cannot be explained by an overall increase in membrane expression of *wt*-receptors in neurons overexpressing the variants, as these cells show a strong reduction in Imax  (see __Fig. 6c and Suppl. 7e__). This is now detailed in the text (lines #270-290).

Minor comments:

5 Looking at the fits in the graph of Figure 2b it appears that the slope on the concentration response curves is less steep for the mixed 2B-diheteromeric NMDA receptors. How much are the Hill coefficients changing and can this be interpreted to provide more mechanistic insight? Wouldn't it make sense to include the Hill coefficients in Table 1?

We agree with the reviewer’s observation. Actually, the mixed di-heteromers have a similar Hill coefficient (nH) as the purely di-heteromeric GluN2Bwt receptors (see Table 1), and these show the typical near nH ~1 (e.g., 14–16). The only diverging groups are the purely di-heteromeric variant-containing channels (G689C/S only containing receptors; nH~2). Although these may suggest positive cooperation between the subunits, we are less inclined to infer insights from the latter owing to the fact that we limited our examination to 10 mM glutamate (we limit exposure of oocytes to 10 mM glutamate due to artifacts arising past this concentration, as discussed in Kellner et al.8: Fig. 2—figure supplement 1). (this description is now mentioned in page lines #149, 318, 319).

6 The authors illustrate the changes in potency by the shift in the concentration response curves, but is there any change in efficacy? A simple way to illustrate this would be also present a simple graph showing the maximum current amplitudes (i.e. to 10 mM glutamate) for each of the receptor complexes.

We now provide these data in (Suppl. 2a, b). We would like to note however that the expression pattern of the tailed-receptors (i.e., subunits with carboxy-termini tagged with C1/C2 tails, see Fig. 1a) are less expressive in general when compared with the native subunits (Suppl. 2c). This description is detailed in lines# 162-166.

#7 The authors characterize the 'apparent' affinity (or potency) of the receptor using concentration-response curves, but numerous points in the manuscript refer to changes in affinity. None of the experiments shown directly measure affinity (which would require ligand-binding assays) and so the use of the word affinity is inaccurate/misleading. I suggest the authors replace the instances of the word 'affinity' with 'potency'.

We apologize for the confusion surrounding our use of the term affinity. In fact, we do initially define this term in introduction (page #4): “apparent glutamate affinity (EC50)” to differentiate from affinity (KD). Regardless, and to avoid confusion, we replaced all terms, as suggested by reviewer to potency.

#8 In the third line of the abstract, the authors wrote, 'for which there are no treatments' in relation to GRINopathies. My understanding is that there are symptomatic treatments but that there are no disease-modifying treatments.

Indeed, all current treatments are supportive, rather than provide a bona fide cure or disease-modifying. These are now better defined in the abstract.

#9 The authors have interchangeably used the terms NMDAR or GluNRs throughout the manuscript. I suggest sticking to one of these terms. I would suggest NMDARs since this is less likely to be misread as a a specific NMDA receptor subunit.

Agreed and corrected throughout manuscript.

#10 Typos: 1) Results paragraph 2 sentence one: 'We thereby produced GluN2B-wt, GluN2B-G689C and GluN2B-G689S subunits tagged with C1 or C2, co-expressed these along with the GluN1a-wt subunits in...') Results paragraph 2: '...but these were mainly noticeable when oocytes are were exposed to high (saturating) glutamate concentrations...'
3) Last sentence in the second to last paragraph of the results section entitled 'Mixed di-and tri-heteromeric channels...': 'This , PS may serve to rescue...'
4) Last sentence in last paragraph of the results section entitled 'Mixed di-and tri-heteromeric channels...': 'Despite the latter, we found no evidence for any direct effect of three different physiologically relevant concentrations of the drug on di- or tri-heteromeric receptors'

All typos corrected.

#11 Figures 1e, 2b, 3b: it would be helpful to add a legend to the graph so that the curves can be interpreted without having to read through the figure legend.

Corrected.

#12 The bar graphs in Figure 6 show individual data points but those in figures 4b and 5b don't. Can the authors please add the data points to these graph.

Individual data points have been added.

#13 It would be helpful to reviewers that future manuscripts by the authors include page numbers and line numbers.

Included.

**Referees cross-commenting**

#14 Reviewers 2 and 3 highlight an important issue concerning figure 6 and the extent to which the overexpressed variants subunits can compete and assemble with endogenous NMDA receptors (unlike the system where the surface expression of specific receptor complexes is controlled). Indeed in the recent paper by the same authors, the two variants differed in their surface expression (in HEK cells), with G689C expressing particularly poorly. With reference to the second minor comment of Reviewer 1, the maximum current amplitudes would of course need to be normalized to cell surface expression of the receptor to gain any insight into efficacy.

We provide maximal current amplitudes (Imax) as a proxy for expression level as typically done (e.g.,8,17). These are now shown in Suppl. 2a, b (and see our response to comment #6, above). We would like to emphasize that we find it challenging to gain insights about efficacy of the variants in neuronal synapses, as we purposefully express non-C1/C2 tagged subunits in neurons (as we covet assembly of the variants with endogenous subunits). Moreover, the C1/C2-tagged subunits (whether wt or variants) are less expressive compared to their non-tagged NMDAR-counterparts. For instance, tagged GluN2B-wt subunits express at ~50% compared to non-tagged GluN2B wt subunits (Suppl. 2c). Thus, we find that efficacy of the C1/C2 tagged-subunits is less relatable to the non-tagged subunits (which are used in neurons and likely more relevant to the disease).

Despite the latter, we deem that we have specifically addressed this issue by measuring miniature EPSCs (mEPSCs) (see our reply to comment #4, Suppl. 7a, b). Briefly, even though the non-tagged G689C expresses at ~40% compared to other subunits (in oocytes and mammalian cells8), in neurons it engenders a robust (and highly significant) negative effect over synaptic currents (mEPSCs), as strong as the G689S-variant which expresses much more robustly (non-tagged G689S expresses to same extent as wt subunits). This demonstrates that the reduced efficacy the tagged subunits is less relatable to the non-tagged subunits and, importantly, it does not hinder the variants’ ability to incorporate within the synapse and affect function (i.e., exert a dominant negative effect). Here, we extend these observations towards the major postnatal channel subtype, namely tri-heteromers (2A/2B*), and therefore demonstrate that the robust dominant negative effect of G689C and G689S variants is likely due to their ability to incorporate within the predominant receptor subtype at the synapse (Suppl. 8).

Reviewer #1 (Significance (Required)):

This study emphasizes the complex pattern of effects that variants can have on glutamate receptor function and pharmacology, especially considering the context of receptor subunit composition. The authors have followed up their previous findings on the same mutants (Kellner et al, 2021, Elife), but used a trafficking control system here to characterize properties of mutated receptor complexes that are most likely to exist in neurons. The authors show that the defective currents mediated by NMDA receptors containing a loss-of-function GluN2B variant can be enhanced by neurosteroids (and in the case of GluN1/2B receptors, polyamines also). Development and approval of neurosteroids for the clinic would be required for the findings to translate to a therapy for patients. Readers should also be aware that neurosteroids act on other receptors too (e.g. GABA receptors), which could complicate the outcome. The expertise of the reviewer is in glutamate receptors and synaptic transmission.

We agree with the reviewer’s comment pertaining to challenges in translating PS to the clinic. Indeed, we explicitly mentioned its inhibitory effect on GABAA receptors (see line #366-367 and reference 18), as well as note its potential negative effect over GluN2C/D-containing receptors (line #365 and reference 19). We further describe alternative neurosteroids and means to bypass the limitations of PS, for instance by use of 24(S)-hydroxycholesterol6,18 or synthetic analogues (SGE-201, SGE-301)6. Lastly, we also propose a novel therapeutic approach, for which we did not find any mentions in the literature with regard to GRINopathies, consisting of the use of the FDA-approved Efavirenz (anti-retroviral compound20) to promote activity of cytochrome P450 46A1 (CYP46A1) to increase amounts of 24-S in the brain (discussion, lines #370-383).

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

#1 The objective of this paper is to assess whether a single mutated subunit of GRIN can affect the function of various forms of NMDA receptors. In particular, this study investigates the functional consequences of a GRIN variant when assembled within tri-heteromers, containing 2 GluN1, 1 GluN2A and 1 GluN2B subunits, the major postnatal receptor type. For this purpose, the authors artificially forced the subunits to associate in predefined complexes, using chimeras of GRIN subunits fused to GABAb receptor retention control sites at the endoplasmic reticulum. This trick allows to control the stoichiometry of the channels at the membrane and thus to focus on the function of a single type of NMDA receptor.The take home message of the paper is that a single GluN2B‐variant, whether assembled with a GluN2B‐wt subunit to form mixed di‐heteromer or with a GluN2A‐wt‐subunit (tri‐heteromer), strongly impairs the receptor functioning, as reported by a decrease of the apparent glutamate affinity of the receptor.

Altogether, this is a straightforward study of great interest for the GRIN community.

We greatly appreciate the reviewer’s comment about the relevance of our work towards the GRIN-community.

2 However, the way the background and purpose of the study (title and abstract) are presented is a bit confusing for non-specialists and could be easily improved. Technical information, which is crucial to validate the conclusions drawn from data analysis, should be added to the article. Some additional experiments are suggested to consolidate the work. Finally, additional discussion points are strongly encouraged.

We apologize for not making the paper more accessible to a broader readership. We did so for the sake of brevity. Nevertheless, we have re-written major parts of the manuscript to address this issue and retitled the report: “Rescuing Tri-Heteromeric NMDA Receptor Function: The potential of Pregnenolone-Sulfate in Loss-of-Function GRIN2B Mutations”.

Specific comments

Abstract / Title:

3 This work shows that a single GRIN variant impairs the function of various forms of NMDA receptors. Several sentences in the title and the abstract are confusing for a non-specialized audience. "Two extreme Loss‐of‐Function GRIN2B‐mutations are detrimental to triheteromeric NMDAR‐function, but rescued by pregnanolone‐sulfate." "Here, we have systematically examined how two de novo GRIN2B variants (G689C and G689S) affect the function of di‐ and tri‐heteromers." The number of variants tested is not of capital importance in the title, especially because one could believe that both are tested at the same time; similarly, when variants are named in the abstract, the fact that only 1 variant is studied at a time should be clarified (G689C OR G689S). Indeed, the problem is obvious to those familiar with GRIN disorders, but if this paper is to be published in a journal reaching a large audience rather than a specialized audience, the title of the paper should be modified.

As noted in our reply to comment #1 of this reviewer, we apologize for not making the paper more accessible and have therefore changed the title and re-written major parts of the manuscript to address this issue. We would like to note that we appreciate the reviewer’s comment and intent to increase the readership of our manuscript.

#4 "We find that the inclusion of a single GluN2B‐variant within mixed di‐ or tri‐heteromeric channels is sufficient to prompt a strong reduction in the receptors' glutamate affinity, but these reductions are not as drastic as in purely di‐heterometric receptors containing two copies of the variants. This observation is supported by the ability of a GluN2B‐selective potentiator (spermine) to potentiate mixed diheteromeric channels." Please, clarify the link between the two sentences. How do spermin potentiation of mixed diheteromeric channels supports the observation that the inclusion of a single GluN2B‐variant has less effect than the inclusion of two variants?

Our intention was to highlight that mixed di-heteromeric channels (2B/2B*) are less “damaged” (this is the link) than purely di-heteromeric channels (2B*/2B*).Explicitly mixed di-heteromers show less reduction in glutamate potency AND are also spermine-responsive, whereas purely mutant di-heteromers (2B*/2B*) show reduced potency, BUT do not respond to spermine at all. We have rephrased the sentences in our current manuscript to be clearer:

For instance: The positive responses of mixed di-heteromers, compared to the null effect over pure di-heteromers, is likely the result of the restored pH-sensitivity of mixed di-heteromers (Suppl. 3). This was surprising as the minimal and essential rules of engagement for potentiation by spermine are not well established, particularly in the case of tri-heteromers21,22 (see discussion, lines #341-353).

Methods

#5 All this study is based on the use of a unique ER‐retention technique to limit expression of a desired receptor‐population at the membrane of cells. According to the ER system retention of GABAb receptor, used in this study, while C1/C1-fused subunits are retained in the ER, C2/C2 reach the cell surface and the association of C1/C2 in the ER enables cell-surface targeting of the heterodimer. However, GB2 does not contain any retention signal and can reach the cell surface in the absence of GB1, as a functionally inactive homo-dimer (doi: 10.1042/BJ20041435). If there is an experimental trick that prevents the addressing of C2/C2 to the cell membrane, it should be specified and explained. This is critically important for understanding which receptor populations the data are derived from: receptors containing C1/C2 fused subunits only as stated by the authors, or C1/C2 and C2/C2 fused subunits?

We base our experiments on two seminal reports—23,24—that have developed this unique method (which we also refer to in the text, lines# 112-116). Briefly, the method employs the binding motifs of GABAB1 (GB1) and GB2 subunits and ER-retention motifs (these are now better detailed in Methods section, line # 448). Previous reports explicitly demonstrate that C1/C1- OR C2/C2-containing receptors do not reach the plasma (or very minimally) and we have reproduced these data with our variants (C1/C1: Suppl. 1a-d; C2/C2: Fig. 1a-c).

Figures #6 NMDA-receptor current amplitude should be normalized by the membrane expression of the receptors. A preliminary experiment should measure the effective cell surface expression of each of the subunits in the different transfection conditions.

To address the effective cell surface expression, we employed Imax as a proxy for functional expression (e.g.,8,17). These are now shown in Suppl. 2a, b (and see our response to reviewer 1, comments #6 and 14). Expectedly, we find significantly reduced efficacy by the varinats compared to wt-receptors, and the purely mutant di-heteromeric receptors exhibit the weakest efficacy. We have also addressed this issue by measuring miniature EPSCs (mEPSCs) (see our reply to reviewer 1, comment #4,). We find the variants to abolish mEPSCNMDA frequency (Suppl. 7a, b). This shows that the variants’ reduced efficacy translates to elimination of synaptic activity (dominant negative effect) (also seen in Suppl. 8).

#7 Fig.1a

The scheme should include C2-C2 complexes and mention whether these complexes are expressed at the cell surface (see previous and following comments).

As noted in our reply to comment #5 of this reviewer (above), C2/C2-containing receptors do not reach the plasma membrane (Fig. 1a-c). To avoid confusion, we have now added this scheme to the cartoon presented in Fig. 1a and have provided a more detailed description of the method and clones produced in the Methods section (line # 448).

#8 Fig.1b and c

Current from cells transfected with GluN2B‐wt‐C1 and GluN2B‐wt‐C2 should be compared to current expressed in cells expressing untagged receptor subunits: GluN2B‐wt Current from cells transfected with GluN2B‐wt‐C1 alone should be shown as well (although expected to be retained in the ER) (as performed for GluN2A‐wt‐C1 GluN2B‐wt‐C1 in suppl Fig. 1a)

Current comparisons of oocytes expressing tagged GluN2B‐wt‐C1 and GluN2B‐wt‐C2 and non-tagged GluN2B‐wt are now demonstrated in Suppl. 2c. The results indicate that the “tags” (C1 and C2) affect the expression of the subunits. We have also added a sample trace of current from a cell expressing the GluN2B‐wt‐C1 alone (Fig. 1b).

9 How could you explain the null current from cells transfected with GluN2B‐wt‐C2 alone (Fig.1b middle, and 1c)? since GB2 does not contain any retention signal and can reach the cell surface in the absence of GB1, GluN2B‐wt‐C2 is supposed to reach the cell surface. This is a very important point to clarify (I am probably missing a technical detail) because if the sub-unit tagged with C2 does reach the cell surface, then all the results and conclusions drawn from the C1-C2 conditions are wrong and could be attributed to a mix of complexes containing either C1-C2 or C2-C2.

We now realize that the reviewer was missing a crucial technical detail, namely how the clones are designed. Briefly, all clones have ER retention motifs and cannot reach plasma membrane unless they necessarily assemble as C1/C223,24. Also, please see our replies to comments #5, 7 to this reviewer (and Methods section, line # 448).

My following comments are based on the assumption that only receptors containing C1-C2 tagged subunits reach the membrane (as assumed by the authors and suggested in Figure 1b middle), but explanations should absolutely be provided to convince the reader. Fig. 4a and 5a (see our above replies to comments #5, 7 and 9; and references 23,24).

#10 Please, keep the current scale constant between all current illustrations within the same figure (4a and 5a). Indeed, not only the Spermin- or SP- induced potentiation is an important data (which is presently quantified on the histograms of fig. 4b and 5b) but also knowing whether the amount of current recorded in cells expressing one mutant subunit in presence of SP (for example GluN2A‐wt‐C1 GluN2B‐G689S‐C2) is comparable to the current recorded in wt receptor-expressing cells (GluN2A‐wt‐C1 GluN2B‐wt‐C2) in absence of SP would be an excellent added value for the paper. A special figure could quantify this rescue effect of SP, measuring and comparing the mean currents recorded in these conditions (one current illustration is not sufficient given variations between similar samples). By the way 5mM glutamate might be an excessive concentration. At 1mM, the expected synaptic concentration of glutamate following action potential, according to figures 3 and Suppl1 the response of the mutated receptor is much lower than that of the WT which is already almost maximal. In these conditions, SP-induced potentiation by a factor of two of GluN2A‐wt‐C1 GluN2B‐G689S‐C2 current could be equivalent to control currents recorded in GluN2A‐wt‐C1 GluN2B‐wt‐C2 cells.

We have rescaled all current amplitudes in Figs. 4 and 5 to be identical in size for easier comparison.

We have added all current amplitudes to try to examine the rescue effect of the two drugs in cell overexpressing a specific channel subtype, as requested (Suppl. 4). We find that; indeed, the potentiated currents of the mutant receptors reach (or even surpass) the basal Imax (i.e., current before potentiation) of the non-mutated receptors (Suppl. 4, dashed statistics bar).

In neurons, we address this in two ways. First, we show that the total NMDA-current is reduced by expression of the variants, and this current is “normalized” by PS (Fig. 6a-c). Similar reductions in Imax (by the variants) are shown in Suppl. 7e (to provide more examples). Secondly, neurotransmission (i.e., 1 mM glutamate25,26) is not sufficient for activating mutant receptors, certainly not pre-di-heteromers (see Table1, EC50 and Suppl. 7a, b- no mEPSCs)27–29. Therefore, 5 mM was required. Together, these strongly suggest that PS may normalize the currents of different receptors that respond to PS (under physiological settings and not 1- or 5mM NMDA). As suggested by the reviewer, there are many subtypes, and some may be activated by ambient glutamate (as suggested by application of PS onto neurons without opening the receptors by NMDA; see Suppl. 7c, d).

#11 Fig. 6

Figure 6 is not convincing because cultured hippocampal neurons do express endogenous NMDA receptors. To what extent the recording currents are affected by endogenous, non-mutated GluN2B subunits? Western Blots showing an extinction of endogenous subunits expression when transfected tagged subunits are competitively expressed would be required.

We have previously shown that the two variants incorporate very efficiently within synapses, causing a very robust elimination of synaptic currents (by measuring miniature NMDA-dependent EPSCs; minis) [see Fig. 8 in Kellner et al. eLIFE, 202127, and see review by Sabo et al.9 ). Change in minis’ frequency can be interpreted as either a presynaptic change or a change in synapse number, however we observed that AMPAR-mEPSC frequency was unaffected by these variants. These imply that synapse number and probability of release are unchanged by the variants. As the experiments are performed in wild-type neurons, (which express wild-type GluN2A and -2B), the dramatic effects we observed on minis suggests a dominant-negative effect of these disease-associated GluN2B variants. These are consistent with our observations that mutant subunits can co-assemble with wild-type GluN2B and/or GluN2A subunits. We have now reproduced this experiment (in fact, we employ this strategy prior each experiment to ensure expression of the variants) (Suppl. 7a, b). This thereby shows that there are no available wt-receptors at the synapse.

As there are various pools of NMDARs at synaptic and extrasynaptic sites, we did not think that a western blot would sufficiently differentiate between the latter, and thereby would not provide insight about extinction of wt-receptors (which could be simply pushed to other sites compared to synapse). Moreover, the intracellular pool of receptors is much larger than the amount of NMDARs that can be detected at the membrane (e.g., 30,31), and therefore electrophysiology seemed to be a better means to monitor membrane receptors only:

Thus, to examine the distribution of the variants between synaptic- and extrasynaptic loci, we employed a standard procedure consisting of the use of the activity-dependent blocker MK-801 (Methods). Briefly, neurons were persistently bathed in TTX during which they were probed for Imax using 100 mM NMDA (to refrain from activating other GluRs), followed by application of MK-801 for 10 minutes to exclusively blocks synaptic receptors (that open following action-potential independent miniature neurotransmission). This thereby spares all extrasynaptic receptors from being blocked by MK-801, which are subsequently revealed by a second application of 100 mM NMDA (Suppl. 8a, inset)12. In neurons overexpressing the GluN2B-wt subunit, we obtained an extrasynaptic fraction of 38%, highly consistent with previous reports12,13. Overexpression of the variants, however, yielded a significantly and higher fraction (~50%) of remaining current (Suppl. 8b, c), but instead of reflecting a larger pool of extrasynaptic receptors, the experiment represents quite the inverse when involving LoF variants. Firstly, 100 mM NMDA does not saturate variant receptors (whether pure, mixed di- or tri-heteromers, see Table 1). Secondly, normal neurotransmission does not open synaptic receptors containing mutant GluN2B-subunits, attested by the complete absence of mEPSCs (see Suppl. 7). Thus, during the 10 minutes exposure to MK-801, only wt receptors are blocked by spontaneous synaptic activity, and thus the second bout of 100 mM NMDA solely exposes the remaining wt-receptors at the extrasynapse. Thus, the observed increase in the fraction of extrasynaptic receptors, in neurons overexpressing the variants, implies that the number of wt-receptors is necessarily decreased from the synapse and increases at the extrasynapse, most likely due to the incorporation of the variants at the synapse. This increase cannot be explained by an overall increase in membrane expression of wt-receptors in neurons overexpressing the variants, as these cells show, yet again, a strong reduction in Imax as seen above (see Fig. 6c and Suppl. 7e) (lLines #270-291). These thereby suggest that purely wt-receptors are not necessarily eliminated from the membrane (extinct), rather pushed outside of the synapse.

12 Fig.6b “PE-S” on the graph should be replaced by “PS”

Typo corrected.

Discussion #13 The authors are surprised by the fact (Fig.2) that 1 variant reduces the apparent glutamate affinity of the receptor, but not as much as 2 variants, despite the fact that "NMDARs opening requires all four subunits to be liganded (i.e., occupied by a ligand) which implies that the least affine subunit should have dominated the final affinity of the receptor". I agree that the difference is noticeable, however the glutamate affinity for receptors containing 1 variant is much closer to that of receptors containing 2 variants than that of wild-type receptors. Hence, the results obtained do not seem so surprising and could result, as rightly explained by the authors from a possible cooperativity between the subunits.

We agree with the reviewer that glutamate potency of receptors containing 1 variant subunit is much closer to that of receptors containing 2 variant subunits. However, we maintain our surprise because we expected it to equal (not just close) to the potency of the least affine subunit (the limiting factor). This is based on the notion that all four subunits need to be liganded for channel opening4,32–34. We gently raise the possibility of potential cooperativity (Table 1, see Hill-coefficient and 33,35,36), as well as mention that this may also stem from the variants’ lower proton sensitivity (Suppl. 3), which has also been shown to promote motions of the ATD (amino terminal domain) and increase open probability (positive cooperativity)36. Nevertheless, we are very careful with interpreting the Hill coefficient , as we limited exposure of oocytes to 10 mM glutamate due to artifacts arising past this concentration (see Kellner et al.8: Fig. 2—figure supplement 1). This description is now mentioned in page lines #149, 318, 319. Thus, even the slightest underestimation of the maximal reposnse would surely affect the slope.

#14 On the other hand, the data in Figure 6 are much more difficult to interpret and reconcile with the nature of the expressed receptor subunits (which this time is not controlled) nor their association within the same receptor. However, this aspect, which is essential to the understanding of the consequences of 1 variant on neuronal signalling, is not discussed: Whatever the stoichiometry of the complexes in the heterozygous disease, the mutated and wild type GluN2B subunits coexist in the same cell: Either within the same di-heteromeric complexes GluN2B-wt + GluN2B-mutant, or in separate complexes but nevertheless expressed in the same cell, in di heteromeric (GluN2B-wt + GluN2B-wt and GluN2B-mutant + GluN2B-mutant); or tri-heteromeric (GluN2A-wt + GluN2B-wt and GluN2A-wt + GluN2B-mutant) complexes. Assuming that half of the complexes remain wild-type, e.g. (GluN2A-wt + GluN2B-wt and GluN2A-wt + GluN2B-mutant) we would expect (Fig. 6) a small decrease in NMDA current (carried only by the half that expresses the mutated subunit, and whose function is not zero but only decreased by about 20% in response to 5 mM Glutamate, Fig. 3b). The same reasoning applies to the di-heteromeric conditions (GluN2B-wt + GluN2B-wt; GluN2B-mutant + GluN2B-mutant), here again the decrease observed Fig. 6b is difficult to reconcile with the responses measured Fig. 2b.

In other words, how to explain a 50% decrease of the currents, instead of the 10% expected by the previous reasoning. In this experiment we do not know which subunits are expressed, their proportions, nor how they are associated in functional complexes, which makes the interpretation of the data impossible. The only explanation, far-fetched, for 50 % decrease would be that the complexes were to contain all (or the vast majority) 1 wild-type subunits associated with 1 variant, then a homogeneous 50% reduction in current could be expected. But this extreme condition could only be possible in the case of di-heteromers, which is unlikely the case in Fig.6 as GluN2A currents are measured in presence of Ifenprodil. To conclude

1) the comparison of the currents in transfected and non-transfected neurons does not make sense in figure 6b which is not convincing because we do not know the nature of the currents actually measured. A comparison in controlled condition would make more sense (as I suggested in the criticism of figures 4, 5).

2) The reality of the combinations of expression and association between subunits within different complexes expressed in the same cell must be considered and taken into account in the interpretation of the data. Undoubtedly, the means of restoring the NMDA current will be different depending on the presence of mutated subunits in all functional channels or not.

Indeed, neurons express a variety of different combinations of channel stoichiometry, including following transfection with the variants. We do find find that the effect on whole-cell current is indeed ~50% (Fig. 6b, c), thereby safe to assume that 50% remain “wt”, but we do not know how they distribute between synaptic and extrasynaptic loci. Our results however argue against 50% remaining receptors at the synapse. First, mEPSCNMDA disappear (Suppl. 7a, b and see reply to comment #11 of this reviewer), but wt-receptors are still at the membrane, and they seem to be moving out of synapse (Suppl. 8). Thus, we can only state with higher certainty that the variant subunits are very efficient in incorporating within mixed or pure receptors, especially at the synapse.

  We also consider that the reduction in the whole-cell current observed in __Fig. 6b, c__ is not due to the remaining 50% GluN2B-*wt*-containing receptors, rather likely due to other variants, notably GluN2A, which are more prominent at postnatal stages37, such as in our case. In support, we see a large remaining current after saturating ifenprodil application (__Suppl. 7 e, f__)38. Thus, the variants incorporate within all 2A/2B membrane receptors, at the synapse and outside it (i.e., extrasynaptic) (see __Suppl. 8, c__).

**Referees cross-commenting**

The referees' comments are highly relevant. In particular, referee 3's comment 1 seems very interesting because it may help to better understand the discrepancy in the results observed in neurons, i.e. a 50% decrease in the currents induced by the expression of the mutant and wild type subunits in the same cells, whereas theoretically one would expect a 10% decrease of this current (cf. referee 2's 2nd comment in the discussion section). This comment 1 of referee 3 indeed stresses the fact that the control (non-transfected neurons) to which the heterozygous condition is compared is not the correct control, which should rather be neurons transfected with wild type receptor subunits. More generally, this comment underlines the importance of monitoring the effective membrane expression of the different subunits in each of the experimental conditions in order to be able to compare conditions and draw conclusions.

We initially did not perform this control as the literature paints a clear picture whereby expression of the GluN2B-subunit (without adding excess of the GluN1 subunit) does not instigate a robust increase in surface expression of NMDARs (and thus current remains the ~same) 4,39–43, and see our reply to comment #14 (above), and reviewer 3 comment 1 (below). Nevertheless, we have now performed this test by overexpressing GluN2B-wt. In support of previous reports, we do not find any statistical difference in current size between non-transfected neurons and neurons solely overexpressing the GluN2B-wt subunit (Fig. 6a, b). Furthermore, application of PS onto naïve or GluN2Bwt expressing neurons yields identical currents (Imax) and potentiation (Fig. 6c, d). These argue that we did not obtain “overexpression”.

We suggest that the 50% reduction in current size between neurons expressing the mutant and wt expressing neurons stems from the integration of mutant subunits and their dominant negative effect. Evidence for this incorporation is provided by the very strong reduction in synaptic currents (suppl 7a, b), and the supposedly higher abundance of wt-containing receptors in extrasynaptic regions (see reviewer 1 comment 4 and suppl 8). This is

Reviewer #2 (Significance required):

The novelty of the study, is to evaluate the consequences of a single mutated subunit within NMDA receptors affected by GRIN variant, to mimic the heterozygous condition of GRIN encephalopathies, this is of potential value for the field and the interest could also be extended to other genetic diseases (at least the experimental way to study the functioning of only one desired stoichiometric configuration). The strength of this paper is precisely to isolate technically and to study the functioning of a desired stoichiometric configuration only. The main limitation of the paper is the interpretation that the authors make of their data in a physio-pathological context. This work could be of interest for general audience, providing the title and summary are slightly modified. My area of expertise could not be closer to the topic of the article: Glutamate receptors; GRIN; molecular tinkering, cell culture, electrophysiology, receptor stoichiometry...

We thank the reviewer for noting the value in our work and its potential contribution and interest to the field and other diseases. Per reviewer’s suggestion, we have modified the title and text to suit a larger audience.

Reviewer #3 (Evidence, reproducibility and clarity (Required):

This paper is a follow up of an earlier paper published by the group (Kellner et al., eLife 2021), which aimed at characterizing the functional properties of two de novo GluN2B mutations in patients suffering from severe pediatric diseases, GluN2B-G689C and -G689S. NMDA receptors (NMDARs) are tetramers composed of two GluN1 and two GluN2 subunits. A single receptor can incorporate either two identical GluN2 subunits (di-heteromers) or two different GluN2 subunits (tri-heteromers), leading to a large diversity of NMDAR subtypes. The main NMDAR subtypes in the adult forebrain are GluN1/GluN2A and GluN1/GluN2B di-heteromers, as well as GluN1/GluN2A/GluN2B tri-heteromers. While the exact proportions of these three subtypes are still contentious, there are evidence that in the adult N1/2A/2B tri-heteromers form the major population of synaptic NMDARs in the adult forebrain. In addition, patients bearing pathogenic mutations are often heterozygous for the mutation, giving rise to mixed NMDARs incorporating one mutated and one intact GluN2 subunit. In their previous paper, Kellner et al. had shown that purely di-heteromeric GluN1/GluN2B-G689C and -G689S mutants display a drastic (> 1,000-fold) decrease of glutamate sensitivity and a decrease of surface expression. In the current paper, the authors characterize the effects of the -G689C and -G689S mutations on N1/2A/2B tri-heteromeric receptors, as well as on mixed di-heteromeric GluN1/GluN2B receptors containing one copy of the wild-type GluN2B subunit and one copy of the mutated GluN2B subunit. They show that one copy of the mutant subunit, either within mixed diheteromeric or tri-heteromeric receptors, is sufficient to decrease drastically glutamate sensitivity, although the shift in glutamate EC50 is not as strong as in pure di-heteromeric receptors (≈ 500-fold). They furthermore explore strategies to counteract the hypofunction induced by these mutations by testing the effect of positive allosteric modulators (PAMs). They show that spermine, a GluN2B-specific PAM, can potentiate the activity of mixed diheteromeric N1/2B but not N1/2A/2B tri-heteromers. However pregnenolone sulfate (a 2A/2B-specific PAM) can potentiate both the activity of mixed diheteromeric and tri-heteromeric NMDAR populations, either in oocytes or cultured neurons.I have very few major comments to make. The experiments are straightforward and the adequate controls have been made. Here are my two only major comments:

We thank the reviewer for the very detailed overview of our work and for appreciating our controls and methods.

#1 About the experiment on cultured neurons. The authors compare the currents of cultured neurons transfected with GluN2B-G689C and -G689S to non transfected neurons. The adequate control is rather neurons transfected with the wild-type GluN2B subunit to even out any phenomenon linked to transfection of the neuron. Given the overexpression that can occur after transfection, the effect of the mutations on the size of NMDAR currents might be even stronger than what the authors show. However in that case PS might not completely rescue mutant NMDAR currents to wild-type levels.

We initially did not perform this control as the literature paints a clear picture whereby expression of the GluN2B-subunit (without adding excess of the GluN1 subunit) does not instigate a robust increase in surface expression of NMDARs (and thus current remains the ~same) 4,39–43, and see our reply to comment #14 (above), and reviewer 3 comment 1 (below). Nevertheless, we have now performed this test by overexpressing GluN2B-wt. In support of previous reports, we do not find any statistical difference in current size between non-transfected neurons and neurons solely overexpressing the GluN2B-wt subunit (Fig. 6a, b). Furthermore, application of PS onto naïve or GluN2Bwt expressing neurons yields identical currents (Imax) and potentiation (Fig. 6c, d). These argue that we did not obtain “overexpression”. Thus, our results and interpretations hold true, and are therefore not underestimation of the effects of PS in neurons.

2 How come high concentrations of glutamate (>100µM) produce additional current on wt GluN1/GluN2B (with retention signals) compared to 100 µM glutamate, which is supposed to be saturating? It does not seem to stem from an osmotic effect since 10 mM glutamate does not produce any current on uninjected oocytes. Knowing that this "artefactual" effect might also occur in the mutant receptors, how do you take this effect into account when calculating the glutamate EC50s of the mutants? Given the drastic shift in EC50 produced by the mutant, taking into account this artefact is not going to change the conclusion, but the actual EC50s will be affected.

GluN1/GluN2B-wt receptors (with or without retention signals) are indeed saturated at 100 mM glutamate. However, excessively large concentrations of glutamate (>100 mM) may yield artefacts even in non-injected oocytes (in 10 mM, this occurs in ~20% of the cells, see Kellner et al 20218—Fig. 2 and Suppl. 1c, d) as well as in GluN2B-wt injected oocytes (supplementary Table 1 in 44). This is not due to osmolarity, as rightly mentioned by the reviewer (and below), rather possibly by endogenous glutamate receptors and transporters that do not readily contribute to current amplitude (these are extremely small currents), but can cause deterioration of the cell (and enhance ‘leak’) when activated for prolonged times by very large concentrations (e.g.,45). In fact, we explicitly report these to highlight potential artefacts, as these are often overlooked in the field. Regardless, most reports do no go past 100 mM glutamate, not even when describing GRIN2 mutations since most mutations do not cause such drastic shifts in potency as we observed (to the best of our knowledge only one report describes such an extreme LoF mutation for a GluN2A variant46). Of note, these effects are not seen when glycine is applied at high concentrations (supporting lack of effect by osmolarity)47. Thus, we refrained from testing concentrations past 10 mM, aware that it may yield a slight underestimation of glutamate potency (and perhaps the reason for the larger Hill coefficient, nH; see our reply to reviewer #1, comment #5). Importantly, despite the potential underestimation of the EC50, it does not change our conclusions as all groups are measured side-by-side (thus, the same underestimation equally applies to all other groups as well). We now mention this more in detail in the methods under the section – “Two Electrode Voltage Clamp recordings in Xenopus Laevis oocytes”.

Minor comments:

3 In the first paragraph of the "Results" section, when describing the design of the constructs used to force a heteromeric stoichiometry in recombinant systems, the authors do as if they had designed the constructs themselves "Briefly, we tagged...are retained in the ER (Fig. 1a)". Please rewrite this paragraph to show that you used constructs that had been previously designed by another group (Hansen et al., 2014).

We apologize. We did not mean to express that we have developed the method and indeed refer readers to the seminal works of those who did (Stroebel et al., 2014 and Hansen et al. 2014, lines #109-116). We did not go into details for the sake of brevity. We have rewritten this part to give proper acknowledgement to the method’s developers (also see Methods, line# 448).

4 I do not see any evidence of "positive cooperativity" between subunits in ref. 32. Ref. 32, to the best of my knowledge, states that in N1/2A/2B tri-heteromers, the 2A subunit sets the biophysical properties of the tri-heteromer. But there is no account of mixed di-heteromers. In addition, the cooperativity between the glutamate and glycine binding sites is negative.

The reviewer is correct, and we apologize for the mis-citation. Indeed, the cooperativity between glutamate- and glycine-binding is typically reported as negative48,49, and our intention was to highlight the strong cooperativity (whether positive or negative) observed between NMDAR-subunits and meant to cite the works of: 33,35,50 (lines . We have now rephrased the sentence: The divergence from this scenario suggests that the slight amelioration in potency could stem from positive cooperativity between the subunits50 (but see Hill coefficients in Table 1). Indeed, mixed receptors show restored proton sensitivity (Suppl. 3), which has been suggested to be coupled to other receptor features, notably increase in open probability.

5 Interpretation of spermine action within the Results section: it is striking indeed to observe that the mutations in the context of a mixed di-heteromer still allow spermine potentiation, while they abolish this potentiation in pure di-heteromers. As rightly said in the discussion, the regain of spermine potentiation in the mixed compared to the pure diheteromers is likely due to a more favorable transduction of spermine signaling to the pore, likely via a higher pH sensitivity of mixed di-heteromers compared to di-heteromers. I would thus avoid the terms of "one single intact interface" for the mixed di-heteromer, since both spermine binding sites are likely intact in this NMDAR configuration. How is pH sensitivity affected in the mixed di-heteromers?

We have performed a detailed pH dose-responses for the various channel types (Suppl 3). We find that GluN2B mixed di-heteromers exhibit similar IC­50 as pure GluN2B-wt di-heteromers, thus explaining their ability to undergo potentiation by spermine via alleviation of proton inhibition. We therefore further suggest that mixed di-heteromers’ have higher pH-sensitivity compared to pure mutant di-heteromers and this mat also contributes to their higher spermine sensitivity. Lastly, we observed that all GluN2A-wt-containing tri-heteromeric receptors were non-responsive to spermine (Fig. 4a). In fact, under our experimental conditions tri-heteromers underwent slight inhibition by spermine, regardless the identity of the GluN2B subunit (whether wt or variant) (Fig. 4b). Thus, as the tri-heteromers used here exhibit identical pH-sensitivity as 2B-di-heteromers, the only diverging aspect is the missing interface between the GluN1a and GluN2B subunits, demonstrating that potentiation by spermine requires at least one GluN2B-subunit with an intact proton sensitivity, and mandates two intact interfaces between GluN1-wt and GluN2B-wt subunits (Table 1)21.

6 In the methods section, the oocyte recording solution (likely Ringer and not Barth) does not contain any potassium. This is probably a typo. Could you correct the composition of your Ringer?

Corrected. We record NMDARs currents by use of a Barth solution containing (in mM): 100 NaCl, 0.3 BaCl2, 5 HEPES, pH 7.3 (adjusted with KOH, at ~2.5 mM) (as in 4,51).

7 There are several typos, especially in the Discussion.

We have corrected the typos throughout the publication.

**Referees cross-commenting**

I overall agree with the comments of reviewers 1 and 2. In particular, I agree that it is pointless to compare the absolute currents of non transfected neurons vs mutant-transfected neurons without an idea of receptor cell-surface expression.

We have performed this experiment (Fig. 6) and please see our reply to this reviewer’s comment #1.

I would like however to give some precisions about some comments of Reviewer 2. About the ER retention technique to express tri-heteromers: I didn't know that the C2 signal could be addressed to the membrane on its own. The lack of leak current stemming from C1-C1 or C2-C2 combinations has been demonstrated in the paper establishing the technique (Hansen et al, 2014), as well as in another paper that developed an analog technique based on GABAB retention signals (Stroebel et al., J Neurosci 2014). So it is fair to consider that the authors were not surprised by the lack of current when co-expressing two GluN2B subunits carrying the C2 signal.

