📜 A Future-Built Foundation 🧠 Ideas, Not Just Institutions Fifty years from then, the world was going to be very different. And they knew it.

The founders weren’t just building a country for their time— they were building for a future they couldn't fully imagine. 🌍

⚙️ Systems Over Statues So they created it not as a frozen monument, but as a living framework—a set of ideas, processes, and experiments designed to adapt. 🔧📐

Not bound to 1776. Not bound to kings, popes, or prophets. Bound only to the question:

“How do we do this better?”

🧪 The Great Experiment They didn’t aim for perfection. They aimed for a system capable of self-correction. 🔁

That’s the genius. That’s the gamble. That’s the reason we’re still arguing—because we can. 🗣️💬

Awakening in the Age of Crisis Before Vietnam, a Mirror Cracked In a time of crisis—just before the Vietnam War—people began to wake up. A large percentage of the population was being oppressed: Native Americans, Japanese Americans, Black Americans. Ethnicity, heritage, language—these became invisible bars in a quiet prison.

This rupture sparked a renewed interest in morality—what the country "stood for." How could we claim to be good, when so much of our foundation was rotten?

Ideals Tested by Time Some ideas held up. Some didn’t. And that’s important.

Education—real education—was one of those ideals that proved resilient. But how do we teach in a way that embraces everything? That compares and contrasts knowledge without filtering it through a single culture’s lens?

Jerome Bruner and the Cultural Blindspot Jerome Bruner pointed out something brutal and honest:

"It’s hard to assess your own culture—because it’s the only thing you see."

We don’t notice the walls until we bump into someone else’s. We only start to see when we encounter others. That’s when we begin to use stats, science, stories—tools that gather local solutions from many places.

Some are good. Some are pretty awful. But the point is: it’s not about always being right. It’s about being aware.

From Local Noise to Global Signal The story of history has always been like this—messy, partial, reactive.

But now, we have a chance to move: From narrow, local views—where we assume we’re always right— To a movement, a new place, where multiple truths can sit side by side.

Where perspective isn’t a threat—it’s a tool.

The language was important invention

Believes in the same type of religions and custums so that people that we ahvnet met are still recognizable wen we run into them it too thousands of years with the invention of art and language before you were talking you were making arting

hi my name is Alan Kay and I like to apologize for having a bit of laryngitis just on the day of this shoot and I've been asked to talk about inventing the future and of course we mostly think of inventing in the realm of technology but I think most people watching this will have been struck by the fact that living in the 21st century in the United States is a vastly different experience than living a hundred thousand years ago anywhere in the world and as far as we know the brains that we have are roughly the same as those brains that belong to the very same species we are mostly lived in small groups of people hunting and gathering and falling in love and telling stories to each other and fighting other people taking revenge caring for the young and gradually building up a culture that they taught to the next generation in their tribe and the first great invention of human beings or of evolution was this idea of culture and it came from a slightly earlier invention of evolution which was language and there's a language that just as a few important ways more different than our primate ancestors and that was enough to be able to deal with sequences of things and portrayals of things and being able to make up things which we no other animals can do but be able to tell our made-up things to other people and get them to believe in it that started to allow us to aggregate together in larger than about a hundred people which is what we can deal with face to face so this notion of culture beliefs in the same kinds of religions beliefs in the same kinds of customs is something that can spread so that people we've never met are still recognizable when we finally do run into them so we can think about that as the first great inventing the future for Humanity and it took many tens of thousands of years with the invention art which may have always been with us in between before we started taking stock of the world around us and starting

For a long time, inventions were accidental. The rock was an accident. The idea of improvement is a modern one. We weren’t built for a world that changes. Society programmed us to resist change—we just found ways to enjoy ourselves as much as possible.

But eventually, we began shifting from accidental inventions to intentional ones. And those early inventions—like writing—started changing everything.

Writing itself was invented for accounting. Gradually, people started writing down what others were saying. Then they began comparing it with what had happened before. And even that took thousands of years.

The real revolution came just in the last few hundred years—when people became self-conscious about how we deal with the rest of the world. We stopped trying to understand and started interpreting everything through our belief systems. That shift was key. The invention of science—true science—was transformational.

DOI: 10.1158/2159-8290.CD-24-0805

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What is this?

aca odria discutir que los cherelles de nuestro estudio se necrosaron y fueron cubiertos por la masa fungica

aqui habla sobre conidios y esporas y justifica que encontraron que mr se produce mediante mitosis

esto me podria servir para justificar la infeccion en mazorcas jovenes y la expresion de los sintomas

aca describe a la masa fungica como un polvo impalpable

reducciones de la produccion

esto me puede servir para mencionar que es mas detructiva que mazorca negra y escoba de bruja

porque en mis resultados algunas estan mas severas que otras, podria justificar que cada edad de mazorcas se cosecharon a la par con sus controles

Util puesto que habla de asintomatico y me puede servir para discutir mis resultados

Reviewer #1 (Public review):

Summary:

The manuscript investigates lipid scrambling mechanisms across TMEM16 family members using coarse-grained molecular dynamics (MD) simulations. While the study presents a statistically rigorous analysis of lipid scrambling events across multiple structures and conformations, several critical issues undermine its novelty, impact, and alignment with experimental observations.

Review on revised version:

The referee notes that the authors, in their response letter, have concurred with most of the concerns originally raised. Specifically, the authors acknowledge the referee's view that the manuscript primarily confirms previously reported findings and does not present a significantly novel advance, particularly regarding the central observation of groove-mediated lipid scrambling in the open Ca²⁺-bound TMEM16 structures. The authors have also acknowledged the potential discrepancies with existing experimental studies and have addressed this point candidly through additional discussion. Furthermore, the referee appreciates that the authors have echoed the concern regarding the limited statistical robustness of the observed scrambling events.<br /> Given that the authors have essentially affirmed the key points raised in the initial review, the referee believes that these acknowledgements reinforce the basis of the original assessment. Therefore, the referee maintains the original opinion that, despite its technical merits and useful discussion made in the revised version, the manuscript does not offer sufficient novelty or mechanistic depth.

Reviewer #3 (Public review):

Summary:

The paper investigates the TMEM16 family of membrane proteins, which play roles in lipid scrambling and ion transport. A total of 27 experimental structures from five TMEM16 family members were analyzed, including mammalian and fungal homologs (e.g., TMEM16A, TMEM16F, TMEM16K, nhTMEM16, afTMEM16). The identified structures were in both Ca²⁺-bound (open) and Ca²⁺-free (closed) states to compare conformations and were preprocessed (e.g., modeling missing loops) and equilibrated. Coarse-grain simulations were performed in DOPC membranes for 10 microseconds to capture the scrambling events. These events were identified by tracking lipids transitioning between the two membrane leaflets and they analysed correlation between scrambling rates, in addition, structural properties such as groove dilation and membrane thinning were calculated. They report 700 scrambling events across structures and the figure 2 elaborates on how open structures show higher activity, also as expected. The authors also address how structures may require open groove, this and other mechanisms around scrambling is a bit controversial in the field.

Strengths:

The strength of this study emerges from comparative analysis of multiple structural starting points and understand global/local motions of the protein with respect to lipid movement. Although the protein is well-studied, both experimentally and computationally, the understanding of conformational events in different family members, especially membrane thickness less compared to fungal scramblases offers good insights.

Weaknesses:

The weakness of the work is to fully reconcile with experimental evidence of Ca²⁺-independent scrambling rates observed in prior studies, but this part is also challenging using coarse-grain molecular simulations. Previous reports have identified lipid crossing, packing defects and other associated events, so it is difficult to place this paper in that context. However, the absence of validation leaves certain claims, like alternative scrambling pathways, speculative.

Author response:

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

We wanted to clarify Reviewer #1’s latest comment in the last round of review, “Furthermore, the referee appreciates that the authors have echoed the concern regarding the limited statistical robustness of the observed scrambling events.” We appreciate the follow up information provided from Reviewer #1 that their comment is specifically about the low count alternative pathway events that we view at the dimer interface, and not the statistics of the manuscript overall as they believe that “the study presents a statistically rigorous analysis of lipid scrambling events across multiple structures and conformations (Reviewer #1)”. We agree with the Reviewer and acknowledge that overall our coarse-grained study represents the most comprehensive single manuscript of the entire TMEM16 family to date.

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

Public Review:

Reviewer #1 (Public review):

Summary:

The manuscript investigates lipid scrambling mechanisms across TMEM16 family members using coarse-grained molecular dynamics (MD) simulations. While the study presents a statistically rigorous analysis of lipid scrambling events across multiple structures and conformations, several critical issues undermine its novelty, impact, and alignment with experimental observations.

Critical issues:

(1) Lack of Novelty:

The phenomenon of lipid scrambling via an open hydrophilic groove is already well-established in the literature, including through atomistic MD simulations. The authors themselves acknowledge this fact in their introduction and discussion. By employing coarse-grained simulations, the study essentially reiterates previously known findings with limited additional mechanistic insight. The repeated observation of scrambling occurring predominantly via the groove does not offer significant advancement beyond prior work.

We agree with the reviewer’s statement regarding the lack of novelty when it comes to our observations of scrambling in the groove of open Ca2+-bound TMEM16 structures. However, we feel that the inclusion of closed structures in this study, which attempts to address the yet unanswered question of how scrambling by TMEM16s occurs in the absence of Ca2+, offers new observations for the field. In our study we specifically address to what extent the induced membrane deformation, which has been theorized to aid lipids cross the bilayer especially in the absence of Ca2+, contributes to the rate of scrambling (see references 36, 59, and 66). There are also several TMEM16F structures solved under activating conditions (bound to Ca2+ and in the presence of PIP2) which feature structural rearrangements to TM6 that may be indicative of an open state (PDB 6P48) and had not been tested in simulations. We show that these structures do not scramble and thereby present evidence against an out-of-the-groove scrambling mechanism for these states. Although we find a handful of examples of lipids being scrambled by Ca2+-free structures of TMEM16 scramblases, none of our simulations suggest that these events are related to the degree of deformation.