We thank you for this addition and support for our observations.

About the comparison about absolute currents wt vs mutants, +/- spermine (Fig. 4a and 5a). I agree with reviewer 2 that being able to compare absolute currents of wt without spermine to mutant + spermine would be very interesting to see if spermine can actually rescue mutant hypofunction. However, to the defense of the authors, comparing absolute current values of recordings from Xenopus oocytes is meaningless. Indeed the variability of currents for the same construct and same day of experiment is too high (there can be up to a ten-fold difference between the lowest and the highest current of oocytes expressing the same construct the same experimental day). A way to investigate this aspect would be to estimate the open probability of the different constructs with or without spermine via the inhibition kinetics of an open channel blocker (e.g. MK801) and measure surface expression by Western blot, but I am not sure these experiments are worth it for the spermine experiment.

We agree with this reviewer about current size. It is quite variable among cells and would therefore introduce an additional variable and variability: the expression of these modified (C1/C2-tagged) subunits is dually affected by the mutation itself (Kellner et al. 2021) and by the introduction of the tagging (which really hampers there trafficking to membrane, Suppl. 2c); with unknown contribution of each variable. We thereby do not think these provide an added value to our conclusions, yet to grant reviewers’ no 2 request we have added __Suppl. 4 __which shows the rescue effect of the different drugs.

Reviewer #3 (Significance (Required)):

This paper is not of high significance since most of the characterization of the 2B-G689C and -G689S de novo mutants found in patients has already been published (Kellner et al., eLife 2021). However, this paper is worth publishing since it brings new data on the effect of the mutations on tri-heteromeric and mixed di-heteromeric NMDAR populations, which are likely the most abundant NMDAR populations in the patient's brain at adult stage. Tri-heteromeric and mixed NMDAR populations have often been overlooked when studying pathogenic NMDAR mutations due to the difficulty to express them specifically in recombinant systems. This paper (in addition to other papers in the field, see for instance Elmasri et al., Brain Sci. 2022; Li et al., Hum. Mutat. 2019) shows that the effect of the mutations on the receptor biophysical and pharmacological properties (but also on trafficking) differ whether the receptor contains one or two copies of the mutant subunit. This paper is of interest to scientists interested in NMDA receptor structure-function and pharmacology, as well as clinicians interested in GRINopathies (pathologies linked to NMDAR mutations).

I, the reviewer, am an expert in NMDAR structure-function and pharmacology. I believe I have sufficient expertise to evaluate the entirety of the paper.

We thank the reviewer for appreciating and acknowledging the merits of our work for publication.

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Reviewer #2 (Public Review):

In the manuscript entitled "Linking the evolution of two prefrontal brain regions to social and foraging challenges in primates" the authors measure the volume of the frontal pole (FP, related to metacognition) and the dorsolateral prefrontal cortex (DLPFC, related to working memory) in 16 primate species to evaluate the influence of socio-ecological factors on the size of these cortical regions. The authors select 11 socio-ecological variables and use a phylogenetic generalized least squares (PGLS) approach to evaluate the joint influence of these socio-ecological variables on the neuro-anatomical variability of FP and DLPFC across the 16 selected primate species; in this way, the authors take into account the phylogenetic relations across primate species in their attempt to discover the influence of socio-ecological variables on FP and DLPF evolution.

The authors run their studies on brains collected from 1920 to 1970 and preserved in formalin solution. Also, they obtained data from the Mussée National d´Histoire Naturelle in Paris and from the Allen Brain Institute in California. The main findings consist in showing that the volume of the FP, the DLPFC, and the Rest of the Brain (ROB) across the 16 selected primate species is related to three socio-ecological variables: body mass, daily traveled distance, and population density. The authors conclude that metacognition and working memory are critical for foraging in primates and that FP volume is more sensitive to social constraints than DLPFC volume.

The topic addressed in the present manuscript is relevant for understanding human brain evolution from the point of view of primate research, which, unfortunately, is a shrinking field in neuroscience. But the experimental design has two major weak points: the absence of lissencephalic primates among the selected species and the delimitation of FP and DLPFC. Also, a general theoretical and experimental frame linking evolution (phylogeny) and development (ontogeny) is lacking.

Major comments.<br /> 1.- Is the brain modular? Is there modularity in brain evolution?: The entire manuscript is organized around the idea that the brain is a mosaic of units that have separate evolutionary trajectories:

"In terms of evolution, the functional heterogeneity of distinct brain regions is captured by the notion of 'mosaic brain', where distinct brain regions could show a specific relation with various socio-ecological challenges, and therefore have relatively separate evolutionary trajectories".

This hypothesis is problematic for several reasons. One of them is that each evolutionary module of the brain mosaic should originate in embryological development from a defined progenitor (or progenitors) domain [see García-Calero and Puelles (2020)]. Also, each evolutionary module should comprise connections with other modules; in the present case, FP and DLPFC have not evolved alone but in concert with, at least, their corresponding thalamic nuclei and striatal sector. Did those nuclei and sectors also expand across the selected primate species? Can the authors relate FP and DLPFC expansion to a shared progenitor domain across the analyzed species? This would be key to proposing homology hypotheses for FP and DLPFC across the selected species. The authors use all the time the comparative approach but never explicitly their criteria for defining homology of the cerebral cortex sectors analyzed.

Contemporary developmental biology has showed that the selection of morphological brain features happens within severe developmental constrains. Thus, the authors need a hypothesis linking the evolutionary expansion of FP and DLPFC during development. Otherwise, the claims form the mosaic brain and modularity lack fundamental support.

Also, the authors refer most of the time to brain regions, which is confusing because they are analyzing cerebral cortex regions.

2.- Definition and delimitation of FP and DLPFC: The precedent questions are also related to the definition and parcellation of FP and DLPFC. How homologous cortical sectors are defined across primate species? And then, how are those sectors parcellated?

The authors delimited the FP:

"...according to different criteria: it should match the functional anatomy for known species (macaques and humans, essentially) and be reliable enough to be applied to other species using macroscopic neuroanatomical landmarks".

There is an implicit homology criterion here: two cortical regions in two primate species are homologs if these regions have similar functional anatomy based on cortico-cortical connections. Also, macroscopic neuroanatomical landmarks serve to limit the homologs across species.

This is highly problematic. First, because similar function means analogy and not necessarily homology [for further explanation see Puelles et al. (2019); García-Cabezas et al. (2022)]. Second, because there are several lissencephalic primate species; in these primates, like marmosets and squirrel monkeys, the whole approach of the authors could not have been implemented. Should we suppose that lissencephalic primates lack FP or DLPFC? Do these primates have significantly more simplistic ways of life than gyrencephalic primates? Marmosets and squirrel monkeys have quite small brains; does it imply that they have not experience the influence of socio-ecological factors on the size of FP, DLPFC, and the rest of the brain?

The authors state that:

"the strong development of executive functions in species with larger prefrontal cortices is related to an absolute increase in number of neurons, rather than in an increase in the ration between the number of neurons in the PFC vs the rest of the brain".

How does it apply to marmosets and squirrel monkeys?

References:<br /> García-Cabezas MA, Hacker JL, Zikopoulos B (2022) Homology of neocortical areas in rats and primates based on cortical type analysis: an update of the Hypothesis on the Dual Origin of the Neocortex. Brain structure & function Online ahead of print. doi:doi.org/10.1007/s00429-022-02548-0

García-Calero E, Puelles L (2020) Histogenetic radial models as aids to understanding complex brain structures: The amygdalar radial model as a recent example. Front Neuroanat 14:590011. doi:10.3389/fnana.2020.590011

Nieuwenhuys R, Puelles L (2016) Towards a New Neuromorphology. doi:10.1007/978-3-319-25693-1

Puelles L, Alonso A, Garcia-Calero E, Martinez-de-la-Torre M (2019) Concentric ring topology of mammalian cortical sectors and relevance for patterning studies. J Comp Neurol 527 (10):1731-1752. doi:10.1002/cne.24650

Reviewer #1 (Public Review):

Nitta et al, in their manuscript titled, "Drosophila model to clarify the pathological significance of OPA1 in autosomal dominant optic atrophy." The novelty of this paper lies in its use of human (hOPA1) to try to rescue the phenotype of an OPA1 +/- Drosophilia DOA model (dOPA). The authors then use this model to investigate the differences between dominant-negative and haploinsufficient OPA1 variants. The value of this paper lies in the study of DN/HI variants rather than the establishment of the drosophila model per se as this has existed for some time and does have some significant disadvantages compared to existing models, particularly in the extra-ocular phenotype which is common with some OPA1 variants but not in humans. I judge the findings of this paper to be valuable with regards to significance and solid with regards to the strength of the evidence.

Suggestions for improvements:

1. Stylistically the results section appears to have significant discussion/conclusion/inferences in each section with reference to existing literature. I feel that this information would be better placed in the separate discussion section. E.g. lines 149-154

2. I do think further investigation as to why a reduction of mitochondria was noticed in the knockdown. There are conflicting reports on this in the literature. My own experience of this is fairly uniform mitochondrial number in WT vs OPA1 variant lines but with an increased level of mitophagy presumably reflecting a greater turnover. There are a number of ways to quantify mitochondrial load e.g. mtDNA quantification, protein quantification for tom20/hsp60 or equivalent. I feel the reliance on ICC here is not enough to draw conclusions. Furthermore, mitophagy markers could be checked at the same time either at the transcript or protein level. I feel this is important as it helps validate the drosophila model as we already have a lot of experimental data about the number and function of mitochondria in OPA+/- human/mammalian cells.

3. Could the authors comment on the failure of the dOPA1 rescue to return their biomarker, axonal number to control levels. In Figure 4D is there significance between the control and rescue. Presumably so as there is between the mutant and rescue and the difference looks less.

4. The authors have chosen an interesting if complicated missense variant to study, namely the I382M with several studies showing this is insufficient to cause disease in isolation and appears in high frequency on gnomAD but appears to worsen the phenotype when it appears as a compound het. I think this is worth discussing in the context of the results, particularly with regard to the ability for this variant to partially rescue the dOPA1 model as shown in Figure 5.

5. I feel the main limitation of this paper is the reliance on axonal number as a biomarker for OPA1 function and ultimately rescue. I have concerns because a) this is not a well validated biomarker within the context of OPA1 variants b) we have little understanding of how this is affected by over/under expression and c) if it is a threshold effect e.g. once OPA1 levels reach x%. I think this is particularly relevant when the authors are using this model to make conclusions on dominant negativity/HI with the authors proposing that if expression of a hOPA1 transcript does not increase opa1 expression in a dOPA1 KO then this means that the variant is DN. The authors have used other biomarkers in parts of this manuscript e.g. ROS measurement and mito trafficking but I feel this would benefit from something else particularly in the latter experiments demonstrated in figure 5 and 6.

6. Could the authors clarify what exons in Figure 5 are included in their transcript. My understanding is transcript NM_015560.3 contains exon 4,4b but not 5b. According to Song 2007 this transcript produces invariably s-OPA1 as it contains the exon 4b cleavage site. If this is true, this is a critical limitation in this study and in my opinion significantly undermines the likelihood of the proposed explanation of the findings presented in Figure 6. The primarily functional location of OPA1 is at the IMM and l-OPA1 is the primary opa1 isoform probably only that localizes here as the additional AA act as a IMM anchor. Given this is where GTPase likely oligomerizes the expression of s-OPA1 only is unlikely to interact anyway with native protein. I am not aware of any evidence s-OPA1 is involved in oligomerization. Therefore I don't think this method and specifically expression of a hOPA1 transcript which only makes s-OPA1 to be a reliable indicator of dominant negativity/interference with WT protein function. This could be checked by blotting UAS-hOPA1 protein with a OPA1 antibody specific to human OPA1 only and not to dOPA1. There are several available on the market and if the authors see only s-OPA1 then it confirms they are not expressing l-OPA1 with their hOPA1 construct.

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Reply to the reviewers

We thank the four reviewers for their generally positive feedback on the manuscript. Below, we provide a point-by-point response to each reviewer.

We are performing new FCS and gradient measurements as suggested by the reviewers. We are confident we can have these completed within three months (accounting for the summer break).

Reviewer #1 (Evidence, reproducibility and clarity (Required)):

*This manuscript reports a very thorough and careful study of the mobility of Bicoid in the early embryo, explored with single-point fluorescence correlation spectroscopy. Although previous groups have looked into this question in the past, the work presented here is novel and interesting because of the different Bicoid mutants and constructs the authors have examined, in particular with the goal of understanding the role of the protein DNA-binding homeodomain. The authors convincingly show that there is a significant increase in Bicoid dynamics from the anterior to the posterior region of the embryo, and that the homeodomain plays an important role in regulating the protein's dynamics. Their experiments are very well designed and carefully analyzed. The authors also modelled gradient formation to see whether this change in dynamics might play a role in setting the shape of the gradient. I am not sure I fully agree with their conclusion that it does, as mentioned in my comment below. However, it is an interesting discussion to have, and I think this paper makes a significant advance in our understanding of Bicoid's behavior in the early embryo. *

We thank the Reviewer for their positive comments and their suggestions for improving the manuscript. We will resolve the concerns raised by the reviewer with clarity in the revision. We will also add additional comment in the Discussion regarding the interpretation of our results.

*Major comments: *

• 1) Gradient profile quantification: Some of the conclusions made by the authors rely on the comparison between their model of gradient formation (as captured in the equations in lines 232 and 233) and the Bcd intensity profile measured in the embryos. Since the differences in gradient shape predicted by the different models are very small (see Fig. 3B, which is on a log scale and therefore emphasize small differences, and Fig. 3C), it is very important to understand how reliable the experimental concentration profiles are.*

This is a fair comment. It is worth noting that the key differences between the 1- and 2-component models are only apparent at large distances (and hence low concentrations) from the source.

We performed the quantification of the gradients in a manner similar to the Gregor lab, whereby the midsagittal plane is analysed. We used 488nm illumination (rather than 2-photon, as the Gregor lab does) so our measurements are likely noisier. However, we are not investigating the variability in the gradient here, but the mean extent. We currently correct background with a uniform subtraction, but we appreciate that is not the optimal method.

In the revised manuscript, we will repeat the above experiments using a 2-photon microscope. Further, we will image lines expressing His::mcherry without eGFP under the same imaging conditions to more accurately estimate the background signal. While we expect this to improve the data quality, we do not envisage significant change to the observed profiles based on prior experience.

At the moment, I do not find the evidence that [Bcd] concentration profile is more consistent with a 2-component diffusion model than a 1-component model very strong. A few comments related to this: * * 1a. Line 249, it is mentioned that: "observations ... incompatible with the SDD model". Which observations exactly are incompatible with the SDD model?

The key points are in the preceding paragraph. We will improve the model presentation in the Results and also include further contextualisation in the Discussion.

1b. In Fig. 3D, only the prediction of the 2-component model is shown. What would the simple 1-component diffusion model look like? Is it really incompatible with the data?

We agree with this comment and will provide the 1-component fit to the gradient profiles. We expect it to fit well for the anterior half of the embryo but fail at larger distances (as has been previously shown).

Regarding the FCS data, we also show one and two component fits. We will show the alternative fits – a 2 particle fit is clearly an improvement (see also related response to reviewer 2).

1c. Line 243: "The increased fraction in the fast form ... consistent with experimental observation of Bcd in the most posterior" (Mir et al.)". I am not sure how this is significant, since the simple model also predicts there will be Bcd in the posterior - the only difference is how much is there (as shown in Fig. 3C), and it's a very small difference.

The absolute differences are not large between the two models, but due to the observed clustering (Mir et al. 2018), even small differences can have very large effects. In the revision we will provide estimates of the actual concentration differences.

We are performing new experiments with the Fritzsche lab at Oxford to estimate if there is clustering of Bcd. We will also repeat our FCS experiments to validate our key conclusion of AP differences in diffusion of Bcd. These should be completed by the end of the summer.

1d. Since the difference between models is in the posterior region where Bcd concentration is very low, when comparing the models to the data the question of background subtraction is essential. How was the subtracted background (mentioned line 612) estimated?

See above response to the first comment.

1e. Along the same line, were the detectors on the Zeiss LSM analog or photon counting detectors, and how confident can we be that signal is exactly proportional to concentration?

We used PMTs and did not directly do photon counting. But the intensity is still proportional to the concentration. It is possible to estimate the absolute concentration value, e.g., Zhang et al., 2021 (https://doi.org/10.1016/j.bpj.2021.06.035). However, our main conclusions – especially regarding the spatially varying Bcd dynamics – are not dependent on this.

1f. Can the gradients created by the two Bcd mutants (FIg. 4B) be quantified as well, and are they any different from the original Bcd gradient?

We agree this would be useful. We will provide the gradient quantifications of the bcd mutants in the revision.

1e. What is the pink line in Figure 5C (I am assuming the green one is the same as in Fig. 3D)? It could be better to not use normalization here, or normalize everything respective to the eGFP::Bcd data to make comparison in relative concentrations in the posterior for different constructs more evident (also maybe different colors for the three different data sets would help clarity).

This is a fair comment, and we will create graphs with new data for better visualisation.

1f. Discussion, lines 402-403: Does the detailed shape of the Bcd in the posterior region matter at all, since the posterior is not a region where Bicoid is active, as far as we know? Could a varying Bcd dynamics have other consequences that would be more biologically relevant?

Bcd is now known to act at 70% EL (Singh et al., Cell Reports 2022). So, the gradient is relevant for a large extent of the embryo length, though it is not known if there is any effect in the most posterior region.

2) Model for gradient formation (lines 231-238): * * 2a. Whether the molecules of Bcd can change from their fast to slow form is never questioned. How do we know (or why might we suspect) they do exchange?

This is a good point. Within the nucleus, and based on our mutant data, we suspect the fast/slow forms correspond to unbound/bound DNA states.

In the cytoplasm, the dynamics are less clear. Bcd can bind to cytoskeletal elements (Cai et al., PLoS One 2017) as well as to Caudal mRNA. Therefore, it seems reasonable to have different effective dynamic modes – yet, how such switching occurs remains unclear.

Ultimately, our model approximates multiple dynamic modes that are integrated to drive Bcd motion. Including switching between states is a reasonable assumption based on what is known about cytoskeletal and protein dynamics, but we do not have a specific mechanism.

It is challenging to estimate a specific kon / koff rate, as the dynamic changes also depend on the diffusion – which itself is changing. For now, we believe our level of abstraction is appropriate given what is known about the system. It will be very interesting to explore the specific interactions underlying such behaviour in the future, but that is beyond this current manuscript.

2b. The values used in the model for alpha, beta_0 and rho_0 should be mentioned. Maybe having a table with all the parameters in the method section, or even in the supplementary section, would help. The exact values of alpha and beta matter, because if they are large (fast exchange) a single exponential gradient is to be expected, if they are 0 (no exchange) a double exponential gradient is to be expected, with intermediate behavior in between. Which case are we in here?

We agree and will add a more complete table in the revision.

3) Discussion about anomalous diffusion (lines 386-388): The 2-component model used by the authors to interpret their FCS data seems very well justified here (excellent fits with very small residuals). I agree with the authors' conclusion that "the dynamics of Bcd within the nucleus are more complicated than a simple model of bound versus unbound Bcd", but I don't see how that can lead to a diagnostic of anomalous diffusion instead. Maybe it is just a matter of exactly explaining what is meant by anomalous diffusion here (since this term is often used to mean different things). A more likely scenario I think, is that there are more than just two Bcd components in the system.

This is a good point, and we can’t easily differentiate two/multi- component fits from anomalous diffusion ones. This is a known problem. But we have recently shown in a collaboration with the Laurent Heliot lab (Furlan et al, Biophys J 2019), that anomalous diffusion is a good stable indicator of changes, even if it might not be the right model. We use anomalous diffusion as it stably predicts changes. We do not claim, however, that diffusion is anomalous. We will improve the discussion of these points in the revised manuscript.

4) Line 440 and after: What is the evidence that the transition between the two forms might vary non-linearly with Bcd concentration? How would that help adapt to different embryo sizes? It would be good to be more explicit here instead of just referring to another paper.

We will improve this discussion. The central point is that the action of Bicoid is unlikely to simply depend linearly on concentration as in that case the ratio of fast to slow forms would be constant across the embryo. Related to the above comment, it is important to emphasise that we are using a phenomenological model, not one based on a specific mechanism.

5) Since an important aspect of this work is the study of different Bcd constructs in vivo, it is important that these constructs are very clearly described, so the section on the generation of the fly lines (Methods) should be expanded. In particular: * * 5a. It seems that the eGFP:: NLS control used here was different from that first described in Ref. 64 (and used for FCS experiments in Ref. 30 and 36)? If so, what NLS sequence was used here, and precisely what type of eGFP was used (in particular, was the A206K mutation that prevents dimerization present in the eGFP used)? If it is the same construct as in Ref. 64, it should be mentioned explicitly. * * 5b. Were the mutant N51A and R54A lines gifts as well, or have they been described before? If so, previous publications should be referenced. If not, how the plasmid was introduced in the embryo should be briefly explained.

We agree and will expand on the fly lines in the revision.

6) Concentration calibration measurements (Methods Fig. 2, line 568 and on). It is well known that background noise is going to interfere with the measurement of N when the signal becomes equivalent to the background noise (Koppel 197, Phys Rev A 10:1938-1945, and for a recent discussion of this effect for morphogens in fly embryos: Zhang et al., 2021, Biophysical Journal 120,4230-4241). It is almost certain that in the low signal regions of the embryo (e.g. posterior cytoplasm) this is affecting the reported concentration, and should be at least acknowledged.

We agree with the reviewer. We will provide the SBR. We will also correct the N values based on the method followed in Zhang et al., 2021, Biophysical Journal 120,4230-4241.

*7) Reference 3 is mis-characterized in two different ways in the manuscript: * * 7a. Line 50: The conclusion in Ref. 3 was not that the gradient was due to a diffusive process, on the contrary Gregor et al. argued that Bcd was too slow to form such a long-range gradient by diffusion. Studies that do present data consistent with a morphogen gradient formation mechanism driven by diffusion are reference 5, reference 30, Zhou et al., Curr. Biol. 2012;22(8):668-75 and Müller et al., Science 336 (2012) 721-724. *

Gregor et al., do not argue against a diffusion process – indeed, they utilise a SDD model in their paper. However, they do extensively discuss how the predicted dynamics from the SDD model are not compatible with gradient formation as observed after n.c. 13. This problem was resolved to some degree by FCS measurements of Bcd (e.g., Dostatni lab, Development 2011) and the use of a Bcd tandem reporter which showed that production and degradation change during n.c. 14 (Durrieu et al., MSB 2018). We will improve the framing of these results in the revision.

7b. The diffusion coefficient estimated from FRAP measurements and reported in Ref. 3 (D = 0.4 micron^2/s) is mentioned a couple of times in the manuscript (line 66, line 395, line 411). However, this number is simply incorrect. When fast components (such as the ones clearly detected here by FCS) are present, they diffuse out of the photobleached area during the photobleaching step. If that is not corrected for during the analysis (and it wasn't in Ref. 3), then the recovery time measured is just equal to the photobleaching time, and has nothing to do with either the fast or slow fraction of the studied molecule - it has no other meaning than to give a lower bound on the value of the actual effective diffusion coefficient of the molecule. This effect (called the halo effect) is well known in the FRAP community (see e.g. Weiss 2004, Traffic 5:662-671), it has been experimental demonstrated to occur for Bcd-eGFP in the conditions used in Ref. 3 (Reference 30), and the actual diffusion coefficient that should have been extracted from the data presented in Ref. 3 has been recalculated by another group to be instead D = 0.9 micron^2/s (Castle et al., 2011, Cell. Mol. Bioeng. 4:116-121). It would therefore be better to report the corrected value from Castle et al. to help the field converge towards an accurate description of Bcd mobility.

We fully agree and will use the improved FRAP estimated value for Bcd.

*Minor comments and suggestions: *

• 8) Figure 1: From panel A, it seems that what is called "Anterior" and "Posterior" is about 150 micron away from the embryo mid-section, i.e. about 100 micron from either the anterior pole or the posterior pole (so not the tip of the embryo, but somewhere in the anterior half or posterior half). Maybe this should be made clear in the text. *

We have made changes in Figure 1A to indicate the region within which the FCS measurements are carried out. We have added the relevant details in the legend of figure 1 lines 137-138.

*9) Fig. 2A; It might be good to put this graph on a log scale, so that cytoplasmic values are seen more clearly. Also, what about reporting on nuclear to cytoplasmic ratios? *

We will rework on this graph and make necessary changes.

*10) Fig. 2: It could be interesting to plot D_effective as a function of the measured concentration of Bicoid in different locations, since the (interesting) suggestion is made several time that [Bcd] could the a determinant of the protein mobility. *

Our work provides an indication that Bcd concentration is connected to the diffusion. We did this by measuring at two locations. To extend this to a rigorous model would require substantial new measurement along the whole length of the embryo. While interesting, this represents a very large investment of time and lies beyond the current manuscript.

*11) Figure 3B&C: Is the curve for 2-component diffusion (without concentration dependence) for steady-state missing? *

We will clarify in the revision.

*12) Lines 78 and 471: What do the authors mean by "new reagents"? The word reagent evokes a chemical reaction, but there are none here. Do the authors mean new constructs? or new mutants? *

We have changed lines 78 and 479 from “new reagents” to new Bcd mutant eGFP lines”.

*13) Lines 57-59: Another good reference for FCS measurements performed to study the dynamics of a morphogen (in this case Dpp) is Zhou et al., Curr. Biol. 2012;22(8):668-75 *

We added this reference in no.70.

*14) Lines 109-111: A word must be missing. Precisely determined what? *

Precisely measure within cytoplasm, and nuclear compartments and also during interphase stages. We have changed to “precisely measure in the cytoplasmic and nuclear regions during the interphase stages of nuclear cycles (n.c.)12-14.” in line no.111-112.

*15) Line 278: The increase in the slow mode is expected. Maybe explicitly mention why. *

In line 286, we have added “due to the loss of Bcd binding to the DNA”.

*16) Line 282: "with the fast component increasing", maybe replace with "with the diffusion coefficient of the fast component increasing" or "with the fraction of the fast component increasing". *

We have changed line 289 “with the diffusion component of fast component increasing towards the posterior”.

*17) Line 517: Is there a reason why the dorsal surface is always placed in the coverslip? *

We have added these details in line 528-529 in Methods.

*18) Line 524 and on: FCS measurements: What was the duration of each individual FCS measurement? It is great that the exact number of measurements are reported in the supplementary! *

Thank you for the complement. Typically, cytoplasmic measurements are 60secs and nuclear measurements are 20-40s. We have added this in line no.528-529. We also added a column to indicate the duration of each of the measurements in the supplementary tables.

*19) An Airy unit of 120 um seems large in combination with an objective with a NA of 1.2, is there a reason for that? What was the radius of the resulting detection volume? *

Olympus microscopes have a 3x magnification stage in their confocals. This leads to the change in the Airy unit. Otherwise, it would be 40 mm.

*20) Thank you for detailing the reasons behind the choice of excitation power, an important and often omitted details. Where in the excitation path were the values of the laser power measured (before or after the objective?)? *

Thank you for the complement. The laser power is measured before the objective. We removed the objective and measured the laser power in the objective path.

*21) Line 585: "since the brightness of eGFP::Bcd..." do the authors mean the molecular brightness of a single eGFP::Bcd molecule, or the total fluorescence signal? *

It is the total fluorescence signal. We have edited line no.592.

*22) It would be good for reference to mention the approximate value of the molecular brightness recorded for these eGFP constructs at the laser power used. *

We will measure and tabulate in the revised manuscript.

*23) Reference 766: The year (and maybe other things) is missing. *

We have corrected this reference.

24) Figure 2 (Methods): The concentrations shown on the figure should be in nM not uM. * * Thanks for noticing – we have changed.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

MAJOR POINTS

• 1) FCS measurements and fits *
• a) Please state the duration of each individual FCS measurement. *

In the cytoplasm, the measurements were carried out for 60 secs and in nuclei it is between 20-40s. We could not measure for 60s in the nuclei as the nuclear position fluctuates from its initial position. We will add another column to indicate the duration of FCS measurements in the supplementary tables.

b) The authors acknowledge potential issues with fluorophore photophysics and use different lag time ranges for the calibration dye Atto-488 (0.001 ms in Method Fig. 2) and eGFP (0.1 ms in the main figures). Given the strong influence of different parameters on data interpretation and conclusions, Method Fig. 2 should be repeated with purified eGFP. This is particularly relevant for the noisy FCS measurements in posterior regions.

Performing the experiment with purified eGFP will be a volume calibration. We routinely performed this before each imaging session, and that should be fluorophore independent. As noted by Reviewer 1, it is also important to be clear about background correction. We will provide brightness data for eGFP and background values in the revised manuscript. We can then use this to estimate the corrected concentrations.

We use 0.1 ms to start, as at that point any contribution from the photo-physics should have decayed (0.1 ms is about 3-5 times the day rate of the photophysical process, Sun et al., Analytical Chem 2015).

c) Please explain why no data is shown for "AN" around 0.1 ms lag time in Fig. 1B in contrast to all other figures.

We will add the data for AN from 0.01 in the revised figures.

d) Please state what the estimated diffusion coefficients with one-component model fits are. Please also explain why the fits in Fig. S1E do not reach a value of 1 and why they plateau higher than the experimental data at long lag times. Please constrain the fits to G=1 at 0.1 ms tau and G=0 at 1 s tau to make a fair comparison.

The experimental ACF curves reach 0 at long lag times as would be expected. The one-component fits, however, don’t describe the data well and as a result they do not reach 1 and 0 at short and long lag times, respectively. The fitting is done using a mean-squared estimation of the best approximation of the particular model function to the data. Fixing the parameters can be done, but it will further reduce fit accuracy and deviations will be larger. We will perform this analysis and tabulate the one component fits in supplementary 1 with necessary corrections.

e) Please assess the validity of all multi-component fits by comparing the relative quality of the models to the number of estimated parameters using the Akaike information criterion or similar approaches.

We will provide the values denoting the quality of the fits in the revision. We will provide the 3D 1 particle fit, the 3D 1 particle fit with triplet, the 3D 2 particle fit and the 3D 2 particle fit with triple and will provide appropriate measures of fit quality.

f) Please also present the Bcd-GFP fits with 0.001 ms that are mentioned in line 590, and present the results for the data that did not give comparable tau_D1 and tau_D2 values mentioned in line 593.

We will provide all the curves from 0.001ms in the supplementary. We did not provide these details as we have followed the methods from Abu Arish et al., 2010. As our cytoplasmic and nuclear TauD values match with Abu Arish et al., 2010 and Porcher et al., 2010, we thought the excess data would be redundant.

3) Bicoid gradient and modeling * a) Little et al. 2011 observed that the Bcd gradient decreases around n.c. 13. Can the authors of the present work observe a similar concentration decrease using FCS? This is important to i) validate the FCS concentration measurements, and ii) to resolve the controversy regarding "previous claims based on imaging the Bcd profile within nuclei, which predicted decrease in Bcd diffusion in later stages".*

This is a good point regarding conclusions from the previous literature. The Little et al. paper inferred that diffusion had to decrease from fitting to the gradient profiles. However, subsequent analysis from our lab (Durrieu et al., MSB 2018 [which uses a different method involving a tandem reporter for Bicoid] and this manuscript) strongly suggest that Bicoid remains dynamic, at least through n.c. 13 and early n.c. 14. One way to test this is to use SPIM-FCS, where longer time courses can be taken (though with slower time resolution in the FCS). We have performed preliminary experiments with SPIM-FCS and we will revisit this data to see if we can find evidence for changes in the diffusion.

We will also extend the Discussion to make the results clearer in terms of previous models and literature.

b) Please explain why the experimental Bcd-GFP gradient data does not reach a value of 1 (e.g. in Fig. 3D) despite normalization. Please also explain why the fits become flatter in Fig. 5B compared to the steep fit in Fig. 3D.

Both lines were measured under identical conditions. Therefore, we normalised to the maximum value of both experiments. We will redo, normalising to each individual experiment. Regarding Fig. 5C, the Bcd::eGFP curve is identical to Fig. 3D. The flatter curve is the line with eGFP tagged to a NLS alone.

c) For modeling, please take into account observations that the Bcd source is graded with a wide distribution (30-40% EL, see Spirov et al. 2009, Little et al. 2011, Cai et al. 2017 etc.). The extent of the source used in the present work (x_s=20 um, line 620) is at least five times too small.

Care must be taken in defining the source extent. The most careful measurements are reported in Little et al., PLoS Biology 2011 who performed single molecule FISH. They conclude “We demonstrate that all but a few mRNA particles are confined to the anterior 20% of the egg”. Further, the peak in the particle density is around 20-30um from the anterior (Figure 3, Little et al., PLoS Biology 2011), with the vast majority of counts being with 10% of the anterior pole. Further, Durrieu et al. MSB 2018, showed using a Bcd tandem reporter that there was unlikely to be an extended gradient of bcd mRNA (maximum extent of around 50um). Here, we used a simple source domain, which was arguable a little narrow, but not significantly so. We will increase the value in the revision, but the claim that there is an extended bcd mRNA gradient (Spirov et al., Development 2009) has not been substantiated by later experiments.

• d) Please discuss in the paper how well the simulations in Fig. 3B agree with the experimental data.*

We will provide these details in the revision.

• e) Please provide a precise estimate for the statement "Even with an effective diffusion coefficient of 7 μm2s-1, few molecules would be expected at the posterior given the estimated Bcd lifetime (30-50 minutes)" to turn this into a quantitative argument. How many molecules are expected to reach posterior in which model, and how does it compare to experimental observations?*

This can be estimated based on the root-mean-square distance for diffusive processes. We will provide this in the revision.

• f) The sentence "we find that a model of Bcd dynamics that explicitly incorporates fast and slow forms of Bcd (rather than a single "effective" dynamic mode) is consistent with a range of observations that are otherwise incompatible with the standard SDD model" needs to be toned down and corrected since a simple SDD appears to be sufficient to account for the observed gradients. If the authors disagree, please specifically point out in the paragraph around line 249 what observations exactly are incompatible with a standard SDD model.*

This is similar to the point raised by Reviewer 1. While the standard SDD model can explain the overall gradient shape, it is not compatible with the observed time scales and Bcd puncta tracked in the posterior pole. We will improve the Discussion around this point to make the distinctions between the models clearer.

• 5) Data presentation *
• a) In line 27 and 122 it would be better to rephrase the wording "find/found" and give credit to previous papers that first made these observations. *

We will edit in the revision.

• b) For the statement "This suggests that the dynamics of the fast fraction were not captured by previous FRAP measurements", please explain why this should not be the case even though the fast fraction is shown to be larger than the slow fraction in the current work.*

We will edit in the revision.

• c) Similarly, the sentence "The dynamics of the slower mode correspond closely to measured Bcd dynamics from FRAP" likely needs to be corrected since it neglects the contribution of the faster mode, which is fluorescent as well and should also contribute to the dynamics from FRAP.*

This is similar to the point raised by Reviewer 1 and we will edit in the revision.

d) In the absence of further evidence (see above), the sentences "We establish that such spatially varying differences in the Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole" and "These results explain how a long- ranged gradient can form while retaining a steep profile through much of its range" in the abstract need to be toned down.

We are not sure here what needs to be toned down. Our results show that there are (at least) two dynamic forms of Bcd and, combined, they are capable of forming a long-ranged gradient while also ensuring the gradient remains steep in the anterior (because the diffusion coefficient itself varies across the embryo). We will go through these statements and make sure the meaning is clear.

e) The authors state that "However, we show that eGFP::Bcd in its fastest form can move quickly (~18 μm2s-1), and the fraction of eGFP::Bcd in this form increases at lower concentrations", but this has not been directly shown. Please tone down this statement or directly test the prediction that Bcd has a higher fraction of the fast form in earlier nuclear cycles when Bcd concentration is smaller.

This is a good suggestion, and we will test whether early nuclear cycles of the anterior domain show faster dynamics.

*MINOR POINTS * * 1) Introduction * * a) Please explain explicitly what exactly the contention in Bcd, Nodal and Wingless dynamics is in the cited references. *

We will add in the revision. b) In line 95, it would be better to state that this is a variation of the SDD model rather than "a new model". * We changed from “a new model” to “an improved version of SDD model” in the current version of the manuscript. 2) Methods * * a) The authors state that "The same software was also used to calculate the cross-correlation function", but I couldn't find any cross-correlation analyses. Please clarify. *

It is line 538. There is no cross correlation. We changed this to the autocorrelation function.

b) Please correct the "uM" typo to "nM" in the legend of Method Fig. 2A.

We have changed this in the current version.

• c) In the sentence "Further, since the brightness eGFP:Bcd in the anterior and posterior cytoplasm is lower compared to the nuclei", "brightness" probably needs to be changed to "concentration" since the molecular brightness is unlikely to change. *

We edited the line no.591.

• d) Please explain the background-correction method mentioned in line 612. Please also state at what temperature the experiments were performed.*

We will add a better background correction in the revision. Currently, it is the non-embryo background as background noise. The measurements are carried out at 25oC.

*3) Results * * a) Please provide labels for anterior, posterior, dorsal and ventral in Fig. 1A. * * b) Please explain the colors in Fig. 5C. * * c) Please explain the dashed lines in Fig. 3C. * We have edited Figure 1A and Figure 5C. We will edit Figure 3C in further revision.

*OPTIONAL * * 1) If possible, it would be helpful to mention whether the transgenic animals have any abnormal phenotypes or whether they can rescue the bcd mutant. * We will update in the revision.

*2) To validate the concentration measurements, it would be ideal if the authors could determine the Bcd concentration gradient using FCS along the anterior-posterior axis. This would also address whether there are further unexpected changes in diffusivity in medial regions and along the anterior-posterior axis that would have to be considered for modeling. * To measure the Bcd concentration using FCS along the whole axis would be a very challenging undertaking. To get the data for the two positions analysed already represents a significant amount of work. We have done SPIM-FCS measurements, and we will be repeating our FCS measurements in the Fritzsche lab at Oxford. Combined, we believe this provides sufficient corroboration of our results.

*3) Local photoconversion experiments, e.g. in Bcd-Dendra2 embryos if available, would provide compelling support for the relevance of the measurements in the current work. * This is a nice idea, but this would represent a substantial project in its own right and lies beyond the current work.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

*In my estimation the experimental work is rigorous and the results fully support the conclusions of the authors. I was surprised, however, that the HD-only form localizes via very different and simpler dynamics than does full-length Bcd, but nevertheless forms at least a qualitatively similar gradient. That leads to the question as to whether the existence of the fast and slow forms and their different ratios in different parts of the embryo actually are physiologically relevant. I don't see a straightforward way to test this experimentally, because the mutations that effect Bcd gradient formation also affect essential functions of the protein that if abrogated produce severe downstream effects on embryonic development and lethality. However I would like to see this point at least addressed in the discussion. The data and the methods are presented in such a manner that they can be reproduced, and the number of replicates and statistical analysis is overall robust. * We thank the Reviewer for the positive and constructive review. They, like both previous reviewers, raise the issue of the model and how it fits with the data. As outlined above, we will improve this part of the data presentation and also the Discussion to make sure the main results are clear.

We agree that the underlying importance of the different dynamic forms of Bicoid – and why they change across the embryo – remains unknown. We believe that our careful characterisation of such behaviour is important nonetheless, as it reveals that: (1) morphogen dynamics are more complicated than typically modelled, and this may be just as relevant for ligands moving through extracellular space; and (2) dynamics can vary in space/time, providing an additional possible mechanism of control for regulating morphogen gradient profiles.

Of course, we would like to explore potential physiological relevance. Further exploration of the homeodomain and its role in regulating dynamics is a potential route, but that belongs in future work.

*Minor comments: *

• The presentation of the graphical data measuring Bcd levels along the a-p axis (Fig 1C, 1D, 4C-F and others) needs to be improved, because the grey lines that represent ACF curves are essentially invisible. This is partly because there is usually extensive overlap between the grey lines and other lines. This may be solved by using a more vivid colour than grey for the ACF curves, or perhaps the ACF lines could be made thicker but with some transparency so that overlapping data can be seen. In any event this aspect of the presentation needs to be improved. * We have made the ACF lines thicker to distinguish from the model fit.

*In Figs 2D and 2I measurements of statistical significance between the proportion of protein in fast and slow modes need to be added. * We will add in the revision.

*Relevant to line 174 and Fig 2, NLS should be defined when first used, the source of the NLS should be given (is it from Bcd?) and the rationale for looking at eGFP::NLS should be made explicit. *

We have added details on how the eGFP::NLS is generated in the methods.

*In Fig 3D the dashed lines need to be defined. I assume these are experimental error bars but this is not stated. *

We now state this in the legends.

*On lines 344-5, shouldn't this conclusion concern the HD rather than the NLS? * Yes, thanks for pointing it out it is related to only NLS not NLSHD. We removed this statement from line 351.

*On line 432, CAP is not an acronym, the correct term is 5' 'cap' or 'cap structure'. Also Cho et al. PMID 15882623 should be added to the references here. * We changed the corresponding section and added the references.

*On lines 446, 456, 469, and throughout: replace 'blastocyst' with 'blastoderm'. The former term is generally used for embryos that undergo full cellular divisions and cleavage in early embryogenesis, not for syncytial embryos such as Drosophila. * We have changed blastocyst to blastoderm throughout the manuscript.

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

Major comments: The averaged autocorrelation curves were fitted to models of diffusion with one and two components. The one-component model was insufficient to reproduce the data and the two-component model seems to fit the data. Have the authors tested models with more than two components? Could it be possible to distinguish more Bcd populations?

While it is possible to fit with further components, it rarely provides useful further insight. In particular, the error in measuring three tau_D’s is typically very large. In addition, the improvement in the fit will be marginal, and thus the extra components cannot be justified statistically. Of course, we cannot exclude a third (or more) possible dynamic modes, but within the resolution of our FCS measurements two components with triplets are in general the maximum that can be accommodated without overfitting. We will provide evidence for this claim in the supplement of the revised manuscript.

In Figure 2E, the same concentration of eGFP::NLS is estimated to exist in the cytoplasm and nucleus. Since the NLS should target eGFP to the nucleus, what is the explanation for this observation? Is it possible that the method used to estimate the concentration of molecules is underestimating the concentration in the nucleus or the opposite in the cytoplasm?

This is a good observation. There are two possible explanations. First, the regular division cycles “reset” the nuclear levels. Therefore, differences may not be so large. Second, FCS measurements of concentration can be noisy, as they depend on the very short time scales in the measurement. We will double check our measurements and clarify this in our revision.

*In the simulation of the SDD model (Figure 3B), simulations at 10 min, 25 min and 120 min are shown. Assuming that 120 min corresponds to early nc14, are simulations at earlier timepoints corresponding to nc12 and nc13 indistinguishable from the profile at 120 min? This demonstration would further support the option to merge the data from all nuclear cycles. *

This is a good point. Here, we were primarily focused on showing the time evolution of the model, rather than directly mapping onto experiment. We will clarify in the revision.

*The results obtained with the BcdN51A mutant show an increase in diffusion speed, while retaining similar proportions of fast and slow populations. In the slow fraction, a new population is found. Assuming that the BcdN51A molecules cannot bind specifically to DNA due to the mutation, what would this newly found population correspond to? Could the authors explore the possibility of nonspecific binding to DNA? The article would also win by discussing more on this aspect or other options. *

This is an interesting question. Dslow for anterior nuclei of N51A mutants increases (Dslow from ~0.2um2/s to ~1.5 um2/s), and the proportion is similar to the slow fraction of WT Bcd in the anterior nuclei (F=50%). The Dslow values of bcdWT suggest that 0.2um2/s is a result of DNA binding. For bcdN51A, Dslow of 1.5 um2/s is suggestive of nonspecific interaction of bcdN51A to the DNA. Such a nonspecific interaction is also noticed in the case of NLS::eGFP, where we see a significant amount (Dslow~ 1-1.5 um2/s , F=20%) of slow form in the anterior nuclei, likely due to non-specific interaction with the DNA.

It is worth noting that the inactive homeodomain of transcription factor sex comb reduced (scr) also interacts non-specifically with DNA at high concentration (Vukojevic et al., PNAS 2010). Non-specific interaction of eGFP fluorophore is also noted to be higher in the nuclei of AT-1 cells that suggest “obstacle-free accessible space” is low in the nuclei (Wachsmuth et al., JMB 2000). Therefore, though we do not understand the specific mechanism, our results for N51 mutants are aligned with previous observations of intra-nuclei dynamics.

The experimental rational behind the BcdMM reporter needs to be better explained as it is not clear. It was previously shown that the N51A mutation disturbs zygotic hb activation and Caudal gradient formation (see Figure 3 in Niessing et al., 2000). Since N51A already causes a strong phenotype by disturbing hb expression and Cad gradient formation, what is the reasoning being adding extra mutations to this background? Since the mutations in the PEST domain and YIRPYL motif are involved in cad translational repression, it would be more interesting to add them to the R54A mutation and further study the repression of cad? It would also shed light on the unexpected no difference or even decrease in diffusion in the cytoplasm of the R54A mutant which should increase if indeed the cad mRNA binding is being repressed.

Our rationale was to remove more elements of Bcd to see if there was some degree of redundancy – at least in terms of the dynamics.

The Bicoid homeodomain N51A mutation is physiologically known to cause de-repression of caudal and inhibit hunchback expression. Mechanistically, nuclear Bcd activates hb transcription. However, in the cytoplasm Bcd interacts with other proteins and forms a complex to de-repress caudal. Bcd binds to caudal mRNA through its HD at one end of the complex. However, in the other end, other proteins in the complex are bound to the 5’cap region caudal mRNA. Our rationale for generating the MM mutation was that the N51A mutation may not be sufficient for Bcd to be released from the protein complex. Therefore, additional mutations to N51A may release Bcd from interactions with either DNA or with other proteins through PEST domain and YIRPYL motif.

*Have the authors confirmed that their BcdR54A indeed inhibits cad translation? *

We have not tested the eGFP:bcdR54A to inhibit cad translation. We will add the data in the revision.

*How many embryos of BcdMM were analysed? The authors should also provide a table with all the values in SI as they have done for all the other reporters. *

We will add this data with the revision.

*The claims with eGFP::NLSBcdHD need to be supported by data from multiple embryos. Even if multiple ACF curves are obtained from one embryo, analysing only one embryo is not sufficient. This would clarify the fact that this reporter seems to be able to reproduce the mobility of Bcd in the nucleus. *

We agree and we are arranging to collect more data. This should be completed by the end of the summer.

*According to the methods, all reporters were expressed in a bcd null background, made with the bcd1 allele. This allele is also known as bcd085 and according to Driever and Nusslein-Volhard, 1988 (PMID: 3383244), this allele only causes an intermediate phenotype. This indicates that a truncated version of the protein probably still exists on the embryo. Do the conclusions obtained here still hold if a truncated version of the Bcd protein exists in addition to their reporters? *

We used the bcdE1 mutant, a null mutant of bcd. This was used by Gregor et al., Cell 2007 in their generation of the original Bcd::eGFP. We have also recently generated a more complete bcdKO mutation (Huang et al., eLife 2017). Our embryos do not have a clear phenotype that we can relate to the specific bcd- background used. Nonetheless, we agree it is an important point to be clear about the genetic background and we will clarify in the revised manuscript.

Minor comments: * * In line 45: "Morphogens are signalling molecules", the authors should consider removing the word "signalling" since not all morphogens are, especially the one being studied, Bicoid. * * In lines 80-81 (and also throughout the text): "We measure the Bcd dynamics at multiple locations along the embryo AP-axis", should be more accurate and changed to anterior and posterior of the embryo. Using "multiple locations along the AP axis" is ambiguous and not exact for what was done.

Yes, this is a fair comment. We have edited these sections in the current manuscript.

*Throughout the article, the authors refer multiple times to "modes for/of Bcd transport". Since they or others have not proven that Bcd is being transported, which would involve at least another factor, the authors should replace transport by movement, diffusion or a similar word with which they are comfortable. *

We have changed transport to movement wherever relevant in the text.

*Suggestion: The authors claim that the Bcd gradient is exponential up to 60% of embryo length. Would this information allow a more precise calculation of the gradient decay length in the exponential region than the 80-100µm stated on line 202? *

This is an interesting point, but our results suggest that the idea of the decay length is not so applicable in the posterior region. There, the Bcd dynamics are generally quicker, thereby increasing l. Of course, we cannot discount possible spatial variation in degradation. However, in previous work, our Bcd tandem reporter (which is sensitive to changes in degradation) did not reveal spatial variation in degradation.

In lines 258-259, the sentence "Further, Bcd binds to caudal mRNA, repressing its expression in the cytoplasm" should be improved to clarify the role of Bcd in caudal mRNA translation repression and references should be added. This should also be corrected in the following paragraph.

We will add the necessary corrections in the revision.

*In line 262, "mutations" should be singular since it corresponds to only one amino acid mutation. *

We have corrected this.

*Figure 4J needs to be corrected as the fractions of the slow and fast populations do not correspond to what is shown in Table 3. For example, Fslow fraction of AC is ~45% in the figure while it is 36% in Table 3. The problem occurs in all fractions. *

We are sorry there is a mislabelling in the corresponding figure. AN is in the place of AC. We have edited figure 4J and removed the mislabelling.

*In the discussion, in lines 379-380, "Given the changing fractions of the fast and slow populations in space, the interactions between the populations are likely non-linear". What is the reasoning for non-linearity and not interchangeability? *

If the interactions between the two populations were linear, then the fraction in each form would be constant across the embryo. Some degree of nonlinearity is required in order to have spatially varying relative populations.

*In line 432 caudal should be italicized. *

We have edited this.

*In the discussion, the authors conclude that "In the nucleus, the two populations can be largely (though not completely) explained by Bcd binding to DNA". The discussion would win by explaining all the possible options. * We will add the necessary changes in the discussion. This is also related to above reviewer comments.

Author Response

Reviewer #1 (Public Review):

This paper studies color vision in anemonefish. The central conclusion of the paper is that anemonefish use signals from their UV cones to discriminate colors that would not otherwise be distinguishable; this differs from other fish in which UV cones extend the range of wavelengths of sensitivity but do not add a dimension to color vision. The work fits into a rich history of studies investigating how color vision fits into an animal's ecological niche. My primary concerns regard the microspectrophotometry data from single cones and some aspects of the presentation of the behavioral data.

Microspectrophotometry

The spectral properties of the cone types are a key issue for interpreting the results. These were measured using MSP, and fits are shown in Figure 2. The raw data shown in Fig. S1 appears more complicated than indicated in the main text. The templates miss the measurements across broad wavelength bands in each cone type. Particularly concerning is the high UV absorbance across cone types and the long-wavelength absorbance in the UV cone. It is not clear how this picture supports the relatively simple description of cone types and spectral sensitivities given in the main text and which forms the basis of the modeling.

Microspectrophotometry is an inherently noise-prone measurement technique, particularly for very small photoreceptor outer segments such as that of single cones, which are also difficult to detect as intact, isolated (nonoverlapping) cells. As such, the absorbance curve fitting and derived lambda max (λmax) values should be treated as estimates. The accuracy of these estimates is adequate for this type of study, and visual modelling results have been shown to be robust against small errors (±10 nm λmax) in photoreceptor sensitivity for multiple species [see Lind, O. & Kelber, A. (2009). Vis Res. 49(15), 1939-1947; and Bitton, PP. et al. (2017). PLOS ONE, 12: e0169810]. We consider it highly unlikely that small shifts in cone λmax from measurement error would make a meaningful difference to the colour discrimination thresholds.

It should be noted that the raw data shown in the original Supplementary Figure 1, included all scans overlain with an average absorbance curve for presentation purposes; however, the actual lambda max values for different cone types were measured and then averaged among individual scans fitted with photopigment absorbance curve templates. For clarity and transparency, we have now provided three multipaned plots (see Figure 1 – figure supplements 1-3) showing the individual pre- and post-bleach scans of absorbance spectra, fitted absorbance curve templates, and R2 values from the best visual pigment template fit.

It is worth noting that most of the cone absorbance spectra found in our study closely resemble those in λmax and quality to those measured in another anemonefish species (Amphiprion akindynos) [see Supplementary Figure 1 in Stieb S. et al. (2019). Sci Rep. 9, 16459]. These cone λmax values can also be reconciled with previous estimates on opsin λmax based on amino acid sequences and cone opsin expression in the A. ocellaris retina characterised in Mitchell LJ et al. (2021). GBE, 13: evab184.

Evidence that the unusual long-wavelength absorbance detected in a couple of the single cone (pre-bleach) measurements were not of visual pigment in origin comes from post-bleach scans, which showed their persistence (i.e., did not show a photobleaching response) and were likely instead contaminants (e.g., blood, RPE pigment). UV absorbance in some of the double cone measurements (above that expected of the prebleached beta peak from chromophore spectral absorption) can be attributed to either noise from scans as is quite typical of MSP and/or partial (accidental) bleaching from stray light sources. Although utmost care was taken to minimise contamination and unintended bleaching sometimes it is unavoidable.

We refer the Reviewer to multiple published studies for further examples of typical MSP measurements that share similar levels of noise to ours e.g., see Figure 1 in Knott B. et al. (2013). JEB, 216:4454-4461; Figure 3 in Schott, RK et al. (2015). PNAS, 113(2): 356-361; Figure 2 in Dalton BE et al. (2014). Proc R Soc B. 281; Figure 5 in Tosetto, JE et al. (2021). Brain Behav Evol. 96: 103-123.

Presentation

The results are not presented in a straightforward way - at least for this reviewer. What is missing for me is a clear link between the psychometric curves in Figure 3A and the discrimination thresholds indicated in Figure 3B and Figure 4. Figure 3A is only discussed in the text on line 289 - after Figure 4 has been introduced and discussed. It would have been very helpful for me if the psychometric curves were first introduced and described, then the relation to Figure 3B was clearly indicated (perhaps with a single psychometric curve as an example). Similarly for Figure 4 the relationship between specific psychometric curves and the threshold plotted would be quite helpful. Currently it takes a careful reading to understand why being below the dashed line in Figure 4 is important.

We have made the following changes, including the introduction of the psychometric curves earlier in the results (lines 236-249) and moved the psychometric function comparison before the mention of Figure 4. Additionally, to make the association between the plotted colour loci and psychometric curves clearer, we have added a smaller psychometric curve plot adjacent to the colour space (in Figure 3B) using red as an example which has an averaged psychometric curve overlying the individual fish curves. The figure caption (lines 250-274) explains that the plotted colour loci and given thresholds are mean values calculated from the individual fish behavioural data.

We have also added a brief reminder that the theoretical limit of colour discrimination is predicted by the RNL model as 1∆S, where in our task fish should be just able to distinguish targets from grey distractors (see lines 222-224). To clarify, the plotted values in Figure 4B are both the individual fish thresholds (points) and average threshold (black bar) per colour set. The individual threshold values are taken at a correct choice probability of 50% from fitted psychometric curves of fish behavioural performance (shown in Figure 3A).

RNL model

The data is fit and interpreted in the context of the receptor noise limited model. The paragraph in the discussion about complementary color pairs suggests that this model is incorrect (text around line 332). Consideration of how the results depend on the RNL model is important, especially given the interpretation here.

The inability of the RNL model to account for the observed asymmetry between color discrimination thresholds implies that they cannot be solely attributed to photoreceptor noise. We can therefore infer from the asymmetry that thresholds are set by a higher-level process, whether that involves post-receptor processes within the inner retina or in the brain remains to be investigated. As explained in lines 396-397 one possibility is that activation of the UV receptor suppresses noise in the visual pathway or enhances the saliency of colors for anemonefish. The high sensitivity to violet-green, which was found in all six of the fish tested, is consistent with the heightened saliency of this color (lines 397-399).

Figure 3B

This is the key figure in the paper. But several issues make seeing the data in this figure difficult. First, the important part of the figure is buried near the origin and hard to see. Can you show a surface that connects the thresholds in the different chromatic directions, or otherwise highlight the regions of discriminable and not discriminable colors?

See previous comment. In short, we have taken the advice of the Reviewer and added highlighted areas around the regions of discriminable colors in Figure 3B to help visually separate them from the non-discriminable regions of colors (from grey). Additionally, we have added an inset showing an enlarged image of the area surrounding the centre of colour space.

Reviewer #2 (Public Review):

Mitchell and colleagues examined the contribution of a UV-sensitive cone photoreceptor to chromatic detection in Amphiprion ocellaris, a type of anemonefish. First, they used biophysical measurements to characterize the response properties of the retinal receptors, which come in four spectrally-distinct subtypes: UV, M1, M2, and L. They then used these spectral sensitivities to construct a 4-dimensional (tetrahedral) color space in which stimuli with known spectral power distributions can be represented according to the responses they elicit in the four cone types. A novel five-LED display was used to test the fish's ability to detect "chromatic" modulations in this color space against a background of random-intensity, "achromatic" distractors that produce roughly equal relative responses in the four cone types. A subset of stimuli, defined by their high positive UV contrast, were more readily detected than other colors that contained less UV information. A well-established model was used to link calculated receptor responses to behavioral thresholds. This framework also enabled statistical comparisons between models with varying number of cone types contributing to discrimination performance, allowing inferences to be drawn about the dimensionality of color vision in anemonefish.

The authors make a compelling case for how UV light in the anemonefish habitat is likely an important ecological source of information for guiding their behavior. The authors are to be commended for developing an elegant behavioral paradigm to assess visual performance and for incorporating a novel display device especially suited to addressing hypotheses about the role of UV light in color perception. While the data are suggestive of behavioral tetrachromacy in anemonefish, there are some aspects of the study that warrant additional consideration:

1) One challenge faced by many biological imaging systems is longitudinal chromatic aberration (LCA) - that is, the focal power of the system depends on wavelength. In general, focal power increases with decreasing wavelength, such that shorter wavelengths tend to focus in front of longer wavelengths. In the human eye, at least, this focal power changes nonlinearly with wavelength, with the steepest changes occurring in the shorter part of the visible spectrum (Atchison & Smith, 2005). In the fish eye, where the visible spectrum extends to even shorter wavelengths, it seems plausible that a considerable amount of LCA may exist, which could in turn cause UV-enriched stimuli to be more salient (relative to the distractor pixels) due to differences in perceived focus rather than due solely to differences in their respective spectral compositions. Such a mechanism has been proposed by Stubbs & Stubbs (2016) as a means for supporting "color vision" in monochromatic cephalopods (but see Gagnon et al. 2016). It would be worth discussing what is known about the dispersive properties of the crystalline lens in A. ocellaris (or similar species), and whether optical factors could produce sufficient cues in the retinal image that might explain aspects of the behavioral data presented in the current study.

This is an interesting point, and we appreciate the reviewer’s thoughtful comment regarding this topic especially as LCA increases exponentially in the UV. Although we certainly cannot disprove such a mechanism in the present study, we are highly sceptical that LCA could be used by reef fish and is involved in the heightened saliency of UV stimuli. Previous work has found that LCA is mostly corrected for in the teleost retina of both marine and freshwater species by graded, multifocal lenses that focus different wavelengths at the same depth as their maximally sensitive cone photoreceptors [e.g., for evidence in African cichlids see Kröger, R. H. H. et al. (1999). J Comp Physiol. A, 184, 361-369; Malkki, P. E. & Kröger, R. H. H. (2005). J Opt. A, 7, 691-700; and for various reef fishes see Karpestam, B. et al. (2007). J Exp Biol., 210, 16: 2923-2931]. In essence, LCA is corrected in the eyes of many teleosts by accurately tuning longitudinal spherical aberration through having a graded density lens. We draw particular attention to the latter reference which comparatively examined the optical properties of reef fish lenses, including diurnal, planktivorous damselfishes (from the same family as anemonefishes, Pomacentridae). They found that not only were the lenses of these species highly UV-transmissive (as we show in anemonefish), but all were multifocal and capable of focusing both visible (non-UV) and UV wavelengths. Considering the coastal cephalopod species examined thus far, all of them contain only one type of visual pigment which is packed in their long photoreceptor (150-450µm long outer segment) across an entire retina (Chung and Marshall 2016, Proceeding B). Theoretically, given these long photoreceptors, the LCA and the resulting differentials of focal length onto different patches of photoreceptors or different depth of the outer segment might provide cues for colour discrimination even though no behavioural evidence exists to prove this hypothesis yet. Unlike the cephalopod case, the four specific spectral cones arranged in a mosaic pattern along with their very short outer segments (5-10µm) in the anemonefish retina likely makes the LCA less effective in this retinal design.

We have added a short paragraph (Lines 400-412) discussing the possibility of an optical mechanism contributing to heightened UV saliency with a particular focus on LCA and our thoughts on why we consider it an unlikely mechanism in anemonefish.

2) The authors provide a quantitative description of anemonefish visual performance within the context of a well-developed receptor-based framework. However, it was less clear to me what inferences (if any) can be drawn from these data about the post-receptoral mechanisms that support tetrachromatic color vision in these organisms. Would specific cone-opponent processes account for instances where behavioral data diverged from predictions generated with the "receptor noise limited" model described in the text? The general reader may benefit from more discussion centered on what is known (or unknown) about the organization of cone-opponent processing in anemonefish and related species.

In short, we do not know the specific opponent interactions of anemonefish cones. The RNL model assumes all possible opponent interactions in its calculations. From our results, very little can be said about the post-receptor mechanisms involved in their putative tetrachromatic vision. We would like to avoid overreaching beyond what our data can show. A future directions section has now been added to the discussion (lines 467-497), which briefly mentions the known UV opponency in larval zebrafish and that future investigation in anemonefish should attempt to disentangle the specific opponent (chromatic) and non-opponent (achromatic) circuits in the anemonefish retina.

Reviewer #3 (Public Review):

The comments below focus mainly on ways that the data and analysis as currently present do not to this reviewer compel the conclusions the authors wish to draw. It is possible that further analysis and/or clarification in the presentation would more persuasively bolster the authors' position. It also seems possible that a presentation with more limited conclusions but clarity on exactly what has been demonstrated and where additional future work is needed would make a strong contribution to the literature.

• Fig 3A. It might be worth emphasizing a bit more explicitly that the x-axis (delta S) is the result of a model fit to the data being shown, since this then means that if RNL model fit the data perfectly, all of the thresholds would fall at deltaS = 1. They don't, so I would like to see some evaluation from the authors' experience with this model as to whether they think the deviations (looks like the delta S range is ~0.4 to ~1.6 in Figure 4B) represent important deviations of the data from the model, the non-significant ANOVA notwithstanding. For example, Figure 4B suggests that the sign of the fit deviations is driven by the sign of the UV contrast and that this is systematic, something that would not be picked up by the ANOVA. Quite a bit is made of the deviations below, but that the model doesn't fully account for the data should be brought out here I think. As the authors note elsewhere, deviations of the data from the RNL model indicate that factors other than receptor noise are at play, and reminding the reader of this here at the first point it becomes clear would be helpful.

We have now stated more explicitly in the figure caption for Figure 3A, that the delta S values presented were calculated by fitting fish behavioral data to the RNL model. To test the overall effect that the sign of the UV contrast had on the discrimination threshold, we have now included ‘contrast’ (positive or negative) as another fixed effect in the linear mixed effects model. We have now included details of this test in the results which shows the systematic effect (lines 338-340). Additionally, as suggested we now briefly introduce in the results the idea that factors other than receptor noise are causing the observed deviations in data from the RNL model.

• Line 217 ff, Figure 4, Supplemental Figure 4). If I'm understanding what the ANOVA is telling us, it is that the deviations of the data across color directions and fish (I think these are the two factors based on line 649) is that the predictions deviate significantly from the data, relative to the inter-fish variability), for the trichromatic models but not the tetrachromatic model. If that's not correct, please interpret this comment to mean that more explanation of the logic of the test would be helpful.

The interpretation of the ANOVA by the Reviewer is mostly correct. We had the variables color set and Fish ID, with threshold delta S as the dependent variable. This showed that deviations from the predicted threshold were significant relative to the inter-fish variability for the trichromatic models. Missing details describing the ANOVA have now been added to the methods (lines 789-798).

Assuming that the above is right about the nature of the test, then I don't think the fact that the tetrachromatic model has an additional parameter (noise level for the added receptor type) is being taken into account in the model comparison. That is, the trichromatic models are all subsets of the tetrachromatic model, and must necessarily fit the data worse. What we want to know is whether the tetrachromatic model is fitting better because its extra parameter is allowing it to account for measurement noise (overfitting), or whether it is really doing a better job accounting for systematic features of the data. This comparison requires some method of taking the different number of parameters into account, and I don't think the ANOVA is doing that work. If the models being compared were nested linear models, than an F-ratio test could be deployed, but even this doesn't seem like what is being done. And the RNL model is not linear in its parameters, so I don't think that would be the right model comparison test in any case.

Typical model comparison approaches would include a likelihood ratio test, AIC/BIC sorts of comparisons, or a cross-validation approach.

If the authors feel their current method does persuasively handle the model comparison, how it does so needs to be brought out more carefully in the manuscript, since one of the central conclusions of the work hinges at least in part on the appropriateness of such a statistical comparison.

Our visual model comparisons were aimed at assessing whether a trichromatic or tetrachromatic model best fit the colour discrimination data. The trichromatic and tetrachromatic models assume two and three opponency pathways, respectively. If the fish were not tetrachromatic, and instead trichromatic, then we would expect that the RNL model should better fit the data with two opponency mechanisms (rather than three). Our reason for making this assessment, is because of the possibility that not all the cones could be contributing to colour vision and could be used exclusively for achromatic tasks (e.g., luminance vision or motion detection). However, according to our finding that the data best fit the tetrachromatic model (i.e., how the behavioural discrimination thresholds more closely fitted the theoretical prediction of 1∆S), it is likely that anemonefish used all four cones for colour vision.

We have also now repeated our analysis using unweighed delta S values which are calculated using general n-dimensional models of colour vision (using the PAVO2 package). These models essentially follow the same initial steps followed by the RNL model (and many others) but omit the receptor noise correction stage. After comparing (using ANOVA, see lines 303-311) the predicted thresholds with the data in this non-RNL space, it was found that again the tetrachromatic model predictions did not deviate significantly from the data relative to individual fish performance; however, we also found that the trichromatic model without M2 cone input no longer differed from the predicted values. In this case, it seems that the extra noise parameter did contribute to the difference in fit. Whether this is a biologically meaningful comparison (as all photoreceptors contain noise) is an open question. We have added a short statement explicitly framing our interpretation of anemonefish having a 3-D colour space to being in accordance with the closeness of RNL model predictions (lines 370-371, 506-508).

• Also on the general point on conclusions drawn from the model fits, it seems important to note that rejecting a trichromatic version of the RNL model is not the same as rejecting all trichromatic models. For example, a trichromatic model that postulates limiting noise added after a set of opponent transformations will make predictions that are not nested within those of RNL trichromatic models. This point seems particularly important given the systematic failures of even the tetrachromatic version of the RNL model.

This is a good point. We have limited our conclusions to specifically address trichromatic models generated within the framework of the RNL model by adding in the conclusion section that fish psychophysical thresholds were best explained by the RNL model when all four cone types contributed to colour vision (see lines 370-371, 506-508). In this same sentence, we have also added in parentheses that “suggesting (but not proving) tetrachromacy” (line 508). We have also edited the abstract to state that our results were “…best described by a tetrachromatic model using all four cone types…”, rather than stating we have shown tetrachromacy (lines 36-37).

• More generally, attempts to decide whether some human observers exhibit tetrachromacy have taught us how hard this is to do. Two issues, beyond the above, are the following. 1) If the properties of a trichromatic visual system vary across the retina, then by imaging stimuli on different parts of the visual field an observer can in principle make tetrachromatic discriminations even though visual system is locally trichromatic at each retinal location. 2) When trying to show that there is no direction in a tetrachromatic receptor space to which the observer is blind, a lot of color directions need to be sampled. Here, 9 directions are studied. Is that enough? How would we know? The following paper may be of interest in this regard: Horiguchi, Hiroshi, Jonathan Winawer, Robert F. Dougherty, and Brian A. Wandell. "Human trichromacy revisited." Proceedings of the National Academy of Sciences 110, no. 3 (2013): E260-E269. Although I'm not suggesting that the authors conduct additional experiments to try to address these points, I do think they need to be discussed. We agree with the reviewer, that colour discriminability achieved by tetrachromatic vision could in theory be achieved by the combined effect of localised, distinct forms of trichromacy. Evidence in other fishes suggests that such multiple forms of trichromacy across the retina likely exist in many species. However, the behavioural effects of this retinal setup remain to be studied likely due to its extremely difficult nature. We have added a new section titled “future directions” (Lines 474-489), in which we discuss the possibility that distinct forms of trichromacy in the anemonefish retina could in theory achieve colour discrimination on par with tetrachromatic vision. We also give suggestions on how this could be investigated.

Although we tried to include as many colour directions as practically possible in our experiment, we have certainly not provided an exhaustive range that completely encompasses anemonefish colour space. Whether 9 colour directions are adequate to assess the dimensionality of their color vision is difficult to say. As addressed in the previous comment, we now acknowledge this limitation by refining our conclusion, saying that our results do not prove tetrachromacy.

• Line 277 ff. After reading through the paper several times, I remain unsure about what the authors regard as their compelling evidence that the UV cone has a higher sensitivity or makes an omnibus higher contribution to sensitivity than other cones (as stated in various forms in the title, Lines 37-41, 56-57, 125, 313, 352 and perhaps elsewhere).

At first, I thought they key point was that the receptor noise inferred via the RNL model as slightly lower (0.11) for the UV cone than for the double cones (0.14). And this is the argument made explicitly at line 326 of the discussion. But if this is the argument, what needs to be shown is that the data reject a tetrachromatic version of the RNL model where the noise value of all the cones is locked to be the same (or something similar), with the analysis taking into account the fewer parametric degrees of freedom where the noise parameters are so constrained. That is, a careful model comparison analysis would be needed. Such an analysis is not presented that I see, and I need more convincing that the difference between 0.11 and 0.14 is a real effect driven by the data. Also, I am not sanguine that the parameters of a model that in some systematic ways fails to fit the data should be taken as characterizing properties of the receptors themselves (as sometimes seems to be stated as the conclusion we should draw).

We have performed various modelling scenarios where receptor noise was adjusted for each channel; however, the UV channel was consistently found to be more sensitive than the other channels. In (the original) Supplementary Figure 6 (now Figure 4 – figure supplements 1 and 2), we show predicted dS values calculated using receptor noise levels in the exact manner that the Reviewer suggests by ranging from 0.05 to 0.15, and most importantly, included scenarios where receptor noise was held equal across cone types and others where it was varied between single cones and double cones. None of the models adjusted the data so that sensitivity was equal across all four channels, which means that by an unknown mechanism, the UV channel is more sensitive, but this is unrelated to noise levels. Our best-fit receptor noise values of 0.11 (for single cones) and 0.14 (for double cones) are estimate values and should be treated as such till actual receptor noise measurements are made.

Then, I thought maybe the argument is not that the noise levels differ, but rather that the failures of the model are in the direction of thresholds being under predicted for discriminations that involve UV cone signals. That's what seems to be being argued here at lines 277 ff, and then again at lines 328 ff of the discussion. But then the argument as I read it more detail in both places switches from being about the UV cones per se to being about postive versus negative UV contrast. That's fine, but it's distinct from an argument that favors omnibus enhanced UV sensitivity, since both the UV increments and decrements are conveyed by the UV cone; it's an argument for differential sensitivity for increments versus decrements in UV mediated discriminations. The authors get to this on lines 334 of the discussion, but if the point is an increment/decrement asymmetry the title and many of the terser earlier assertions should be reworked to be consistent with what is shown.

To clarify our argument, we found that the colour discrimination thresholds were systematically lower than predicted by the RNL model for colours which elicited higher UV cone stimulation relative to other cone types. These colours we refer to as UV positive based on the sign direction of their contrast against grey distractors produced by higher UV/V LED channel (i.e., in a positive direction). Whereas colours with UV negative chromatic contrast had lower UV cone stimulation relative to the other cone types. Therefore, our interpretation of the importance of UV cone signals for colour discrimination are congruent with the results. In the discussion, we suggest a possibility that activation of the UV receptor suppresses noise downstream in the visual pathway or enhances the saliency of colours (see lines 397-398). This activation of the UV receptor would, of course, be at its highest for colours with positive UV chromatic contrast.

Note that we have added to the discussion the possibility that colour preferences or a difference in attentiveness might have contributed to differences in discrimination thresholds (see discussion lines 412-413, 427-428, 433-435, 456-466, and 469-473). However, we consider it a less likely explanation due to a couple of reasons, including 1) a lack of difference in responsiveness across colour sets in their timing to peck the target, and 2) any non-learnt bias would have likely been overridden or at least weakened by training prior to the experiment where colours were rewarded equally (see lines 462-466).

We have edited the results (lines 334-352) to make our point clearer and by changing the subtitle to be more explicit: “Lower discrimination thresholds induced by positive UV contrast”. The subsection begins by explaining the different types of UV chromatic contrast by elevation angle and, finally, how this division among colour sets was a major determinant of colour discrimination thresholds.

Perhaps the argument with respect to model deviations and UV contrast independent of sign could be elaborated to show more systematically that the way the covariation with the contrasts of the other cone stimulations in the stimulus set goes, the data do favor deviations from the RNL in the direction of enhanced sensitivity to UV cone signals, but if this is the intent I think the authors need to think more about how to present the data in a manner that makes it more compelling than currently, and walk the reader carefully through the argument.

We have added to the results the linear mixed-effects model output with ‘contrast’ (positive/negative) added as a fixed effect. This analysis shows that the sign direction of UV contrast was a strong predictor of threshold (see address to previous comments and lines 399-401, 790-799).

• On this point, if the authors decide to stick with the enhanced UV sensitivity argument in the revision, a bit more care about what is meant by "the UV cone has a comparatively high sensitivity (line 313 and throughout)" needs more unpacking. If it is that these cones have lower inferred noise (in the context of a model that doesn't account for at least some aspects of the data), is this because of properties of the UV cones, or the way that post-receptoral processing handles the signals from these cones mimicking a cone effect in the model. And if it is thought that it is because of properties of the cones, some discussion of what those properties might be would be helpful. As I understand the RNL model, relative numbers of cones of each type are taken into account, so it isn't that. But could it be something as simple as higher photopigment density or larger entrance aperture (thus more quantum catches and higher SNR)?

It is unknown what aspect of the cone morphology or physiology sets the activation or inactivation threshold. Electrophysiological data collected from the UV cones of other fish species e.g., in goldfish and zebrafish [see Hawryshyn & Beauchamp (1985). 25, Vis Res.; and Yoshimatsu et al. (2020). 107, Neuron.] show that they have exceptionally high sensitivity. What has not been shown is that having a UV cone can improve colour discrimination.

Previous quantitative cone opsin gene expression analysis showed that the single cone opsins (SWS1 and SWS2B) are expressed at lower levels than all double cone opsin genes. This difference in expression combined with the smaller size of single cone outer segments than the double cones make it unlikely that a larger photoreceptor size, higher volume or packing density of visual pigment is responsible. Contrary to our findings, these aspects of the different cone types (if they had an effect) would instead predict that double cones have a higher SNR, and non-UV colours would be more discriminable. We have now added these details to the discussion (see lines 391-397).

• Line 288 ff. The fact that the slopes of the psychometric functions differed across color directions is, I think, a failure of the RNL model to describe this aspect of the data, and tells us that a simple summary of what happens for thresholds at delta S = 1 does not generalize across color directions for other performance levels. Since one of the directions where the slope is shallower is the UV direction, this fact would seem to place serious limits on the claim that discrimination in the UV direction is enhanced relative to other directions, but it goes by here without comment along those lines. Some comment here, both about implications for fit of RNL model and about implications for generalizations about efficacy of UV receptor mediated discrimination and UV increment/decrement asymmetries, seems important.

The variation in the psychometric functions is difficult to interpret and cannot be explained by the RNL model. What the RNL model predicts is delta S based on low level factors (namely receptor noise). In the discussion, we completely agree with the notion that the asymmetry in thresholds from predicted values, and the variation in psychometric slopes cannot be explained by the RNL model, e.g., this is heavily implied by “colour discrimination thresholds cannot be directly attributed to noise in the early stages of the visual pathway…” (lines 388-390). To clarify the inability of the RNL model to account for this aspect of the data, we have included a statement (see line 390).

It is a good point that this could be an indication of heterogeneity in colour space. Heterogeneity in discrimination thresholds across animal colour space (both surrounding the threshold area and for more saturated regions) has been explored in detail using trichromatic triggerfish by Green N. F. et al. (2022). JEB, 7(225):jeb243533. We have added this idea to the discussion (see lines 490-498). For UV, it seems that two of the five fish (#34 and 20) had noticeably shallower curves than the others tested for UV (fish #19, 33, 36). Both also varied more in their ability to distinguish targets, as shown by their wider confidence intervals. One of these two fish (#34) was retested for UV at the end of the experiment, and in the secondary assessment had a steeper psychometric curve more in line with the other fish in the experiment (see Figure 3 – figure supplement 1 and added lines 247-250). Based on this discrepancy in performance between assessments, it is also possible that individual learning effects had a role in impacting the shape of the psychometric curve. Note, this had minimal effect on colour discrimination thresholds and any differences were in the direction of change observed across colour sets in the experiment (i.e., lower dS for UV positive directions).

• Line 357 ff. Up until this point, all of the discussion of differences in threshold across stimulus sets has been in terms of sensitivity. Here the authors (correctly) raise the possibility that a difference in "preference" across stimulus sets could drive the difference in thresholds as measured. Although the discussion is interesting and germaine, it does to some extent further undercut the security of conclusions about differential sensitivity across color directions relative to the RNL model predictions, and that should be brought out for the reader here. The authors might also discuss about how a future experiment might differentiate between a preference explanation and a sensitivity explanation of threshold differences.

We have now added a paragraph (see lines 469-473) discussing that future work should test for color preferences and suggest how this could be done using a similar foraging task. We also include our thoughts immediately prior on why it is unlikely that a colour preference was a major contribution towards the results. In short, we consider it unlikely as fish showed no evidence of reduced latency for pecking at targets across the colour sets and because the training regime prior to the experiment equally rewarded fish for all colours and would likely have overridden a strong preference (at least in this specific foraging context).

• RNL model. The paper cites a lot of earlier work that used the RNL model, but I think many readers will not be familiar with it. A bit more descriptive prose would be helpful, and particularly noting that in the full dimensional receptor space, if the limiting noise at the photoreceptors is Gaussian, then the isothreshold contour will be a hyper-ellipsoid with its axes aligned with the receptor directions.

There is now added explanation of the RNL model (see lines 141-151), particularly on its assumptions that it only receives chromatic input and that discrimination is limited by noise arising in the photoreceptors and not by any specific opponent mechanisms. We also added the mention of the expected hyper-ellipsoid shape of isothreshold contours if receptor noise is Gaussian. Note, while we appreciate the importance of the reader to understand the basic functionality of the model, we wanted to avoid overloading the introduction with details on the RNL model which is not the focus of the paper. The RNL model is well-established in the field of visual ecology and animal vision research for well over a decade and has been thoroughly dissected by previous methodological reviews. We refer to one of these more recent reviews by Olsson et al. (2018) Behav Ecol. 29(2):273-282, and direct the reader to the methods section for further details on the RNL model.

• Use of cone isolating stimuli? For showing that all four cone classes contribute to what the authors call color discrimination, a more direct approach would seem to be to use stimuli that target stimulation of only one class of cone at a time. This might require a modified design in which the distractors and target were shown against a uniform background and approximately matched in their estimated effect on a putative achromatic mechanism. Did the authors consider this approach, and more generally could they discuss what they see as its advantages and disadvantages for future work.

The Reviewer is correct in that a targeted approach of isolated cone stimulation would be the optimal approach to demonstrating tetrachromatic colour vision. However, the extreme spectral overlap in the absorption curves of anemonefish cones, particularly in the mid-wavelength region makes this problematic in using the current LED display. We added to the discussion ways that this could be studied in the future (see lines 474-489). This might be possible (but still challenging) using a monochromator, but such technology severely limits the diversity of stimuli which can be created and usually restricts experiments to a simple paired choice design (or grey card experiment). The traditional paired choice experiment requires animals to be trained to distinguish a specific colour, while the Ishihara-like task trains animals to distinguish targets using an odd-one-out approach. This latter approach is highly efficient, as it does not require retraining when testing a new colour (i.e., fish learnt the task not a specific colour). Here, we wanted to assess colour discrimination in multiple directions to compare performance, and the flexible LED display combined with a generalisable task was important.