(2) Redundancy Across Systems:

The manuscript explores multiple TMEM16 family members in activating and non-activating conformations, but the conclusions remain largely confirmatory. The extensive dataset generated through coarse-grained MD simulations primarily reinforces established mechanistic models rather than uncovering fundamentally new insights. The effort, while statistically robust, feels excessive given the incremental nature of the findings.

Again, we agree with the reviewer’s statement that our results largely confirm those published by other groups and our own. We think there is however value in comparing the scrambling competence of these TMEM16 structures in a consistent manner in a single study to reduce inconsistencies that may be introduced by different simulation methods, parameters, environmental variables such as lipid composition as used in other published works of single family members. The consistency across our simulations and high number of observed scrambling events have allowed us to confirm that the mechanism of scrambling is shared by multiple family members and relies most obviously on groove dilation.

(3) Discrepancy with Experimental Observations:

The use of coarse-grained simulations introduces inherent limitations in accurately representing lipid scrambling dynamics at the atomistic level. Experimental studies have highlighted nuances in lipid permeation that are not fully captured by coarse-grained models. This discrepancy raises questions about the biological relevance of the reported scrambling events, especially those occurring outside the canonical groove.

We thank the reviewer for bringing up the possible inaccuracies introduced by coarse graining our simulations. This is also a concern for us, and we address this issue extensively in our discussion. As the reviewer pointed out above, our CG simulations have largely confirmed existing evidence in the field which we think speaks well to the transferability of observations from atomistic simulations to the coarse-grained level of detail. We have made both qualitative and quantitative comparisons between atomistic and coarse-grained simulations of nhTMEM16 and TMEM16F (Figure 1, Figure 4-figure supplement 1, Figure 4-figure supplement 5) showing the two methods give similar answers for where lipids interact with the protein, including outside of the canonical groove. We do not dispute the possible discrepancy between our simulations and experiment, but our goal is to share new nuanced ideas for the predicted TMEM16 scrambling mechanism that we hope will be tested by future experimental studies.

(4) Alternative Scrambling Sites:

The manuscript reports scrambling events at the dimer-dimer interface as a novel mechanism. While this observation is intriguing, it is not explored in sufficient detail to establish its functional significance. Furthermore, the low frequency of these events (relative to groove-mediated scrambling) suggests they may be artifacts of the simulation model rather than biologically meaningful pathways.

We agree with the reviewer that our observed number of scrambling events in the dimer interface is too low to present it as strong evidence for it being the alternative mechanism for Ca2+-independent scrambling. This will require additional experiments and computational studies which we plan to do in future research. However, we are less certain that these are artifacts of the coarse-grained simulation system as we observed a similar event in an atomistic simulation of TMEM16F.

Conclusion:

Overall, while the study is technically sound and presents a large dataset of lipid scrambling events across multiple TMEM16 structures, it falls short in terms of novelty and mechanistic advancement. The findings are largely confirmatory and do not bridge the gap between coarse-grained simulations and experimental observations. Future efforts should focus on resolving these limitations, possibly through atomistic simulations or experimental validation of the alternative scrambling pathways.

Reviewer #2 (Public review):

Summary:

Stephens et al. present a comprehensive study of TMEM16-members via coarse-grained MD simulations (CGMD). They particularly focus on the scramblase ability of these proteins and aim to characterize the "energetics of scrambling". Through their simulations, the authors interestingly relate protein conformational states to the membrane's thickness and link those to the scrambling ability of TMEM members, measured as the trespassing tendency of lipids across leaflets. They validate their simulation with a direct qualitative comparison with Cryo-EM maps.

Strengths:

The study demonstrates an efficient use of CGMD simulations to explore lipid scrambling across various TMEM16 family members. By leveraging this approach, the authors are able to bypass some of the sampling limitations inherent in all-atom simulations, providing a more comprehensive and high-throughput analysis of lipid scrambling. Their comparison of different protein conformations, including open and closed groove states, presents a detailed exploration of how structural features influence scrambling activity, adding significant value to the field. A key contribution of this study is the finding that groove dilation plays a central role in lipid scrambling. The authors observe that for scrambling-competent TMEM16 structures, there is substantial membrane thinning and groove widening. The open Ca2+-bound nhTMEM16 structure (PDB ID 4WIS) was identified as the fastest scrambler in their simulations, with scrambling rates as high as 24.4 {plus minus} 5.2 events per μs. This structure also shows significant membrane thinning (up to 18 Å), which supports the hypothesis that groove dilation lowers the energetic barrier for lipid translocation, facilitating scrambling.

The study also establishes a correlation between structural features and scrambling competence, though analyses often lack statistical robustness and quantitative comparisons. The simulations differentiate between open and closed conformations of TMEM16 structures, with open-groove structures exhibiting increased scrambling activity, while closed-groove structures do not. This finding aligns with previous research suggesting that the structural dynamics of the groove are critical for scrambling. Furthermore, the authors explore how the physical dimensions of the groove qualitatively correlate with observed scrambling rates. For example, TMEM16K induces increased membrane thinning in its open form, suggesting that membrane properties, along with structural features, play a role in modulating scrambling activity.

Another significant finding is the concept of "out-of-the-groove" scrambling, where lipid translocation occurs outside the protein's groove. This observation introduces the possibility of alternate scrambling mechanisms that do not follow the traditional "credit-card model" of groove-mediated lipid scrambling. In their simulations, the authors note that these out-of-the-groove events predominantly occur at the dimer interface between TM3 and TM10, especially in mammalian TMEM16 structures. While these events were not observed in fungal TMEM16s, they may provide insight into Ca2+-independent scrambling mechanisms, as they do not require groove opening.

Weaknesses:

A significant challenge of the study is the discrepancy between the scrambling rates observed in CGMD simulations and those reported experimentally. Despite the authors' claim that the rates are in line experimentally, the observed differences can mean large energetic discrepancies in describing scrambling (larger than 1kT barrier in reality). For instance, the authors report scrambling rates of 10.7 events per μs for TMEM16F and 24.4 events per μs for nhTMEM16, which are several orders of magnitude faster than experimental rates. While the authors suggest that this discrepancy could be due to the Martini 3 force field's faster diffusion dynamics, this explanation does not fully account for the large difference in rates. A more thorough discussion on how the choice of force field and simulation parameters influence the results, and how these discrepancies can be reconciled with experimental data, would strengthen the conclusions. Likewise, rate calculations in the study are based on 10 μs simulations, while experimental scrambling rates occur over seconds. This timescale discrepancy limits the study's accuracy, as the simulations may not capture rare or slow scrambling events that are observed experimentally and therefore might underestimate the kinetics of scrambling. It's however important to recognize that it's hard (borderline unachievable) to pinpoint reasonable kinetics for systems like this using the currently available computational power and force field accuracy. The faster diffusion in simulations may lead to overestimated scrambling rates, making the simulation results less comparable to real-world observations. Thus, I would therefore read the findings qualitatively rather than quantitatively. An interesting observation is the asymmetry observed in the scrambling rates of the two monomers. Since MARTINI is known to be limited in correctly sampling protein dynamics, the authors - in order to preserve the fold - have applied a strong (500 kJ mol-1 nm-2) elastic network. However, I am wondering how the ENM applies across the dimer and if any asymmetry can be noticed in the application of restraints for each monomer and at the dimer interface. How can this have potentially biased the asymmetry in the scrambling rates observed between the monomers? Is this artificially obtained from restraining the initial structure, or is the asymmetry somehow gatekeeping the scrambling mechanism to occur majorly across a single monomer? Answering this question would have far-reaching implications to better describe the mechanism of scrambling.

The main aim of our computational survey was to directly compare all relevant published TMEM16 structures in both open and closed states using the Martini 3 CGMD force field. Our standardized simulation and analysis protocol allowed us to quantitatively compare scrambling rates across the TMEM16 family, something that has never been done before. We do acknowledge that direct comparison between simulated versus experimental scrambling rates is complicated and is best to be interpreted qualitatively. In line with other reports (e.g., Li et al, PNAS 2024), lipid scrambling in CGMD is 2-3 orders of magnitude faster than typical experimental findings. In the CG simulation field, these increased dynamics due to the smoother energy landscape are a well known phenomenon. In our view, this is a valuable trade-off for being able to capture statistically robust scrambling dynamics and gain mechanistic understanding in the first place, since these are currently challenging to obtain otherwise. For example, with all-atom MD it would have been near-impossible to conclude that groove openness and high scrambling rates are closely related, simply because one would only measure a handful of scrambling events in (at most) a handful of structures.

Considering the elastic network: the reviewer is correct in that the elastic network restrains the overall structure to the experimental conformation. This is necessary because the Martini 3 force field does not accurately model changes in secondary (and tertiary) structure. In fact, by retaining the structural information from the experimental structures, we argue that the elastic network helped us arrive at the conclusion that groove openness is the major contributing factor in determining a protein’s scrambling rate. This is best exemplified by the asymmetric X-ray structure of TMEM16K (5OC9), in which the groove of one subunit is more dilated than the other. In our simulation, this information was stored in the elastic network, yielding a 4x higher rate in the open groove than in the closed groove, within the same trajectory.