The above assumes that anemonefish do not use multiple trichromatic systems. In which case, the use of standard experimental stimuli (e.g., a monochromator, an LED display) would be unsuitable as they illuminate the whole retina. To definitively test the range of opponent interactions, it would be necessary to make electrophysiological measurements targeting the transmitting neurons using a retinal multielectrode array (MEA) approach or by in-vivo calcium imaging (lines 484-486).

We understand that our results are not a direct test of the dimensionality of anemonefish colour vision and should not be interpreted as such, as we do not have direct evidence of tetrachromacy. To recognize this limitation of our data, we have drawn back some of our conclusive statements that claimed to have demonstrated tetrachromacy.

Author Response

Reviewer #2 (Public Review):

Here, a simple model of cerebellar computation is used to study the dependence of task performance on input type: it is demonstrated that task performance and optimal representations are highly dependent on task and stimulus type. This challenges many standard models which use simple random stimuli and concludes that the granular layer is required to provide a sparse representation. This is a useful contribution to our understanding of cerebellar circuits, though, in common with many models of this type, the neural dynamics and circuit architecture are not very specific to the cerebellum, the model includes the feedforward structure and the high dimension of the granule layer, but little else. This paper has the virtue of including tasks that are more realistic, but by the paper’s own admission, the same model can be applied to the electrosensory lateral line lobe and it could, though it is not mentioned in the paper, be applied to the dentate gyrus and large pyramidal cells of CA3. The discussion does not include specific elements related to, for example, the dynamics of the Purkinje cells or the role of Golgi cells, and, in a way, the demonstration that the model can encompass different tasks and stimuli types is an indication of how abstract the model is. Nonetheless, it is useful and interesting to see a generalization of what has become a standard paradigm for discussing cerebellar function.

We appreciate the Reviewer’s positive comments. Regarding the simplifications of our model, we agree that we have taken a modeling approach that abstracts away certain details to permit comparisons across systems. We now include an in-depth discussion of our simplifying assumptions (Assumptions & Extensions section in the Discussion) and have further noted the possibility that other biophysical mechanisms we have not accounted for may also underlie differences across systems.

Our results predict that qualitative differences in the coding levels of cerebellum-like systems, across brain regions or across species, reflect an optimization to distinct tasks (Figure 7). However, it is also possible that differences in coding level arise from other physiological differences between systems.

Reviewer #3 (Public Review):

1) The paper by Xie et al is a modelling study of the mossy fiber-to-granule cell-to-Purkinje cell network, reporting that the optimal type of representations in the cerebellar granule cell layer depends on the type task. The paper stresses that the findings indicate a higher overall bias towards dense representations than stated in the literature, but it appears the authors have missed parts of the literature that already reported on this. While the modelling and analysis appear mathematically solid, the model is lacking many known constraints of the cerebellar circuitry, which makes the applicability of the findings to the biological counterpart somewhat limited.

We thank the Reviewer for suggesting additional references to include in our manuscript, and for encouraging us to extend our model toward greater biological plausibility and more critically discuss simplifying assumptions we have made. We respond to both the comment about previous literature and about applicability to cerebellar circuitry in detail below.

2) I have some concerns with the novelty of the main conclusion, here from the abstract: ’Here, we generalize theories of cerebellar learning to determine the optimal granule cell representation for tasks beyond random stimulus discrimination, including continuous input-output transformations as required for smooth motor control. We show that for such tasks, the optimal granule cell representation is substantially denser than predicted by classic theories.’ Stated like this, this has in principle already been shown, i.e. for example: Spanne and Jo¨rntell (2013) Processing of multi-dimensional sensorimotor information in the spinal and cerebellar neuronal circuitry: a new hypothesis. PLoS Comput Biol. 9(3):e1002979. Indeed, even the 2 DoF arm movement control that is used in the present paper as an application, was used in this previous paper, with similar conclusions with respect to the advantage of continuous input-output transformations and dense coding. Thus, already from the beginning of this paper, the novelty aspect of this paper is questionable. Even the conclusion in the last paragraph of the Introduction: ‘We show that, when learning input-output mappings for motor control tasks, the optimal granule cell representation is much denser than predicted by previous analyses.’ was in principle already shown by this previous paper.

We thank the Reviewer for drawing our attention to Spanne and Jo¨rntell (2013). Our study shares certain similarities with this work, including the consideration of tasks with smooth input-output mappings, such as learning the dynamics of a two-joint arm. However, our study differs substantially, most notably the fact that we focus our study on parametrically varying the degree of sparsity in the granule cell layer to determine the circumstances under which dense versus sparse coding is optimal. To the best of our ability, we can find no result in Spanne and J¨orntell (2013) that indicates the performance of a network as a function of average coding level. Instead, Spanne and Jo¨rntell (2013) propose that inhibition from Golgi cells produces heterogeneity in coding level which can improve performance, which is an interesting but complementary finding to ours. We therefore do not believe that the quantitative computations of optimal coding level that we present are redundant with the results of this previous study. We also note that a key contribution of our study is mathemetical analysis of the inductive bias of networks with different coding levels which supports our conclusions.

We have included a discussion of Spanne and Jo¨rntell (2013) and (2015) in the revised version of our manuscript:

"Other studies have considered tasks with smooth input-output mappings and low-dimensional inputs, finding that heterogeneous Golgi cell inhibition can improve performance by diversifying individual granule cell thresholds (Spanne and J¨orntell, 2013). Extending our model to include heterogeneous thresholds is an interesting direction for future work. Another proposal states that dense coding may improve generalization (Spanne and Jo¨rntell, 2015). Our theory reveals that whether or not dense coding is beneficial depends on the task."

3) However, the present paper does add several more specific investigations/characterizations that were not previously explored. Many of the main figures report interesting new model results. However, the model is implemented in a highly generic fashion. Consequently, the model relates better to general neural network theory than to specific interpretations of the function of the cerebellar neuronal circuitry. One good example is the findings reported in Figure 2. These represent an interesting extension to the main conclusion, but they are also partly based on arbitrariness as the type of mossy fiber input described in the random categorization task has not been observed in the mammalian cerebellum under behavior in vivo, whereas in contrast, the type of input for the motor control task does resemble mossy fiber input recorded under behavior (van Kan et al 1993).

We agree that the tasks we consider in Figure 2 are simplified compared to those that we consider elsewhere in the paper. The choice of random mossy fiber input was made to provide a comparison to previous modeling studies that also use random input as a benchmark (Marr 1969, Albus 1971, Brunel 2004, Babadi and Sompolinsky 2014, Billings 2014, LitwinKumar et al., 2017). This baseline permits us to specifically evaluate the effects of lowdimensional inputs (Figure 2) and richer input-output mappings (Figure 2, Figure 7). We agree with the Reviewer that the random and uncorrelated mossy fiber activity that has been extensively used in previous studies is almost certainly an unrealistic idealization of in vivo neural activity—this is a motivating factor for our study, which relaxes this assumption and examines the consequences. To provide additional context, we have updated the following paragraph in the main text Results section:

"A typical assumption in computational theories of the cerebellar cortex is that inputs are randomly distributed in a high-dimensional space (Marr, 1969; Albus, 1971; Brunel et al., 2004; Babadi and Sompolinsky, 2014; Billings et al., 2014; Litwin-Kumar et al., 2017). While this may be a reasonable simplification in some cases, many tasks, including cerebellumdependent tasks, are likely best-described as being encoded by a low-dimensional set of variables. For example, the cerebellum is often hypothesized to learn a forward model for motor control (Wolpert et al., 1998), which uses sensory input and motor efference to predict an effector’s future state. Mossy fiber activity recorded in monkeys correlates with position and velocity during natural movement (van Kan et al., 1993). Sources of motor efference copies include motor cortex, whose population activity lies on a lowdimensional manifold (Wagner et al., 2019; Huang et al., 2013; Churchland et al., 2010; Yu et al., 2009). We begin by modeling the low dimensionality of inputs and later consider more specific tasks."

4) The overall conclusion states: ‘Our results....suggest that optimal cerebellar representations are task-dependent.’ This is not a particularly strong or specific conclusion. One could interpret this statement as simply saying: ‘if I construct an arbitrary neural network, with arbitrary intrinsic properties in neurons and synapses, I can get outputs that depend on the intensity of the input that I provide to that network.’ Further, the last sentence of the Introduction states: ‘More broadly, we show that the sparsity of a neural code has a task-dependent influence on learning...’ This is very general and unspecific, and would likely not come as a surprise to anyone interested in the analysis of neural networks. It doesn’t pinpoint any specific biological problem but just says that if I change the density of the input to a [generic] network, then the learning will be impacted in one way or another.

We agree with the Reviewer that our conclusions are quite general, and we have removed the final sentence as we agree it was unspecific. However, we disagree with the Reviewer’s paraphrasing of our results.

First, we do not select arbitrary intrinsic properties of neurons and synapses. Rather, we construct a simplified model with a key quantity, the neuronal threshold, that we vary parametrically in order to assess the effect of the resulting changes in the representation on performance. Second, we do not vary the intensity/density of inputs provided to the network – this is fixed throughout our study for all key comparisons we perform. Instead, we vary the density (coding level) of the expansion layer representation and quantify its effect on inductive bias and generalization. Finally, our study’s key contribution is an explanation of the heterogeneity in average coding level observed across behaviors and cerebellum-like systems. We go beyond the empirical statement that there is a dependence of performance on the parameter that we vary by developing an analytical theory. Our theory describes the performance of the class of networks that we study and the properties of learning tasks that determine the optimal expansion layer representation.

To clarify our main contributions, we have updated the final paragraph of the Introduction. We have also removed the sentence that the Reviewer objects to, as it was less specific than the other points we make here.

"We propose that these differences can be explained by the capacity of representations with different levels of sparsity to support learning of different tasks. We show that the optimal level of sparsity depends on the structure of the input-output relationship of a task. When learning input-output mappings for motor control tasks, the optimal granule cell representation is much denser than predicted by previous analyses. To explain this result, we develop an analytic theory that predicts the performance of cerebellum-like circuits for arbitrary learning tasks. The theory describes how properties of cerebellar architecture and activity control these networks’ inductive bias: the tendency of a network toward learning particular types of input-output mappings (Sollich, 1998; Jacot et al., 2018; Bordelon et al., 2020; Canatar et al., 2021; Simon et al., 2021). The theory shows that inductive bias, rather than the dimension of the representation alone, is necessary to explain learning performance across tasks. It also suggests that cerebellar regions specialized for different functions may adjust the sparsity of their granule cell representations depending on the task."

5) The interpretation of the distribution of the mossy fiber inputs to the granule cells, which would have a crucial impact on the results of a study like this, is likely incorrect. First, unlike the papers that the authors cite, there are many studies indicating that there is a topographic organization in the mossy fiber termination, such that mossy fibers from the same inputs, representing similar types of information, are regionally co-localized in the granule cell layer. Hence, there is no support for the model assumption that there is a predominantly random termination of mossy fibers of different origins. This risks invalidating the comparisons that the authors are making, i.e. such as in Figure 3. This is a list of example papers, there are more: van Kan, Gibson and Houk (1993) Movement-related inputs to intermediate cerebellum of the monkey. Journal of Neurophysiology. Garwicz et al (1998) Cutaneous receptive fields and topography of mossy fibres and climbing fibres projecting to cat cerebellar C3 zone. The Journal of Physiology. Brown and Bower (2001) Congruence of mossy fiber and climbing fiber tactile projections in the lateral hemispheres of the rat cerebellum. The Journal of Comparative Neurology. Na, Sugihara, Shinoda (2019) The entire trajectories of single pontocerebellar axons and their lobular and longitudinal terminal distribution patterns in multiple aldolase C-positive compartments of the rat cerebellar cortex. The Journal of Comparative Neurology.

6) The nature of the mossy fiber-granule cell recording is also reviewed here: Gilbert and Miall (2022) How and Why the Cerebellum Recodes Input Signals: An Alternative to Machine Learning. The Neuroscientist. Further, considering the re-coding idea, the following paper shows that detailed information, as it is provided by mossy fibers, is transmitted through the granule cells without any evidence of re-coding: Jo¨rntell and Ekerot (2006) Journal of Neuroscience; and this paper shows that these granule inputs are powerfully transmitted to the molecular layer even in a decerebrated animal (i.e. where only the ascending sensory pathways remains) Jo¨rntell and Ekerot 2002, Neuron.

We agree that there is strong evidence for a topographic organization in mossy fiber to granule cell connectivity at the microzonal level. We thank the Reviewer for pointing us to specific examples. We acknowledge that our simplified model does not capture the structure of connectivity observed in these studies.

However, the focus of our model is on cerebellar neurons presynaptic to a single Purkinje cell. Random or disordered distribution of inputs at this local scale is compatible with topographic organization at the microzonal scale. Furthermore, while there is evidence of structured connections at the local scale, models with random connectivity are able to reproduce the dimensionality of granule cell activity within a small margin of error (Nguyen et al., 2022). Finally, our finding that dense codes are optimal for learning slowly varying tasks is consistent with evidence for the lack of re-coding – for such tasks, re-coding may absent because it is not required.

We have dedicated a section on this issue in the Assumptions and Extensions portion of our Discussion:

"Another key assumption concerning the granule cells is that they sample mossy fiber inputs randomly, as is typically assumed in Marr-Albus models (Marr, 1969; Albus, 1971; LitwinKumar et al., 2017; Cayco-Gajic et al., 2017). Other studies instead argue that granule cells sample from mossy fibers with highly similar receptive fields (Garwicz et al., 1998; Brown and Bower, 2001; J¨orntell and Ekerot, 2006) defined by the tuning of mossy fiber and climbing fiber inputs to cerebellar microzones (Apps et al., 2018). This has led to an alternative hypothesis that granule cells serve to relay similarly tuned mossy fiber inputs and enhance their signal-to-noise ratio (Jo¨rntell and Ekerot, 2006; Gilbert and Chris Miall, 2022) rather than to re-encode inputs. Another hypothesis is that granule cells enable Purkinje cells to learn piece-wise linear approximations of nonlinear functions (Spanne and J¨orntell, 2013). However, several recent studies support the existence of heterogeneous connectivity and selectivity of granule cells to multiple distinct inputs at the local scale (Huang et al., 2013; Ishikawa et al., 2015). Furthermore, the deviation of the predicted dimension in models constrained by electron-microscopy data as compared to randomly wired models is modest (Nguyen et al., 2022). Thus, topographically organized connectivity at the macroscopic scale may coexist with disordered connectivity at the local scale, allowing granule cells presynaptic to an individual Purkinje cell to sample heterogeneous combinations of the subset of sensorimotor signals relevant to the tasks that Purkinje cell participates in. Finally, we note that the optimality of dense codes for learning slowly varying tasks in our theory suggests that observations of a lack of mixing (J¨orntell and Ekerot, 2002) for such tasks are compatible with Marr-Albus models, as in this case nonlinear mixing is not required."

7) I could not find any description of the neuron model used in this paper, so I assume that the neurons are just modelled as linear summators with a threshold (in fact, Figure 5 mentions inhibition, but this appears to be just one big lump inhibition, which basically is an incorrect implementation). In reality, granule cells of course do have specific properties that can impact the input-output transformation, PARTICULARLY with respect to the comparison of sparse versus dense coding, because the low-pass filtering of input that occurs in granule cells (and other neurons) as well as their spike firing stochasticity (Saarinen et al (2008). Stochastic differential equation model for cerebellar granule cell excitability. PLoS Comput. Biol. 4:e1000004) will profoundly complicate these comparisons and make them less straight forward than what is portrayed in this paper. There are also several other factors that would be present in the biological setting but are lacking here, which makes it doubtful how much information in relation to the biological performance that this modelling study provides: What are the types of activity patterns of the inputs? What are the learning rules? What is the topography? What is the impact of Purkinje cell outputs downstream, as the Purkinje cell output does not have any direct action, it acts on the deep cerebellar nuclear neurons, which in turn act on a complex sensorimotor circuitry to exert their effect, hence predictive coding could only become interpretable after the PC output has been added to the activity in those circuits. Where is the differentiated Golgi cell inhibition?

Thank you for these critiques. We have made numerous edits to improve the presentation of the details of our model in the main text of the manuscript. Indeed, granule cells in the main text are modeled as linear sums of mossy fiber inputs with a threshold-linear activation function. A more detailed description of the model for granule cells can now be found in Equation 1 in the Results section:

"The activity of neurons in the expansion layer is given by: h = φ(Jeffx − θ), (1) where φ is a rectified linear activation function φ(u) = max(u,0) applied element-wise. Our results also hold for other threshold-polynomial activation functions. The scalar threshold θ is shared across neurons and controls the coding level, which we denote by f, defined as the average fraction of neurons in the expansion layer that are active."

Most of our analyses use the firing rate model we describe above, but several Supplemental Figures show extensions to this model. As we mention in the Discussion, our results do not depend on the specific choice of nonlinearity (Figure 2-figure supplement 2). We have also considered the possibility that the stochastic nature of granule cell spikes could impact our measures of coding level. In Figure 7-figure supplement 1 we test the robustness of our main conclusion using a spiking model where we model granule cell spikes with Poisson statistics. When measuring coding level in a population of spiking neurons, a key question is at what time window the Purkinje cell integrates spikes. For several choices of integration time windows, we show that dense coding remains optimal for learning smooth tasks. However, we agree with the Reviewer that there are other biological details our model does not address. For example, our spiking model does not capture some of the properties the Saarinen et al. (2008) model captures, including random sub-threshold oscillations and clusters of spikes. Modeling biophysical phenomena at this scale is beyond the scope of our study. We have added this reference to the relevant section of the Discussion:

"We also note that coding level is most easily defined when neurons are modeled as rate, rather than spiking units. To investigate the consistency of our results under a spiking code, we implemented a model in which granule cell spiking exhibits Poisson variability and quantify coding level as the fraction of neurons that have nonzero spike counts (Figure 7-figure supplement 1; Figure 7C). In general, increased spike count leads to improved performance as noise associated with spiking variability is reduced. Granule cells have been shown to exhibit reliable burst responses to mossy fiber stimulation (Chadderton et al., 2004), motivating models using deterministic responses or sub-Poisson spiking variability. However, further work is needed to quantitatively compare variability in model and experiment and to account for more complex biophysical properties of granule cells (Saarinen et al., 2008)."

A second concern the Reviewer raises is our implementation of Golgi cell inhibition as a homogeneous rather than heterogeneous input onto granule cells. In simplified models, adding heterogeneous inhibition does not dramatically change the qualitative properties of the expansion layer representation, in particular the dimensionality of the representation (Billings et al., 2014, Cayco-Gajic et al., 2017, Litwin-Kumar et al., 2017). We have added a section about inhibition to our Discussion:

"We also have not explicitly modeled inhibitory input provided by Golgi cells, instead assuming such input can be modeled as a change in effective threshold, as in previous studies (Billings et al., 2014; Cayco-Gajic et al., 2017; Litwin-Kumar et al., 2017). This is appropriate when considering the dimension of the granule cell representation (Litwin-Kumar et al., 2017), but more work is needed to extend our model to the case of heterogeneous inhibition."

Regarding the mossy fiber inputs, as we state in response to paragraph 3, we agree with the Reviewer that the random and uncorrelated mossy fiber activity that has been used in previous studies is an unrealistic idealization of in vivo neural activity. One of the motivations for our model was to relax this assumption and examine the consequences: we introduce correlations in the mossy fiber activity by projecting low-dimensional patterns into the mossy fiber layer (Figure 1B):

"A typical assumption in computational theories of the cerebellar cortex is that inputs are randomly distributed in a high-dimensional space (Marr, 1969; Albus, 1971; Brunel et al., 2004; Babadi and Sompolinsky, 2014; Billings et al., 2014; Litwin-Kumar et al., 2017). While this may be a reasonable simplification in some cases, many tasks, including cerebellumdependent tasks, are likely best-described as being encoded by a low-dimensional set of variables. For example, the cerebellum is often hypothesized to learn a forward model for motor control (Wolpert et al., 1998), which uses sensory input and motor efference to predict an effector’s future state. Mossy fiber activity recorded in monkeys correlates with position and velocity during natural movement (van Kan et al., 1993). Sources of motor efference copies include motor cortex, whose population activity lies on a low-dimensional manifold (Wagner et al., 2019; Huang et al., 2013; Churchland et al., 2010; Yu et al., 2009). We begin by modeling the low dimensionality of inputs and later consider more specific tasks.

We therefore assume that the inputs to our model lie on a D-dimensional subspace embedded in the N-dimensional input space, where D is typically much smaller than N (Figure 1B). We refer to this subspace as the “task subspace” (Figure 1C)."

The Reviewer also mentions the learning rule at granule cell to Purkinje cell synapses. We agree that considering online, climbing-fiber-dependent learning is an important generalization. We therefore added a new supplemental figure investigating whether we would still see a difference in optimal coding levels across tasks if online learning were used instead of the least squares solution (Figure 7-figure supplement 2). Indeed, we observed a similar task dependence as we saw in Figure 2F. We have added a new paragraph in the Discussion under Assumptions and Extensions describing our rationale and approach in detail:

"For the Purkinje cells, our model assumes that their responses to granule cell input can be modeled as an optimal linear readout. Our model therefore provides an upper bound to linear readout performance, a standard benchmark for the quality of a neural representation that does not require assumptions on the nature of climbing fiber-mediated plasticity, which is still debated. Electrophysiological studies have argued in favor of a linear approximation (Brunel et al., 2004). To improve the biological applicability of our model, we implemented an online climbing fiber-mediated learning rule and found that optimal coding levels are still task-dependent (Figure 7-figure supplement 2). We also note that although we model several timing-dependent tasks (Figure 7), our learning rule does not exploit temporal information, and we assume that temporal dynamics of granule cell responses are largely inherited from mossy fibers. Integrating temporal information into our model is an interesting direction for future investigation."

Finally, regarding the function of the Purkinje cell, our model defines a learning task as a mapping from inputs to target activity in the Purkinje cell and is thus agnostic to the cell’s downstream effects. We clarify this point when introducing the definition of a learning task:

"In our model, a learning task is defined by a mapping from task variables x to an output f(x), representing a target change in activity of a readout neuron, for example a Purkinje cell. The limited scope of this definition implies our results should not strongly depend on the influence of the readout neuron on downstream circuits."

8) The problem of these, in my impression, generic, arbitrary settings of the neurons and the network in the model becomes obvious here: ‘In contrast to the dense activity in cerebellar granule cells, odor responses in Kenyon cells, the analogs of granule cells in the Drosophila mushroom body, are sparse...’ How can this system be interpreted as an analogy to granule cells in the mammalian cerebellum when the model does not address the specifics lined up above? I.e. the ‘inductive bias’ that the authors speak of, defined as ‘the tendency of a network toward learning particular types of input-output mappings’, would be highly dependent on the specifics of the network model.

We agree with the Reviewer that our model makes several simplifying assumptions for mathematical tractability. However, we note that our study is not the first to draw analogies between cerebellum-like systems, including the mushroom body (Bell et al., 2008; Farris, 2011). All the systems we study feature a sparsely connected, expanded granule-like layer that sends parallel fiber axons onto densely connected downstream neurons known to exhibit powerful synaptic plasticity, thus motivating the key architectural assumptions of our model. We have constrained anatomical parameters of the model using data as available (Table 1). However, we agree with the Reviewer that when making comparisons across species there is always a possibility that differences are due to physiological mechanisms we have not fully understood or captured with a model. As such, we can only present a hypothesis for these differences. We have modified our Discussion section on this topic to clearly state this.

"Our results predict that qualitative differences in the coding levels of cerebellum-like systems, across brain regions or across species, reflect an optimization to distinct tasks (Figure 7). However, it is also possible that differences in coding level arise from other physiological differences between systems."

9) More detailed comments: Abstract: ‘In these models [Marr-Albus], granule cells form a sparse, combinatorial encoding of diverse sensorimotor inputs. Such sparse representations are optimal for learning to discriminate random stimuli.’ Yes, I would agree with the first part, but I contest the second part of this statement. I think what is true for sparse coding is that the learning of random stimuli will be faster, as in a perceptron, but not necessarily better. As the sparsification essentially removes information, it could be argued that the quality of the learning is poorer. So from that perspective, it is not optimal. The authors need to specify from what perspective they consider sparse representations optimal for learning.

This is an important point that we would like to clarify. It is not the case that sparse coding simply speeds up learning. In our study and many related works (Barak et al. 2013; Babadi and Sompolinsky 2014; Litwin-Kumar et al. 2017), learning performance is measured based on the generalization ability of the network – the ability to predict correct labels for previously unseen inputs. As our study and previous studies show, sparse codes are optimal in the sense that they minimize generalization error, independent of any effect on learning speed. To communicate this more effectively, we have added the following sentence to the first paragraph of the Introduction:

"Sparsity affects both learning speed (Cayco-Gajic et al., 2017), and generalization, the ability to predict correct labels for previously unseen inputs (Barak et al., 2013; Babadi and Sompolinsky, 2014; Litwin-Kumar et al., 2017)."

10) Introduction: ‘Indeed, several recent studies have reported dense activity in cerebellar granule cells in response to sensory stimulation or during motor control tasks (Knogler et al., 2017; Wagner et al., 2017; Giovannucci et al., 2017; Badura and De Zeeuw, 2017; Wagner et al., 2019), at odds with classic theories (Marr, 1969; Albus, 1971).’ In fact, this was precisely the issue that was addressed already by Jo¨rntell and Ekerot (2006) Journal of Neuroscience. The conclusion was that these actual recordings of granule cells in vivo provided essentially no support for the assumptions in the Marr-Albus theories.

In our reading, the main finding of J¨orntell and Ekerot (2006) is that individual granule cells are activated by mossy fibers with overlapping receptive fields driven by a single type of somatosensory input. However, there is also evidence of nonlinear mixed selectivity in granule cells in support of the re-coding hypothesis (Huang et al., 2013; Ishikawa et al., 2015). Jo¨rntell and Ekerot (2006) also suggest that the granule cell layer shares similar topographic organization as mossy fibers, organized into microzones. The existence of topographic organization does not invalidate Marr-Albus theories. As we have suggested earlier, a local combinatorial expansion can coexist with a global topographic organization.

We have described these considerations in the Assumptions and Extensions portion of the Discussion:

"Another key assumption concerning the granule cells is that they sample mossy fiber inputs randomly, as is typically assumed in Marr-Albus models (Marr, 1969; Albus, 1971; LitwinKumar et al., 2017; Cayco-Gajic et al., 2017). Other studies instead argue that granule cells sample from mossy fibers with highly similar receptive fields (Garwicz et al., 1998; Brown and Bower, 2001; J¨orntell and Ekerot, 2006) defined by the tuning of mossy fiber and climbing fiber inputs to cerebellar microzones (Apps et al., 2018). This has led to an alternative hypothesis that granule cells serve to relay similarly tuned mossy fiber inputs and enhance their signal-to-noise ratio (Jo¨rntell and Ekerot, 2006; Gilbert and Chris Miall, 2022) rather than to re-encode inputs. Another hypothesis is that granule cells enable Purkinje cells to learn piece-wise linear approximations of nonlinear functions (Spanne and J¨orntell, 2013). However, several recent studies support the existence of heterogeneous connectivity and selectivity of granule cells to multiple distinct inputs at the local scale (Huang et al., 2013; Ishikawa et al., 2015). Furthermore, the deviation of the predicted dimension in models constrained by electron-microscopy data as compared to randomly wired models is modest (Nguyen et al., 2022). Thus, topographically organized connectivity at the macroscopic scale may coexist with disordered connectivity at the local scale, allowing granule cells presynaptic to an individual Purkinje cell to sample heterogeneous combinations of the subset of sensorimotor signals relevant to the tasks that Purkinje cell participates in. Finally, we note that the optimality of dense codes for learning slowly varying tasks in our theory suggests that observations of a lack of mixing (J¨orntell and Ekerot, 2002) for such tasks are compatible with Marr-Albus models, as in this case nonlinear mixing is not required."

We have also included the Jo¨rntell and Ekerot (2006) study as a citation in the Introduction:

"Indeed, several recent studies have reported dense activity in cerebellar granule cells in response to sensory stimulation or during motor control tasks (Jo¨rntell and Ekerot, 2006; Knogler et al., 2017; Wagner et al., 2017; Giovannucci et al., 2017; Badura and De Zeeuw, 2017; Wagner et al., 2019), at odds with classic theories (Marr, 1969; Albus, 1971)."

11) Results: 1st para: There is no information about how the granule cells are modelled.

We agree that this should information should have been more readily available. We now more completely describe the model in the main text. Our model for granule cells can be found in Equation 1 in the Results section and also the Methods (Network Model):

"The activity of neurons in the expansion layer is given by: h = φ(Jeffx − θ), (2)

where φ is a rectified linear activation function φ(u) = max(u,0) applied element-wise. Our results also hold for other threshold-polynomial activation functions. The scalar threshold θ is shared across neurons and controls the coding level, which we denote by f, defined as the average fraction of neurons in the expansion layer that are active."

12) 2nd para: ‘A typical assumption in computational theories of the cerebellar cortex is that inputs are randomly distributed in a high-dimensional space.’ Yes, I agree, and this is in fact in conflict with the known topographical organization in the cerebellar cortex (see broader comment above). Mossy fiber inputs coding for closely related inputs are co-localized in the cerebellar cortex. I think for this model to be of interest from the point of view of the mammalian cerebellar cortex, it would need to pay more attention to this organizational feature.

As we discuss in our response to paragraphs 5 and 6, we see the random distribution assumption at the local scale (inputs presynaptic to a single Purkinje cell) as being compatible with topographic organization occurring at the microzone scale. Furthermore, as discussed earlier, we specifically model low-dimensional input as opposed to the random and high-dimensional inputs typically studied in prior models.

"A typical assumption in computational theories of the cerebellar cortex is that inputs are randomly distributed in a high-dimensional space (Marr, 1969; Albus, 1971; Brunel et al., 2004; Babadi and Sompolinsky, 2014; Billings et al., 2014; Litwin-Kumar et al., 2017). While this may be a reasonable simplification in some cases, many tasks, including cerebellumdependent tasks, are likely best-described as being encoded by a low-dimensional set of variables. For example, the cerebellum is often hypothesized to learn a forward model for motor control (Wolpert et al., 1998), which uses sensory input and motor efference to predict an effector’s future state. Mossy fiber activity recorded in monkeys correlates with position and velocity during natural movement (van Kan et al., 1993). Sources of motor efference copies include motor cortex, whose population activity lies on a low-dimensional manifold (Wagner et al., 2019; Huang et al., 2013; Churchland et al., 2010; Yu et al., 2009). We begin by modeling the low dimensionality of inputs and later consider more specific tasks. We therefore assume that the inputs to our model lie on a D-dimensional subspace embedded in the N-dimensional input space, where D is typically much smaller than N (Figure 1B). We refer to this subspace as the “task subspace” (Figure 1C)."

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Reviewer #2 (Public Review):

Theta-nested gamma oscillations (TNGO) play an important role in hippocampal memory and cognitive processes and are disrupted in pathology. Deep brain stimulation has been shown to affect memory encoding. To investigate the effect of pulsed CA1 neurostimulation on hippocampal TNGO the authors coupled a physiologically realistic model of the hippocampus comprising EC, DG, CA1, and CA3 subfields with an abstract theta oscillator model of the medial septum (MS). Pathology was modeled as weakened theta input from the MS to EC simulating MS neurodegeneration known to occur in Alzheimer's disease. The authors show that if the input from the MS to EC is strong (the healthy state) the model autonomously generates TNGO in all hippocampal subfields while a single neurostimulation pulse has the effect of resetting the TNGO phase. When the MS input strength is weaker the network is quiescent but the authors find that a single CA1 neurostimulation pulse can switch it into the persistent TNGO state, provided the neurostimulation pulse is applied at the peak of the EC theta. If the MS theta oscillator model is supplemented by an additional phase-reset mechanism a single CA1 neurostimulation pulse applied at the trough of EC theta also produces the same effect. If the MS input to EC is weaker still, only a short burst of TNGO is generated by a single neurostimulation pulse. The authors investigate the physiological origin of this burst and find it results from an interplay of CAN and M currents in the CA1 excitatory cells. In this case, the authors find that TNGO can only be rescued by a theta frequency train of CA1 pulses applied at the peak of the EC theta or again at either the peak or trough if the MS oscillator model is supplemented by the phase-reset mechanism.

The main strength of this model is its use of a fairly physiologically detailed model of the hippocampus. The cells are single-compartment models but do include multiple ion channels and are spatially arranged in accordance with the hippocampal structure. This allows the understanding of how ion channels (possibly modifiable by pharmacological agents) interact with system-level oscillations and neurostimulation. The model also includes all the main hippocampal subfields. The other strength is its attention to an important topic, which may be relevant for dementia treatment or prevention, which few modeling studies have addressed.

The work has several weaknesses. First, while investigations of hippocampal neurostimulation are important there are few experimental studies from which one could judge the validity of the model findings. All its findings are therefore predictions. It would be much more convincing to first show the model is able to reproduce some measured empirical neurostimulation effect before proceeding to make predictions. Second, the model is very specific. Or if its behavior is to be considered general it has not been explained why. For example, the model shows bistability between quiescence and TNGO, however what aspect of the model underlies this, be it some particular network structure or particular ion channel, for example, is not addressed. Similarly for the various phase reset behaviors that are found. We may wonder whether a different hippocampal model of TNGO, of which there are many published (for example [1-6]) would show the same effect under neurostimulation. This seems very unlikely and indeed the quiescent state itself shown by this model seems quite artificial. Some indication that particular ion channels, CAN and M are relevant is briefly provided and the work would be much improved by examining this aspect in more detail. In summary, the work would benefit from an intuitive analysis of the basic model ingredients underlying its neurostimulation response properties. Third, while the model is fairly realistic, considerable important factors are not included and in fact, there are much more detailed hippocampal models out there (for example [5,6]). In particular, it includes only excitatory cells and a single type of inhibitory cell. This is particularly important since there are many models and experimental studies where specific cell types, for example, OLM and VIP cells, are strongly implicated in TNGO. Other missing ingredients one may think might have a strong impact on model response to neurostimulation (in particular stimulation trains) include the well-known short-term plasticity between different hippocampal cell types and active dendritic properties. Fourth the MS model seems somewhat unsupported. It is modeled as a set of coupled oscillators that synchronize. However, there is also a phase reset mechanism included. This mechanism is important because it underlies several of the phase reset behaviors shown by the full model. However, it is not derived from experimental phase response curves of septal neurons of which there is no direct measurement. The work would benefit from the use of a more biologically validated MS model.

[1] Hyafil A, Giraud AL, Fontolan L, Gutkin B. Neural cross-frequency coupling: connecting architectures, mechanisms, and functions. Trends in neurosciences. 2015 Nov 1;38(11):725-40.

[2] Tort AB, Rotstein HG, Dugladze T, Gloveli T, Kopell NJ. On the formation of gamma-coherent cell assemblies by oriens lacunosum-moleculare interneurons in the hippocampus. Proceedings of the National Academy of Sciences. 2007 Aug 14;104(33):13490-5.

[3] Neymotin SA, Lazarewicz MT, Sherif M, Contreras D, Finkel LH, Lytton WW. Ketamine disrupts theta modulation of gamma in a computer model of hippocampus. Journal of Neuroscience. 2011 Aug 10;31(32):11733-43.

[4] Ponzi A, Dura-Bernal S, Migliore M. Theta-gamma phase-amplitude coupling in a hippocampal CA1 microcircuit. PLOS Computational Biology. 2023 Mar 23;19(3):e1010942.

[5] Bezaire MJ, Raikov I, Burk K, Vyas D, Soltesz I. Interneuronal mechanisms of hippocampal theta oscillations in a full-scale model of the rodent CA1 circuit. Elife. 2016 Dec 23;5:e18566.

[6] Chatzikalymniou AP, Gumus M, Skinner FK. Linking minimal and detailed models of CA1 microcircuits reveals how theta rhythms emerge and their frequencies controlled. Hippocampus. 2021 Sep;31(9):982-1002.

This makes me think of how we view everyday religion. We know it's there, and it's a huge part of everyday life for a lot of people while not for others. Socially, both are acceptable depending on which social circle you surround yourself.

That those should be capable to provide an opinion on the subject matter so long they have tools available to aid them. A sound idea to say the least.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Summary

Ge et al. defined the role of Gli1 in M1 macrophage activation and osteoclast differentiation in physiological conditions and inflammatory arthritis. The authors found that Gli1 expression is elevated in human RA synovial tissue relative to that in healthy donor controls. Moreover, the authors showed that the administration of GANT58, a Gli1 inhibitor, ameliorates inflammation and bone erosion in CIA mice. Gli1 expression is suppressed by LPS/IFN-____γ stimulation in Raw264.7 cells while being induced by RANKL stimulation in Raw264.7 cells. However, GANT58 suppressed LPS/IFN-____ɣ -induced expression of inflammatory cytokines and iNOS and osteoclastogenesis. The authors also identified DNMT1 and DNMT3a as downstream effectors of Gli1. Transcriptomic analysis of GANT58 treated Raw264.7 cells identified diminished protein expression of DNMT1 and DNMT3a by GANT58. Gli1 also directly interacts with DNMT1. Intriguingly, DNMT1 overexpression restores the effect of GANT58 on LPS/IFN-____ɣ-mediated activation, while DNMT3a overexpression reverses the effect of GANT58 on RANKL-induced osteoclastogenesis. Since this study defines the role of Gli1 in the function and differentiation of myeloid cells, this is interesting. In addition, GANT58 nearly completely protects mice from arthritis, suggesting a therapeutic potential of Gli1 targeting in RA. However, the details of experiments are not clearly described, and the authors present the mixed data from Raw264.7 cells and BMMs without any explanations.

Reply: Many thanks for your recognition and constructive comments on our research. In this study, used mouse macrophage-like cell line RAW264.7 and primary bone marrow-derived macrophages (BMMs). The RAW264.7 is the most commonly used mouse macrophage cell line in medical research, and it is one of the most commonly used in vitro models for osteoclasts and inflammation research. In addition, compared with cell lines, primary cells have the characteristics of unchanged genetic material and biological characteristics closer to cell physiology in vivo. Therefore, in addition to cell lines, we also extracted primary macrophages from bone marrow for experiments to improve the reliability of this study. According to your comments, we have revised the manuscript, and our point-by-point responses are shown as follows.

Major comments

Comment 1. Figs 1h and i. The author should show the histological score.

Reply: Thanks for the constructive comment. According to your suggestion, we have scored the results of H&E staining histologically and added quantitative results.

Comment 2. Pharmacological inhibitors often show non-specific effects. To complement their findings showing the effect of GANT58 on M1 macrophage activation and osteoclastogenesis, the authors should utilize Gli1-deficient cells that can be obtained by siRNAs-mediated knock down or Gli1 deletion.

Reply: Thanks for the professional and constructive comment. To make the results more reliable, we have synthesized siRNA and supplemented the related experiments to verify the role of GLI1 in M1 macrophage activation and osteoclastogenesis, which showed the same trend as GANT58 intervention. In the revised manuscript, the relevant results were shown in the Response to Reviewer File.

Comment 3. Figure 4d: The authors should measure DNMT1 and DAMT3a RNA expression in LPS/IFN-____ɣ- treated (Fig 2c and d) or RANKL treated Raw264.7 cells.

Reply: Thanks for your constructive comment. According to the suggestion, we have added the RNA expression of DNMT1 and DAMT3a to the revised Figure 4. At the same time, the corresponding contents are also described in the Results part.

__ detailed information of RNA-seq including how many genes are regulated by GANT58 and what is their cutoff (fold induction and FDR). The authors should deposit their RNA seq data in the public databases repository such as GEO.__

Reply: Thanks for the professional and constructive comment. In the revised manuscript, we have made a more detailed analysis of the sequencing results and the detail information of RNA-seq have been added in the supplementary information.