Notably, the manuscript does not explore the impact of membrane composition on scrambling rates. While the authors use a specific lipid composition (DOPC) in their simulations, they acknowledge that membrane composition can influence scrambling activity. However, the study does not explore how different lipids or membrane environments or varying membrane curvature and tension, could alter scrambling behaviour. I appreciate that this might have been beyond the scope of this particular paper and the authors plan to further chase these questions, as this work sets a strong protocol for this study. Contextualizing scrambling in the context of membrane composition is particularly relevant since the authors note that TMEM16K's scrambling rate increases tenfold in thinner membranes, suggesting that lipid-specific or membrane-thickness-dependent effects could play a role.

Considering different membrane compositions: for this study, we chose to keep the membranes as simple as possible. We opted for pure DOPC membranes, because it has (1) negligible intrinsic curvature, (2) forms fluid membranes, and (3) was used previously by others (Li et al, PNAS 2024). As mentioned by the reviewer, we believe our current study defines a good, standardized protocol and solid baseline for future efforts looking into the additional effects of membrane composition, tension, and curvature that could all affect TMEM16-mediated lipid scrambling.

Reviewer #3 (Public review):

Strengths:

The strength of this study emerges from a comparative analysis of multiple structural starting points and understanding global/local motions of the protein with respect to lipid movement. Although the protein is well-studied, both experimentally and computationally, the understanding of conformational events in different family members, especially membrane thickness less compared to fungal scramblases offers good insights.

We appreciate the reviewer recognizing the value of the comparative study. In addition to valuable insights from previous experimental and computational work, we hope to put forward a unifying framework that highlights various TMEM16 structural features and membrane properties that underlie scrambling function.

Weaknesses:

The weakness of the work is to fully reconcile with experimental evidence of Ca²⁺-independent scrambling rates observed in prior studies, but this part is also challenging using coarse-grain molecular simulations. Previous reports have identified lipid crossing, packing defects, and other associated events, so it is difficult to place this paper in that context. However, the absence of validation leaves certain claims, like alternative scrambling pathways, speculative.

Answer: It is generally difficult to quantitatively compare bulk measurements of scrambling phenomena with simulation results. The advantage of simulations is to directly observe the transient scrambling events at a spatial and temporal resolution that is currently unattainable for experiments. The current experimental evidence for the precise mechanism of Ca2+-independent scrambling is still under debate. We therefore hope to leverage the strength of MD and statistical rigor of coarse-grained simulations to generate testable hypotheses for further structural, biochemical, and computational studies.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

The findings are largely confirmatory and do not bridge the gap between coarse-grained simulations and experimental observations. Future efforts should focus on resolving these limitations, possibly through atomistic simulations or experimental validation of the alternative scrambling pathways.

While we agree with what the reviewer may be hinting at regarding limitations of coarse-grained MD simulations, we believe that our study holds much more merit than this comment suggests. We have provided something that has yet to be done in the field: a comprehensive study that directly compares the scrambling rates of multiple TMEM16 family members in different conformations using identical simulation conditions. Our work clearly shows that a sufficiently dilated grooves is the major structural feature that enables robust scrambling for all TMEM16 scramblases members with solved structures. While all TMEM16s cause significant distortion and thinning of the membrane, we assert that the extreme thinning observed around open grooves is significantly enhanced by the lipid scrambling itself as the two leaflets merge through lipid exchange.  We saw no evidence that membrane thinning/distortion alone, in the absence of an open groove, could support scrambling at the rates observed under activating conditions or even the low rates observed in Ca2+-independent scrambling. Moreover, our handful of observations of scrambling events outside of the groove, which has not yet been reported in any study, opens an exciting new direction for studying alternative scrambling mechanisms. That said, we are currently following up on many of the observations reported here such as: scrambling events outside the groove, the kinetics of scrambling, the possibility that lipids line the groove of non-scramblers like TMEM16A, etc. This is being done experimentally with our collaborators through site directed mutagenesis and with all-atom MD in our lab. Unfortunately, it is well beyond the scope of the current study to include all of this in the current paper.

Reviewer #2 (Recommendations for the authors):

Major comments and questions:

(1) Line 214 and Figure 1- Figure Supplement 1: why have you only compared the final frame of the trajectory to the cryo-EM structure? Even if these comparisons are qualitative, they should be representative of the entire trajectory, not a single frame.

We thank the reviewer for this suggestion and replaced the single-frame snapshots in Figure 1-figure supplement 1 for ensemble-averaged head groups densities. The overall agreement between membrane shapes in CGMD and cryo-EM was not affected by this change.

(2) Lines 228-231: You comment 'Residues in this site on nhTMEM16 and TMEMF also seem to play a role in scrambling but the mechanism by which they do so is unclear.' This is something you could attempt to quantify in the simulations by calculating the correlation between scrambling and protein-membrane interactions/contacts in this site. Can you speculate on a mechanism that might be a contributing factor?

We probed the correlation between these residues and scrambling lipids, as suggested by the reviewer, and interestingly not all scrambling lipids interact with these residues. Yet there is strong lipid density in this vicinity (see insets in Figure 1 and Figure 4-figure supplement 2). These observations lead us to suspect these residues impact scrambling indirectly through influencing the conformation of the protein or flexibility and shape of the membrane. This interpretation fits with mutagenesis studies highlighting a role for these residues in scrambling (see refs 59, 62, and 67). Specifically, Falzone et al. 2022 (ref 59) suggested that they may thin the membrane near the groove, but this has not been tested via structure determination and a detailed model of how they impact scrambling is missing. We could address this question with in silico mutations; however, CG simulation is not an appropriate method to study large scale protein dynamics, and AA simulations are likely best, but beyond the scope of this paper.

(3) Lines 240-245 and Figure 1B: This section discusses the coupling between membrane distortions and the sinusoidal curve around the protein, however, Figure 1B only shows snapshots of the membrane distortions. Is it possible to understand how these two collective variables are correlated quantitatively (as opposed to the current qualitative analysis)?

We believe that it may be possible to quantitatively capture these two key features of the membrane, as we did previously with nhTMEM16 using our continuum elasticity-based model of the membrane (Bethel and Grabe 2016). Our model agreed with all atom MD surfaces to within ~1 Å, hence showing good quantitative agreement throughout the entire membrane. However, we doubt that we could distill the essence of our model down to a simple functional relationship between the sinusoidal wave and pinching, which we think the reviewer is asking. Rather, we believe that the large-scale sinusoidal distortion (collective variable 1) and pinching/distortion (collective variable 2) near the groove arise from the interplay of the specific protein surface chemistry for each protein (patterning of polar and non-polar residues) and the membrane. This is why we chose to simply report the distinct patterns that the family members impose on the surrounding membrane, which we think is fascinating. Specifically, Fig. 1B shows that different TMEM16 family members distort the membrane in different ways. Most notably, fungal TMEM16s feature a more pronounced sinusoidal deformation, whereas the mammalian members primarily produce local pinching. Then, in Fig. 3A we show that the thinning at the groove happens in all structures and is more pronounced in open, scrambling-competent conformations. In other words, proteins can show very strong thinning (e.g. TMEM16K, 5OC9) even though the membrane generally remains flat.

(4) Lines 257-258: Authors comment that TMEM16A lacks scramblase activity yet can achieve a fully lipid-lined groove (note the typo - should be lipid-lined, not lipid-line). Is a fully lipid-lined groove a prerequisite for scramblase activity? Are lipid-lined grooves the only requirement for scramblase activity? Could the authors clarify exactly what the prerequisite for scramblase activity is to avoid any confusion; this will be useful for later descriptions (i.e. line 295) where scrambling competence is again referred to. Additionally, the associated figure panel (Figure 1D) shows a snapshot of this finding but lacks any statistical quantifications - is a fully lipid-lined groove a single event? Perhaps the additional analyses, such as the groove-lipid contacts, may be useful here.

The definition of lipid scrambling is that a lipid fully transitions from one membrane leaflet to the other. While a single lipid could transition through the groove on its own, it is well documented in both atomistic and CG MD simulations, that lipid scrambling typically happens through a lipid-lined groove, as shown in Fig. 1A-B. The lipids tend to form strong choline-to-phosphate interactions with nearest neighbors that make this energetically favorable. That said, lipid-lined grooves are not sufficient for robust scrambling, which is what we show in Fig. 1D where the non-scrambler TMEM16A did in fact feature a lipid-lined groove. As suggested, we performed contact analysis and found that residue K645 on TM6 in the middle of the groove contacts lipids in 9.2% of the simulation frames.

To get a better understanding of how populated the TM4-TM6 pathway is with lipids across all simulated structures, we determined for every simulation frame how many headgroup beads resided in the groove. This indicates that the ion-conductive state of TMEM16A (5OYB*, Fig. 1D) only had 1 lipid in the pathway, on average, meaning that the configuration shown Fig. 1D is indeed exceptional. As a reference, our strongest scrambler nhTMEM16 4WIS, had an average of 2.8 lipids in the groove. We added a table containing the means and standard deviations that resulted from this analysis as Figure 1-Table supplement 1.

(5) Lines 295-298 : The scrambling rates of the Ca²⁺-bound and Ca²⁺-free structures fall within overlapping error margins, it becomes difficult to definitively state that Ca²⁺ binding significantly enhances scrambling activity. This undermines the claim that the Ca²⁺-bound structure is the strongest scrambler. The authors should conduct statistical analyses to determine if the difference between the two conditions is statistically significant.

In contrast to the reviewer’s comment, we do not claim that Ca2+-binding itself enhances lipid scrambling. Instead, what we show is that WT structures that are solved in an open confirmation (all of which are Ca2+-bound, except 6QM6) are robust scramblers. For nhTMEM16, we did not observe any scrambling events for the closed-groove proteins, making further statistical analysis redundant.