Revised in the manuscript:

2.4. GLI1 regulates the expression of DNMTs in distinct ways during the different fates of macrophages

As a nuclear transcription factor, GLI1 exerts an active effect through nuclear entry. In order to explore the potential downstream regulation mechanism of GLI1, RNA sequencing (RNA-seq) on the macrophages before and after GLI1 intervention was performed then to observe gene expression changes. The seq data showed that more genes were down-regulated (143) than up-regulated (74) in GANT58 treated cells (Fig. S7a, b). Among these differentially altered genes, we revealed through Gene Ontology (GO) analysis that GANT58's intervention in GLI1 affected multiple biological processes including macrophage chemotaxis and macrophage cytokine production (Fig. 4a). What’s more, the results of the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the pathway team enrichment was then performed and we showed the TOP30 enriched pathway. In these pathways, we classified them into cellular processes (red), human diseases (blue) and organismal systems (green) respectively. It showed that these down-regulated genes were involved in the development of human diseases such as rheumatoid arthritis, as well as organismal systems such as osteoclast differentiation (Fig. S8c; ____Fig. 4b). These evidences confirmed our previous results. Specifically, GANT58 reduced some of the osteoclast and inflammation-related genes in the cell resting state.

Comment 5. Figure 5c. The authors should add non-stimulating condition as a control.

__Reply: __Thanks for your constructive comment. We have re-conducted the experiment and added the control group.

Comment 6. Figure 6C: DNMT3a deficiency regulates limited number of genes such as IRF8. The authors should measure IRF8 RNA or protein expression in RANKL-treated cells.

Reply: Thanks for your constructive comment. It is reported that DNMT3a can affect the activity of IRF8 and regulate the formation of osteoclasts. Thus, according to your suggestion, we have added IRF8 gene expression detection in the revised manuscript. As shown below, the gene expression of Irf8 was decreased after being treated by RANKL. However, the expression of Irf8 was reversed by Dnmt3a knock down.

Comment 7. Although the effects of Gli1 on bone metabolism in the literature are inconclusive, Gli1 is expressed on other cell types in bone. Gli1 haplodeficiency in mice decreased bone mass with reduced bone formation and enhanced bone resorption compared to control mice (PMID:25313900). Gli1 is also used as a marker for osteogenic progenitors which are precursors of chondrocytes and osteoblasts (PMID: 29230039). Thus, the beneficial effect of GANT58 on inflammation and bone erosion in CIA mice may result from the effects of GANT58 on multiple cell types other than F4/80+ cells. The authors should include these references in the discussion on pg.9 and expand their discussion.

__Reply: __Thank you for your constructive comments. Indeed. there have been some divergent conclusions about the function of hedgehog and GLI1 in bone metabolism, which suggests that GLI1 may have multiple roles. According to your suggestion, we have expanded the relevant discussion and added related references in the Discussion part.

Discussion:

… … Although we have demonstrated that the inhibition of GLI1 by GANT58 can reduce the inflammatory response and inhibit osteoclast formation and that this mechanism is achieved through the downregulation of DNMTs, these findings also raise new questions. In the previous research report, Gli1 haplodeficiency in mice decreased bone mass with reduced bone formation compared to control mice, which was due to the osteoblasts with weakened function [44]. In this process, the osteogenic differentiation of mesenchymal stem cells also affected the function of osteoclasts. In addition, GLI1 is also used as a marker for osteogenic progenitors which are precursors of chondrocytes and osteoblasts [45]. These studies suggest that the regulation of GLI1 on bone metabolism is complex, and the therapeutic effect of GANT58 on RA may be more than just affecting the inflammatory reaction mediated by macrophages and the bone destruction mediated by osteoclasts. In addition to macrophages and osteoclasts, the functions of synovial fibroblasts and osteoblasts play essential roles in the RA microenvironment. These cells are also closely linked to each other. Synovial fibroblasts OPG and RANKL secreted by osteoblasts are important factors that regulate osteoclasts. Therefore, in a follow-up study, we will extend the study of GLI1 to its regulatory mechanism in osteoblasts.

Reference:

[44] Y. Kitaura, H. Hojo, Y. Komiyama, T. Takato, U.I. Chung, S. Ohba, Gli1 haploinsufficiency leads to decreased bone mass with an uncoupling of bone metabolism in adult mice, PLoS One 9(10) (2014) e109597.

[45] Y. Shi, G. He, W.C. Lee, J.A. McKenzie, M.J. Silva, F. Long, Gli1 identifies osteogenic progenitors for bone formation and fracture repair, Nat Commun 8(1) (2017) 2043.

Minor comments

Comment 1. CIA model: The experiment design of CIA model is not clearly described. The author should specify the time point of GANT58 injection.

__Reply: __Thank you for your comment and we are sorry for the confusion caused by vague method descriptions about animal experiments. We have added the specific design and method description of related experiments in the revised manuscript.

Revised in the manuscript:

Materials and Methods:

… … An emulsion of bovine type II collagen (Chondrex, Redmond, WA, USA) and an equal amount (1:1, v/v) of complete Freund’s adjuvant (Chondrex) was prepared to establish the CIA mouse model. First, 0.1 ml of the emulsion was injected intradermally into the base of the tail on day 0. On day 21, 0.1 mg of bovine type II collagen mixed with incomplete Freund’s adjuvant (Chondrex) was injected. From the 21st day, mice began to receive injection intervention treatment. For vehicle group, mice were injected with the same volume of placebo daily. For treatment groups, mice were injected with GANT58 or 5-AzaC solution daily. All interventions began the day after the second injection of bovine type II collagen. Arthritis score was given every three days from the second immunization. On day 49, all mice were sacrificed (in accordance with the guidelines of the Animal Welfare and Ethics Committee of the Soochow University) for the collection of specimens. … …

Comment 2. Joint inflammation of RA can be caused by many different cells. Abstract needs to be revised.

Reply: Thanks for your constructive comment. According to the suggestion, we have revised relevant descriptions in the abstract.

Abstract:

Rheumatoid arthritis (RA) is characterized by joint synovitis and bone destruction, the etiology of which remains to be explored. Many types of cells are involved in the progress of RA joint inflammation, among which the overactivation of M1 macrophages and osteoclasts has been thought an essential cause of joint inflammation and bone destruction. Glioma-associated oncogene homolog 1 (GLI1) has been revealed to be closely linked to bone metabolism. In this study, GLI1-expression in synovial tissue of RA patients showed to be positively correlated with RA-related scores and was highly expressed in collagen-induced arthritis (CIA) mouse articular macrophage-like cells. The decreased expression and inhibition of nuclear transfer of GLI1 downregulated macrophage M1 polarization and osteoclast activation, the effect of which was achieved by modulation of DNA methyltransferases (DNMTs) via transcriptional regulation and protein interaction ways. By pharmacological inhibition of GLI1, the proportion of proinflammatory macrophages and the number of osteoclasts were significantly reduced, and the joint inflammatory response and bone destruction in CIA mice were alleviated. This study clarified the mechanism of GLI1 in macrophage phenotypic changes and activation of osteoclasts, suggesting potential applications of GLI1 inhibitor in the clinical treatment of RA.

Comment 3. Figure 4g, h: are these experiments done in the resting states?

Reply: Thank you for your comment. This part of the experiments was carried out during the induction of M1 macrophage or induction of osteoclast. In this work, we found that GANT58 can inhibit GLI1 and at the same time reduce the gene expression of DNMT3a but not DNMT1 in the resting state. However, during M1 macrophage and osteoclast induction, GANT58 seemed to be able to inhibit both DNMT1 and DNMT3a protein expression. In view of the discovery that the expression of DNMT1 increased during the polarization of M1 macrophages, while the expression of DNMT3a increased during the activation of osteoclasts, we performed the binding experiment of GLI1 with DNMT1 in the process of LPS/IFN-γ induction, while the binding experiment with DNMT3a in the process of RANKL induction. We have added a detailed description to the revised manuscript.

Reviewer #1 (Significance (Required)):

Strengths: Hedgehog (hh) signaling has been implicated in the differentiation of osteogenic progenitors. Gli1+ mesenchymal progenitors are responsible for both normal bone formation and fracture repair. This study defines a new role of Gli1 in the function and differentiation of myeloid cells. In addition, GANT58 nearly completely protects mice from arthritis, suggesting a therapeutic potential of Gli1 targeting in RA.

Reply: Thank the reviewer for your recognition of our research work.

Limitations: This study mainly uses a pharmacological inhibitor to study the mechanism underlying Gli1's action. In addition, the details of experiments are not clearly described, and the authors present the mixed data from Raw264.7 cells and BMMs without any explanations. Advance: This study provides conceptual advancement for hh signaling research by demonstrating the function of Gli1 in myeloid cells.

Reply: Thank the reviewer for your constructive comments and help us to further improve the manuscript.

Audience: Basic research

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

Summary:

The paper by Ge et al seeks to identify a role for GLI1 in rheumatoid arthritis, as GLI1 is upregulated in the synovium of patients with rheumatoid arthritis. Inhibition of GLI1 by the GANT58 limited inflammation and destructive bone loss in a murine model of arthritis (Collagen Induced Arthritis). Inhibition of GLI1 increased expression of pro-inflammatory cytokines and M1 macrophage differentiation. Inhibition of GLI1 also blocked osteoclast formation. As has been shown in other settings, the function of GLI1 in M1 and osteoclast differentiation was linked to regulation by DNMTs.

Major comments:

Comment 1. There are several main problems with the text. Overall, the authors show an intriguing set of data implicating the use of GANT58 as a means to limit rheumatoid arthritis inflammation and bone destruction. The authors directly link the functions of GANT58 with loss of GLI1 activity by showing that GLI1 protein is reduced or translation to the nucleus blocked. It would be compelling if the authors would leverage a genetic model (either GLI1 knockout, or a CRISPR/siRNA approach) to see if it recapitulates key findings in vitro and in vivo. These data could further their claims that their findings are in fact directly due to GLI1.

Reply: Thanks for the professional and constructive comment. To make the results more reliable, we have synthesized siRNA and supplemented the related experiments to verify the role of GLI1 in M1 macrophage activation and osteoclastogenesis. Related experiments have been updated in the revised manuscript, which are shown in the Response to Reviewer File.

Comment 2. Overall, the paper lacks methodologic clarity that limits thorough interpretation of the data. Multiple experiments are missing from the Materials and Methods, including descriptions of the definition of trabecular bone and its analysis in micro-CT, the means by which cytoplasmic and nuclear fractions were generated, and the timing and dosing of GANT58 in vitro studies. In addition, key details regarding the reagents include the sources of primary antibodies used in the western blots and immunoprecipitation studies. Important methodologies are not well explained, which include the treatment of the Sham animals (presumably healthy) are not explained, that is, whether they receive injections of vehicle or are truly naïve. Finally, there is no statistical methodology, minimal explanation of the RNA-sequencing analyses, and no statement about how the RNA-sequencing data will be made available. This lack of detail makes a thorough assessment of the quality and interpretations of the data challenging and replication of the results impossible.

Reply: Thanks for your careful reading and constructive comments. We are sorry for the lack of some detailed methodological descriptions in the manuscript. In order to better explain how our experiment is carried out and improve the repeatability of the experiment, we have comprehensively improved the description of the experimental method in the revised manuscript.

Materials and Methods:

4.1. Experimental animals and human synovial tissue. Male DBA mice aged 6-8 weeks and weighing 15-20 g were randomly selected and fed in a specific pathogen-free (SPF) environment at a room temperature of 25℃, a relative humidity of 60%, and 12 hours of alternating light. All animal experiments were approved by the Animal Ethics Committee of the Soochow University (201910A354). The animals were divided randomly into groups (6 per group): sham group (healthy mice not received any treatment), vehicle control group (CIA model mice treated with solvent), and GANT58 (GLI1 specific inhibitor; MedChemExpress, New Jersey, USA) group (mice treated with 20 mg/kg GANT58) or 5-AzaC (DNMTs specific inhibitor; MedChemExpress) group (mice treated with 2 mg/kg 5-AzaC). An emulsion of bovine type II collagen (Chondrex, Redmond, WA, USA) and an equal amount (1:1, v/v) of complete Freund’s adjuvant (Chondrex) was prepared to establish the CIA mouse model. First, 0.1 ml of the emulsion was injected intradermally into the base of the tail on day 0. On day 21, 0.1 mg of bovine type II collagen mixed with incomplete Freund’s adjuvant (Chondrex) was injected. For vehicle group, mice were injected with the same volume of placebo daily. For treatment groups, mice were injected with GANT58 or 5-AzaC solution daily. All interventions began the day after the second injection of bovine type II collagen. Arthritis score was given every three days from the second immunization. On day 49, all mice were sacrificed (in accordance with the guidelines of the Animal Welfare and Ethics Committee of the Soochow University) for the collection of specimens. … …

4.3. Micro-CT analysis. The fixed bone samples of mice were collected. The joint samples were placed in a SkyScan 1174 Micro-CT scanning warehouse (Belgium). The parameters were set as follows: voltage 50 kV, current 800 μA, scanning range 2 cm × 2 cm, and scanning layer thickness 8 μm. The scan data were then entered into computer to conduct three-dimensional reconstruction with NRecon software (Bruker, Germany), and the bone tissue parameters were analysed with CTAn software (Bruker, Germany) after data conversion. During this procedure, we performed an analysis of bone parameters including BMD (Bone Mineral Density), BV/TV (Percentage Trabecular Area), Tb.N (Trabecular Number) and Tb.Sp (Trabecular Separation) by selecting the small joint of paws as the region of interest (ROI) in CTAn software. The three-dimensional reconstruction images were exhibited by Mimics Research software (Version 21.0; Materialise, Belgium).

4.11. Western blotting. Cells were seeded in 6-well plates at a density of 1 × 106/well with stimulation with RANKL (50 ng/ml) or LPS (100 ng/ml) + IFN-γ (20 ng/ml). First, cells were collected to extract total protein, and the BCA (Beyotime) method was used to adjust the protein concentration. Total protein was mixed with 5× loading buffer (Beyotime) and boiled at 95 °C for 10 minutes. For cytoplasmic/nucleus isolation, cells were collected and protein was extracted according to the instructions using the nuclear protein and cytoplasmic protein extraction kit (Beyotime). The proteins were separated by SDS polyacrylamide gel electrophoresis (SDS–PAGE; EpiZyme, Shanghai, China) based on their different molecular weights. Electrophoresis was performed using Bio–Rad (California, USA) equipment at 180 V for 40 minutes. Then, the proteins were transferred to a nitrocellulose membrane at 350 mA for 70 minutes using membrane transfer equipment (Bio–Rad). The membrane was removed and placed into western blot blocking buffer for 1 hour at room temperature. The diluted primary antibodies (GLI1, Abclonal, A14675; β-actin, Beyotime, AF5003; Lamin-B1, Abcam, ab16048; NFATc1, Abclonal, A1539; CTSK, Abclonal, A5871; MMP9, Abclonal, A11147; DNMT1, Abclonal, A16729; DNMT3a, Cell Signaling Technology, D23G1; GAPDH, Abclonal, A19056) were placed on the membrane and incubated at 4 ℃ for 12 hours, and then the corresponding secondary antibody was added and incubated for 1 hour at room temperature. Finally, a chemiluminescence detection system (Bio–Rad) was used to observe the results.

4.12. High-throughput sequencing (RNA-seq). To further screen for differential genes, we first subjected RAW264.7 cells to a 24-hour adaptive culture, followed by the addition of GANT58 at a final concentration of 10 μM to the GANT58 intervention group and cultured for a total of 24 h. After the cell treatment was completed, cells of the control group and GANT58 treated group were collected respectively, and RNA-seq detection and analysis were entrusted to a professional biological company (Azenta Life Sciences, Suzhou, China). Briefly, for differential expression gene analysis, the differential expression conditions were set as fold change (FC) > 1.5 and false discovery rate (FDR) 4.14. Statistical analysis. All data are presented as the mean ± standard deviation (SD). Statistical analysis was performed with an unpaired two-tailed Student’s t test for single comparisons with GraphPad Prism 8 (GraphPad Software, CA, USA). One-way analysis of variance (ANOVA) was used to compare data from more than two groups. p values less than 0.05 were considered statistically significant.

The specific statistical methods are marked in Figure legends as well.

Data Availability: The authors declare that all data supporting the findings of this study are available within this paper and its Supplementary Information and raw data are available on request from the corresponding author.

Comment 3. The authors should expand their introduction and Discussion to include a description of the history of other GLI inhibitors (such as GANT61) in rheumatoid arthritis. Further, the authors failed to cite current studies showing that GLI1 is upregulated in RA patients (DOI: 10.1007/s10753-015-0273-3 amongst others).

Reply: Many thanks to your thoughtful reading and constructive comment. According to your suggestion, we have added some revisions, including the description of GLI1 inhibitors, in the introduction and discussion sections. At the same time, we have also added descriptions and citations of GLI1 and RA-related research in corresponding positions.

Introduction:

… … To date, three mammalian GLI proteins have been identified, among which GLI1 usually acts as a transcriptional activator. On the basis of these studies, small molecular compounds such as GANT58 (selective inhibitor of GLI1) and GANT61 (inhibitor of GLI1 and GLI2) are often used as pharmacological interventions of GLI1, so as to achieve the purpose of inhibiting GLI1 activity and regulating the molecular biological process [13, 14]. Many of the physiopathological processes involved with GLIs are complex and worth discussing. Relevant studies have shown that GLI1-activated transcription promotes the development of inflammatory diseases such as gastritis, and antagonizing GLI1 transcription can alleviate the inflammatory degradation of articular cartilage [15, 16]. … …

Discussion:

… … In previous studies, GLI1 signal transduction and other pathways, including the NF-κB signaling pathway, were usually studied in tumor-associated diseases and are considered a response network that promotes cancer development [21, 22]. Qin. et al. found that the content of SHH in RA patients serum increased significantly by comparing with healthy patients [23]. At the same time, our study also showed that GLI1 was more expressed in the joint tissue of RA patients. These results suggest that HH-GLI signaling pathway may be involved in the regulation of the pathological process of RA. However, the research results of the hedgehog pathway in bone metabolism are complex. … …

Reference:

[13] X. Chen, C. Shi, H. Cao, L. Chen, J. Hou, Z. Xiang, K. Hu, X. Han, The hedgehog and Wnt/beta-catenin system machinery mediate myofibroblast differentiation of LR-MSCs in pulmonary fibrogenesis, Cell Death Dis 9(6) (2018) 639.

[14] R.K. Schneider, A. Mullally, A. Dugourd, F. Peisker, R. Hoogenboezem, P.M.H. Van Strien, E.M. Bindels, D. Heckl, G. Busche, D. Fleck, G. Muller-Newen, J. Wongboonsin, M. Ventura Ferreira, V.G. Puelles, J. Saez-Rodriguez, B.L. Ebert, B.D. Humphreys, R. Kramann, Gli1(+) Mesenchymal Stromal Cells Are a Key Driver of Bone Marrow Fibrosis and an Important Cellular Therapeutic Target, Cell Stem Cell 23(2) (2018) 308-309.

[23] S. Qin, D. Sun, H. Li, X. Li, W. Pan, C. Yan, R. Tang, X. Liu, The Effect of SHH-Gli Signaling Pathway on the Synovial Fibroblast Proliferation in Rheumatoid Arthritis, Inflammation 39(2) (2016) 503-12.

Comment 4. The antibody for GLI1 seems poor and inconsistent. Knockdown studies to show its specificity, and an example of the whole membrane stained for GLI1 would provide important validation of the reagent.

Reply: Thanks for your comment and we are sorry for showing the western blot results with poor quality. In the revised manuscript, we used the newly purchased antibody (Abclonal, Catalog: A14675) and rearranged the groupings for better comparison of protein expression and replaced the results with clearer blot images. Original images of all western blot results can be uploaded subsequently.

Comment 5. Regarding Figure S1:

The studies of RA patients are underpowered. With only three RA patients and three healthy synovial the distribution of DAS28 scores is clustered at healthy and active disease, and the correlation study is unconvincing.

Reply: Thanks for your constructive comment. We are sorry that the studies of RA patients might not be convincing enough due to the small sample size. In order to avoid controversial conclusions, we left out the results of correlation analysis between GLI1 expression and DAS28. In the follow-up study, we will collect additional clinical pathology data for statistical analysis and quantified the expression of GLI1 in healthy control patients and RA patients.

Comment 6. Regarding Figure 1 f-g and Figure 4j-k:

However, the information on inflammatory bone loss are incomplete. The methodology for the assessment of BMD and trabecular bone parameters in the hind paw is not explained. The 3D reconstructions are of the whole bone hind paw, but the anatomical region where trabecular bone is assayed not defined. It would be convincing if the authors added erosion scores in the hind paws or knees to show that the erosion in the synovium, which contributes to inflammatory arthritis, mirrors what occurs in the trabeculae.

Reply: Thanks for your constructive comment. We are sorry for incomplete description on in vivo experiments, including the micro-CT analysis and histological analysis. In the revised manuscript, we further supplemented and improved the relevant methods. The Inflammatory cell infiltration score and bone erosion score were also added according to your suggestion.

Materials and Methods:

4.3. Micro-CT analysis. The fixed bone samples of mice were collected. The joint samples were placed in a SkyScan 1174 Micro-CT scanning warehouse (Belgium). The parameters were set as follows: voltage 50 kV, current 800 μA, scanning range 2 cm × 2 cm, and scanning layer thickness 8 μm. The scan data were then entered into computer to conduct three-dimensional reconstruction with NRecon software (Bruker, Germany), and the bone tissue parameters were analysed with CTAn software (Bruker, Germany) after data conversion. During this procedure, we performed an analysis of bone parameters including BMD (Bone Mineral Density), BV/TV (Percentage Trabecular Area), Tb.N (Trabecular Number) and Tb.Sp (Trabecular Separation) by selecting the small joint of paws as the region of interest (ROI, bone tissue from ankle joint to toe) in CTAn software. The three-dimensional reconstruction images were exhibited by Mimics Research software (Version 21.0; Materialise, Belgium).

Comment 7. Regarding Figure 2:

-The methods and text do not state the dose of GANT58 used in these assays. Nor do they specify the timing of the GANT58 application in relationship to LPS and IFNg stimulation.

Reply: Thanks for your thoughtful reading and constructive comment. We apologize for not expressing the detailed dose and intervention time of GANT58 in some experiments in detail. In the revised manuscript, we have added drug dose and intervention time cutoff points in the parts of Methods, Results, and Figure Legends.

-The authors conclude that GLI1 limits the differentiation of M1 macrophages and also directly blocks the production of pro-inflammatory cytokines. The data are difficult to parse in that the directionality is not clear. If GLI1 promotes M1 macrophages, there would be less proinflammatory cytokines due to the reduction of their proliferation. To evaluate the role of GLI1 in regulating the cytokines, additional studies showing a transcriptional regulation of these cytokines is warranted.

Reply: Thank you for your professional and constructive comment. We totally agree with you that the release of inflammatory cytokines is affected not only by gene expression but also by the number of cells that proliferate. Therefore, to exclude this interference, we further examined transcriptional expression of cytokines responsible for cellular inflammation under the same conditions. The results shown in the Response to Reviewer File confirmed the inhibition of GANT58 on the expression of pro-inflammatory cytokine mRNAs, which further supported our conclusion.

-To show that the fractionation of the cytoplasm and nuclear compartments was complete, the westerns for GLI1, lamin-B1 and beta actin should be shown in the same blot.

Reply: Thank you for your professional and constructive comment. According to your suggestion, we have rearranged the groupings to show the westerns for GLI1, lamin-B1 and β-actin in the same blot for better comparison in the revised manuscript.

-In Section 2.3 ("the expression of and intranuclear transport..."), the authors state that their previous studies showed GLI was expressed in macrophages (line 80-81). It is unclear whether the authors are referring to studies in this manuscript or a previously published study and a citation is needed.

Reply: Thank you for your careful reading and helpful comment. We are sorry that the description in this part is confusing. In fact, what we want to refer to is the in vivo results described in the first section of the results part. We have changed this description in the revised manuscript.

2.3. The expression and intranuclear transport of GLI1 is involved in osteoclast activation

The over activation of osteoclast is the direct cause of bone destruction in RA. As described of the in vivo experimental results in the first part, we have found that GLI1 is highly expressed in macrophage-like cells in the subchondral bone of the joints, which raised our concerns about GLI1 and osteoclasts. … …

In response to Figure 3:

-The authors show that GANT58 has a potent impact in limiting osteoclast formation. The text states that GANT58 is a pretreatment, but the timing of this is not stated.

Reply: Thanks for your constructive comment. In order to reach the working concentration of drugs at the beginning of some experiments, we usually pretreated cells for 6-8 hours. We have added the specific time in the parts of Materials and Methods or Figure legends.

-It would be interesting to see whether there is a dose-response effect of GANT58.

Reply: Thanks for your comment. According to your comment, we set the concentration of GANT58 to 0, 1, 5 and 10 μM to intervene the induction of M1 macrophages and osteoclasts respectively. As shown in the Response to Reviewer File, with the increase of GANT58 concentration, the mean fluorescence intensity of iNOS in macrophages seems to decrease gradually, but there is no statistical significance when the concentration is below 5 μM. Similarly, when the concentration reached 10 μM, GANT58 significantly inhibited the formation of osteoclasts.

-It is not stated how long the cells are RANKL treated prior to nuclear/cytoplasmic fractionation? (3a, b, c and i).

Reply: Thanks for your constructive comment. For osteoclast induction and intervention, we treated cells for 48 h as cell transcription regulation usually occurs in the early and middle stages of osteoclast differentiation. According to your comment, we have added the description of specific intervention time information in Figure legends and other parts.

-The "Zoom" images in Figure 3j do not have a box to delineate where the higher magnification images are taken from in the top panes. The images appear to be from serial sections. This should be clarified.

Reply: Thanks for your constructive comment. In the revised Figure, we have boxed the area represented by the Zoom images. We can ensure that these images come from different groups of specimen slices. In order to better observe the number of osteoclasts, we chose a larger shooting multiple, which might make the pictures look similar. The revised images are shown in the Figure 3n, o in the Response to Reviewer File.

In Figure 3 and Figure 6e and 6f:

Although the data in BMM showed that there was no impact on cell survival was limited at low concentrations, showing that the differentiating osteoclasts are not more sensitive to apoptosis by GANT58 would be compelling. The large difference in cellularity in the presence of GANT58 provokes this question.

Reply: Thank you for your careful reading and helpful comment. As shown of the CCK8 result, GANT58 had no significant inhibitory effect neither on BMMs nor RAW264.7 cells until the concentration reached 40 μM. In the process of changing the polarization phenotype of macrophages, the cell morphology will also change to some extent. In our research results, the change of cell morphology after GANT58 intervention might be due to the inhibition of M1 macrophages. In order to observe the effect of GANT58 on BMM cell death and apoptosis, we further performed living/dead staining and apoptosis detection by fluorescence after GANT58 intervention. The results showed that GANT58 did not change the level of apoptosis nor increase the number of dead cells at the concentration of 10 μM. However, when the concentration increased to 30μM, the number of apoptotic cells increased. These results suggest that we should pay strict attention to the control of drug concentration in experimental intervention and transformation application. The supplementary results are shown in the Response to Reviewer File.

In Figure 4:

-The IP studies (4g and 4h) lack showing successful pull-down of GLI1 by western blotting as a critical control for the study.

Reply: Thanks for your constructive comment. During the performance of CO-IP experiment, we simultaneously detected the expression of GLI1 to verify the effectiveness of the antibodies used. In the revised Figure 4g and h, we have updated the corresponding results.

Revised Figure 4:

-Details about the steps involved in RNA-sequencing analyses need to be provided.

__Reply: __Thanks for your constructive comment. According to your suggestion, we have provided the steps involved in RNA-sequencing analyses in the Methods.

4.12. High-throughput sequencing (RNA-seq). To further screen for differential genes, we first subjected RAW264.7 cells to a 24-hour adaptive culture, followed by the addition of GANT58 at a final concentration of 10 μM to the GANT58 intervention group and cultured for a total of 24 h. After the cell treatment was completed, cells of the control group and GANT58 treated group were collected respectively, and RNA-seq detection and analysis were entrusted to a professional biological company (Azenta Life Sciences, Suzhou, China). Briefly, for differential expression gene analysis, the differential expression conditions were set as fold change (FC) > 1.5 and false discovery rate (FDR)

-Studies have previously shown a reduction of inflammatory arthritis by 5'-Azac and should be cited.

__Reply: __Thank you for your careful reading and helpful comment. In the discussion part of the revised manuscript, we have cited the related articles, which is shown as below.

Discussion:

… … In addition to normal physiological development, the abnormal expression of DNMTs causes the development of tumors and other diseases [35]. Through the treatment of DNMTs inhibitors, the inflammatory arthritis in mice was significantly relieved, which was consistent with the previous studies [36]. These results suggested that DNMTs might be involved in the inflammatory reaction and bone destruction of RA. Reports have suggested that the absence of DNMT3a inhibits the formation of osteoclasts, which may be due to the methylation of downstream IRF8 by DNMT3a [37]. In our study, we also verified this finding through pharmacological and genetic intervention. … …

Reference:

[36] D.M. Toth, T. Ocsko, A. Balog, A. Markovics, K. Mikecz, L. Kovacs, M. Jolly, A.A. Bukiej, A.D. Ruthberg, A. Vida, J.A. Block, T.T. Glant, T.A. Rauch, Amelioration of Autoimmune Arthritis in Mice Treated With the DNA Methyltransferase Inhibitor 5'-Azacytidine, Arthritis Rheumatol 71(8) (2019) 1265-1275.

-What is the proposed functional consequence for GLI1 binding to DNMT3a? Does GLI1 inhibition lead to hypomethylation of DNA by DNMT?

Reply: Many thanks for your constructive comment. In this study, it is interesting to find that GLI1 can affect the expression of Dnmt3a at the level of gene transcription, and affect the expression of DNMT3a and DNMT1 both in the process of protein expression. Through the CO-IP experiment, we confirmed that GLI1 protein can bind to DNMT1 instead of DNMT3a protein. These results suggested that GLI1 may regulate the expression of DNMT3a and DNMT1 at genetic level and post-translation proteinic level, respectively. Patricia Gonz á lez Rodr í Guez's latest research showed that during autophagy induction, GLI1 is upregulated, phosphorylated, translocated to the nucleus and recruited to the regions closer to the Transcription Start Site (TSS) of the Dnmt3a gene. This may be the direct mechanism of GLI1 regulating the expression of DNMT3a [1]. Theoretically, the expression of DNMTs affects the degree of methylation of related genes [2]. Thus, in the follow-up study, we will further verify the degree of genomic methylation caused by GLI1's regulation of DNMTs, and further explore more possible ways of GLI1's regulation of DNMTs and its potential role in other cell models.

Reference:

[1] P. Gonzalez-Rodriguez, M. Cheray, L. Keane, P. Engskog-Vlachos, B. Joseph, ULK3-dependent activation of GLI1 promotes DNMT3A expression upon autophagy induction, Autophagy (2022) 1-12.

[2] Dura M, Teissandier A, Armand M, Barau J, Lapoujade C, Fouchet P, Bonneville L, Schulz M, Weber M, Baudrin LG, Lameiras S, Bourc'his D. DNMT3A-dependent DNA methylation is required for spermatogonial stem cells to commit to spermatogenesis, Nat Genet 54(4) (2022) 469-480.

Figure 5:

-The groups in 5g are not well defined.

Reply: Thank you for your careful reading and comment. We're sorry that we didn’t clearly show the grouping information. In the revised Figure 5g, we have added the complete information of the groups.

-DNMT1 and DNMT3a reduction by siRNA, CRISPR or knockout would strengthen the inhibitor studies.

Reply: Thanks for your constructive comment. In the revised manuscript, we knocked down the expression of DNMT1 and DNMT3a by siRNA, and supplemented the related experimental results, which are shown in the Response to Reviewer File.

Regarding Figure 5 and 6:

-What is the impact of DNMT1 and DNMT3a overexpression on their own (not in the presence of GANT58)?

Reply: Thanks for your constructive comment. According to your comment, we observed and compared the differences in the polarization of macrophages M1 and the activation of osteoclasts between the DNMTs overexpression group and the control group. The results showed that overexpression of DNMT1 seemed to have no obvious effect on the formation of M1 macrophages. During the osteoclast activation, at day 4 of RANKL induction, the TRAP positive stained osteoclast number seemed to be no significance between WT group and Dnmt3aOE group. However, at day 3, there was more osteoclast in Dnmt3aOE group, which suggested that overexpression of Dnmt3a might accelerate the activation of osteoclasts to some extent. The results are shown in the Response to Reviewer File.

Minor comments:

Comment 1. The authors do not include a description of DNMTs in the introduction.

Reply: Thanks for your constructive comment. According to your suggestion, we have added a description of DNMTs in the Introduction.

Introduction:

DNA methylation is an important epigenetic marker playing an important role in regulating gene expression, maintaining chromatin structure, gene imprinting, X chromosome inactivation and embryo development an important epigenetic modification way to regulate gene expression, which is activated by DNA methyltransferases (DNMTs) [17]. As reported, DNMT1 and DNMT3a are involved in the progress of many physiological disorders, such as immune response and cell differentiation [18, 19]. In this study, … …

Reference:

[17] E. Li, Y. Zhang, DNA methylation in mammals, Cold Spring Harb Perspect Biol 6(5) (2014) a019133.

[18] Y. Fu, X. Zhang, X. Liu, P. Wang, W. Chu, W. Zhao, Y. Wang, G. Zhou, Y. Yu, H. Zhang, The DNMT1-PAS1-PH20 axis drives breast cancer growth and metastasis, Signal Transduct Target Ther 7(1) (2022) 81.

[19] R. Ramabadran, J.H. Wang, J.M. Reyes, A.G. Guzman, S. Gupta, C. Rosas, L. Brunetti, M.C. Gundry, A. Tovy, H. Long, T. Gu, S.M. Cullen, S. Tyagi, D. Rux, J.J. Kim, S.M. Kornblau, M. Kyba, F. Stossi, R.E. Rau, K. Takahashi, T.F. Westbrook, M.A. Goodell, DNMT3A-coordinated splicing governs the stem state switch towards differentiation in embryonic and haematopoietic stem cells, Nat Cell Biol 25(4) (2023) 528-539.

Comment 2. The descriptions of the groups are often unclear. In Figure 2, the label "GANT58" (blue bars) is presumably for a group that is treated for LPS+IFNg+GANT58 but this is not clarified.

Reply: Thanks for your careful reading and we are sorry for the ambiguous labeling. We have checked the whole manuscript and changed the related labeling information.

Comment 3. The distinction of Figure 3g as multinuclear giant cells (vs TRAP+ OCs in panel 3d) should be explained.

Reply: Thanks for your comment. Osteoclast is defined as a multinucleated giant cell with bone absorption function, which is composed of multiple monocytes/macrophages [1]. As osteoclasts mature, their cytoskeleton will undergo drastic reorganization. Filamentous actin (F-actin) firstly constitutes a podosomes with a highly dynamic structure, thereby completing the cell adhesion, migration, dissolution of bone minerals and digestion of organic matrix [2]. Therefore, in addition to observing the formation of osteoclasts by TRAP staining, we also carried out immunofluorescence staining to observe the F-actin ring formation to further evaluate the functional maturity of osteoclasts. Osteoclasts usually have 2-50 nuclei, so we mainly regarded multinucleated giant cells with complete F-actin rings as mature osteoclasts during the quantification process.

Reference:

[1] da Costa CE, Annels NE, Faaij CM, Forsyth RG, Hogendoorn PC, Egeler RM, Presence of osteoclast-like multinucleated giant cells in the bone and nonostotic lesions of Langerhans cell histiocytosis. J Exp Med 7;201(5) (2005) 687-93.

[2] Portes M, Mangeat T, Escallier N, Dufrancais O, Raynaud-Messina B, Thibault C, Maridonneau-Parini I, Vérollet C, Poincloux R, Nanoscale architecture and coordination of actin cores within the sealing zone of human osteoclasts, Elife (11) (2022) e75610.

Comment 4. The labels in 4C of "R1, R2, R3" standing for GANT58 is confusing

__Reply: __We are sorry for the confusing labeling. In the revised manuscript, we have added specific grouping information in the Figure legend, as shown below.

*Figure 4. DNA methyltransferases might be a regulatory target downstream of GLI1. a Biological process GO analysis of RNA-seq results for macrophages with or without GANT58 treatment. b KEGG rich analysis of RNA-seq results. c Heat map of parts of the relevant gene transcriptional expressions (C = control group; R = GANT58 treated group; red: increased expression; blue: decreased expression). d Relative mRNA expression of Gli1, Dnmt1 and Dnmt3a in macrophages with or without GANT58 treatment. Statistical analysis was performed using two-way ANOVA test. e RAW264.7 cells were stimulated by LPS and IFN-γ for 24 h, with or without GANT58 co-intervention. Western blot results of DNMT1 and DNMT3a protein expression and grayscale value ratio to β-actin of western blot results. n = 3. f RAW264.7 cells were stimulated by RANKL for 3 days, with or without GANT58 co-intervention. Western blot results of DNMT1 and DNMT3a protein expression and grayscale value ratio to β-actin of western blot results. n = 3. Statistical analysis was performed using two-way ANOVA test. g, h Co-IP detection of protein binding between GLI1 and DNMT1/DNMT3a. n = 3. i Protein–protein interface interaction of GLI1 and DNMT1 with PyMOL. j Micro-CT scanning and 3D reconstruction of mouse paws. k Bone parameters of BV/TV, BMD, Tb.N, Tb.Th. n = 6. Statistical analysis was performed using one-way ANOVA test. Data shown represent the mean ± SD. *p

Comment 5. In Figure S8, the numbers between the western blots are not explained.

__Reply: __Many thanks for your careful reading and comment. The numbers between the blots represent the ratio of the gray value of DNMT1 and DNMT3a immunoblot to the gray value of β-actin immunoblot, so as to reflect the relative expression of proteins. In order to avoid confusion, we made a statistical chart of the results and added it to revised Figure S8.

Comment 6. In Figure S9 there are references to asterisks which do not appear in the figure.

__Reply: __We are sorry for the mistake. We have deleted the relevant information in the revised Supplementary information. Thanks again.

Reviewer #2 (Significance (Required)):

The paper presented by Ge et al present interesting data suggesting that a GLI1 inhibitor (GANT58) has a strong impact on inflammatory arthritis in a murine model. Interesting data are presented whose novelty need better contextualization with other published studies, as previously published studies which are not cited in this manuscript include the finding that GLI1 is upregulated in patients with rheumatoid arthritis, that other GLI inhibitors have been utilized in murine models of rheumatoid arthritis, and that GLI1 has been shown to regulate DNMT expression in cancer settings. The authors connect GLI1 inhibition with DNMT activation in limiting M1 macrophage and osteoclast differentiation. However, several important controls are needed to in the in vitro studies as outlined above.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary

The manuscript by Ge et al. describes the possible roles of GLI1 in macrophage and osteoclast activation in rheumatoid arthritis via its functional interaction with DNA methyltransferases. The authors found that the GLI1 expression was elevated in RA synovial tissues and GLI1-specific inhibitor, GANT58, ameliorated arthritis in CIA mice. GLI1 expression in F4/80-positive macrophages in CIA synovial tissues led the authors to assess the roles of GLI1 in macrophages and osteoclasts. GANT58 suppressed M1 macrophage polarization by IFNg+LPS and osteoclastogenesis by RANKL. RNA-seq analysis of GANT58-treated macrophages revealed that DNA methyltransferases, DNMT1 and DNMT3a were possible targets of GLI1, and the studies with small inhibitors or overexpression of DNMTs suggest that GLI1 enhanced M1 polarization and osteoclastogenesis through DNMTs. The manuscript is well-written, the methods are accurate, and the results and data interpretation are consistent and clearly presented. This work deserves publication in Research Commons after addressing the following questions:

Major comments

Comment 1. GANT58 may inhibit GLI2 in addition to GLI1 and have off-target effects. Major findings with GANT58 in vitro, the suppressive effects on M1 polarization, osteoclastogenesis, and DNMT3a expression should be assessed with siRNA/shRNA knockdown or CRISPR/Cas9 knockout of GLI1.

Reply: Many thanks for your careful reading and constructive comment. According to your comment, we have constructed Gli1 knock-down cells and carried out related experiments. The results have been added in the revised manuscript, which are shown in the Response to Reviewer File.

Comment 2. In CIA with GANT58, the author performed only preventive treatment, not therapeutic treatment. Does GANT58 suppress adaptive immune responses via inhibiting APC function (ex. anti-CII IgG production)? Alternatively, the inhibitory effects of GANT58 on the effecter phase of RA (M1 macrophage and osteoclast activation) can be assessed using the serum-transfer arthritis models.

__Reply: __Many thanks for your constructive comments. Your question is indeed a direction worthy of attention. In our study, GANT58 was given during the stage of model establishment, showing a good effect of relieving arthritis, which was proved to come from the direct inhibition of inflammatory phenotype macrophages and osteoclasts. However, as autoimmune diseases, the enhancement of antigen presenting function and anti-Col II IgG production can enhance the immune response of the body [1]. The regulatory effect of GANT58 on macrophages suggests that it may have a potential impact on APC function. Despite this, whether GANT58 can regulate the pathological process of RA by influencing this pathway is inconclusive. Therefore, according to your suggestion, we will improve the relevant experiments in our follow-up research, and apply GANT58 to various animal models of RA to further explore the possible mechanism of GANT58 in the treatment of RA and provide more reliable theoretical support for its transformation and application.

Reference:

[1] Tsark EC, Wang W, Teng YC, Arkfeld D, Dodge GR, Kovats S, Differential MHC class II-mediated presentation of rheumatoid arthritis autoantigens by human dendritic cells and macrophages, J Immunol 1;169(11) (2002) 6625-33.

Minor comments

Comment 1. GANT58 is a water insoluble agent. Can you please include how to dissolve GANT58, administration route, and rationale of 20 mg/kg, for CIA?

__Reply: __Thank you for your professional comment. In this work, GANT58 was ordered from MedChemExpress (MCE; Cat. No.: HY-13282) Company. According to the instructions for use, we prepared 20 mg/ml ethanol solution of GANT58 into 2 mg/ml working solution for injection in vivo according to the following ratio: 10% EtOH + 90% (20% SBE-β-CD in PBS); Clear solution; Need ultrasonic. During the experiment, GANT58 was injected i.p. at a dose of 20 mg/kg daily for 28 days. With regard to the choice of drug injection concentration, according to the previous literature, most studies used a dose of 50 mg/kg for daily injection [1, 2]. Hereby, we set up concentration gradient intervention (0, 10, 20, and 50 mg/kg) in the preliminary experiment and found that 20 and 50 both had good therapeutic effects. Therefore, according to the consideration of economy and safety, we chose 20 mg/kg as our final intervention concentration.

Reference:

[1] Li G, Deng Y, Li K, Liu Y, Wang L, Wu Z, Chen C, Zhang K, Yu B, Hedgehog Signalling Contributes to Trauma-Induced Tendon Heterotopic Ossification and Regulates Osteogenesis through Antioxidant Pathway in Tendon-Derived Stem Cells, Antioxidants (Basel) 16;11(11) (2022) 2265.

[2] Lauth M, Bergström A, Shimokawa T, Toftgård R, Inhibition of GLI-mediated transcription and tumor cell growth by small-molecule antagonists. Proc Natl Acad Sci U S A. 15;104(20) (2007) 8455-60.

Comment 2. Zoom photos in Fig 1j are not clear. Is GLI1 exclusively expressed in F4/80+ macrophages in synovial tissues?

__Reply: __Many Thanks for your comment. In the revised manuscript, we have improved the resolution of the image for better observation. According to the results, although GLI1 is more expressed in F4/80 positive cells, not all GLI1 proteins are expressed in macrophages, and we can find that some GLI1 positive staining is expressed in other cells. In the follow-up study, we will continue to explore this phenomenon and study the relationship between GLI1 and cells like synovial fibroblasts in RA.

Comment 3. In Fig 2 and 3, the treatment of macrophages with IFNg+LPS and RANKL enhanced the nuclear translocation of GLI1, suggesting that these stimuli may activate hedgehog signals. Recent studies, however, suggest various non-canonical activation pathways of GLI1. Does hedgehog inhibitor (ex. SMO inhibitor) also suppress M1 polarization and osteoclastogenesis?

__Reply: __Thank you for your constructive comment. We agree with that the activation of GLI1 is regulated by many various pathways. According to your comment, we additionally used Cyclopamine, a selective inhibitor of SMO, to intervene during the polarization of M1 macrophages and the activation of osteoclasts. The results are shown in the Response to Reviewer File: Cyclopamine could also inhibit the proinflammatory polarization of macrophages to a certain extent, and a significant inhibition of the osteoclast formation could be observed as well. These results may further confirm the important role of HH/GLI1 in regulating macrophage caused inflammation and osteoclast activation.

Comment 4. In Fig 6, the overexpression of DNMT3a reversed the inhibitory effects of GANT58 in osteoclastogenesis. This supports the author's conclusion that GLI1 may enhance osteoclastogenesis via DNMT3a upregulation. However, this conclusion should be carefully evaluated by examining effects of the overexpression of DNMT3a without GANT58. Does the overexpression of DNMT3a by itself enhance osteoclastogenesis or just reverse the GANT58-mediated suppression?

Reply: Thanks for your constructive comment. According to your comment, we observed and compared the differences in the activation of osteoclasts between the DNMT3a overexpression group and the control group. The results showed that at day 4 of induction, the TRAP positive stained osteoclast number seemed to be no significance between WT group and Dnmt3aOE group. However, at day 3, there was more osteoclast in Dnmt3aOE group, which suggested that overexpression of Dnmt3a might accelerate the activation of osteoclasts to some extent. The results are shown in the Response to Reviewer File.

Comment 5. Is RNA-seq data with GANT58 compatible with known target genes of GLI1 reported in previous studies?

Reply: Thanks for your constructive comment. By consulting and comparing with other research articles, most of the data trends in RNA sequencing results are the same as those in other studies. In addition, the expression of some genes is different from other studies (MMP13 increased in our data but decreased in other study [1]), which may be caused by different cell lines and different intervention methods.

Reference:

[1] Akhtar N, Makki MS, Haqqi TM, MicroRNA-602 and microRNA-608 regulate sonic hedgehog expression via target sites in the coding region in human chondrocytes, Arthritis Rheumatol 67(2) (2015) 423-34.

Reviewer #3 (Significance (Required)):

Significance

The main limitation of this paper is the lack of siRNA knockdown study of GLI1 and DNMTs. Another limitation of this paper is that the direct in vivo data demonstrating the inhibitory effects of GANT58 on M1 macrophage and osteoclast activation in CIA is lacking. The strength is the promising activity of GLI inhibitor, GANT58 as an anti-rheumatic drug on monocyte/macrophage-associated inflammation and bone destruction. The roles of hedgehog/GLI signals in macrophage function are largely unknown, and the findings of this study may contribute to this research field. This study will be interesting to rheumatologists and immunologists.

Reply: Thanks again for your constructive comments, which helped us to improve the quality of the manuscript.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Reply to the Reviewers

I thank the Referees for their...

Referee #1

1. The authors should provide more information when...

Responses + The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A). + Though this is not stated in the MS 2. Figure 6: Why has only...

Response: We expanded the comparison

Minor comments:

1. The text contains several...

Response: We added...

Referee #2

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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Referee #1

Evidence, reproducibility and clarity

Summary:

In zebrafish embryos, progenitor cells for both the prechordal plate and anterior endoderm reside at the dorsal margin in early gastrulation. Both cell populations are induced via signaling through the Nodal signaling pathway, however the mechanisms that send Nodal-exposed cells to one fate versus the other remain a matter of debate. Cheng et al use single-cell RNA sequencing to investigate the mechanistic origins of this developmental decision. They argue that both populations emerge from a common progenitor pool marked by the prechordal-plate marker gene goosecoid (gsc). By adding single-cell ATACseq analysis, they go on to argue that Nodal signaling encourages open chromatin states at target genes, and that this may underly the distinction between prechordal plate and endodermal fates. Finally, they suggest two potential regulators (gsc and ripply1) that may repress commitment to the endodermal fate.

Major Comments:

1. In lines 128-136, the authors describe a live imaging experiment to support the argument that anterior endodermal cells emerge from a gsc+ progenitor pool. The claim is that sox17+ cells (marked by RFP fluorescence) arise in gsc+ cells (marked by GFP fluorescence). From the presented data, I find it very hard to evaluate this claim. The GFP signal appears quite close to background in the highlighted cell. Additionally, the argument- as presented-turns on the behavior of a single highlighted cell. I think that this analysis should be clarified and extended to support the claim.

I suggest that the authors (1) plot average cell fluorescence over time rather than a 'line scan' across the cell, (2) draw cell borders from the mask used in each frame for clarity of presentation, and (3) plot the trajectories of gsc+/sox17+, gsc-/sox17- and gsc+/sox17-cells for comparison.

Alternatively, it could be helpful to extract fluorescence intensities for each cell in the field of view and scatter the RFP vs. GFP intensity for each cell. If the claim is true, three distinct subpopulations should be visible (i.e. gsc+/sox17-, gsc-/sox17- and gsc+/sox17+). Statistical analysis supporting the significance of these differences (e.g. comparing the means of each reporter within the populations) would be clarifying.

OPTIONAL: The live imaging experiment the authors present is quite ambitious, but perhaps overly difficult for the task at hand. I think this point could be more easily and clearly demonstrated by using two-color fluorescent in situs or HCR staining for gsc and sox17. Using an endpoint measurement would allow for deeper sampling across multiple embryos, and would likely yield clearer signals for cell type quantifications.<br /> 2. In the same section, I suggest that the authors address the possibility that the sox17+ cells observed don't go on to become part of the anterior endoderm. I commend the authors experimental work to support their scRNA-Seq data, however observation of the expression of a reporter gene (injected on a plasmid) is not equivalent to demonstrating that those cells adopt a given fate in the end. Is it not possible that the sox17 expression is transient, and these cells revert to prechordal plate fate? This point would be sealed by a formal fate mapping study (e.g. photoconversion of sox17::kaede cells), but I don't think this is a necessary bar for publication.<br /> 3. In Figure 1 M, the explant data does not seem to clearly support the claim that higher Nodal signaling intensities favor prechordal plate over endoderm. It appears that, for the endodermal panel, 2/3 replicates for 6 pg and 10 pg injections resulted in no endodermal cells observed. Could the authors clarify how this reflects the certainty of the conclusion? No statistical analysis is indicated on this panel or the one below.<br /> 4. OPTIONAL: The analysis presented in Fig. 1M strikes me as rather indirect (i.e. deconvolution of bulk RNA-Seq data to infer cell population proportions), and not strongly compelling. I think a stronger support of this point would be to inject Nodal into embryos and measure positive cell counts for gsc and an endodermal marker (e.g. sox32 or sox17) via HCR or in situ hybridization. This would yield a direct measurement of the cell counts in question. I think this would greatly support the claim, but I don't think should be considered a requirement for publication.<br /> 5. In Fig. 2H, the authors analyze responses to ectopic Nodal gradients in order to corroborate the results of their LIANA analysis. This experiment is a welcome addition to the argument, but has weak points that should be addressed.<br /> - a. The description of image analysis procedures used to construct the quantification plots are inadequate. It seems likely that the nuclei were segmented from the DAPI images, but this was not clear from the methods section. The authors should completely describe the segmentation pipeline and include sample code in the supplementary material.<br /> - b. The methods section seems to suggest that the analysis was performed exclusively on maximum intensity projections. I think this procedure may make the data hard to interpret and should be modified/support with additional analysis. For example, there is no reason that, at any given position in the image, the brightest DAPI and pSmad2 channel pixels occur in the same plane. Segmentation boundaries may therefore not reliably match between channels in the maximum intensity projection. The segmentation should be performed using the full Z-stack images. This can be done using widely-available software packages (e.g. CellProfiler).<br /> - c. The fluorescence images in 2H (specifically for the pSmad2 channel) look like they may contain some artifacts that carry through into the quantification. Specifically, there appears to be substantial non-specific background (both hazy and punctate) in the lft1 mutant that may artificially elevate the quantified intensity. This is evident in the quantification as a larger 'offset' to which the gradient decays than in the other presented images. This may be another explanation for the observation that pSmad2 staining is stronger in this background. I suggest that the authors (a) present all fluorescence images from the dataset in the supplement to allow for visual inspection, and (b) estimate the effect of fluorescence background on their quantifications to ensure that this artifact is not the source of the claimed difference.<br /> 6. In lines 267-284 and Fig. 4 L, the authors make the argument that ripply1 acts as a cell-autonomous repressor of endodermal fate. I find the argument for the cell autonomous character of its function hard to follow. Specifically, the authors lean on the experiment in which a plasmid with a sox17 promoter-ripply1 construct is injected, resulting in a decrease in endodermal cell count. Could the authors elaborate on how this proves a cell autonomous effect? Is it not possible that ripply1 expressed from this construct induces a signal that influences neighboring cells?<br /> 7. The suggestion that prechordal plate fate is favored (over endodermal fate) by higher Nodal signaling levels is interesting. This claim is supported by the derivation of a 'Nodal score' from RNAseq data. However, I don't see where the score is defined in the Methods section or in the supplementary materials. If this was accidentally omitted (my apologies if I am just missing it), it should be added. Additionally, I found the description in the main text to be opaque, and the paper would benefit from a more intuitive/friendly explanation of this metric.

Additionally, could the authors comment on what they believe-in terms of Nodal signaling history for a given cell- this score represents? Does it correlate with integrated Nodal exposure? Nodal exposure duration? Peak Nodal exposure? Given the results of Sako et al-that Nodal exposure duration is a critical determinant of prechordal plate fate- it would be useful to know if the authors believe their Nodal score findings point toward a different mechanism.

Minor Comments:

1. Line 84: The authors refer to the prechordal plate cells being 'more mature' than endoderm. It is unclear what the claim is here; some elaboration would be helpful.
2. The fluorescence images in Fig. S2 are virtually invisible in the PDF. The images should be rescaled to make them visible.
3. Fig. 2H would be easier to make sense of if the image panels were labeled. Please indicate which color corresponds to which stain.

Significance

I believe that this study fills in some details on the process of anterior endoderm specification that will be of interest to specialists in zebrafish Nodal signaling. I believe that the strongest and most novel section is the combined scRNA-Seq/ATAC-Seq analysis. This dataset is likely to be of interest to researchers who want to dig into potential mechanisms for the separation anterior endoderm and prechordal plate. Further, the singling out of ripply1 as a potential regulator of endodermal specification is interesting, and I hope that the authors follow this promising lead in future work.

While this study does provide a useful single-cell view of the specification of anterior endoderm, I didn't feel that it came to a concrete conclusion about the mechanism of separation of the anterior endoderm and prechordal plate. A few interesting processes/players are suggested by the findings- for example, Nodal/Lefty signaling between the populations or ripply1 expression could tip the balance- but I don't believe these hypotheses were tested clearly. The authors correctly point out that models for Nodal-driven endoderm/mesoderm separation have recently emerged in the literature, however the findings presented here don't rule out either of these models or compellingly support an alternative. I don't believe that this should preclude publication, however I do think it will limit the reach of the paper. Experiments that more concretely test the possible mechanisms hinted at here- for example, studying the separation of the two lineages in ripply1 mutants- would strengthen the paper's reach.

My enthusiasm for the paper is also somewhat reduced by the fact that some key findings of the paper can be found in earlier work. Acknowledgement of this prior work in the relevant sections could be improved. Specifically:

1. The finding that anterior endoderm cells emerge from a gsc-expressing population in the dorsal margin was strongly suggested in the classic Warga et al paper on the origin of zebrafish endoderm. There, fate mapping experiments demonstrate that dorsal marginal cells (in the first two cell tiers) in the late blastula can go on to form both endoderm and mesoderm. This strongly implies that anterior endoderm cells emerge from a gsc+ population, given that these cells are firmly within the gsc expression domain. I also note that the scRNAseq data from Fig. 2 in Farrell et al directly demonstrates that some sox17+ endoderm cells express gsc in their developmental trajectory. The findings in this paper are a welcome confirmation of these earlier observations, however this context should be discussed.
2. The observation that squint and lefty single mutants (either lefty1 or lefty2) can alter the propensity to adopt endodermal or mesodermal fates has also been observed previously. See for example Fig.1 in Norris et al, Figs 3 and 4 in Rogers et al, or Fig.1 in Chen et al. Acknowledging some of these earlier findings would benefit the paper.

As a reviewer, I feel most qualified to comment on the embryological aspects of the presented work. While I am generally familiar with the single-cell genomics toolkit, I am not in a position to rigorously assess the technical merit of that side of this work. Accordingly, I have tried to restrict my comments to the embryology side.

References:

1. Warga, R.M. and Nüsslein-Volhard, C., 1999. Origin and development of the zebrafish endoderm. Development, 126(4), pp.827-838.
2. Farrell, J.A., Wang, Y., Riesenfeld, S.J., Shekhar, K., Regev, A. and Schier, A.F., 2018. Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis. Science, 360(6392), p.eaar3131.
3. Norris, M.L., Pauli, A., Gagnon, J.A., Lord, N.D., Rogers, K.W., Mosimann, C., Zon, L.I. and Schier, A.F., 2017. Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling. Elife, 6, p.e22626.
4. Rogers, K.W., Lord, N.D., Gagnon, J.A., Pauli, A., Zimmerman, S., Aksel, D.C., Reyon, D., Tsai, S.Q., Joung, J.K. and Schier, A.F., 2017. Nodal patterning without Lefty inhibitory feedback is functional but fragile. Elife, 6, p.e28785.
5. Chen, Y. and Schier, A.F., 2002. Lefty proteins are long-range inhibitors of squint-mediated nodal signaling. Current Biology, 12(24), pp.2124-2128.
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Author Response

Reviewer #1 (Public Review):

This study by Park et al. describes an interesting approach to disentangle gene-environment pathways to cognitive development and psychotic-like experiences in children. They have used data from the ABCD study and have included PGS of EA and cognition, environmental exposure data, cognitive performance data and self-reported PLEs. Although the study has several strengths, including its large sample size, interesting approach and comprehensive statistical model, I have several concerns:

• The authors have included follow-up data from the ABCD Study. However, it is not very clear from the beginning that longitudinal paths are being explored. It would be very helpful if the authors would make their (analysis) approach clearer from the introduction. Now, they describe many different things, which makes the paper more difficult to read. It would be of great help to see the proposed path model in a Figure and refer to that in the Method.

We clarified the specific longitudinal paths explored in our study in the end of the Introduction section (line 149~160). We also added a figure of the proposed path model (Figure 1) and refer to it in the Method section (line 232~239).

• There is quite a lot of causal language in the paper, particularly in the Discussion. My advice would be to tone this down.

We corrected and tone-downed all causal languages used in our manuscript. Per your suggestion, we deleted statements like ‘unbiased estimates’ and used expressions such as ‘adjustment for observed/unobserved confounding’ instead.

• I feel that the limitation section is a bit brief, and can be developed further.

We specified additional potential constraints of our study, including limited representativeness, limited periods of follow-up data, possible sample selection bias, and the use of non-randomized, observational data. These corrections can be found in line 518~538.

• I like that the assessment of CP and self-reports PEs is of good quality. However, I was wondering which 4 items from the parent-reported CBCL were used and how did they correlate with the child-reported PEs? And how was distress taken into account in the child self-reported PEs measurement? Which PEs measures were used?

We believe that the Reviewer #1’s comment for the correlations between PLEs derived from PQ-BC (total score and distress score PLEs) and from CBCL (parent-rated PLEs) might have been due to the fact that she/he was referring to the prior version of our manuscript submitted to a different journal. We obtained Pearson’s correlation coefficients between the PLEs (baseline year: r = 0.095~0.0989, p<0.0001; 1-year follow-up: r = 0.1322~0.1327, p<0.0001; 2-year follow-up: r = 0.1569~0.1632, p<0.0001) and added this information in the Method section for PLEs (line 198~201).

• What was the correlation between CP and EA PGSs?

We also added the Pearson’s correlation between the two PGSs (r =0.4331, p<0.0001) in the Methods section for PGS (line 214~215).

• Regarding the PGS: why focus on cognitive performance and EA? It should be made clearer from the introduction that EA is not only measuring cognitive ability, but is also a (genetic) marker of social factors/inequalities. I'm guessing this is one of the reasons why the EA PGS was so much more strongly correlated with PEs than the CP PGS. See the work bij Abdellaoui and the work by Nivard.

We thank the reviewer for the feedback to clarify that educational attainment (EA) is not only a genetic marker of cognitive ability but also that of socioeconomic outcomes. Per your suggestion, we included the associations of EA PGS with multiple biological and socioeconomic outcomes found in prior studies (e.g., Abdellaoui et al., 2022) in the Introduction (line 131~142).

Abdellaoui, A., Dolan, C. V., Verweij, K. J. H., & Nivard, M. G. (2022). Gene–environment correlations across geographic regions affect genome-wide association studies. Nature Genetics. doi:10.1038/s41588-022-01158-0

• Considering previous work on this topic, including analyses in the ABCD Study, I'm not surprised that the correlation was not very high. Therefore, I don't think it makes a whole of sense to adjust for the schizophrenia PGS in the sensitivity analyses, in other words, it's not really 'a more direct genetic predictor of PLEs'.

We conducted this adjustment considering that PLEs often precede the onset of schizophrenia. In addition, prior studies found that schizophrenia PGS is significantly associated with cognitive intelligence within psychosis patients (Shafee et al., 2018) and individuals at-risk of psychosis (He et al., 2021), and that significant distress psychotic-like experiences had greater positive correlation with schizophrenia PGS than PGS for psychotic-like experiences (Karcher et al., 2018).

For these reasons, we thought that it is necessary to assess whether the effects of cognitive phenotypes PGS (i.e., CP PGS and EA PGS) in the linear mixed model are significant after adjusting for schizophrenia PGS. We believe our results from the mixed linear model showed the sensitivity and specificity of the association between cognitive phenotype PGS and PLEs.

He, Q., Jantac Mam-Lam-Fook, C., Chaignaud, J., Danset-Alexandre, C., Iftimovici, A., Gradels Hauguel, J., . . . Chaumette, B. (2021). Influence of polygenic risk scores for schizophrenia and resilience on the cognition of individuals at-risk for psychosis. Translational Psychiatry, 11(1). doi:10.1038/s41398-021-01624-z

Karcher, N. R., Paul, S. E., Johnson, E. C., Hatoum, A. S., Baranger, D. A. A., Agrawal, A., . . . Bogdan, R. (2021). Psychotic-like Experiences and Polygenic Liability in the Adolescent Brain Cognitive Development Study. Biological Psychiatry: Cognitive Neuroscience and Neuroimaging. doi:https://doi.org/10.1016/j.bpsc.2021.06.012

Shafee, R., Nanda, P., Padmanabhan, J. L., Tandon, N., Alliey-Rodriguez, N., Kalapurakkel, S., . . . Robinson, E. B. (2018). Polygenic risk for schizophrenia and measured domains of cognition in individuals with psychosis and controls. Translational Psychiatry, 8(1). doi:10.1038/s41398-018-0124-8

• How did the FDR correction for multiple testing affect the results?

For all analysis results presented in our study, False Discovery Rate (FDR) correction for multiple testing compared p-values of nine key study variables: PGS (cognitive performance or educational attainment), family income, parental education, family’s financial adversity, Area Deprivation Index, years of residence, proportion of population below -125% of the poverty line, positive parenting behavior, and positive school environment. An exception was the sensitivity analysis that included schizophrenia PGS in the linear mixed model for adjustment: with another PGS variable added, FDR correction compared p-values of ten key variables. Overall, the effects of FDR correction on the results were limited; i.e., the majority of associations between the key variables and the outcomes, which were deemed highly significant, remained unchanged after the FDR correction.

Overall, I feel that this paper has the potential to present some very interesting findings. However, at the moment the paper misses direction and a clear focus. It would be a great improvement if the readers would be guided through the steps and approach, as I think the authors have undertaken important work and conducted relevant analyses.

We express our appreciation to the reviewer for the constructive feedback and guidance, which has significantly contributed to the improvement of our manuscript. As addressed in the preceding sections, we have implemented the necessary corrections and clarifications in response to the reviewer's suggestions. We remain open to making further amendments as needed, and thus invite any additional comments should any aspect of our revisions be deemed inadequate or inappropriate.

Reviewer #2 (Public Review):

This paper tried to assess the link between genetic and environmental factors on psychotic-like experiences, and the potential mediation through cognitive ability. This study was based on data from the ABCD cohort, including 6,602 children aged 9-10y. The authors report a mediating effect, suggesting that cognitive ability is a key mediating pathway in the link between several genetic and environmental (risk and protective) factors on psychotic-like experiences.

While these findings could be potentially significant, a range of methodological unclarities and ambiguities make it difficult to assess the strength of evidence provided.

Strengths of the methods:

The authors use a wide range of validated (genetic, self- and parent-reported, as well as cognitive) measures in a large dataset with a 2-year follow-up period. The statistical methods have the potential to address key limitations of previous research.

We sincerely thank the reviewer for recognizing these methodological strengths of our study. The reviewer’s positive comments are highly supportive and encouraging for us.

Weaknesses of the methods:

The rationale for the study is not completely clear. Cognitive ability is probably a more likely mediator of traits related to negative symptoms in schizophrenia, rather than positive symptoms (e.g., psychosis, psychotic-like symptom). The suggestion that cognitive ability might lead to psychotic-like symptoms in the general population needs further justification.

We sincerely thank and highly appreciate the concerns that the reviewer has raised regarding our proposal that cognitive ability may serve as a mediator of psychotic-like experiences. To the best of our knowledge, it has been proposed that cognitive ability can be a mediator of positive symptoms in schizophrenia (including psychotic-like experiences), as well as negative symptoms. This mediating role of cognitive ability was proposed in several prior studies on cognitive model of schizophrenia/psychosis. Per your suggestion, we included further justification in the Introduction section of our study (line 104~107). Specifically, we highlighted that cognitive ability has been theoretically proposed as a potential mediator of genetic & environmental influence on positive symptoms of schizophrenia such as psychotic-like experiences. We refer to studies conducted by Howes & Murray (2014) and Garety et al. (2001).

Howes, O. D., & Murray, R. M. (2014). Schizophrenia: an integrated sociodevelopmental-cognitive model. The Lancet, 383(9929), 1677-1687. doi:https://doi.org/10.1016/S0140-6736(13)62036-X

Garety, P. A., Kuipers, E., Fowler, D., Freeman, D., & Bebbington, P. E. (2001). A cognitive model of the positive symptoms of psychosis. Psychological Medicine, 31(2), 189-195. doi:10.1017/S0033291701003312

Terms are used inconsistently throughout (e.g., cognitive development, cognitive capacity, cognitive intelligence, intelligence, educational attainment...). It is overall not clear what construct exactly the authors investigated.

Thank you for your comment. We corrected the term ‘cognitive capacity’ to ‘cognitive phenotypes’ throughout our manuscript. We also added in the Introduction (line 141~143) that we will collectively refer to these two PGSs of focus as ‘cognitive phenotypes PGSs’, which is similar to the terms used in prior research (Joo et al., 2022; Okbay et al., 2022; Selzam et al., 2019).

Joo, Y. Y., Cha, J., Freese, J., & Hayes, M. G. (2022). Cognitive Capacity Genome-Wide Polygenic Scores Identify Individuals with Slower Cognitive Decline in Aging. Genes, 13(8), 1320. doi:10.3390/genes13081320

Okbay, A., Wu, Y., Wang, N., Jayashankar, H., Bennett, M., Nehzati, S. M., . . . Young, A. I. (2022). Polygenic prediction of educational attainment within and between families from genome-wide association analyses in 3 million individuals. Nature Genetics, 54(4), 437-449. doi:10.1038/s41588-022-01016-z

Selzam, S., Ritchie, S. J., Pingault, J.-B., Reynolds, C. A., O’Reilly, P. F., & Plomin, R. (2019). Comparing Within- and Between-Family Polygenic Score Prediction. The American Journal of Human Genetics, 105(2), 351-363. doi:https://doi.org/10.1016/j.ajhg.2019.06.006

Not the largest or most recent GWASes were used to generate PGSes.

Thank you for mentioning this point. The reason why we were not able to use the largest GWAS for cognitive intelligence, educational attainment and schizophrenia is because (unfortunately) our study started earlier than the point when the GWAS studies by Okbay et al. (2022) and Trubetskoy et al. (2022) were published. We corrected that our study used ‘a GWAS of European-descent individuals for educational attainment and cognitive performance’ instead of the largest GWAS (line 206~208).

It is not fully clear how neighbourhood SES was coded (higher or lower values = risk?). The rationale, strengths, and assumptions of the applied methods are not fully clear. It is also not clear how/if variables were combined into latent factors or summed (weighted by what). It is not always clear when genetic and when self-reported ethnicity was used. Some statements might be overly optimistic (e.g., providing unbiased estimates, free even of unmeasured confounding; use of representative data).

Consistent with the illustration of neighborhood SES in the Methods section, higher values of neighborhood SES indicate risk. In the original Figure 2, higher values of neighborhood SES links to lower intelligence (direct effects: β=-0.1121) and higher PLEs (indirect effects: β=-0.0126~ -0.0162). We think such confusion might have been caused by the difference between family SES (higher values = lower risk) neighborhood SES (higher values = higher risk). Thus, we changed the terms to ‘High Family SES’ and ‘Low Neighborhood SES’ in the corrected figure (Figure 3) for clarification.

Considering that shorter duration of residence may be associated with instability of residency, it may indicate neighborhood adversity (i.e., higher risk). This definition of the ‘years of residence’ variable is in line with the previous study by Karcher et al. (2021).

We represented PGSs, family SES, neighborhood SES, positive family and school environment, and PLEs as composite indicators (derived from a weighted sum of relevant observed variables). To the best of our knowledge, it has been suggested from prior studies that these variables are less likely to share a common factor and were assessed as a composite index during analyses. For instance, Judd et al. (2020) and Martin et al. (2015) analyze genetic influence of educational attainment and ADHD as composite indicators. Also, as mentioned in Judd et al. (2020), socioenvironmental influences are often analyzed as composite indicators. Studies on psychosis continuum (e.g., van Os et al., 2009) suggest that psychotic disorders are likely to have multiple background factors instead of having a common factor, and notes that numerous prior research uses composite indices to measure psychotic symptoms. These are the reasons why we used components for these constructs instead of generating latent factors (which is done in the standard SEM method). On the contrary, we represented general intelligence as a common factor that determines the underlying covariance pattern of fluid and crystallized intelligence, based on the classical g theory of intelligence. We added this explanation in line 269~285.

Moreover, during estimation, the IGSCA determines weights of each observed variable in such a way as to maximize the variances of all endogenous indicators and components. We added this explanation in the description about the IGSCA method (line 266~268).

We deleted overly optimistic statements like ‘unbiased estimates’ and used expressions such as ‘adjustment for observed/unobserved confounding’ instead, throughout our manuscript.

Judd, N., Sauce, B., Wiedenhoeft, J., Tromp, J., Chaarani, B., Schliep, A., ... & Klingberg, T. (2020). Cognitive and brain development is independently influenced by socioeconomic status and polygenic scores for educational attainment. Proceedings of the National Academy of Sciences, 117(22), 12411-12418.

Karcher, N. R., Schiffman, J., & Barch, D. M. (2021). Environmental Risk Factors and Psychotic-like Experiences in Children Aged 9–10. Journal of the American Academy of Child & Adolescent Psychiatry, 60(4), 490-500. doi:10.1016/j.jaac.2020.07.003

Martin, J., Hamshere, M. L., Stergiakouli, E., O'Donovan, M. C., & Thapar, A. (2015). Neurocognitive abilities in the general population and composite genetic risk scores for attention‐deficit hyperactivity disorder. Journal of Child Psychology and Psychiatry, 56(6), 648-656.

van Os, J., Linscott, R., Myin-Germeys, I., Delespaul, P., & Krabbendam, L. (2009). A systematic review and meta-analysis of the psychosis continuum: Evidence for a psychosis proneness–persistence–impairment model of psychotic disorder. Psychological Medicine, 39(2), 179-195. doi:10.1017/S0033291708003814

It appears that citations and references are not always used correctly.

We thoroughly checked all citations and specified the references for each statement. We deleted Plomin & von Stumm (2018) and Harden & Koellinger (2020) and cited relevant primary studies (e.g., Lee et al., 2018; Okbay et al., 2022; Abdellaoui et al., 2022) instead. We also specified the references supporting the statement that educational attainment PGS links to brain morphometry (Judd et al., 2020; Karcher et al., 2021). As Okbay et al. (2022) use PGS of cognitive intelligence (which mentions the analyses results in their supplementary materials) as well as educational attainment, we decided to continue citing this reference. These corrections can be found in line 131~141.

Strengths of the results:

The authors included a comprehensive array of analyses.

We thank the reviewer for the positive comment.

Weaknesses of the results:

Many results, which are presented in the supplemental materials, are not referenced in the main text and are so comprehensive that it can be difficult to match tables to results. Some of the methodological questions make it challenging to assess the strength of the evidence provided in the results.

As you rightly identified, we inadvertently failed to reference Table S2 in the main text. We have since corrected this omission in the Results section for the IGSCA (SEM) analysis (line 375). The remainder of the supplementary tables (Table S1, S3~S7) have been appropriately cited in the main manuscript. We recognize that the quantity of tables provided in the supplementary materials is substantial. However, given the comprehensiveness and complexity of our analyses, which encompass a wide array of study variables, these tables offer intricate results from each analysis. We deem these results, which include valuable findings from sensitivity analyses and confound testing, too significant to exclude from the supplementary materials. That said, we are open to, and would greatly welcome, any further suggestions on how to present our supplementary results in a more accessible and digestible format. We are ready and willing to implement any necessary modifications to ensure clarity and ease of comprehension. Your guidance in this matter is highly valued.

Appraisal:

The authors suggest that their findings provide evidence for policy reforms (e.g., targeting residential environment, family SES, parenting, and schooling). While this is probably correct, a range of methodological unclarities and ambiguities make it difficult to assess whether the current study provides evidence for that claim.

Impact:

The immediate impact is limited given the short follow-up period (2y), possibly concerns for selection bias and attrition in the data, and some methodological concerns.

We added as study limitations (line 518~538) that the impact of our findings for understanding cognitive and psychiatric development during later childhood may be limited due to the relatively short follow-up period, the possibility of sample selection bias, and the problems of interpreting analyses results from an observational study as causality (despite the novel causal inference methods, designed for non-randomized, observational data, that we used).

As responded above, we made necessary corrections and clarifications for the points suggested by the reviewer. As we are willing to make additional revisions, please feel free to give comments if you feel that our corrections are insufficient or inappropriate.

Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.

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Reply to the reviewers

Reviewer #1

Evidence, reproducibility and clarity____:

Summary: the paper suggested a new approach to study in vivo possible interaction between glioblastoma cells and glioblastoma associated macrophages. By using single cells transcriptome profiling and in vitro and in vivo functional experiments the authors also suggested LGALS1 as possible key factor in the suppression of the immune system and a new target for immune modulation in glioma patients. The experimental plan is well described, and the results are beautifully presented using images, clear drawings, and videos.

Major comments: none

Minor comments:

• The number of zebrafish embryos analyzed after the xenograft is highly variable (e.g. 3-18; 4-22 in Figure 6). These numbers can be reported in the results section (not only in the legends) and the authors may comment on them in the discussion. The reproducibility of thexenotransplant experiments is always challenging as it is quite difficult to inject the same number of cells in every embryo and to have the same survival rate of injected cells and of transplanted embryos. For these reasons the volume of each xenograft can vary significantly in different embryos and in different experimental session. Accordingly, the number of macrophages associated to the tumor can vary and the statistical analysis can be deeply influenced by the number of replicates for each experimental group (a group with 3 embryos is very different in term of quality and quantity of information in respect to a group of 18 embryos). It could be useful for the reader, who has no experience in this technique, be aware of the advantages and disadvantages of the procedure including the possible influence of the temperature (34°C instead of 37°C) on the embryo survival and the replication rate of glioma cells or macrophages behavior. Comment on these aspects does not weaken the power and the relevance of the model but unveil the critical aspects that every scientist has to evaluate before planning these kinds of experiments.

__Response: __We agree with Reviewer #1 that the zebrafish avatar model is challenging, and it is difficult to obtain reproducible tumor sizes and survival rates. To be even more transparent about this, we have added a few sentences about the variable n number in the Results section and a critical comment about it in the Discussion section.

• An aspect that could be interesting to address, to further validate the avatar model, is to monitor the level of pro-inflammatory cytokines (Tumor Necrosis Factor and Interleukin 1, 6, and 8) that are expressed at basal level in the early developing zebrafish embryos. Do their expression level increase after the xenotransplantation? Can the zebrafish cytokines affect the behavior of glioma associated macrophages (i.e. macrophages polarization)?

__Response: __This is an interesting point, indeed. We have injected murine melanoma (B16) cells into Tg(mpeg1:mCherry-F); Tg(TNFa:eGFP-F) embryos, a TNFa reporter line. Some (but not all) macrophages expressed TNFa and their expression decreased over time, which is consistent with previous reports (Póvoa et al, 2021). We further observed that TNFa-expressing macrophages mostly had a round, “tumor-attacking” phenotype. This is in line with our hypothesis that the tumor induces a phenotype switch in GAMs. Of note, we did not see TNFa expression in the rest of the brain tissue. We would be happy to add this data if deemed useful.

We did not investigate other cytokines in the developing zebrafish, but we believe this is not essential for the following reasons: We are mainly interested in the differences between the patient-derived GBM stem cell cultures (GSSCs), and since they are all used in the same avatar model, we expect that if zebrafish cytokines would have an effect on GAMs and their polarization, this effect would be consistent in all avatars, and can thus be ignored when comparing different GSCCs. More importantly, our findings in the zebrafish avatar model were consistent with those in the in vitro model. We observed the same phenotype switch in the co-culture model, indicating that the key interaction is between tumor cells and macrophages.

Significance____:

Strengths and limitation. The manuscript is the result of a well-orchestrated effort to dissect a biological problem by complementary approaches and provide new data with high impact translational value. The image processing pipeline developed by the authors is a step forward in the in vivo analysis of cells interaction in living embryos. The identification of LGALS1 as a potential target for immune modulation can support the development of new therapeutical strategy implementing chemo- or immunotherapy protocols. The described zebrafish avatar can represent a new tool for personalized drug testing recapitulating in a in vivo model the heterogeneity of GBM found in patients.

Audience: All the scientist interested in cell biology, cancer cell biology, imaging techniques, translational medicine, in vivo models for cancer research, precision medicine.

Reviewer expertise: applied developmental biology

Reviewer #2

Evidence, reproducibility and clarity____:

Finotto et al aim to address the polarisation of macrophages within GBM in their study. To do this, they have developed two different models. The first model is an in-vitro co-culture model of patient derived GSC lines and human monocyte derived macrophages. This model was used for single cell sequencing to understand the transcriptomic changes of macrophages upon contact to GBM cells. The second model is a zebrafish xenograft model. Here GFP labeled GBM cells were transplanted into the larval zebrafish ventricle. These experiments were done in the transgenic mpeg zebrafish which allowed to monitor responses of macrophages in vivo.

In my opinion both models are not sophisticated enough to draw solid conclusions on macrophage polarisation in GBM. The in vitro model is highly artificial and is far from the complex situation in GBM. Within GBM the GAM population represents a heterogenous mix of resident microglia and infiltrating macrophages. These are influenced by the heterogeneous environment (which consists of tumour cells but also other host cells) and show diverse transcriptomic adaptations as shown in rodent models as well as sequencing studies of patient derived tumour samples. Studying monocyte derived macrophages in vitro does not provide any reliable insight.