(6) The authors claim that the scrambling rates derived from their MD simulations are in "excellent agreement" with experimental findings (lines 294-295), despite significant discrepancy between simulated and experimentally measured rates. For example, the simulated rate of 24.4 {plus minus} 5.2 events/µs for the open, Ca²⁺-bound fungal nhTMEM16 (PDB ID 4WIS) corresponds to approximately 24 million events per second, which is vastly higher than experimental rates. Experimental studies have reported scrambling rate constants of ~0.003 s⁻¹ for TMEM16 family members in the absence of Ca²⁺, measured under physiological conditions (https://doi.org/10.1038/s41467-019-11753-1 ). Even with Ca²⁺ activation, scrambling rates remain several orders of magnitude lower than the rates observed in simulations. Moreover, this highlights a larger problem: lipid scrambling rates occur over timescales that are not captured by these simulations. While the authors elude to these discrepancies (lines 605-606), they should be emphasised in the text, as opposed to the table caption. These should also be reconducted to differences between the membrane compositions of different studies.

We agree with the spirit of the reviewer’s comment, and because of that, we were very careful not to claim that we reproduce experimental scrambling rates, just that the trends (scrambling-competent, or not) are correct. On lines 294-295, we actually said that the scrambling rates in our simulations excellently agree with “the presumed scrambling competence of each experimental structure”, which is true. 

As explained extensively in the discussion section of our paper (and by many others), direct comparison between MD (e.g., Martini 3, but also atomistic force fields) dynamics and experimental measurements is challenging. The primary goal of our paper is to quantify and compare the scrambling capacity of different TMEM16 family members and different states, within a CGMD context.

That said, we agree with the reviewer that we may have missed rare or long-timescale events (as is the case in any MD experiment) and added this point to the discussion.

(7) To address these discrepancies, the authors should: i) emphasize that simulated rates serve as qualitative indicators of scrambling competence rather than absolute values comparable to experimental findings and ii) discuss potential reasons for the divergence, such as simulation timescale limitations or lipid bilayer compositions that may favor scrambling and force field inaccuracies.

Please see our answer to question 6. Within the context of our CGMD survey, we confidently call our results quantitative. However, we agree with the reviewer that comparison with experimental scrambling rates is qualitative and should be interpreted with caution. To reflect this, we rewrote the first sentence of the relevant paragraph in the discussion section.

(8) Line 310: Can the authors provide a rationale as to why one monomer has a wider groove than the other? Perhaps a contact analysis could be useful. See the comment above about ENM.

The simulation of Ca2+-bound TMEM16K was initiated from an asymmetric X-ray structure in which chain B features a more dilated groove than chain A (PDB 5OC9). The backbones of TM4 and TM6 in the closed groove (A) are close enough together to be directly interconnected by the elastic network. In contrast, TM4 and TM6 in the more dilated subunit (B) are not restricted by the elastic network and, as a consequence, display some “breathing” behavior (Fig. 3B and Fig. 3-Suppl. 6A), giving rise to a ~4x higher scrambling rate. We explicitly added the word “cryo-EM” and the PDB ID to the sentence to emphasize that the asymmetry stems from the original experimental structure.

When answering this question, we also corrected a mislabeled chain identifier which was in the original manuscript ‘chain A’ when it is actually ‘chain B’ in Fig.2-Suppl. 3A.

(9) Line 312: Authors speculate that increased groove width likely accounts for increased scrambling rates. For statistical significance, authors should attempt to correlate scrambling rates and groove width over the simulation period.

The Reviewer is referring to our description of scrambling rates we measured for TMEM16K where we noted that on average the groove with the highest scrambling rate is also on average wider than the opposite subunit which is below 6 Å. We do not suggest that the correlation between scrambling and groove width is continuous, as the Reviewer may have interpreted from our original submission, but we think it is a binary outcome – lipids cannot easily enter narrow grooves (< 6 Å) and hence scrambling can only occur once this threshold is reached at which point it occurs at a near constant rate. We showed this for 4 different family members in the original Fig. 3B, where scrambling events (black dots) were much more likely during, or right after, groove dilation to distances > 6 Å. 

(10) Line 359: Authors have plotted the minimum distance between residues TM4 and TM6 in Fig. 3A/B, claiming that a wide groove is required for scrambling. Upon closer examination, it is clear that several of these distributions overlap, reducing the statistical significance of these claims. Statistical tests (i.e. KS-tests) should be performed to determine whether the differences in distributions are significant.

The Reviewer appears to be asking for a statistical test between the six distance distributions represented by the data in Fig. 3A for the scrambling competent structures (6QP6*, 8B8J, 6QM6, 7RXG, 4WIS, 5OC9), and we think this is being asked because it is believed that we are making a claim that the greater the distance, the greater the scrambling rate. If we have interpreted this comment correctly, we are not making this claim. Rather, we are simply stating that we only observe robust scrambling when the groove width regularly separates beyond 6 Å. The full distance distributions can now be found in Figure 3-figure supplement 6B, and we agree there is significant overlap between some of these distributions. However, the distinguishing characteristic of the 6 distributions from scrambling competent proteins is that they all access large distances, while the others do not. Notably, TMEM16F proteins (6QP6*, 8B8J) are below the 6 Å threshold on average, but they have wide standard deviations and spend well over ¼ of their time in the permissive regime (the upper error bar in the whisker plots in Fig. 3A is the 75% boundary).

(11) Line 363-364: The authors state that all TMEM16 structures thin the membrane. Could the authors include a description of how membrane thinning is calculated, for instance, is the entire membrane considered, or is thinning calculated on a membrane patch close to the protein? Do membrane patches closer to the transmembrane protein increase or decrease thickness due to hydrophobic packing interactions? The latter question is of particular concern since Martini3 has been shown to induce local thinning of the membrane close to transmembrane helices, yielding thicknesses 2-3 Å thinner than those reported experimentally (https://doi.org/10.1016/j.cplett.2023.140436). This could be an important consideration in the authors' comparison to the bulk membrane thickness (line 364). Finally, how is the 'bulk membrane thickness' measured (i.e., from the CG simulations, from AA simulations, or from experiments)?

Regarding the calculation of thinning and bulk membrane thickness, as described in Method “Quantification of membrane deformations”, the minimal membrane thickness, or thinning, is defined as the shortest distance between any two points from the interpolated upper and lower leaflet surfaces constructed using the glycerol beads (GL1 and GL2). Bulk membrane thickness is calculated by taking the vertical distance between the averaged glycerol surfaces at the membrane edge.

The concern of localized membrane deformation due to force field artifacts is well-founded. However, the sinusoidal deformations shown here are much greater than 2-3 Å Martini3 imperfections, and they extend for up to 10 Å radially away from the protein into the bulk membrane (see Figure 3-figure supplement 1-5 for more of a description). Most importantly, the sinusoidal wave patterns set up by the proteins is very similar to those described in the previous continuum calculation and all-atom MD for nhTMEM16 (https://www.pnas.org/doi/full/10.1073/pnas.1607574113).

(12) Line 374: The authors state a 'positive correlation' between membrane thinning/groove opening and scrambling rates. To support this claim, the authors should report. the correlation coefficients.

We have removed any discussion concerning correlations between the magnitude of the scrambling rate and the degree of membrane thinning/groove opening. Rather we simply state that opening beyond a threshold distance is required for robust scrambling, as shown in our analysis in Fig. 3A.

Concerning the relation between thinning and scrambling: Instantaneous membrane thinning is poorly defined (because it is governed by fluctuations of single lipids), and therefore difficult to correlate with the timing of individual scrambling events in a meaningful way.  Moreover, as we state later in that same section, “we argue that the extremely thin membranes are likely correlated with groove opening, rather than being an independent contributing factor to lipid scrambling”.

(13) Line 396: It is stated that TMEM16A is not a scramblase but the simulating scrambling activity is not zero. How can you be sure that you are monitoring the correct collective variable if you are getting a false positive with respect to experiments?

We only observe 2 scrambling events in 10 ms, which is a very small rate compared to the scrambling competent states. In a previous large survey Martini CG simulation study that inspired our protocol (Li et al, PNAS 2024), they employed a 1 event/ms cut-off to distinguish scramblers from non-scramblers. Hence, they would have called TMEM16A a non-scrambler as well. We expect that false negatives in this context might be an artifact of the CG forcefield, or it could be that TMEM16A can scramble but too slowly to be experimentally detected. Regarding the collective variable for lipid flipping, it is correct, and we know that this lipid actually flipped.

(14) Line 402: Distance distributions for the electrostatic interactions between E633 and K645 should be included in the manuscript. This is also the case for the interactions between E843-K850 (lines 491-492).

Our description of interactions between lipid headgroups and E633 and K645 in TMEM16A (5OYB*) are based on qualitative observations of the MD trajectory, and we highlight an example of this interaction in Figure 3-video 4. The video clearly shows that the lipid headgroups in the center of the groove orient themselves such that the phosphate bead (red) rests just above K645 (blue) and at other times the choline bead (blue) rests just below E633 (red). We do not think an additional plot with the distance distributions between lipids and these residues will add to our understanding of how lipids interact residues in the TMEM16A pore.

We made a similar qualitative observation for the interaction between the POPC choline to E843 and POPC phosphate to K850 while watching the AAMD simulation trajectory of TMEM16F (PDB ID 6QP6). Given that this was a single observation, and the same interactions does not appear in CG simulation of the same structure (see simulation snapshots in Figure 4-figure supplement 5) we do not think additional analysis would add significantly to our understanding of which residues may stabilize lipids in the dimer interface.

(15) Lines 450-451: 'As the groove opens, water is exposed to the membrane core and lipid headgroups insert themselves into the water-filled groove to bridge the leaflets.' Is this a qualitative observation? Could the authors report the correlation between groove dilation and the number of water permeation events?

Yes, this is qualitative, and it sketches the order of events during scrambling, and we revised the main text starting at line 450 to indicate this. As illustrated by the density isosurfaces in Appendix 1-Figure 2A, the amount of water found in the closed versus open grooves is striking – there is a significant flood of water that connects the upper and lower solutions upon groove opening. Moreover, Appendix 1-Figure 2B shows much greater water permeation for open structures (4WIS, 7RXG, 5OC9, 8B8J, …) compared to closed structures (6QMB, 6QMA, 8B8Q, and many of the non-labeled data in the figure that all have closed grooves and near 0 water permeation). A notable exception is TMEM16A (7ZK3*8), which has water permeation but a closed groove and little-to-no lipid scrambling.

Minor Comments:

(1) Inconsistent use of '10' and 'ten' throughout.

We like to kindly point out that we do not find examples of inconsistent use.

(2) Line 32: 'TM6 along with 3, 4 and 5...' should be 'TM6 along with TM3, TM4 and TM5...'. Same in line 142. Naming should stay consistent.

Changes are reflected in the updated manuscript.

(3) Line 141: do you mean traverse (i.e. to travel across)? Or transverse (i.e. to extend across the membrane)?

This is a typo. We meant “traverse”. Thanks for pointing it out.

(4) Line 142: 'greasy' should be 'strongly hydrophobic'.

Changes are reflected in the updated manuscript.

(5) Line 143-144: "credit card mechanism" requires quotation marks.

Changes are reflected in the updated manuscript.

(6) Line 144: state if Nectria haematococca is mammalian or fungal, this is not obvious for all readers.

Changes are reflected in the updated manuscript.

(7) Line 147-148: Is TMEM16A/TMEM16K fungal or mammalian? What was the residue before the mutation and which residue is mutated? Perhaps the nomenclature should read as TMEM16X10Y where X=the residue prior to the mutation, 10 is a placeholder for the residue number that is mutated and Y=the new residue following mutation.

“TMEM16” is the protein family. “A” denotes the specific homolog rather than residue.  

(8) Lines 157-158: same as 10, it is unclear if these are fungal or mammalian.

Clarifications added.

(9) Line 184: "...CGMD simulation" should be "...CGMD simulations".

Changes made.

(10) Line 191-192: It would help to create a table of all of the mutants (including if they are mammalian or fungal) summarizing the salt concentrations, lipid and detergent environments, the presence of modulators/activators, etc.

We added this information to Appendix 1-Table 1 in the supplemental information. We did not specify NaCl concentrations, because they all experimental procedures used standard physiological values for this (100-150 mM).

(11) Line 210: inconsistencies with 'CG' and 'coarse-grain'.

Changes made.

(12) Figure 1 caption: '...totaling ~2μs (B)...' is missing the fullstop after 2μs.

Changes made.

(13) Figure 1B: it may be useful to label where the Ca2+ ion binds or include a schematic.

We updated Fig. 1A to illustrate where Ca2+ binds.

(14) Line 311: Are these mean distances? The authors should add standard deviations.

Yes, they are. We added the standard deviations to the text.

(15) Line 321-322: Perhaps a schematic in Figure 2 would be useful to visualize the structural features described here.

We would kindly refer interested readers to reference [60].

(16) Line 377: '...are likely a correlate of groove opening...' should read as: '...are likely correlated to groove opening...'.

Thank you for pointing it out. Changes made.

(17) Line 398: the '...empirically determined 6Å threshold for scrambling.' Was this determined from the simulations or from experiments? What does "empirically" mean here? Please state this.

This value was determined from the simulations. Based on our analysis of the correlation between scrambling rate and groove dilation, we found that the minimal TM4/6 distance of 6 Å can distinguish between the high and low activity scramblers. The exact numerical value is somewhat arbitrary as there is a range of values around 6 Å that serve to distinguish scramblers from non-scramblers.

(18) Figure 4: This figure should be labelled as A, B, C and D, with the figure caption updated accordingly.

We updated Figure 4 and its caption.

Reviewer #3 (Recommendations for Authors):

The authors must do additional simulations to further validate their claim with different lipids and further substantiate dimer interface independent of Ca2+ ions.

Thank you for the suggestion. We completely agree that studying scrambling in the context of a diverse lipid environment is an exciting area to explore. We are indeed actively working on a project that shares the similar idea. We decided not to include that study because we think the additional discussion involved would be excessive for the current manuscript. We, however, look forward to publishing our findings in a separate manuscript in the near future. In terms of Ca2+-independent scrambling, we are planning with our experimental collaborator for mutagenesis studies that target the residues we identified along the dimer interface.

Since calcium ions are critical for the stability of these structures, authors should show that they were placed throughout the simulations consistently.

As stated in the method section “Coarse-grained system preparation and simulation detail”, all Ca2+ ions are manually placed into the coarse-grained structure from the beginning of the simulation at their identical corresponding position in the experimental structure and harmonically bonded to adjacent acidic residues throughout the duration of simulation. We have also added a label to Fig 1A to indicate where the two Ca2+ ions are located.

The comparison with experimental structures should be consistent with complete simulation, and not the last structure of the trajectory. Depending on the conformational variability, this might be misleading.

We agree and updated Fig. 1-supplement figure 1 accordingly. The overall agreement between membrane shapes in CGMD and cryo-EM was not affected by this change.

ha, and then

I think this was mentioned in a previous chapter, maybe when building stand and stock tables. Perhaps it's a good idea to mention it again, but I thought I'd point it out.

Reestruturar sessão para dar uma visão de que esse é o valor original, mas que faremos uma grande oferta na abertura da turma no dia 07

Trocar a imagem, já usamos ela muitas vezes

Podemos retirar essa parte da sessão

• Posicionamento da sessão
• Os ícones deram uma cara de LP de cursinho (Não passam seriedade)

Não curti essa diagramação e a cor azul

Tirar a parte em roxo

Acharmos uma forma de colocar "Uma nova versão" da pós-graduação

Colocaria o "também" na frente de "podemos"

Reviewer #3 (Public review):

Summary:<br /> Membrane-bound pyrophosphatases (mPPases) are homodimeric proteins that hydrolyze pyrophosphate and pump H+/Na+ across membranes. They are an attractive drug target against protist pathogens. Non-hydrolysable PPi analogue bisphosphonates such as risedronate (RSD) and pamidronate (PMD) serve as primary drugs currently used. Bisphosphonates have a P-C-P bond, with their central carbon can accommodate up to two substituents, allowing a large compound variability. Here authors solved two TmPPase structures in complex with the bisphosphonates etidronate (ETD) and zoledronate (ZLD) and monitored their conformational ensemble using DEER spectroscopy in solution. These results reveal the inhibition mechanism by these compounds, which is crucial for developing future small-molecule inhibitors.

Strengths:<br /> Authors show that seven different bisphosphonates can inhibit TmPPase with IC50 values in the micromolar range. Branched aliphatic and aromatic modifications showed weaker inhibition. High-resolution structures for TmPPase with ETD (3.2 Å) and ZLD (3.3 Å) are determined. These structures reveal the binding mode and shed light on the inhibition mechanism. The nature of modification on the bisphosphonate alters the conformation of the binding pocket. The conformational heterogeneity is further investigated using EPR/DEER spectroscopy under several conditions. Altogether, this provides convincing evidence for a distinct conformational equilibrium of TmPPase in solution and further supports the notion of asymmetric inhibitor binding at the active site, while maintaining a symmetric conformation at the periplasmic interface.

Proposta: Com participo en un procés? * Assemblees: identifica el teu grup de debat. * Debats: Debat i discuteix els documents, comparteix les teves opinions i enriqueix els temes rellevants. * Propostes: Realitza propostes, dóna suport a les ja existents i promociona els canvis que vols veure. * Més informació sobre Decidim CoARA-UOC

• A teoria da culpa do serviço, também chamada de culpa administrativa, ou teoria do acidente administrativo, procura desvincular a responsabilidade do Estado da ideia de culpa do funcionário. Passou a falar em culpa do serviço público.

• Distinguia-se, de um lado, a culpa individual do funcionário, pela qual ele mesmo respondia, e, de outro, a culpa anônima do serviço público; nesse caso, o funcionário não é identificável e se considera que o serviço funcionou mal; incide, então, a responsabilidade do Estado.

• Essa culpa do serviço público ocorre quando: o serviço público não funcionou (omissão), funcionou atrasado ou funcionou mal. Em qualquer dessas três hipóteses, ocorre a culpa (faute) do serviço ou acidente administrativo, incidindo a responsabilidade do Estado independentemente de qualquer apreciação da culpa do funcionário.

• Sem abandonar essa teoria, o Conselho de Estado francês passou a adotar, em determinadas hipóteses, a teoria do risco, que serve de fundamento para a responsabilidade objetiva do Estado.

• Essa doutrina baseia-se no princípio da igualdade de todos perante os encargos sociais e encontra raízes no art. 13 da Declaração dos Direitos do Homem, de 1789, segundo o qual “para a manutenção da força pública e para as despesas de administração é indispensável uma contribuição comum que deve ser dividida entre os cidadãos de acordo com as suas possibilidades”. O princípio significa que, assim como os benefícios decorrentes da atuação estatal repartem-se por todos, também os prejuízos sofridos por alguns membros da sociedade devem ser repartidos. Quando uma pessoa sofre um ônus maior do que o suportado pelas demais, rompe-se o equilíbrio que necessariamente deve haver entre os encargos sociais; para restabelecer esse equilíbrio, o Estado deve indenizar o prejudicado, utilizando recursos do erário.

• Nessa teoria, a ideia de culpa é substituída pela de nexo de causalidade entre o funcionamento do serviço público e o prejuízo sofrido pelo administrado. É indiferente que o serviço público tenha funcionado bem ou mal, de forma regular ou irregular. Constituem pressupostos da responsabilidade objetiva do Estado: (a) que seja praticado um ato lícito ou ilícito, por agente público; (b) que esse ato cause dano específico (porque atinge apenas um ou alguns membros da coletividade) e anormal (porque supera os inconvenientes normais da vida em sociedade, decorrentes da atuação estatal); (c) que haja um nexo de causalidade entre o ato do agente público e o dano.