Response: We understand the reviewer’s concern about the complexity of our in vitro model. However, these simple models are needed to gain more insight into the complex in vivo situation. Others have demonstrated their usefulness in the past (C. Jayakrishnan et al, 2019; Zhou et al, 2022; Hubert et al, 2016; Chen et al, 2020; Coniglio et al, 2016; Li et al, 2022). Moreover, it may be advantageous to look at only two different cell types and unravel their reciprocal interaction, without the influence of other cell types, making it too complex to draw conclusions. We acknowledge that GAMs are a heterogeneous mix of both microglia and bone marrow-derived macrophages. Considering that bone marrow-derived macrophages have been shown to play an important role in tumor progression and are by far the most abundant immune cell population in GBM tumors (which even increases in recurrent GBM) (Pombo Antunes et al, 2021; Abdelfattah et al, 2022), we chose to focus initially on bone marrow-derived macrophages. Notably, it has already been reported that microglia were associated with significantly better survival, suggesting that they are anti-tumorigenic, whereas macrophages were associated with worse survival, suggesting that they are pro-tumorigenic (Pombo Antunes et al, 2021; Abdelfattah et al, 2022). This justifies our approach to focus on this cell type. Furthermore, although this model may be rather simplistic, it allowed us to screen different GSCCs side by side in a standardized way, through which we found an apparent phenotype switch within the macrophages, even without the complex interplay with other cell types. Because the results obtained using the in vitro model were also confirmed in GBM patient material and KO experiments in the zebrafish avatar model, our work shows that reliable and important insights can be derived. This, combined with its simplicity, makes our co-culture model an exceptionally relevant model that is scalable, screenable and allows us to study the effect of perturbations. Finally, the immunosuppressive role of the target we identified using this model, LGALS1, has been previously demonstrated by others (Verschuere et al, 2014; Van Woensel et al, 2017; Chen et al, 2019), which proves our approach is valid.

Although the zebrafish can be a great model to understand the progression of tumours and the role of immune cells, I don't think that the model developed by the authors is suitable to address their questions. Transplantation of GBM cells into the the ventricle of larval zebrafish doesn't seem to be the right approach here. The poor survival of the transplanted cells is a clear indication of that. Many other groups have reported growth and proliferation of human cancer cells in the larval zebrafish. Direct transplantation into the brain parenchyma would be the better approach here. The brain parenchyma would provide the right environment for the GBM cells including a resident microglial population. This would also allow to study the complex mix of microglia and infiltrating macrophages in the context of GBM.

Response: The reviewer does not specify which articles have reported growth and proliferation of human cancer cells in zebrafish larvae. Most research groups reporting this, did not follow tumor growth/proliferation over time or used immortalized cell lines (Vargas-Patron et al, 2019; Pan et al, 2020; Pudelko et al, 2018; Breznik et al, 2017; Vittori et al, 2017; Hamilton et al, 2016), which obviously have a much higher proliferation rate than the patient-derived cell lines used in this work. Second, although the number of patient-derived tumor cells decreases over time, we observed a clear invasive and migratory behavior, indicating that the human tumor cells reside well in the zebrafish microenvironment. Furthermore, it is important to note that the zebrafish avatars are grown at 34°C, a temperature that is suboptimal for tumor cell growth. The tumor cells still proliferate, albeit at a lower rate than at 37°C.

To our knowledge, there is only one publication that reports the growth of patient-derived GBM tumors over time (Almstedt et al, 2022). However, here, zebrafish embryos were grown at 33°C. Also, prior to injection, patient-derived GBM cells were resuspended in medium containing polyvinylpyrrolidone, a polymer that enhances extracellular matrix deposition and cell proliferation. Furthermore, the authors observed substantial differences in proliferative capacity, ranging from growth to decline of signal, and represented only two patient-derived cell lines with growing tumors. Similar to our findings, another article has demonstrated that injected patient-derived GBM tumor cells progressively underwent mitotic arrest, while maintaining an invasive and aggressive growth pattern (Rampazzo et al, 2013).

Although the tumor cells are injected into the hindbrain ventricle, they end up in the brain parenchyma, as evidenced by the presence of the typical brain vasculature of the zebrafish embryo. Notably, in Tg(mpeg1:mCherryF)ump2 zebrafish embryos, both macrophages and microglia are labeled with mCherry, meaning that we have studied both cell types in our zebrafish avatar model. Therefore, we consider the reviewer’s comment to be unfounded.

Reviewer #3

__ Evidence, reproducibility and clarity: __

In this study, Finotto and colleagues developed patient-derived Glioblastoma (GBM) stem cell cultures from 7 patients. These GBM stem cell cultures were either co-cultured in vitro with human macrophages combined with single-cell RNA sequencing or injected into the orthotopic zebrafish xenograft to study live GBM-macrophage/microglia interactions. Authors aimed at studying tumor heterogeneity and GBM-associated macrophages (GAMs) which often exhibit immunosuppressive features that promote tumor progression. Their analyses revealed substantial heterogeneity across GBM patients in GBM-induced macrophages polarization and the ability to attract and activate GAMs - features that correlated with patient survival. Also authors show 3 distinct macrophage subclusters (MC1-3), highlighting that the simple M1/M2 polarization phenotypes is too reductive and there are no clear "markers". Authors associate these profiles with morphology and macrophage behaviour. Differential gene expression analysis, immunohistochemistry on original tumor samples, and knock-out experiments in zebrafish subsequently identified / confirmed that LGALS1 as a primary regulator of immunosuppression.

Cheng et ( DOI: 10.1002/ijc.32102) had previously shown the immunosuppression effect of LGALS1 - but this work shows as a proof of concept that the authors approach is a valuable and interesting approach to find immune regulators.

Response: We fully agree with Reviewer #3. In fact, the immunosuppressive role of LGALS1 has already been described by several research groups (Van Woensel et al, 2017; Verschuere et al, 2014), which indeed proves that our approach is valid. The reference cited by the reviewer was already included in the manuscript, along with other references.

Major comments:

In general claims are supported by date - very carefully presented and well characterized data with numbers, stats. It is an interesting descriptive study that illustrates the complexity and diversity of glioblastoma and the induced TME. I just have a few comments or clarifications that I would like to have elucidated:

• I did not understand why not single cell sequence the original tumor - without in vitro passaging and have the original patient population of MACs/microglia and monocytes sequenced? In other words why sequence the in vitro system-with its inherent caveats of in vitro culturing and not the original tumor? Can you please clarify.

Response: We agree with Reviewer #3 that our in vitro model does indeed have caveats inherent to patient-derived cell culture models. However, we chose this model to specifically focus on the reciprocal interaction between GBM tumor cells and macrophages in a way that also allows us to investigate how perturbations affect these interactions. This is not possible when using original tumors (e.g. we cannot make KO cells, as we did for LGALS1, and study the effects of genes of interest). (See also the response to the comment of Reviewer #2)

We do have scRNAseq data from one original tumor sample (LBT123) that is currently being analyzed. Unfortunately, scRNAseq is not available for the other tumor samples. Also, for some of the patients, there is no original material left to use for sequencing. For LBT123, we will compare the scRNAseq data from the original tumor with the in vitro data from the co-culture model.

• Mac signatures - out of curiosity- authors could not find TNFa and IFN signatures in any population?

Response: Our analyses did not reveal TNF or IFN as cluster signature genes. However, we did find that TNF expression was slightly higher in MC2, the pro-inflammatory macrophages, although still at low levels. We did not find IFN expression in the macrophage subclusters, but we did find low expression of some IFN receptors. We found a gradient for IFNGR1 with the highest expression in MC3, followed by MC1 and the lowest expression in MC2. IFNGR2 was expressed at slightly higher levels in MC1 compared to the other subclusters. IFNAR1 and IFNAR2 were expressed at comparable low levels in all subclusters. Finally, IFNLR1 expression was higher in MC3 compared to the other two macrophage subclusters. Considering the overall low expression of IFN receptors, we believe that the differences in expression are rather negligible. Furthermore, it has been previously shown that IFN exerts its anti-tumor effect primarily through the responsiveness of endothelial cells and not of myeloid cells, such as macrophages (Kammertoens et al, 2017). Since vascular cells were not present in the co-culture model, low IFN receptor expression is not surprising. We are happy to investigate this in more detail and include it if deemed useful.

• 8 please show controls side by side with the KO

Response: We thank Reviewer #3 for this comment. We are not quite sure which panel the reviewer is referring to. If it is panel F, we agree with Reviewer #3 and have changed the order of the bars in the revised version. If it is panel E, the corresponding control images are shown in Figure 5I. Since we believe that these images should not be repeated, we have added a figure reference to Figure 5I in the figure legend of Figure 8, in addition to the figure reference already provided in the text. Furthermore, images of all embryos are presented side by side in Figure S8D-E.

• Figure 5: if each pair of images are separated and have the legend on top would be easier to *read and follow. *

Response: We appreciate the comment that the figure should be intuitively easy to read and follow. However, we have chosen a compromise between overview and visibility of details (e.g. morphological features of GAMs). Since this figure already has the maximum width, the images would become smaller if they needed to be separated. Reducing the size would compromise the visibility of important details.

Significance:

It is a very interesting study, carefully designed and performed that highlights the heterogeneity of glioblastoma and how GBM can modulate the macrophage population into 3 different subsets. This study constitutes a proof of concept of the combination of and in vitro approach and an in vivo approach to find new players and treatments in glioblastoma. I believe that it would be important and interesting to have a the original tumor sequenced to compare to the in vitro platform and understand how the in vitro selection impacts on the tumor biology and even if it changes the heterogeneity and differential composition of the tumor and macrophage profiles.

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Author Response

Reviewer #1 (Public Review):

Bacterial carboxysomes are compartments that enable the efficient fixation of carbon dioxide in certain types of bacteria. A focus of the current work is on two protein components that provide spatial regulation over carboxysomes. The McdA system is an ATPase that drives the positioning of carboxysomes. The McdB system is essential for maintaining carboxysome homeostasis, although how this role is achieved is unclear. Previous studies, by the lead author's lab, showed that the McdB system is a driver of phase separation in vitro and in cells. They proposed a putative connection between McdB phase separation and carboxysome homeostasis. The central premise of the current work is as follows: In order to understand if and how phase separation of McdB impacts carboxysome homeostasis, it is important to know how the driving forces for phase separation are encoded in the sequence and architecture of McdB. This is the central focus of the current work. The picture that emerges is of a protein that forms hexamers, which appears to be a trimer of dimers. The domains that drive that the dimerziation and trimerization appear to be essential for driving phase separation under the conditions interrogated by the authors. The N-terminal disordered region regulates the driving forces for phase separation - referred to as the solubility of McdB by the authors. To converge upon the molecular dissections, the authors use a combination of computational and biophysical methods. The work highlights the connection between oligomerization via specific interactions and emergent phase behavior that presumably derives from the concentration (and solution condition) dependent networking transitions of oligomerized McdB molecules.

Having failed to obtain specific structural resolution for the full-length McdB as a monomer or oligomer, the authors leverage a combination of computational tools, the primary one being iTASSER. This, in conjunction with disorder predictors, is used to identify / predict the domain structure of McdB. The domain structure predictions are tested using a limited proteolysis approach and, for the most part, the predictions stand up to scrutiny affirming the PONDR predictions. SEC-MALS data are used to pin down the oligomerization states of McdB and the consensus that emerges, through the investigations that are targeted toward a series of deletion constructs, is the picture summarized above.

Is the characterization of the oligomerization landscape complete and likely perfect? Quite possibly, the answer is no. Deletion constructs pose numerous challenges because they delete interactions and inevitably impose a modularity to the interpretation of the totality of the data.

This is a good point and always a possibility with truncations – the protein McdB may not be as modular in nature as it seems in our tripartite model. But the deletion constructs were more so intended to be tools for identifying key regions of oligomerization and condensate formation as others have done, and for this, they were indeed useful. Additionally, we were able to strategically aim our substitution mutations based on data from the deletion constructs. These substitutions provided data consistent with the deletions, but in the context of the full-length protein (see Fig. 5 vs. Figs. 2, 4). However, we ultimately agree with the reviewer that this is always a possibility with truncations, and we have therefore mentioned this caveat in the discussion.

Line 415 “Truncated proteins have been useful in the study of biomolecular condensates. But it is important to note that using truncation data alone to dissect modes of condensate formation can lead to erroneous models since entire regions of the protein are missing. However, data from our truncation and substitution mutants were entirely congruent. For example, deletion of the CTD or substitutions to this region caused destabilization of the hexamer to a dimer, and deletion of the IDR or substitutions to this region caused solubilization of condensates without affecting hexamer formation.”

Accordingly, we are led to believe that the N-terminal IDR plays no role whatsoever in the oligomerization.

Our updated data still strongly supports this interpretation. Both truncation of the IDR (Fig. 2) and the six-Q-substitution mutant in the IDR (Fig. 5) form a monodispersed hexamer in solution via SEC-MALS, as does wild-type McdB.

Close scrutiny, driven by the puzzling choice of nomenclature and the Lys to Gln titrations in the N-terminal IDR raise certain unresolved issues. First, the central dimerization domain is referred to as being Q-rich. This does not square with the compositional biases of this region. If anything is Q/L or just L-rich. This in fact makes more sense because the region does have the architecture of canonical Leu-zippers, which do often feature Gln residues. However, there is nothing about the sequence features that mandates the designation of being Q-rich nor are there any meaningful connections to proteins with Q-rich or polyQ tracts. This aspect of the analysis and discussion is a serious and erroneous distraction.

We changed the language here, and no longer refer to the central region as “Q-rich”. However, we would like to note that the second half of the McdB central domain is indeed enriched in glutamines (14/53 = 26.4%) to a comparable extent as the region of FUS, which has been shown to help drive condensate formation via glutamine H-bonding (14/44 = 31.8%; Murthy et al 2019). We were simply proposing that, at a molecular level, there was some insight to be gained from this comparison. We agree, however, that there is no functionally meaningful comparison between McdB and polyQ-tract proteins, as we may have previously alluded to in our discussion, and that text has been removed.

Back to the middle region that drives dimerization, the missing piece of the puzzle is the orientation of the dimers. One presumes these are canonical, antiparallel dimers. However, this issue is not addressed even though it is directly relevant to the topic of how the trimer of dimers is assembled.

Indeed, we were unable to resolve the orientation issue, despite much effort. The story we present is not a complete and final model of McdB structure, nor its molecular modes of oligomerization or condensate formation. However we now provide a discussion section “McdB homologs have polyampholytic properties between their N- and C-termini” that highlights this issue. We also mention the remaining dimer orientation issue at the end of the results section “Se7942 McdB forms a trimer-of-dimers hexamer”. However, we believe the data presented still provides useful initial models, which for example, allowed us to create a series of substitutions that tune McdB condensate solubility and verify that they do not affect oligomerization. We would like to further add that for other condensate forming proteins in bacteria, like the PopZ protein we mention in the text, there remains no detailed structural model beyond the resolution we provide here for McdB; despite PopZ being first identified in 2008. Over 40 publications on PopZ have progressively provided useful and more detailed models that are only now being used to develop PopZ as a tool for condensate technologies that are furthering our understanding of the biological implications of condensate formation across all cell types. The intention with our current report is therefore not to generate a finalized molecular model of this entirely unstudied class of McdB proteins. But instead, to generate useful insight into McdB biochemistry that can advance our understanding of this class of protein’s function in vivo. To this end, we now add in vivo data based on these initial models where we specifically link cellular phenotypes to McdB condensate solubility (Fig. 8). Of course, there are several follow-up studies that come from the current report, but we believe that speaks to the value of the presented research in advancing this field.

If the trimer is such that all binding sites are fully satisfied (with the binding sites presumably being on the C-terminal pseudo-IDR), then the hexamer should be a network terminating structure, which it does not seem to be based on the data. Instead, we find that only the full-length protein can undergo phase separation (albeit at rather high concentrations) in the absence of crowder. We also find that the driving forces for phase separation are pH dependent, with pH values above 8.5 being sufficient to dissolve condensates. Substitution of Lys to Gln in the N-terminal IDR leads to a graded weakening of the driving forces for phase separation. The totality of these data suggest a more complex interplay of the regions than is being advocated by the authors.

Thank you and we agree. As we discuss above in response #4 and below in response #7, we have changed the focus and tone of our report to say that, while the models we have generated are useful, we are aware they are incomplete at a molecular level. Furthermore, as we describe in response #6, we have added several new McdB mutants to investigate more deeply the role of the CTD, but this region was not amenable to mutagenesis as these mutants affected McdB oligomerization. Lastly, while network forming interactions are certainly important for condensate formation as the reviewer describes, so are solvent interactions. We have added new text and data related to Figs. 3, 4 that address these issues.

Almost certainly, there are complementary electrostatic interactions among the N-terminal IDR and C-terminal pseudo IDR that are important and responsible for the networking transition that drives phase separation, even if these interactions do not contribute to hexamer formation. The net charge per residue of the 18-residue N-terminal IDR is +0.22 and the NCPR of the remainder is ≈ -0.1. To understand how the N-terminal IDR is essential, in the context of the full-length protein, to enable phase separation (in the absence of crowder), it is imperative that a model be constructed for the topology of the hexamer. It is also likely that the oligomer does not have a fixed stoichiometry.

We agree and thank the reviewer for these comments. We have added several new substitution mutants aimed at addressing this (Figs. 5, S6). However, the C-terminus was not amenable to substitutions as the trimer-of-dimers was significantly destabilized in these mutants (Figs. 5, S7). Therefore, in this report we were unable to determine specifically how the basic residues in the IDR contribute to condensate formation. However, with the addition of new data in Fig. 8, we think we adequately show that the IDR mutants can be used to investigate McdB condensate formation in vivo, and that follow-up studies will be aimed at investigating these details. We have also added an new discussion section “McdB homologs have polyampholytic properties between their N- and C-termini” that highlight this very likely possibility suggested by the reviewer.

Therefore, the central weakness of the current work is that it is too preliminary. A set of interesting findings are emerging but by fixating on Lys to Gln titrations within the N-terminal IDR and referring to these titrations as impacting solubility, a premature modular and confused picture emerges from the narrative that leaves too many questions unanswered.

The work itself is very important given the growing interest in bacterial condensates. However, given that the focus is on understanding the molecular interactions that govern McdB phase behavior - a necessary pre-requisite in the authors minds for understanding if and how phase separation impacts carboxysome homeostasis - it becomes imperative that the model that emerges be reasonably robust and complete. At this juncture, the model raises far too many questions.

We agree that our previous report was focused mainly on the molecular basis of McdB condensate biochemistry, and in that report we left the model short. In this revised version, we have added several pieces of new data that strengthen the model (Figs. 3-5), although it is still incomplete. However, in this revised version, we have also shifted the focus from a complete biochemical understanding of McdB condensates to a study that links McdB condensate formation in vitro to phenotypes in vivo. In this regard, we have added the in vivo data in Fig. 8 and somewhat changed the focus in the text.

The MoRF analysis is distraction away from the central focus.

The MoRF analysis has been removed.

The problem, as I see it, is that the authors have gone down the wrong road in terms of how they have interpreted the preliminary set of results. Further, the methods used do not have the resolution to answer all the questions that need to be answered. Another issue is that a lot of standard tropes are erected and they become a distraction. For example, it is simply not true that in a protein featuring folded domains and IDRs it almost always is the case that the IDR is the driver of phase transitions. This depends on the context, the sequence details of the IDRs, and whether the interactions that contribute to the driving forces for phase separation are localized within the IDR or distributed throughout the sequence. In McdB it appears to be the latter, and much of the nuance is lost through the use of specific types of deletion constructs.

Thank you. We have removed much of this and changed the diction on how our current model of McdB condensate formation fits into the literature in the discussion.

Overall, the work represents a good beginning but the data do not permit a clear denouement that allows one to connect the molecular and mesoscales to fully describe McdB phase behavior. Significantly more work needs to be done for such a picture to emerge.

Reviewer #2 (Public Review):

In this work, Basalla et al. study the biochemical properties of the carboxysome positioning protein, McdB. Using in vitro experiments, the authors characterize McdB oligomeric states and the domains driving and modulating its phase separation. Based on bioinformatics analysis, the authors identify a putative binding recognition motif between McdB and its two-component system counterpart McdA. As McdAB-like systems emerge as spatial regulators of bacterial compartments, the data presented here may be of general interest. The study is well executed and provides exciting hypotheses to be tested in vivo.

The authors found that McdB from S. elongatus PCC 7942 consists of three domains: an N-terminal 18 aa disordered region, a Q-rich helical domain, and a helical C-terminal domain (CTD). Analyzing these domains, the authors present three key results: (i) The Q-rich domains form dimers, and the CTD drives the formation of trimers of dimers (ii) Phase separation is pH sensitive, driven by the Q-rich domain, and modulated by basic residues in the IDR, (iii) The IDR contains a putative recognition motif that binds McdA. While these three sets of results are rich in data, they are disjointed. Relating the three datasets (oligomeric states of the protein, its phase separation behavior, and its ability to bind McdA) is required to provide a complete picture of the molecular mechanism driving McdB condensation.

Specific comments:

1) The main limitation of this manuscript is the lack of integration between the three areas of results. In particular: how do the IDR basic residues disrupt phase separation? Is that through interference with either the dimer or timer interface? Does the McdB IDR regulate phase separation behavior when bound to McdA? Or, in other words, is the MoRF acting both as a binding interface and as a solubility regulator, and if so, can both functions be achieved simultaneously? It seems like the MoRF includes at least three basic residues.

Indeed, we were unable to fully resolve the specific molecular interactions that give rise to condensates versus those that give rise to oligomers, and how these two modes of self-association contribute to one another. One limitation was that, as shown in our new data, the CTD was not amenable to mutagenesis, as it caused destabilization of the trimer-of-dimers (Fig. 5, Fig. S7). Therefore, we could not dissect how the CTD contributes to oligomerization versus driving condensates. However, we did include in vivo data showing how the IDR mutations allowed us to specifically link phenotypes to McdB condensate solubility (Fig. 8). As we discuss above in responses #4, #6, and #7, we changed the focus of the revised manuscript from the molecular basis of McdB condensate formation to linking McdB condensate formation in vitro and its functionality in vivo. To this end, we think the IDR mutation set has been useful, and follow-up studies will be done to further the molecular model of McdB condensate formation. Reviewers 1 and 3 deemed the MoRF section a distraction. Therefore, MoRF analysis and discussions of McdA interactions with this potential MoRF have been removed.

Finally, what is the effective concentration of McdB in cells, and how does that translate to the in vitro studies?

In our previous version, we used McdB concentrations between 50-100 µM. We do not know the in vivo concentration of McdB. We have tried several antibodies against McdB, and a few were good enough to detect the presence of McdB, but not quantifiably. We therefore believe in vivo McdB levels are low (sub-micromolar), and definitely lower than the range we previously used in our in vitro studies. In our revised manuscript, we include a titration of McdB at lower concentrations, and see condensates at McdB concentrations lower than 2 µM.

2) How general are the conclusions made here to other McdBs? The authors have published nice work surveying the commonalities and differences between homologous McdB proteins. Can you comment on the applicability of your findings to other McdB proteins?

This is a great point, which we have added to a new discussion section titled “McdB homologs have polyampholytic properties between their N- and C-termini”.

Additional issues:

3) Using SEC and SEC-MALS, the authors demonstrated that the Q-rich domain forms a stable dimer and that the full-length protein forms hexamers, suggesting trimers of dimers assembly. The authors also suggest that the CTD is responsible for forming those trimers of dimers based on SEC-MALS measurements. However, Figure 2D shows that while the full length runs at 6.6x the monomer, the Q-rich+CTD runs at 5.4x the monomer. First, I could not find SEC-MALS of the full-length protein, and it is not clear whether SEC-MALS was used for all or a fraction of the constructs discussed in Figure 2D. Second, could it be that the Q-rich domain+CTD is an ensemble of hexamers and dimers? Perhaps the IDR is playing a secondary role in stabilizing the hexamer?

We have repeated the SEC-MALS experiments and included the full-length protein (Fig. 2). Furthermore, we have included SEC-MALS for some of the key substitution mutants (Figs. 5, S7). With the additional findings, our conclusions remain the same as in our previous version of the manuscript.

4) The analysis of the phase separation results needs to have some extra quantification. The authors show that at 100 uM protein with 10% PEG the full-length phase separates as well as IDR+Q-rich. Lines 176-178: "The CTD, on the other hand, has no effect on the Q-rich domain condensates; Q-rich+CTD condensates formed at the same protein concentration and with identical droplet morphologies at the Q-rich domain alone." It is hard to draw this conclusion solely based on the data presented in Figure 3. An alternative interpretation might be that Q-rich+CTD reduces csat. I suggest the authors include turbidity assays (as shown for pH effect) to quantitively determine csat for these different constructs and perhaps perform FRAP to determine the mobility of these different constructs. In addition, how long after the addition of PEG were these droplets imaged?

We now include an additional figure where we characterize condensates for full-length McdB (Fig. 3), including FRAP as suggested by the reviewer. We also include additional experiments for the truncations as requested (Fig. 4), and relate the truncation data to the model we propose for the full-length protein. All condensate samples were incubated for 30 mins prior to imaging unless otherwise stated, which we have added to the methods section “Microscopy of protein condensates”.

5) Solubility assays shown in Figures 4A, B, D, and 5C are missing error bars. Without replicates, it is difficult to assess, for example, the effect of KCl.

We have included replicates and error bars. Apologies for the omission.

Also, please indicate the physiological ranges of KCl and pH in Figure 6. The phase separation sensitivity to pH is intriguing. By changing basic residues to glutamines, the authors conclude that the positive charge of the IDR modulates solubility. The Q-rich domain, however, is negatively charged. Can the authors comment on the role of acidic residues in the Q-rich domain? Are they required for phase separation? Also - based on your previous bioinformatics analysis, are the charges of the IDR and the Q-rich domains conserved across McdB homologs?

Data from this report, and as described by reviewer #1, suggest that charge in the CTD, and not the central region, may be important. Our previous report (MacCready et al., Mol Biol Evol. 2020) touches on the conservation of charge in the NTD and CTD, which we have now added to the discussion section titled ““McdB homologs have polyampholytic properties between their N- and C-termini””. However, we were unable to experimentally verify electrostatic associations between the NTD and CTD because the CTD was not amenable to mutagenesis, as shown in our new data added to the manuscript (Figs. 5, S7).

6) In previous work, the authors showed a conserved RKR segment in the IDR is highly conserved and missing in S. elongatus PCC 7942 (MacCready et al., Mol Biol Evol. 2020). Given the current finding, it would be important to understand whether the RKR deletion carries functional implications for phase separation behavior.

The RKR segment is not missing, but likely relates to the KKR residues from S. elongatus PCC 7942. We describe this in more detail elsewhere (MacCready et al., Mol Biol Evol. 2020). However, as we show here, these specific residue locations do not seem to be especially important for condensate formation, but instead the overall net charge of the IDR mediates condensate solubility regardless of the specific residues mutated (Fig. 6).

7) McdB proteins with 2Q left mutated vs. 2Q middle and 2Q right seem to result in condensates with different material properties (e.g., DIC pictures show different droplet morphologies for the different constructs). Is that the case? And if so, can you comment on that?

We have included a brief mention of this in the text. However, the overall interpretation of these results remains that regardless of the residues mutated, there is a comparable degree of condensate solubilization for constructs with the same IDR net charge (Fig. 6).

Reviewer #3 (Public Review):

Through a series of rigorous in vitro studies, the authors determined McdB's domain architecture, its oligomerization domains, the regions required for phase separation, and how to fine-tune its phase separation activity. The SEC-MALS study provides clear evidence that the α-helical domains of McdB form a trimer-of-dimers hexamer. Through analysis of a small library of domain deletions by microscopy and SDS-PAGE gels of soluble and pellet fractions, the authors conclude that the Q-rich domain of McdB drives phase separation while the N-terminal IDR modulates solubility. A nicely executed study in Figure 4 demonstrated that McdB phase separation is highly sensitive to pH and is influenced by basic residues in the N terminal IDR. The study demonstrates that net charge, as opposed to specific residues, is critical for phase separation at 100 micromolar. In addition, the experimental design included analysis of McdB constructs that lack fluorescent proteins or organic dyes that may influence phase separation. Therefore, the observed material properties have full dependence on the McdB sequence.

Thank you for the kind words and this perspective. We have added a brief mention to it in the discussion section titled “McdB condensate formation follows a nuanced, multi-domain mechanism”: “Furthermore, it should be noted that the McdB constructs used in our in vitro assays were free from fluorescent proteins, organic dyes, or other modification that may influence phase separation. Therefore, the observed material properties of these condensates have full dependence on the McdB sequence.”

Studies of proteins often neglect short, disordered segments at the N- or C- terminus due to unclear models for their potential role. This study was interesting because it revealed a short IDR as a critical regulator of phase separation. This includes experiments that remove the IDR (Fig 2 & 3) and mutate the basic residues to show their importance towards McdB phase separation. In a nice set of SDS-PAGE experiments, the authors showed that as the net charge of the IDR decreased the construct became more soluble.

One challenge is in the experimental design when mutating residues is to assess their impact on phase separation. The author's avoided substitutions to alanine, as alanine substitutions have synthetically stimulated phase separation in other systems. The authors, therefore, have a good rationale for selecting potentially milder mutations of lysine/arginine to glutamine. A potential caveat of mutation to glutamine is that stretches of glutamines have been associated with amyloid/prion formation. So, the introductions of glutamines into the IDR may also have unexpected effects on material properties. Despite these caveats, the authors show mutation of six basic residues in the short IDR abolished phase separation at 100 mM.

Thank you for the thoughtful consideration, and appreciation of our work! Reviewer 1 had reservations for the Gln substitutions as well. We also used Alanine in new data added to the manuscript. But as the reviewer notes, the alanine mutations artificially drove further phase separation activity, and even aggregation. We show that mutants with the introduction of glutamines, however, remain soluble in vitro and in E. coli even at very high concentrations. Furthermore, we now include SEC-MALS of the McdB variant with 6 glutamines introduced in the IDR and show that there is no impact on oligomeric state. Together the data show no amylogenic properties of these glutamine enriched mutants.

We have added a note to this potential caveat in the discussion section “McdB condensate formation follows a nuanced, multi-domain mechanism”: “Glutamine-rich regions are known to be involved in stable protein-protein interactions such as in coiled-coils and amyloids (52, 53), and expansion of glutamine-rich regions in some proteins lead to amylogenesis and disease (54, 55). However, when we introduced glutamines into the IDR of McdB solubility was increased both in vitro and in vivo, and without any impact on hexamerization. Together, the data show that increasing the glutamine content in the IDR of McdB did not lead to amylogenesis, but rather increased solubility. Our findings therefore underpin the importance of positive charge in the IDR specifically for stabilizing McdB condensates.”

Computational studies (Fig 7) also suggest that this short N-IDR region may play a role as a MORF upon potential binding to a second protein McdA. The formulation of this hypothesis is strengthened by the fact that for other ParA/MinD-family ATPases, the associated partner proteins have also been shown to interact with their cognate ATPase via positively charged and disordered N-termini. This aspect of understanding McdB's N-IDR as a MORF is at a very early stage. This study lacks experimental evidence for an N-IDR: McdA interaction and experimental data showing conformational change upon McdA binding. However, the computation study sets up the future to consider whether and how the phase separation activity of McdB is related to its structural dynamics and interactions with McdA.

Based off of these comments and from Reviewer 1 comments, we have removed the MoRF analyses entirely. The MoRF analysis will be coupled to another study in the lab focused on McdB interactions with McdA.

In summary, this study provides a strong foundation for the contribution of domains to McdB's in vitro phase separation. This knowledge will inform and impact future studies on McdB regulating carboxysomes and how the related family of ParA/MinD-family ATPases and their cognate regulatory proteins. For example, it is unknown if and how McdB's phase separation is utilized in vivo for carboxysome regulation. However, the revealed roles of the Q-rich domain and N-IDR will provide valuable knowledge in developing future research. In addition, the systematic domain analysis of McdB can be combined with a similar analysis of a broad range of other biomolecular condensates in bacteria and eukaryotes to understand the design principles of phase separating proteins.

Reviewer #2 (Public Review):

In this manuscript, the authors describe the role of cibarial mechanosensory neurons in fly ingestion. They demonstrate that pumping of the cibarium is subtly disrupted in mutants for piezo, TMC, and nomp-C. Evidence is presented that these three genes are co-expressed in a set of cibarial mechanosensory neurons named md-C. Silencing of md-C neurons results in disrupted cibarial emptying, while activation promotes faster pumping and/or difficulty filling. GRASP and chemogenetic activation of the md-C neurons is used to argue that they may be directly connected to motor neurons that control cibarial emptying.

The manuscript makes several convincing and useful contributions. First, identifying the md-C neurons and demonstrating their essential role for cibarium emptying provides reagents for further studying this circuit and also demonstrates the important of mechanosensation in driving pumping rhythms in the pharynx. Second, the suggestion that these mechanosensory neurons are directly connected to motor neurons controlling pumping stands in contrast to other sensory circuits identified in fly feeding and is an interesting idea that can be more rigorously tested in the future.

At the same time, there are several shortcomings that limit the scope of the paper and the confidence in some claims. These include:

a) the MN-LexA lines used for GRASP experiments are not characterized in any other way to demonstrate specificity. These were generated for this study using Phack methods, and their expression should be shown to be specific for MN11 and MN12 in order to interpret the GRASP experiments.

b) There is also insufficient detail for the P2X2 experiment to evaluate its results. Is this an in vivo or ex vivo prep? Is ATP added to the brain, or ingested? If it is ingested, how is ATP coming into contact with md-C neuron if it is not a chemosensory neuron and therefore not exposed to the contents of the cibarium?

c) In Figure 3C, the authors claim that ablating the labellum will remove the optogenetic stimulation of the md-L neuron (mechanosensory neuron of the labellum), but this manipulation would presumably leave an intact md-L axon that would still be capable of being optogenetically activated by Chrimson.

d) Average GCaMP traces are not shown for md-C during ingestion, and therefore it is impossible to gauge the dynamics of md-C neuron activation during swallowing. Seeing activation with a similar frequency to pumping would support the suggested role for these neurons, although GCaMP6s may be too slow for these purposes.

e) The negative result in Figure 4K that is meant to rule out taste stimulation of md-C is not useful without a positive control for pharyngeal taste neuron activation in this same preparation.

In addition to the experimental limitations described above, the manuscript could be organized in a way that is easier to read (for example, not jumping back and forth in figure order).

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Reviewer #1:

Major comments:

1. • The relevance of these findings to human biology remains unclear. In Figures 1-4, the authors present data showing that AATBC is enriched in thermogenic fat, and they argue that it regulates thermogenesis and mitochondrial biology. However, in Figures 6-7, where the authors look at AATBC in different human cohorts, they actually find that it is enriched in visceral fat, which is thought of as being the least thermogenic fat depot. The authors do not explain this seeming paradox, and thus, the role of AATBC in fat remains uncertain. *

RESPONSE: We thank the reviewer for this comment and have clarified the discussion to address this point. It has been recently shown (PMID: 28529941) that the pattern of browning genes in human white adipose tissue depots is actually inverted to mice, making visceral adipose tissue in humans actually more thermogenic than subcutaneous. This aligns well with our findings of AATBC is predominantly expressed in thermogenic adipose tissue.

• In many of the experiments, insufficient controls are provided, or the data are not at all convincing. For example:*

(a) The first four figures rely on in vitro adipocyte models, but the authors do not present data to show these cells differentiate properly and equally. This is especially relevant for the gain and loss of function studies.

RESPONSE: We agree with the reviewer that equal differentiation is necessary for in vitro adipocyte models. Therefore, we added Oil-red-O stainings and the corresponding quantifications to Supp. Fig. 4 (see below) for the differentiation of hMADS in the absence of AATBC. We also want to emphasize, that the expression levels of PLIN1, a surrogate marker for differentiation was unchanged in our experiments, as already shown in the initial draft of the manuscript. On top of that, in all experiments presented in the original draft of the manuscript, AATBC gene expression was only altered in mature adipocytes.

(b) Some of the experiments in Figure 1 (K-L) seem to only show an N of 1.

RESPONSE: Figure 1 highlights a screening process to find new lncRNA regulated during thermogenesis. The forskolin sample was included to achieve an additional dimension in the filtering process. The displayed values in K&L demonstrate the validity of the sample. The validation of AATBC as a target was performed with statistical power in the work displayed in the following figures.

(c) The RNAscope data in Figure 2 is not at all convincing for nuclear localization

RESPONSE: We respectfully disagree. In our opinion, the RNAScope is convincing for nuclear localization of the lncRNA. However, we have repeated the experiments with different probes that strengthen our data (see figure for the reviewer)

(d) The ASO mediated knockdown of AATBC in Figure 3 only reduced expression slightly. A more complete knockdown or deletion may elicit a stronger phenotype.

RESPONSE: We thank the reviewer for the feedback. We have repeated the knockdown experiments but were not able to reduce the expression further, even after designing additional ASOs. However, already with current approach, the reduction in AATBC expression elicited a phenotype, highlighting the importance of AATBC in a dose-dependent manner.

(e) In Figure 4, OPA1 is shown as a single band in panel E and a doublet in panel N. Based on this, are the authors certain they are detecting OPA,1 or could this be a nonspecific band?

RESPONSE: We thank the reviewer for this comment. Protein extraction has been performed at different research institutes with slightly different buffers. Multiple bands (cleaved/uncleaved) have been described for OPA1 in the past, therefore we are certain that the correct protein has been detected.

*(f) The correlations in Figure 6 I-L and Figure 7 do not include any statistical analysis. *

REPONSE: For better readability, the statistical analysis is being mentioned in the figure legend. The reviewer might have overlooked this information.

• The gain of function studies in mice are problematic. The authors have performed a large amount of invasive studies in a short period of time. The animals will undoubtedly lose weight after each study and with insufficient time to recover, this could influence the subsequent studies.*

RESPONSE: These general concerns are valid, but all controls are in place and the animals gained weight during the experiments, as one would have been expected with animals of that age (see below).

*In addition, since the authors present data in Figures 1-4 arguing that AATBC overexpression is associated with increased thermogenesis, it is surprising that the authors never looked at this in Figure 5 (aside from measuring Ucp1 mRNA). It would be interesting to measure energy expenditure by indirect calorimetry and cold tolerance. *

RESPONSE: We agree with the reviewer on this point but are due to animal protocol limitations in conjunction with the viral approach are unable to perform these experiments.

• The authors do not provide any mechanistic insights into how AATBC may be acting.*

RESPONSE: Certainly, more mechanistic insight into the direct mode of action of AATBC would be interesting. To address this point, over the past year we performed multiple attempts to perform pulldown of AATBC using the ChIRP technology. However, we were unable to achieve a sufficient enrichment, which would have allowed us to give further information about direct interaction partners of AATBC. However, we believe that our data regarding mitochondrial dynamics, which we now also have confirmed in in vivo experiments, explain the connection of AATBC and thermogenicity. In future, we aim to work on this point further but for multiple reasons have decided to close this chapter here.

Minor comments:

• The introduction is rather long and would benefit from being condensed.*

RESPONSE: We have edited the text for better readability.

Reviewer #2:

Major Comments:

1. • The key conclusion that AATBC is a novel obesity-linked regulator of adipocyte plasticity is made relatively clear with the comparison between various stages of adipocytes and the loss and gain of function with AATBC. - Figure 1 H and J do not seem to be consistent with the data in Figure 1F in LINC00473 level-There is no difference in Control vs NE in the heatmap but in Figure1J, the difference seems to be quite obvious; Figure 1K does not seem to be consistent with AATBC level-The measurement in Control VS Fsk group showed no difference in AATBC in heatmap, but in Figure K, there seem to be a dramatic increase. Therefore, the claims that there is a difference in these two lncRNA expression in these cell groups needs further clarification. *

RESPONSE: To combine the different approaches to identify novel lncRNA into one heatmap the data need to be normalized over experiments. As the fold change of the expression of AATBC in BAT compared to WAT (on average ~100x) is higher than with forskolin (~4x), this will stand out in the heatmap and will to some extent overshadow the smaller fold changes. The same holds true for LINC00473, which is drastically induced with forskolin, which to some extent masks the higher expression in the other approaches. Therefore, we decided to show both the heatmap to represent the general approach and the “zoomed in” versions to show the consistent increases. We are confident this clarifies the issue.

• Figure 4H and I, the difference in the representative immunoblot seem to be minimal and inconsistent with the decrease shown in the bar graph. *

RESPONSE: We agree with the reviewer and have removed the claim from our manuscript.

• In Figure 5, after overexpressing human AATBC in murine adipose tissue , is it possible to look at the mitochondria changes that were seen before in cell lines? If there are similar changes in murine adipose tissue, then it would prove the changes in vitro hold up with the in vivo model. But if the mitochondria changes were not seen, then it would indicate the changes in leptin, triglyceride levels may due to other mechanisms. The length of the suggested experiment to look into the mitochondrial differences in mice may vary depending on whether there are preserved samples from previous experiments. If there are, then the time period would be couple of weeks for immunblot and analysis. If there are no samples preserved, then the estimated period for the suggested experiments may be around 1.5 to 2 months at least .*

RESPONSE: We thank the reviewer for the suggestion. We performed Western Blot analysis on the tissues from the in vivo study and have included them in Fig. 5, further strengthening the link between AATBC and mitochondrial dynamics (please see figure on the right).