• É chamada teoria da responsabilidade objetiva, precisamente por prescindir da apreciação dos elementos subjetivos (culpa ou dolo); é também chamada teoria do risco, porque parte da ideia de que a atuação estatal envolve um risco de dano, que lhe é inerente. Causado o dano, o Estado responde como se fosse uma empresa de seguro em que os segurados seriam os contribuintes que, pagando os tributos, contribuem para a formação de um patrimônio coletivo (cf. Cretella Júnior, 1970, v. 8, p. 69-70).

• O Código Civil acolheu expressamente a teoria da responsabilidade objetiva, ligada à ideia de risco. Em consonância com o art. 927, parágrafo único, “haverá obrigação de reparar o dano, independentemente de culpa, nos casos especificados em lei, ou quando a atividade normalmente desenvolvida pelo autor do dano implicar, por sua natureza, risco para os direitos de outrem”.

• Segundo Hely Lopes Meirelles (2003:623), a teoria do risco compreende duas modalidades: a do risco administrativo e a do risco integral; a primeira admite (e a segunda não) as causas excludentes da responsabilidade do Estado: culpa da vítima, culpa de terceiros ou força maior.

(PIETRO, Maria Sylvia Zanella D. Direito Administrativo - 38ª Edição 2025. 38. ed. Rio de Janeiro: Forense, 2025. E-book. p.741. Acesso em: 02 jun. 2025.)

• a responsabilidade do concessionário por prejuízos causados a terceiros, em decorrência da execução de serviço público, é objetiva, nos termos do art. 37, § 6º, da Constituição vigente, que estendeu essa norma às pessoas jurídicas de direito privado prestadoras de serviços públicos; o poder concedente responde <u>subsidiariamente</u>, em caso de insuficiência de bens da concessionária; mas essa responsabilidade subsidiária somente se aplica em relação aos prejuízos decorrentes da execução do serviço público; eventualmente, pode haver responsabilidade solidária, por má escolha da concessionária ou omissão quanto ao dever de fiscalização;

(PIETRO, Maria Sylvia Zanella D. Direito Administrativo - 38ª Edição 2025. 38. ed. Rio de Janeiro: Forense, 2025. E-book. p.307. ISBN 9788530995935. Acesso em: 11 jun. 2025.)

• A teor do disposto no artigo 37, § 6º, da Constituição Federal, a ação por danos causados por agente público deve ser ajuizada contra o Estado ou a pessoa jurídica privada prestadora de serviço público, sendo parte ilegítima passiva o autor do ato. [RE 1.027.633, voto do rel. min. Marco Aurélio, j. 14-8-2019, P, DJE de 6-12-2019, Tema 940]
• O inadimplemento dos encargos trabalhistas dos empregados do contratado não transfere automaticamente ao poder público contratante a responsabilidade pelo seu pagamento, seja em caráter solidário ou subsidiário, nos termos do art. 71, § 1º, da Lei 8.666/1993. [RE 760.931, red. do ac. min. Luiz Fux, j. 26-4-2017, P, DJE de 12-9-2017, Tema 246.]
• Para que fique caracterizada a responsabilidade civil do Estado por danos decorrentes do comércio de fogos de artifício, é necessário que exista a violação de um dever jurídico específico de agir, que ocorrerá quando for concedida a licença para funcionamento sem as cautelas legais ou quando for de conhecimento do poder público eventuais irregularidades praticadas pelo particular. [RE 136.861, red. do ac. min. Alexandre de Moraes, j. 11-3-2020, P, DJE de 13-8-2020 Tema 366.]

• Informativo nº 740
• 13 de junho de 2022.
• SEGUNDA TURMA
• Processo: REsp 1.708.325-RS, Rel. Min. Og Fernandes, Segunda Turma, por unanimidade, julgado em 24/05/2022.

Ramo do Direito DIREITO ADMINISTRATIVO

Responsabilidade civil do Estado por omissão. Morte em decorrência de disparo de arma de fogo no interior de hospital público. Ausência de vigilância. Falha específica no dever de agir. Excludente de ilicitude. Fato de terceiro. Não ocorrência.

DESTAQUE - O hospital que deixa de fornecer o mínimo serviço de segurança, contribuindo de forma determinante e específica para homicídio praticado em suas dependências, responde objetivamente pela conduta omissiva.

INFORMAÇÕES DO INTEIRO TEOR - A responsabilidade civil estatal é, em regra, objetiva e decorre do risco administrativo, a respeito da qual não se exige perquirir sobre existência de culpa, conforme disciplinado pelos arts. 14 do Código de Defesa do Consumidor; 186, 192 e 927 do Código Civil; e 37, § 6º, da Constituição Federal. O dualismo ocorre diante dos atos omissivos, para os quais, embora a lei não tenha feito distinção, há os que entendem que, para o ente público, a responsabilidade se reveste do caráter subjetivo.

• Na toada, entretanto, de que, conforme assevera a doutrina, "não é dado ao intérprete restringir onde o legislador não restringiu, sobretudo em se tratando de legislador constituinte", esta Corte, em diversos julgados, tem procurado alinhar-se ao entendimento do Excelso Pretório de que - inclusive por atos omissivos - o Poder Público responde de forma objetiva, quando constatada a precariedade/vício no serviço decorrente da falha no dever legal e específico de agir.

• No caso, trata-se de responsabilidade civil atribuída a hospital, em que a atividade pública exercida, por sua natureza, inclui, além do serviço técnico-médico, o serviço auxiliar de estadia e, por tal razão, está o ente público obrigado a disponibilizar equipe/pessoal e equipamentos necessários e eficazes para o alcance dessa finalidade.

• A inação estatal está atrelada ao mau funcionamento dos trabalhos auxiliares e estruturas operacionais (ausência de serviço/pessoal de vigilância), razão pela qual entende-se que o ente público, em virtude da natureza da atividade pública exercida, responde de forma objetiva, uma vez que, inegavelmente, tem o dever de atuar, ao menos minimamente, para impossibilitar a ocorrência do evento nocivo.

• A omissão do Estado no presente feito revela-se específica e contribuiu decisivamente para a morte da vítima, pois o hospital público não ofereceu nenhuma ou sequer a mínima garantia de integridade aos que se utilizam do serviço e pela qual, em razão do risco da atividade prestada, tem o dever de zelo e proteção.

• Ocorre que a responsabilidade civil do Estado, todavia - seja de ordem subjetiva, seja objetiva - depende, para a configuração da ocorrência de seus pressupostos, do ato ilícito, do dano sofrido e do nexo de causalidade entre o evento danoso e a ação ou omissão do agente público.

• Estão descritos na sentença e no acórdão, a saber: (a) o hospital não possui nenhum serviço de vigilância; e (b) o evento morte decorreu de um disparo com arma de fogo contra a vítima dentro do hospital.

• O Tribunal regional - a despeito de a vítima ter sido baleada e o óbito ter ocorrido no interior do hospital -, não considerou o fato de não existir serviço de vigilância; ao contrário, a Corte local afirma, categoricamente, que o serviço do hospital é somente o atendimento médico, razão pela qual estaria desobrigado de prestar segurança aos pacientes.

• Concluiu-se, assim, que a morte da vítima deu-se por fato de terceiro.

• Como observa-se, a Corte regional - embora tenha considerado não existir equipe responsável pela integridade física dos pacientes e transeuntes no local - afastou a responsabilidade civil, consignando, com base na teoria da causalidade adequada, que a ação de alguém mal intencionado, dentro do hospital público, teria o condão de romper o nexo de causalidade entre a conduta do hospital e o evento danoso.

• Acaso se estivesse diante de um atentado de grandes proporções, não seria difícil concluir que, não obstante todo o empenho, o ente público não pudesse, de fato, impedir o resultado. Esta, entretanto, não é a situação narrada no acórdão, que traz, ao contrário, contexto e narrativa simples e bem menos eloquente.

• Neste caso, a causalidade decorre da própria lógica hermenêutica e análise holística das disposições civis e constitucionais mencionadas, devendo ser examinada à luz dos referidos dispositivos.

• A causalidade no âmbito da responsabilidade civil objetiva deve ser entendida de forma normativa, uma vez que a relevância jurídica do não-fazer está inserida na própria norma se encontra perfectibilizado o liame subjetivo entre a conduta omissa do hospital e o evento morte.

• Há de se ressaltar, contudo, que esse entendimento não se aplica indistintamente a qualquer ato derivado de conduta omissiva da administração pública. Neste feito, sob as lentes do bom senso, o não-fazer do ente público no seu dever de cuidado é sobremaneira significativo. Mostra-se lógico concluir que uma mínima ação de vigilância e cuidado poderia efetivamente ter evitado a morte da vítima.

• A análise da responsabilidade civil, no contexto desafiador dos tempos modernos, em que se colocam a julgamento as consequências tão impactantes das omissões estatais, impõe o ônus, indispensável, de que o exame dos dispositivos civis referidos ocorra sob o olhar dos direitos e garantias fundamentais do cidadão.

• Logo, é de se concluir que a conduta do hospital que deixa de fornecer o mínimo serviço de segurança e, por conseguinte, despreza o dever de zelar pela incolumidade física dos seus pacientes contribuiu de forma determinante e específica para o homicídio praticado em suas dependências, afastando-se a alegação da excludente de ilicitude, qual seja, fato de terceiro.

• RE 1209429
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. MARCO AURÉLIO
• Redator(a) do acórdão: Min. ALEXANDRE DE MORAES
• Julgamento: 10/06/2021
• Publicação: 20/10/2021

EMENTA. CONSTITUCIONAL. RESPONSABILIDADE CIVIL DO ESTADO. PROFISSIONAL DE IMPRENSA FERIDO, EM SITUAÇÃO DE TUMULTO, DURANTE COBERTURA JORNALÍSTICA. CULPA EXCLUSIVA DA VÍTIMA. INOCORRÊNCIA. PROVIMENTO DO RECURSO EXTRAORDINÁRIO. 1. O Estado responde civilmente por danos causados a profissional de imprensa ferido pela polícia, durante cobertura jornalística de manifestação popular. A apuração da responsabilidade dá-se na forma da teoria do risco administrativo, pacificamente aceita pela jurisprudência e pela doutrina. 2. Admite-se a invocação da excludente de responsabilidade civil da culpa exclusiva da vítima, nas hipóteses em que em que o profissional de imprensa I - descumpra ostensiva e clara advertência sobre o acesso a áreas delimitadas em que haja grave risco à sua integridade física; ou II - participe do conflito com atos estranhos à atividade de cobertura jornalística. 3. No caso concreto, o Tribunal de origem reconheceu a referida excludente de responsabilidade, sem identificar quaisquer destas circunstâncias - mas unicamente pelo fato de o fotógrafo estar presente na manifestação. 4. A atuação dos profissionais de imprensa na apuração de informações relevantes para a sociedade é tutelada pela Constituição, não podendo ser alegada pela afastar a responsabilidade civil do Estado. 5. O pedido de pensão mensal vitalícia merece ser atendido, em face do grave comprometimento do exercício da atividade de fotojornalismo, após ter o autor perdido 90% da visão em um dos olhos. Já o valor fixado a título de indenização pelos danos morais mostra-se alinhado aos parâmetros adotados pela jurisprudência brasileira em casos análogos, não cabendo sua elevação. 6. Recurso Extraordinário a que se dá provimento. Tema 1055, fixada a seguinte tese de repercussão geral: "“É objetiva a Responsabilidade Civil do Estado em relação a profissional da imprensa ferido por agentes policiais durante cobertura jornalística, em manifestações em que haja tumulto ou conflitos entre policiais e manifestantes. Cabe a excludente da responsabilidade da culpa exclusiva da vítima, nas hipóteses em que o profissional de imprensa descumprir ostensiva e clara advertência sobre acesso a áreas delimitadas, em que haja grave risco à sua integridade física".

Tese - É objetiva a Responsabilidade Civil do Estado em relação a profissional da imprensa ferido por agentes policiais durante cobertura jornalística, em manifestações em que haja tumulto ou conflitos entre policiais e manifestantes. Cabe a excludente da responsabilidade da culpa exclusiva da vítima, nas hipóteses em que o profissional de imprensa descumprir ostensiva e clara advertência sobre acesso a áreas delimitadas, em que haja grave risco à sua integridade física.

• ADI 6148
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. CÁRMEN LÚCIA
• Redator(a) do acórdão: Min. ANDRÉ MENDONÇA
• Julgamento: 05/05/2022
• Publicação: 15/09/2022

ODS 3 - Saúde e Bem-Estar ODS 11 - Cidades e comunidades sustentáveis ODS 12 - Consumo e produção responsáveis ODS 15 - Vida terrestre

AÇÃO DIRETA DE INCONSTITUCIONALIDADE. CONSTITUCIONAL, ADMINISTRATIVO E AMBIENTAL. PADRÕES DE QUALIDADE DO AR. CONSELHO NACIONAL DO MEIO AMBIENTE (CONAMA): COMPETÊNCIA PARA EXERCER JUÍZO TÉCNICO DISCRICIONÁRIO DE NORMATIZAÇÃO DA MATÉRIA. PRINCÍPIO DEMOCRÁTICO. AUTOCONTENÇÃO JUDICIAL. RESOLUÇÃO CONAMA Nº 491, DE 2018: NORMA CONSTITUCIONAL EM VIAS DE SE TORNAR INCONSTITUCIONAL. CONCESSÃO DO PRAZO DE 24 (VINTE E QUATRO) MESES PARA EDIÇÃO DE NOVA RESOLUÇÃO: OBSERVÂNCIA DA ATUAL REALIDADE FÁTICA.

1. O Conselho Nacional do Meio Ambiente (CONAMA) é órgão colegiado criado pela Lei nº 6.938, de 1981, dotado de capacidade institucional e responsabilidade, para, a partir de estudos e debate colegiado, dispor sobre “normas e padrões compatíveis com o meio ambiente ecologicamente equilibrado e essencial à sadia qualidade de vida”.

2. Diante das múltiplas vicissitudes e peculiaridades do caso, cabe, prioritariamente, ao CONAMA, como órgão regulador e no exercício da sua capacidade institucional, aquilatar, com devida atenção e aprofundado rigor técnico, qual o melhor conjunto de medidas apto a orientar a política de controle da qualidade do ar.

3. Impropriedade do Poder Judiciário em adentrar, ou mesmo substituir, o juízo técnico discricionário realizado na elaboração e no aprimoramento da política pública em foco.

4. Não se afigura salutar a conduta judicial de permanente e minudente escrutínio incidente sobre a condução das políticas públicas selecionadas pelo Administrador.

5. Em se tratando de tema de complexa e controvertida natureza técnico-científica, cabe ao Poder Judiciário atuar com ainda maior deferência em relação às decisões de natureza técnica tomadas pelos órgãos públicos com maior capacidade institucional para o tratamento e solução da questão.

6. Eventual atuação desta Suprema Corte no sentido de rever os critérios que redundaram na opção empreendida pelo CONAMA dependeria de manifesta falta de razoabilidade, de ausência de justificação ou de evidente abusividade na escolha empreendida pelo Administrador, não sendo este o caso dos autos.

7. A Organização Mundial da Saúde (OMS) indica que as diretrizes por ela traçadas não devem ser aplicadas automática e indistintamente, devendo cada país levar em conta os riscos à saúde, sua viabilidade tecnológica, questões econômicas e fatores políticos e sociais peculiares, além do nível de desenvolvimento e da capacidade de cada ente competente para atuar na gestão da qualidade do ar.

8. Sob a ótica do desenvolvimento sustentável, é necessário que sejam consideradas, pelo órgão regulador, o estágio mais atual da realidade nacional, das peculiaridades locais, bem como as possibilidades momentâneas de melhor aplicação dos primados da livre iniciativa, do desenvolvimento social, da redução da pobreza e da promoção da saúde pública, como elementos de indispensável consideração para construção e progressiva evolução da norma, de forma a otimizar a proteção ambiental, dentro da lógica da maior medida possível.

9. Reconhecimento de que a Resolução CONAMA nº 491, de 2018, afigura-se “ainda constitucional”. Determinação ao CONAMA de edição de nova resolução sobre a matéria que considere (i) as atuais orientações da Organização Mundial de Saúde sobre os padrões adequados da qualidade do ar; (ii) a realidade nacional e as peculiaridades locais; e (iii) os primados da livre iniciativa, do desenvolvimento social, da redução da pobreza e da promoção da saúde pública.

10. Se decorrido o prazo de 24 (vinte e quatro) meses, sem a edição de novo ato que represente avanço material na política pública relacionada à qualidade do ar, passarão a vigorar os parâmetros estabelecidos pela Organização Mundial de Saúde enquanto perdurar a omissão administrativa na edição da nova Resolução.

11. Ação Direta de Inconstitucionalidade julgada improcedente.

• ADPF 651
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. CÁRMEN LÚCIA
• Julgamento: 28/04/2022
• Publicação: 29/08/2022

ARGUIÇÃO DE DESCUMPRIMENTO DE PRECEITO FUNDAMENTAL. DIREITO CONSTITUCIONAL AMBIENTAL. MEDIDA CAUTELAR. DECRETO PRESIDENCIAL N. 10.224, DE 5.2.2020. EXCLUSÃO DA SOCIEDADE CIVIL DO CONSELHO DELIBERATIVO DO FUNDO NACIONAL DO MEIO AMBIENTE. DECRETO PRESIDENCIAL N. 10.239, DE 11.2.2020. EXCLUSÃO DOS GOVERNADORES DO CONSELHO NACIONAL DA AMAZÔNIA. DECRETO PRESIDENCIAL N. 10.223, DE 5.2.2020. EXTINÇÃO DO COMITÊ ORIENTADOR DO FUNDO AMAZÔNIA. ALEGADA AFRONTA À PROTEÇÃO AO MEIO AMBIENTE E PROIBIÇÃO AO RETROCESSO AMBIENTAL. ARGUIÇÃO DE DESCUMPRIMENTO DE PRECEITO FUNDAMENTAL JULGADA PROCEDENTE.

1. Proposta de conversão de julgamento de medida cautelar em julgamento definitivo de mérito: ausência de complexidade da questão de direito e instrução dos autos. Precedentes.

2. Nas normas impugnadas, a pretexto de reorganizar a Administração Pública federal quanto à composição do Conselho Deliberativo do Fundo Nacional do Meio Ambiental, do Conselho Nacional da Amazônia e do Comitê Orientador do Fundo Amazônia, frustra-se a participação da sociedade civil e dos Governadores dos Estados integrantes da Amazônia Legal na formulação das decisões e no controle da sua execução em matéria ambiental.

3. A exclusão da participação popular na composição dos órgãos ambientais frustra a opção constitucional pela presença da sociedade civil na formulação de políticas públicas ambientais. Contrariedade ao princípio da participação popular direta em matéria ambiental, à vedação do retrocesso e ao princípio da isonomia.

4. A eliminação da paridade na composição dos órgãos ambientais confere ao Poder Executivo federal o controle das suas decisões, neutralizando-se o caráter crítico e diversificado da fiscalização, que deve permear a condução dos trabalhos e políticas públicas.