• The data are convincing overall in that the replicates are clearly marked with dots in many figures. Some immune blot and expression level are inconsistent with other data showing the same results however. *

RESPONSE: We thank the reviewer and have removed the necessary quantifications.

• Figure 6 and 7 are provocative and significant, reporting strong associations of AATBC with well-known markers of metabolism in adipocytes. The sex difference for adiponectin and AATBC expression is particularly intriguing. Further discussion of this point would be interesting. However, there is no information provided about the medication status of the obese subjects that were consented for samples used in the analysis. Specifically, many of the obese subjects (mean BMI 45 or more with a range going up to 97.3) would be expected also to have metabolic diagnoses and to be treated with numerous medications, including Metformin, GLP1 agonists, Orlistat, Liraglutide, Bupropion/Naltrexone and combinations. It is unreasonable to ignore possible effects of major medications on AATBC expression. Please comment on the strengths and weaknesses of the analysis that ignores medications, or if some annotations of clinical data are available, perhaps to explain outliers in the plots, please discuss. *

RESPONSE: We thank the reviewer for this suggestion. Unfortunately, we are unable to exclude additional diagnoses and medication of our patients due to the points the reviewer stated. However, given the large size of the cohorts we are confident that such effects are being compensated for. We have added a part on weaknesses of the study in the discussion.

Minor Comments:

• The labeling of figure 2 A-K is not clear because the use of the same color of bars is easily misunderstood as the same source of cells, but it is in fact not. For example, the grey color that appeared in 2B and 2C are not the same source but can be misunderstood. *

RESPONSE: The coloring of Fig.2A&G has been changed.

• Figure 3 ASO-AATBC has two repeats #1 and #2, and over-expression of AATBC has one, even though there are enough repeats. It would be less confusing to present all of the repeats in ASO_AATBC together in one bar.*

RESPONSE: The two different ASO target different areas of AATBC. In line with general guidelines for ASO use, those are not pooled but used separately, which is why the results are also split up. As the overexpression is additional genomic information of AATBC, it is impossible to use different variants in this case, therefore only one bar for overexpression is shown.

• The experimental outline can be a bit more detailed and explain some of the words like Thermo versus Browning.*

RESPONSE: The manuscript has been revised regarding this point.

• Some of the panels in Figure 7 could be put into supplementary if space is at a premium, and present the representative graph would be enough*

RESPONSE: We think that all our data of Fig. 7 warrants enough attention to be considered in a main figure, but if space is sparse, we are very happy to oblige. We would kindly ask the editors for input on this matter.

Reviewer #3:

1. • Throughout the study, the data provided are mainly correlative and in some cases not robust. In Fig. 2, AATBC expression is described to be elevated in the so-called "thermogenic condition", which contained prolonged PPARg agonist treatment (rosiglitazone) known to promote adipogenesis. Consistent with this notion, adipogenic markers, such as PLIN1 and FABP4, are higher in "thermogenic adipocytes" (Suppl Fig. 2). As such, the result may only suggest that AATBC has higher expression in mature adipocytes vs pre-adipocytes. *

RESPONSE: We thank the reviewer for the suggestion. We have added Oil-Red-O-Stainings to Suppl. Fig. 2 to show unchanged lipid content upon modulation of AATBC gene expression, which can be seen as a surrogate for differentiation. Concerning the use of rosiglitazone as a browning agent, we want to emphasize that rosiglitazone was used during the entirety of differentiation until day 9, where it was removed in the “non-thermogenic” group. At this point we already observe fully differentiated adipocytes. This is an established protocol. Furthermore, the data is in line with using norepinephrine or forskolin as a short-term inducer of browning, making it very likely that the effect seen is due to the “more thermogenic” character of the adipocytes.

• Along the same vein, whether and how AATBC affects adipogenesis is unclear. Suppl Fig. 3H and 3L (misplaced as Suppl Fig. 4) show the adipocyte differentiation marker FABP4 is down-regulated by both ASO- and AV-AATBC. Since mitochondrial respiration (and other parameters including UCP1 expression) is tightly linked to adipogenic efficiency, the authors need to address whether these manipulations affect adipocyte differentiation. *

RESPONSE: We agree with the reviewer that differences in differentiation capacity would falsify our data on mitochondrial dynamics. We have added Oil-Red-O-Staining to Suppl. Fig. 2 to show that no significant difference in lipid content exists during modulation of AATBC gene expression, which can be seen as a surrogate for differentiation. Furthermore, in all experiments presented in the manuscript, the modulation of AATBC occurs in already fully differentiated adipocytes. Accordingly, we are confident that AATBC does not influence differentiation but mainly acts through the modulation of mitochondrial dynamics.

• The data in Fig. 4 supporting a role for AATBC in regulating mitochondrial dynamics are superficial and not robust. Fig. 4A/4J do not have high enough resolution to provide accurate assessment of the mitochondrial network.*

RESPONSE: We respectfully disagree with the reviewer on this point. State of the art methods and algorithms were used to image and analyze the mitochondrial network. Furthermore, we have used multiple established markers of mitochondrial dynamics in western blot analysis to further strengthen our assessments of the immunofluorescence. In summary, we feel like have given enough evidence for an accurate assessment of the mitochondrial network.

• The level of loading control TUBB is clearly lower in siAATBC in Fig. 4H. In addition, OPA1 should have multiple isoforms and Fig. 4E/4N show inconsistent patterns. As such, mitochondrial dynamics is not likely an underlying mechanism. *

RESPONSE: We agree with the reviewer on the assessment of the expression of complex 5 and have removed this claim from the manuscript. Regarding the expression of OPA1, protein extraction has been performed at different research institutes with slightly different buffers. Multiple bands (cleaved/uncleaved) have been described for OPA1 in the past, therefore we are certain that the correct protein has been detected.

• Notably, RNAseq data in Suppl Fig. 4 (misplaced as Suppl Fig. 3) seem to indicate that AATBC over-expression promotes TG synthesis, while AATBC knockdown modulates cell death. The authors should consider exploring the leads from RNAseq analysis?*

RESPONSE: We thank the reviewer for the feedback. The small number of altered genes in the RNASeq make us believe in a rather post-transcriptional role of AATBC. We investigated cell death and oxidative stress response as GO terms were highlighted in the analysis, but we were unable to detect any differences in the absence of AATBC, pointing to a minimal effect on transcriptional level (See figure below for the reviewers).

• In Fig. 5, the AV-AATBC transduction in WAT/BAT is localized, transient and not homogeneous. Not surprisingly, this manipulation does not produce any robust effects. The difference in circulating leptin/leptin expression appears to be driven by 4-5 mice in the control group (Fig. 5H/5N). The correlation data in Fig. 6 and Fig. 7, although relevant, do not provide additional mechanistic insights. Unfortunately, the efforts in Fig. 5-7 fail to lead to information related to the biological function of adipose AATBC.*

RESPONSE: We agree with the reviewer on the limitations of the AV model, but we have performed these experiments with the highest technical standard. As the reviewer states, the overexpression, especially in WAT, has different magnitudes depending on the individual mouse, but the overexpression is present and consistently high in every animal. We would expect even bigger alterations in a genetic model, which, however, is beyond the scope of this first manuscript on AATBC in adipocytes. We are disappointed that the reviewer does not value the human data presented, as it very strongly hints to a relevant function of our human lncRNA in vivo by robust correlations with established biomarkers mirroring the effects seen in vitro and in the mouse model. A limitation of human studies is in virtually every case that it is based on correlations, as manipulation of gene expression, which would be necessary to delineate a biological process as requested by the reviewer, is not possible in humans. We do not concur on dismissing our human data on that behalf.

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Referee #1

Evidence, reproducibility and clarity

The manuscript by Giroud et al. describes a role for the human-specific lncRNA AATBC in adipocyte plasticity. By overlaying datasets from tissues (white vs. brown fat) and cell lines (treated with norepinephrine or forskolin), the authors identified a limited number of lncRNAs demonstrating coordinate regulation. One of these lncRNAs is AATBC, which has not previously been studied in adipocytes. The authors show that AATBC is enriched in thermogenic adipose tissues/cells. They then perform gain and loss of function studies in cellular models and argue that AATBC is involved in thermogenesis and appears to be associated with the state of the mitochondrial network. The authors then explain that modulating AATBC has minimal effects on global transcription, and so they argue it mainly works via post-transcriptional mechanisms, though these are not defined. The authors then expressed AATBC in adipose tissue of mice and observed a decrease in plasma leptin levels and an increase in triglyceride levels, while other metabolic phenotypes were unchanged. Finally, the authors analyzed associations between adipose tissue AATBC and a variety of metabolic parameters in a few human cohorts. While the identification of a novel lncRNA involved in adipocyte biology and systemic metabolism would be of great interest, the data presented here does not convincingly support the conclusions made. Substantial additional experiments are needed to support the claims in this paper.

Major comments:

1. The relevance of these findings to human biology remains unclear. In Figures 1-4, the authors present data showing that AATBC is enriched in thermogenic fat, and they argue that it regulates thermogenesis and mitochondrial biology. However, in Figures 6-7, where the authors look at AATBC in different human cohorts, they actually find that it is enriched in visceral fat, which is thought of as being the least thermogenic fat depot. The authors do not explain this seeming paradox, and thus, the role of AATBC in fat remains uncertain.
2. In many of the experiments, insufficient controls are provided or the data are not at all convincing. For example: (a) The first four figures rely on in vitro adipocyte models, but the authors do not present data to show these cells differentiate properly and equally. This is especially relevant for the gain and loss of function studies. (b) Some of the experiments in Figure 1 (K-L) seem to only show an N of 1. (c) The RNAscope data in Figure 2 is not at all convincing for nuclear localization. (d) The ASO mediated knockdown of AATBC in Figure 3 only reduced expression slightly. A more complete knockdown or deletion may elicit a stronger phenotype. (e) In Figure 4, OPA1 is shown as a single band in panel E and a doublet in panel N. Based on this, are the authors certain they are detecting OPA1 or could this be a nonspecific band?( f) The correlations in Figure 6 I-L and Figure 7 do not include any statistical analysis.
3. The gain of function studies in mice are problematic. The authors have performed a large amount of invasive studies in a short period of time. The animals will undoubtedly lose weight after each study, and with insufficient time to recover, this could influence the subsequent studies. In addition, since the authors present data in Figures 1-4 arguing that AATBC overexpression is associated with increased thermogenesis, it is surprising that the authors never looked at this in Figure 5 (aside from measuring Ucp1 mRNA). It would be interesting to measure energy expenditure by indirect calorimetry and cold tolerance.
4. The authors do not provide any mechanistic insights into how AATBC may be acting. The manuscript contains some potentially interesting observations, but without some mechanistic insight, it is hard to understand how AATBC might regulate adipocyte plasticity.

Minor comments:

1. The introduction is rather long and would benefit from being condensed.

Significance

This manuscript may represent an interesting advance in terms of highlighting a new lncRNA with a role in adipocyte biology. These findings would be of broad interest to researchers interested in obesity and metabolism. I myself am in this field of research, so feel quite qualified to evaluate this manuscript. However, as noted above, major concerns would need to be addressed in order to justify the conclusions made here.

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Reviewer #1 (Evidence, reproducibility and clarity (Required)):

*The current manuscript by Shiryaev et al describes their observation of the new function of zika NS2B-NS3 proteases. They have shown that NS2B-NS3 protease lacking the helicase domain binds to RNA and the interaction can be affected by protease inhibitors. Main two new findings are presented in the manuscript: super open conformation of the protease; RNA binding activity of the protease region. Although the manuscript is interesting, the design of the experiments is not convincing. *

Major issues:

1. • the claim of a super open confirmation is problematic. Using an artificial construct lacking the C-terminal portion of NS2B will of course generate the open conformation. This is a wrong definition unless you observe such a conformation in living cells.*

2. We understand the skepticism towards a less known super-open confutation of flavivirus NS2B-NS3pro complex. In addition to our own structure of ZIKV NS2B-NS3pro (PDB ID 7M1V), the crystal structure of another orthologous flavivirus Japanese encephalitis virus (JEV) NS2B-NS3pro (PDB ID 4R8T) was discovered in 2015 1. However, no functional analysis was provided for this crystal structure resulting in the lack of attention paid by the research community. We computed the overlay of the ZIKV NS2B-NS3 protease structures in the super-open conformation (PDB ID 7M1V, deposited by us in 2021) with the crystal structure of JEV protease (PDB ID 7M1V ) (Rebuttal Figure 1). We observed an almost identical organization of the critical NS3pro C-terminal loop between these two structures (RMSD 0.6A). Polypeptides with over 35% identity are very likely to have a similar fold2. Given over 50% identity(!) between flaviviral proteases across the family3,4, we posit that the super-open conformation demonstrated for JEV and ZIKV NS2B-NS3pro is a common feature of the Flaviviridae family. Further, NS2B peptide is always tightly associated with NS3pro via a three-strand beta-barrel (aa 49-58 of NS2B), which remains intact in all NS3Pro conformations. The C-terminal portion of NS2B progressively loses association with NS3pro, being mostly associated in the closed conformation, less so in the open, and even less in the super-open conformation. The G4SG4 linker between NS2B and NS3pro remains unstructured in all conformations. The native C-terminal portion of NS2B (TGKR) is equally unstructured when competed out of the protease active site by another substrate. It is unclear to us why “lacking the C-terminal portion of NS2B will of course generate the open conformation”.

3. It is odd that authors made homology model to generate open conformation structures. the authors did not cite the two papers of eZiPro (Phoo et al 2016 NC) and bZiPro (Zhang et al 2016, Science). these two structures show the closed conformation of protease in the absence and presence of a natural substrate.*

4. We agree with the reviewer that in both constructs eZiPro5 and bZiPro6 of ZIKV NS2B-NS3pro are likely to exist in the closed conformation as documented by the crystal structures. However, in both cases, the active center of ZIKV NS2B-NS3pro is occupied with a short peptide fragment, which is sufficient to induce the closed conformation of NS2B-NS3 protease. We superimposed eZiPro (PDB ID 5GJ4) with bZiPro (PDB ID 5GPI) to better demonstrate that the active center in both structures is occupied either by tetrapeptide TGKR (T127-G128-K129-R130 ) originating from the NS2B C-terminus (eZiPro) or by a tetrapeptide KKGE (K14-K15-G16-E17) originating from a neighboring NS3 molecule (bZiPro) (Rebuttal Figure 2). Indeed, Zheng et al., 2016 6 stated that: “the structure (bZiPro) does capture the protease in complex with a reverse peptide. The tetrapeptide K14K15G16E17 folds into a small hairpin loop to occupy the active site.” Further, Phoo et al., 2016 5 stated that “binding of the ‘TGKR’ peptide to the catalytic site stabilizes the protease (eZiPro)”. To the best of our knowledge, so far there are no crystal structures of flaviviral NS2B-NS3 proteases in the closed conformation without peptide/inhibitor in the active center. We take it as a hint that the closed conformation is always induced by a substrate present in the active center.

Finally, we would like to draw the attention of this reviewer to the fact that the 15N R2 NMR signal from NS2B residues 65-85 is missing in bZiPro alone but re-appears when AcKR is added. This is consistent with the idea that without AcKR, bZiPro exists in the open conformation where much of the C-terminal part of NS2B is dissociated from NS3Pro and remains unstructured, thus resulting in the lack of NMR signal.

• RNA binding is novel, but is it observed in cells? only one method was used for testing the interactions, not other biophysical methods are used.*

• Given a complex network of protein-RNA interactions and the fact that NS3pro and NS3hel are connected by a single polypeptide, separating dynamically bound 11kB RNA to NS3pro from that to NS3hel in a native cell is a major technical challenge beyond the scope of this work. We employed a fluorescent polarization assay to demonstrate ssDNA and ssDNA binding to ZIKV NS2B-NS3pro. Subsequently, we employed a proteolytic activity assay with labeled peptide mimicking natural substrate for protease to demonstrate that the presence of ssRNA and ssDNA can efficiently inhibit proteolytic activity. To the best of our knowledge, this is the first indication that ssRNA or ssDNA could block proteolytic activity for any serine proteases, let alone a viral protease. Therefore, we consider the proteolytic activity assay used in the current work an orthogonal biochemical method supporting ssRNA binding to ZIKV NS2B-NS3pro.

• binding studies with RNA used artificial construct, how about the one with KTGR present like eZiPro. Keep in mind that the P1-P4 residues are present under native conditions.*

__- __As mentioned by the reviewer, TGKR peptide was found in the active center in the eZiPro crystal. Indeed, the junction region between NS2B and NS3 protease contains native cleavage sites for the NS2B-NS3Pro and is naturally cleaved by protease during the viral polyprotein processing. However, the TGKR peptide representing P1-P4 positions will have to leave the active center after the cleavage to ensure enzyme processivity/cleaving additional targets (otherwise, the protease would get stacked after the first cleavage). Proteolytic activity assay utilizes the fluorogenic peptide labeled with FAM (such as TGKR-FAM; where FAM is a group representing P1’ position in this case). TGKR-FAM peptide will compete and easily replace cleaved TGKR peptide from the active center in proteolytic activity assay. In sum, the C-terminal end of NS2B will be competed out of the protease active center by the next substrate, and there is no evidence that it will be naturally placed back in the active center after each round of protease proteolytic activity. Indeed, several crystal structures of flaviviral NS2B-NS3Pro in open conformation lack the C-terminal part of NS2B in the active center. Our unpublished NMR studies demonstrated that the C-terminal part of NS2B is unstructured in solution if the substrate peptide or small molecule inhibitor are not present in the active center of the protease.

• authors built up nice models, it is great to consider the full length NS2B, but authors haven't taken into account the effect of NS2B on the open or closed conformation of the protease. *

- __ All crystal structures of flavivirus NS2B-NS3pro in the closed, open, or super-open conformations have NS2B associated withNS3pro via a beta-barrel (__Rebuttal Figure 3), which is located at the opposite side from the RNA binding site. The transition from the closed to the open and to the super-open conformation is associated with the progressive dissociation of NS2B from NS3pro. Therefore, the effect of NS2B on NS3Pro is progressively diminished. In the closed conformation of NS3Pro, the negatively charged C-terminal part of NS2B is associated with the same positively charged grove as the RNA in the open conformation of NS3Pro. The C-terminal part of NS2B is dissociated from NS3Pro in the open conformation.

Minor issues:

*This manuscript shows the novel function of zika protease and conclude that protease binds to RNA. This is a novel finding, but the conclusion needs to be further confirmed, to avoid misinterpretations by future readers *

• closed, and super open conformations. But the definition was not carefully compared with current literatures. I am surprised that the two important papers are not cited. It is well known the G4SG4 linker affect the conformation of the protease.*

• The crystal structures and the proteolytic activities of gZiPro, eZiPro, and bZiPro are rather similar. In fact, Km (μM) are 2.86 ± 0.90 for gZiPro, 6.332 ± 2.41 for bZiPro, and the IC 50s of BPTI inhibition for gZiPro, eZiPro and bZiPro are 350, 76 and 12 nM respectively. NS2B and NS3pro have a large binding area in the closed conformation. Upon changing the conformation to the open conformation (and even more so to the super-open conformation), the C-terminal part of NS2B is progressively dissociated from NS3Pro. Therefore, possible minor effects introduced by the G4SG4 linker is unlikely to affect any of the conclusions in our work.

• Authors need to show super open conformation is present in nature e.g. the model in which full length NS2B and NS3pro.*

• A full-length NS2B has 2 transmembrane domains, which tether the NS2B-NS3pro complex to the cell membrane (we have modeled the presence of such transmembrane domains to account for the orientation of NS2B-NS3pro with respect to the cell membrane). The full-length complex has never been crystallized or tested in any assay due to the major technical challenges associated with the modeling of complex transmembrane proteins.

• RNA is a charged molecule under some conditions, NS3 also have charged residues, it is important to show whether the binding between RNA-protease is relevant to the function{Luo, 2010 #9270;Chernov, 2008 #9275;Xu, 2019 #10006}, or is this due to the application of the artificial constructs used in this study. Why so many mutants are used? *

• The requirement of NS3pro for the helicase function was shown by several investigators 7–9. Given the structural independence of NS3pro and NS3hel, which mostly rules out the allosteric effect, RNA binding by NS3pro is a newly proposed function of NS3pro for the helicase activity. We demonstrated biochemically that RNA-bound to NS3pro inhibits its protease function. A variety of mutants were used to constrain the conformations of NS2B-NS3pro (e.g. enforce the super-open confirmation) for crystallization studies.

• Using a construct close to the native protease, at least the P1-P4 residues should be present. Using a peptide in the assay is also useful.*

• We were unable to interpret this critique.

• Test binding of RNA with protease using another method such as biophysical methods, or even gel shift assay*

• We thank the reviewer for this suggestion. Although the gel-shift assay seems to be a reasonable method to test the binding, given the ease of spontaneous conformational change (i.e. into the super-open conformation), this assay could result in a progressive loss of bound RNA during migration in the gel.

• I don't know the correlation between Figure 7 and Figure 6. The authors describe ploy A binding to protease, while Figure 7 is talking about Helicase binds to dsRNAs. *

• There is no correlation. Figure 6 describes the models for NS2B-NS3pro binding to ssRNA. Figure 7 describes a separate point, the direction of dsRNA processing by NS3hel.

• I am glad to see the consideration of full length NS2B, NS3 in the models Figure 8, 9 and 11, but there is no data to support any of the model proposed. *

• There is no experimental data. We have modeled the N-terminal and C-terminal parts of full NS2B, which are predicted to be inserted into the cell membrane due to their characteristic amphipathic helical structure.

• Is the linker a ploy G not G4SG4? *

The linker is GGGGSGGGG (G4SG4) as stated in Materials and Methods of the manuscript.

• Do the mutant sustain their protease activity? *

• All mutants with intact catalytic centers have protease activity, except the mutants with a disulfide bridge that fixes the polypeptides in the super-open conformation.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

*The manuscript by Shiryaev et al., submitted to BioRXiv is an exploration of the ability of NS2B-NS3protease to bind RNA and its subsequent role in NS3 helicase processivity. The authors first utilize fluorescence polarization assays to demonstrate that NS2B-NS3protease can bind ssRNA with a strong affinity (and also ssDNA with lower affinity). They subsequently utilize mutational and small molecule inhibitor strategies in these assays to force the NS2B-NS3protease into different conformations, with the associated results inferring that the "open" conformation is responsible for ssRNA binding affinity. Furthermore, they demonstrate that ssRNA binding impairs protease activity, suggesting these roles may be exclusive in the viral life cycle. They also identified a number of small molecule ligands that target the putative ssRNA binding channel, and demonstrate that these ligands inhibit ssRNA binding by NS2B-NS3protease, providing potential inhibitor candidates for ZIKV. Finally, the authors utilized their crystal structures and others for the various conformations of NS2B-NS3protease to model ssRNA binding by the domain and the full NS3 protein, and used these models to propose a reverse inchworm model for NS3 travelling along ssRNA as it unwinds the dsRNA duplex. Overall, the authors utilize a comprehensive approach to demonstrate a number of novel findings (ssRNA binding by NS2B-NS3protease, small molecule ligands that inhibit this interaction) that would be of interest to both virologists and structural biologists. However, there are some important experimental design limitations and viral life cycle considerations that the authors should address before acceptance of the manuscript. Major and minor comments intended to improve the manuscript are outlined in more detail below. *

Major Comments:

1. • While the quantity of indirect data (ruled out closed and super-open, inhibitors of ssRNA binding pocket) suggest that the open conformation of NS2B-NS3protease is associated with ssRNA binding, the argument would be greatly strengthened by direct experimental data. Is there a mutational or small molecule approach to locking the NS2B-NS3 protease in the open conformation? If so, the authors should perform such experiments to strengthen the foundation of their argument.*

2. Unfortunately, despite significant efforts, mutations or small molecules locking the NS2B-NS3 protease in the open conformation have not been identified for the ZIKV protease. However, several structures for NS2B-NS3 proteases have been documented in other flaviviruses (i.e., DENV PDB IDs 2FOM and 5T1V; WNV PDB ID 2GGV). Polypeptides with over 35% identity are very likely to have a similar fold2. Given over 50% identity(!) between flaviviral proteases across the family3,4, there is little doubt that ZIKV NS2-NS3 protease adopts an open conformation similar to all flaviviral proteases. Our modeling demonstrated that there are no sterically/structural problems in folding NS2B-NS3 protease into the open conformation.

3. A negative control should be used in Figure 4A to strengthen the claim that ssRNA binding in the open conformation impairs protease activity (ie. include a curve for dsRNA). Such an experiment would lend support to ssRNA inhibition being due to specific binding instead of some other non-specific effect of increasing local nucleic acid concentration.*

4. To address this critique, we have conducted the modeling of dsRNA binding to the open conformation of NS2B-NS3Pro. The model revealed that dsRNA could not be accommodated by the open conformation of the NS2B-NS3Pro complex (Rebuttal Figure 4). Indeed, dsRNA has a very different rigid structure compared to the extended form of the ssRNA chain. The dsRNA is unable to provide continuous interactions between negatively RNA backbone and positively charged side chain amino acids in NS3pro. The continuous interface on NS2B-NS3 protease interacting with ssRNA is an extension of the exit groove for one of the ssRNA strands exiting the NS3 Helicase after unwinding. Therefore, the ssRNA, but not dsRNA is naturally always present in close proximity of the NS2B-NS3Pro complex.

5. *

6. Due to the highly coupled roles of NS5 and NS3 in replication, the authors should include some more consideration of the role of NS5 in their complex. They very briefly address this interplay in the fifth paragraph of the discussion, but then neglect to discuss the implications any further. In particular (perhaps in a brief comparison to an NS3/NS5 modeling approach such as Brands et al., 2017; WIRES), the authors should consider some of the following questions: could the channel on protease domain lead to ssRNA entry site on RdRp?*

7. Indeed, our model suggests that the negative strand (-)ssRNA exits from NS2B-NS3protease facing the ER membrane in the area where the protease is anchored to the ER membrane via the NS2B transmembrane domains. It is possible that NS3pro interacts with NS5 polymerase and “handles” (-)ssRNA to the NS5 polymerase. This scenario would modify Brands et al., 2017 model to add NS2B-NS3Pro complex between NS3Hel and NS5. However, at present, the NS3-NS5 (or NS2B-NS3-NS5) complex together has not been crystallized. It would be logical for NS5 polymerase to access the (-)ssRNA strand after it is released from NS2B-NS3Pro since the (-)ssRNA strands are used as a template for the (+)ssRNA which is used for polyprotein synthesis and packaging into viral particles.

8. would NS5 interaction constrain or augment inchworm model of NS2B/NS3 translocation? *

9. Yes, integrating NS5 interaction with the NS2B-NS3pro handling (-)ssRNA will augment the utility of the suggested reverse inchworm model.

10. how does increased activity of NS3 when complexed with NS5 (**Xu et al. 2019) align with proposed inchworm model? *

11. We appreciate the reviewer's question. We think that NS2, NS3, NS4, and NS5 work in concert as one coordinated complex where various subunits of NS2 and NS4 may provide anchoring of the entire complex to the ER membrane. Indeed, such a complex has recently been proposed6. Also, see our response to the previous reviewer’s point (#4). We have incorporated this discussion into the revised manuscript.

Minor Comments: 1. Introduction, 4th paragraph, NS3-NS4 should read NS3-NS4A.

• We corrected this sentence.

* ** Throughout the manuscript, the authors should denote some key amino acid residues in each figure to help orient the reader better to the observed structural changes and rotations. Inclusion, at least in the supplement, of the crystal structures of mutants solved herein should **also be included. *

• We annotated the key residues in all figures (e.g. catalytic residues, loop interacting with the membrane, position of NS2B, and other elements) and kept the same orientation of complexes in all figures.

• Section: RNA binding inhibits the proteolytic activity of ZIKV NS2B-NS3pro, last sentence, NS2N-NS3pro should be NS2B-NS3pro*.

• We corrected this sentence.

• Section: Allosteric inhibitors of NS2B-NS3 protease interfere with RNA binding- first sentence: "The open conformation of NS2B-NS3pro is achieved by the rearrangement of NS2B cofactor (its dissociation from the C-terminal half of NS3pro) leading to a loss of proteolytic activity [32]. - the reference is not correct. I could not find the reference the authors refer to here and had not heard before that NS2B cofactor was able to disassociate from the C-terminal half of NS3pro; hence, this really needs to be appropriately referenced. *

• We have revised this sentence and added additional references. “The open conformation of NS2B-NS3pro is achieved by the rearrangement of NS2B cofactor (partial dissociation from NS3pro), leading to a loss of proteolytic activity4,11.”

• Section: Modeling RNA binding to ZIKV NS2B-NS3, first sentence - unwinds should be unwind*.

• We corrected this sentence.

• With respect to the results of Figure 3A, the authors should address that adding the linker alone to the NS3 protease may not be an accurate examination of its role/importance. The linker in this scenario is only constrained at its N-terminus, while it is always constrained at both termini during infection (and even more so by the interactions of those two linked domains [protease and helicase] with each other). As such, the authors statement that "observations suggests that the 12-aa linker region modulates RNA binding to NS2B-NS3pro" should be more strongly qualified to this effect. In addition, it would be interesting to see the effects of the linker mutations on ssRNA binding in the context of the full NS3 protein, albeit admittedly more complex due to the confounding ssRNA binding by the helicase domain.*

• We agree with this reviewer that the protease-helicase linker is also restrained at both termini. We have rephrased the statement in the revised manuscript. The goal of the experiment shown in Figure 3A was to examine whether a negatively charged linker is able to compete with ssRNA binding as we expected from the structural model. The mutational analysis of the protease helicase linker is, indeed, a very interesting subject that is, however, beyond the scope of this work.

7. The NS#hel should be changed to NS3hel in part (C) of figure legend for Figure 11. - We corrected this mishap.

• The authors data in Figure 4A (and even more so the nature of the viral life cycle where 1000s of viral polyproteins are created from the first genome during infection) disputes the depiction in the inchworm model of how NS3 protease cleaves the polyprotein while the helicase binds ssRNA. At minimum, the authors need to discuss this discrepancy, and it is recommended that they modify the cartoon in their model to not include the ssRNA binding on the protease side of the equation (or show as alternative on that side to the existing cartoon).*

• Indeed, as proposed by our reverse inchworm model, ssRNA is not bound to NS3Pro in the closed conformation, while NS2B-NS3pro has a protein substrate in the active center (Figure 11A). We agree that NS2B-NS3Pro in the closed conformation cannot bind ssRNA as we demonstrated in competitive cleavage assay. Only large amounts of ssRNA can shift the balance towards the open conformation which binds ssRNA. We think that most of the time NS2B-NS3Pro cycles between the open and the super conformations handling ssRNA (Figure 11(B-C_D), but as soon as protein substrate becomes available (typically a loop from a transmembrane viral polypeptide), NS2B-NS3Pro quickly switches to the closed proteolytically active conformation to act as protease.

• In the third paragraph of the discussion, the authors state "An alternative model of coupled transcription and translation where viral RNA is associated with ribosomes right after the release from NS2B-NS3 is also possible". Considering there is abundant evidence that translation and replication are exclusive and that translation does not take place in ROs, it would be prudent to remove such statements from the discussion. Without any supporting evidence, these statements will be misleading to readers by providing a false equivalency. The preceding discussion of RFs would be sufficient to contextualize your inchworm model in the broader viral life cycle (which was done quite well). *

• We have adjusted the discussion in the revised manuscript to avoid a false equivalency.

10. There were a number of aspects I appreciated about the manuscript and will briefly list a few here: ** i) the focus on how different non-structural proteins effect the structure and function of ** each other during the viral life cycle, which forms a more comprehensive and informative model ** ii) the use of structural and functional assays as complementary approaches to studying the intra- and inter-protein relationships of NS3 ** iii) the depiction of the forks in Figure 10, which effectively demonstrated the channels and oriented the reader to the conservation data ** *iv) the use of small molecule inhibitors to modify structure and function of NS3, which greatly deepened the richness of the story from both a basic and applied science view point *

• We are very grateful to the Reviewer for these kind remarks.

Reviewer #2 (Significance (Required)): ** Strengths and limitations: ** - provides some experimental and modeling data to provide a new model for RNA interactions with the NS3pro-hel; may help inform models for enzyme function, mostly consistent with previous literature ** - leaves out the NS5 RdRp, known to contribute to NS3 activity. ** - some suggestions are made which might strengthen the conclusions and inclusions of additional controls would improve the data. ** Advance ** - conceptual, perhaps may provide some insight into mechanism; although limited by the lack of NS5 RdRp which is crucial to helicase activity. It is unclear if the ssRNA would be oriented this way given interactions with NS5 RdRp and MT domains (is the ssRNA routed to NS5 or along NS3, or potentially are both happening?) ** Audience: ** - quite specialist, but may include structural biologists and virologist alike. ** Expertise of the reviewer(s): ** *- molecular virologists, RNA viruses - including flaviviruses; replication complex biogenesis, protein-RNA and RNA-RNA interactions. While comfortable with the concepts regarding complex formation, the appropriateness of computational modeling and RNA docking tools as well as structural biology is out of our area of expertise. *

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

*This paper investigates the nucleic acid binding properties of zika virus protease. In particular the data suggest that single stranded RNAs and DNAs are capable of binding to and inhibiting ZIKV protease at micromolar concentrations. With the use of active site inhibitors and mutants that lock the protease in closed and super-open conformation, the authors concluded that RNA binds to the open conformation. Through extensive modeling of the protease and helicase domains, this manuscript provides a model of how ssRNAs can bind to all conformations of the proteas, but the open conformation provides two positively charged forks that should be available to bind RNA. *

* SECTION A - Evidence, reproducibility, and clarity ** Major comments: **

*·The main conclusions of this paper rely on the existence of the super-open conformation, however this conformation has not been reported in the scientific literature previously. Structures deposited in the pdb are referenced in this manuscript, however no citation for an accompanying publication is provided. This calls into question the biological relevance of this super open conformation. This is of particular concern because in other highly-homologous flaviviral proteases, structures that have been observed crystallographically (e.g. the open conformation of dengue virus protease) appear to be only very sparsely populated in solution. What is the evidence that the super-open conformation exists in solution.

• Please, see our reply to question #1 from Reviewer 1.

• The activity of each of the constructs used was not reported making it impossible to directly compare the impact of these changes on intrinsic activity. In particular, the NS2B-NS3 long construct is predicted to exist in the super-open conformation. If this is correct, it should show no activity against a peptide substrate. *

• We appreciate these concerns. The NS2B-NS3pro-long construct is proteolytically active (only NS2B-NS3pro-short construct is proteolytically inactive because its NS3pro C-terminal part is too short to fold into the closed conformation). It is unconstrained and likely capable of adopting all possible conformations (closed, open, super open). As we suspected, the negatively charged linker interferes with RNA binding, potentially via direct competition. Investigating the role of the protease-helicase linker is an exciting subject of a separate manuscript in preparation.

• This paper reports that the IC50 is much weaker than the Kd for binding of ssRNA to ZIKV NS2B-NS3pro. Are orthogonal assays, such as thermal shift assay, available which could distinguish between the reported IC50 and the Kd. *

• Binding of ssRNA occurs in an area distinct from the protease active center. We think that there is a constant competition between C-terminal NS2B binding/release versus ssRNA binding/release from NS3pro. We think that ssRNA “catches” the moment when protease has the open conformation and freezes that conformation by blocking the C-terminal of NS2B from binding to NS3Pro. In terms of thermal shift assay, the structure of NS3Pro is changed, only the C-terminal of NS2B is affected. Note that the 15N R2 NMR signal from NS2B residues 65-85 is missing in bZiPro alone but re-appears when AcKR is added6. This is consistent with the idea that without AcKR, bZiPro exists in the open conformation where much of the C-terminal part of NS2B is dissociated from NS3Pro and remains unstructured, thus resulting in the lack of NMR signal. Taken together, these observations suggest that thermal shift assay is unlikely to be of much help.

• *This paper suggests that ssRNA binds to the open conformation of ZIKV NS2B-NS3pro, however no experimental evidence, only modeling has been used to suggest binding to the open conformation. In Dengue virus protease, the M84P variant has been reported to lock the protease into the open conformation. How does the F84P variant of ZIKV NS2B-NS3pro impact ssRNA binding? *

• We appreciate this question. Indeed, M84P mutation shifts Dengue NS3Pro to the open conformation, which is proteolytically inactive12, consistent with our reverse inchworm model. We have not investigated the effect of this mutation on ZIKV NS3pro. We expect this mutation has a similar effect in ZIKV NS3pro in Dengue NS3Pro.

• The relevance of the discussion on the co-crystallization of NSC86314 with the Mut7was not clear. What point was being made?

• We provide a proof-of-principle for a novel class of allosteric inhibitors that specifically target newly identified druggable pockets present in the open and super-open conformations of ZIKV NS2B-NS3pro. Our results suggest that such allosteric inhibitors can interfere with the RNA-binding activities of NS2B-NS3pro in addition to blocking the protease activity. The co-crystallization of NSC86314 with the Mut7 confirms a novel pocked bound by NSC86314.

*- These data show that both active site and allosteric inhibitors block binding of ssRNA to the protease. The paper also suggests that ssRNA only binds to the open conformation. What is the evidence that the allosteric inhibitors do not enable or promote formation of the open conformation? *

• We thank this reviewer for an interesting question. Indeed, we have no evidence of whether allosteric inhibitors enable or promote the formation of the open conformation. This is formally possible and will need to be investigated.

• This paper makes two claims about the function of the protease. The title should specify what those dual functions are (proteolytic activity and ssRNA-recruitment).*

• We appreciate this reviewer's suggestions for the title.

• The discussion of Figures 6 and 9 are highly similar. The main takeaway points for both figures seem to be nearly identical: the presence of two positively charged pitchfork on the open conformation. The distinction between these two figures should be more significantly and explicitly stated. *
• Figure 6 presents several models that provide evidence for the open conformation of ZIKV NS2B-NS3pro being uniquely suitable to bind RNA. Figure 9 presents several models of the entire RNA-NS2B-NS3pro-NS3hel complex anchored into the ER membrane. Figure 9 illustrates that the open conformation of NS2B-NS3pro provides two positively charged/polar forks, contiguous with the positively charged groove on NS3hel. Figure 6 does not illustrate that point.

*- Mention explicitly in the materials and methods if the 12-amino acid linker is present in all the mutants used. *

• This is mentioned explicitly and shown in Supplementary Figure 2A.

Minor comments: ** · Figure 1. The rotation that promotes the transitions from orientation in panel A to that in panel B should be drawn. ** · FAM should be defined in the legend of Figure 2. ** · The term Cold should be changed to unlabeled. ** · Please check labels for the supplementary Figure 2. For example one label states 1-1 but it ** should be 1-170. ** · Figure 1C does not exist and it is referenced in the results section under "NS2B-NS3pro substrate-mimicking inhibitors compete with RNA binding." ** · As discussed above, if the super open conformation is going to be addressed in this paper, then either a reference for the manuscript describing those structures should be included, or this manuscript should include in the materials and methods the procedure on crystallization, data collection, structure determination, refinement, and analysis as well as a table for crystallographic data and refinement statistics. ** · Adjust figure arrangement (ABCED to ABCDE) in Figure 11.

• We thank this reviewer for all minor comments. We corrected the above-mentioned errors in the manuscript.

Reviewer #3 (Significance (Required)): ** It is well established that the flaviviral proteases exist in different conformations but most of the structures published are concentrated on the closed conformation which is the one required for effective substrate processing. The open conformation has recently been the subject of increased interest, especially with the discovery of allosteric inhibitors for which modeling suggests that these compounds result in the dissociation of the C-terminal region of NS2B from the NS3. This paper adds important insights into the function of the open conformation and in general implicitly shows the importance of the dynamic nature of ZIKV NS2B-NS3pro. In addition to these insights, this paper aptly demonstrates that ssRNA can bind and inhibit these proteases as has not been shown previously. ** I am a senior graduate student working on characterizing and understanding the mechanism of action of allosteric compounds against viral proteases, specifically proteases from Zika and dengue viruses.

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