5. A organização administrativa em matéria ambiental está protegida pelo princípio de proibição do retrocesso ambiental, o que restringe a atuação do administrador público, de forma a autorizar apenas o aperfeiçoamento das instituições e órgãos de proteção ao meio ambiente.

6. Arguição de descumprimento de preceito fundamental julgada procedente para a) declarar inconstitucional a norma prevista no art. 5º do Decreto n. 10.224/2020, pela qual se extinguiu a participação da sociedade civil no Conselho Deliberativo do Fundo Nacional do Meio Ambiente, restabelecendo-se quanto ao ponto o disposto no Decreto n. 6.985/2009, pelo qual alterado o art. 4º do Decreto n. 3.524/2000; b) declarar a inconstitucionalidade do Decreto n. 10.239/2020, especificamente no ponto em que se excluiu a participação de Governadores no Conselho Nacional da Amazônia Legal; e c) declarar a inconstitucionalidade do art. 1º, CCII, do Decreto nº 10.223/2020, especificamente no ponto em que se extinguiu o Comitê Orientador do Fundo Amazônia.

Localização de reator nuclear pode ser veiculada por lei ordinária federal. Isto é, não se demanda lei complementar para determinar a localização do retor nuclear. A inexistência de tal lei desautoriza a instalação da usina nuclear.

• ADO 59
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ROSA WEBER
• Julgamento: 03/11/2022
• Publicação: 16/08/2023

ODS 13 - Ação contra a mudança global do clima ODS 15 - Vida terrestre ODS 16 - Paz, Justiça e Instituições Eficazes ODS 17 - Parcerias e meios de implementação

Ação direita de inconstitucionalidade por omissão. Omissão inconstitucional da União quanto à implementação das prestações normativas e materiais de proteção da área compreendida como Amazônia Legal. O inadimplemento dos deveres constitucionais de tutela do meio ambiente pela União Federal, materializado na ausência de políticas públicas adequadas para a proteção da Amazônia Legal e na desestruturação institucional das formuladas em períodos antecedentes, configura estado normativo desestruturante e desestruturado em matéria ambiental na região. Omissão normativa quanto às obrigações referentes à ativação do Fundo Amazônia, cuja causa principal consiste na extinção dos mecanismos normativos essenciais para a gestão do Fundo. A consequência da paralisação do Fundo Amazônia consiste na suspensão dos ativos financeiros doados, atualmente na ordem de mais de R$ 3.000.000.000,00 (três bilhões de reais), fato que impossibilita a contratação de projetos voltados às ações de prevenção, combate e controle do desmatamento na Amazônia Legal. Classificação do Fundo Amazônia como instrumento de política pública financeira necessária ao adimplemento dos deveres de proteção ao meio ambiente na região da Amazônia Legal. Vedação do retrocesso em tutela ambiental. Procedência parcial dos pedidos.

1. A controvérsia constitucional objeto da deliberação do Supremo Tribunal Federal é um dos temas jurídicos e sociais mais relevantes da atualidade, tanto na perspectiva nacional quanto internacional. A questão subjacente à controvérsia assume caráter humanitário, cultural e econômico de abrangente impacto na tessitura social e na estrutura constitucional, notadamente no núcleo normativo do art. 225, caput, §§ 1º e 4º, da Constituição Federal.

2. O comportamento omissivo de desrespeito à Constituição por parte dos Poderes Públicos, seja legislador, administrador ou jurisdicional, produz como resultado quadro de inexistência de tutela dos direitos fundamentais e do arcabouço normativo constitucional ou de insuficiência no adimplemento dos deveres fundamentais de proteção.

3. O como concretizar os direitos fundamentais integra o espaço de conformação prática dos Poderes Públicos, em especial do Legislativo e do Executivo. Todavia, a liberdade decisória inerente à formulação da política normativa tem como vetor de atuação o dever de tutela dos direitos fundamentais. A proteção não é discricionária, mas sim as formas de sua implementação, desde que observado o postulado da proporcionalidade em sua dupla face proibitiva: do excesso da intervenção na esfera de proteção de direitos fundamentais e da insuficiência de sua tutela.

4. Os pedidos como formulados no sentido da adoção de providências administrativas enquadram-se na categoria de prestações normativas e fáticas derivadas da estrutura e necessidades da órbita de proteção do direito fundamental alegado.

5. A audiência pública produziu aportes informativos e argumentativos essenciais, com esclarecimentos de questões fáticas e jurídicas necessárias para a contextualização e elucidação do problema posto.

6. O quadro normativo e fático da Amazônia Legal traduz a realidade de um autêntico estado de coisas inconstitucional na Amazônia Legal, a revelar um cenário de tutela insuficiente e deficiente dos biomas patrimônios nacionais por parte do Estado brasileiro.

7. O retrato contemporâneo da Amazônia Legal não responde aos deveres de tutela assumidos pelo Estado constitucional brasileiro, expressamente desenhado no art. 225 da Constituição e na arquitetura legislativa, como prescreve a Lei n. 12.187/2009, que instituiu a Política Nacional sobre Mudança do Clima – PNMC. Tampouco responde à normativa internacional, devidamente ratificada e promulgada pelo Estado brasileiro, a demonstrar seu comprometimento político e jurídico com a centralidade e importância da tutela do meio ambiente, em particular a proteção contra o desmatamento e as mudanças climáticas, a saber a Convenção-Quadro sobre Mudanças Climáticas de 1992 (Decreto n. 2.652 de 01 de julho de 1998); o Protocolo de Kyoto, de 2005 (Decreto n. 5.445 de 12 de maio de 2015); e o Acordo de Paris, aprovado no final de 2015 e em vigor desde 2016 (Decreto n. 9.073, de 05 de junho de 2017).

8. A importância e a centralidade do Fundo Amazônia, como principal política pública financeira em vigor de apoio às ações de prevenção, controle e combate ao desmatamento, conservação das florestas e desenvolvimento sustentável, restou comprovada. Nesse sentido, os resultados fáticos obtidos com a implementação do PPCDAm e os depoimentos das organizações não-governamentais, dos secretários de Estado do Meio Ambiente, dos entes federados da Amazônia Legal e dos órgãos de controle e fiscalização envolvidos. Todavia, a centralidade do Fundo Amazônia como política pública financeira não significa inércia estatal, inclusive dos entes subnacionais, em formular outros instrumentos financeiros necessários ao financiamento das ações e planos de concretização da tutela do meio ambiente. Não é compatível com o modelo de federalismo cooperativo, em matéria ambiental, e com a normativa climática, a exclusividade de atuação da União Federal. Aos Estados igualmente compete concretizar objetivos de tutela dos seus biomas por meio de apresentação de resultados suficientes de redução do desmatamento para lograr políticas financeiras alternativas.

9. A alteração na governança do Fundo Amazônia, com a extinção dos seus comitês, Comitê Orientador – COFA e Comitê Técnico-científico – CTFA, por meio da edição dos Decretos n. 9759/2019, n. 10.144/2020 e n. 10.223/2020, acarretou a suspensão da avaliação e aprovação de novos projetos no âmbito do Fundo e, por conseguinte, da aplicação dos recursos disponíveis em caixa. Coube apenas, em termos de operação do Fundo, a continuidade de execução dos projetos aprovados anteriormente.

10. Da leitura e interpretação do art. 225 da Constituição Federal, fundamento normativo do Estado de Direito e da governança ambiental, infere-se estrutura jurídica complexa decomposta em duas direções normativas. A primeira, voltada ao direito fundamental, e a segunda relacionada aos deveres de proteção de responsabilidade dos poderes constituídos, atores públicos e da sociedade civil.

11. A omissão inconstitucional configurada reside no comportamento comissivo do administrador, que instaurou marco normativo desestruturante do antecedente, sem as salvaguardas jurídicas necessárias para a manutenção de um quadro mínimo de adimplemento dos deveres de proteção ao direito fundamental ao meio ambiente equilibrado, mais especificamente de proteção dos patrimônios nacionais, tal como categorizados pelo art. §4º do art. 225 da Constituição Federal, e de cumprimento das obrigações climáticas firmadas.

12. Apresenta-se, como medida jurisdicional adequada para a solução do problema posto, a invalidação dos dispositivos normativos que alteraram o modelo de governança do Fundo Amazônia. Como consequência, cabe à União tomar as providências administrativas necessárias para a reativação do Fundo, no que lhe compete.

13. A omissão inconstitucional do Poder Executivo no que diz respeito ao funcionamento da política pública do Fundo Amazônia traz consequências em distintas atividades e operações do seu funcionamento, como recebimento de novos recursos, análise de novos projetos a serem financiados com valores já recebidos, em resposta aos resultados obtidos pelo Estado brasileiro na redução do desmatamento em momentos anteriores.

14. As providências administrativas relacionadas as atividades de operação do Fundo é de competência do BNDES, responsável e gestor do Fundo. Por esse motivo, os pedidos b), c), d) e) formulados na inicial carecem de respaldo jurídico, porquanto fora da competência da União Federal e da abordagem constitucional desta demanda. O pedido de declaração de inconstitucionalidade do art. 1º, CCII, do Decreto nº 10.223/2020, no ponto em que extinguiu o Comitê Orientador do Fundo Amazônia, foi resolvido no julgamento da ADPF 651, de relatoria da Ministra Cármen Lúcia, quando o Tribunal, ao deferir o aditamento à inicial, declarou sua inconstitucionalidade.

15. Procedência dos pedidos “a” e “f” para (i) declarar a inconstitucionalidade do art. 12, II, do Decreto nº 10.144/2019 e do art. 1º do Decreto nº 9.759/2019, no que se refere aos colegiados instituídos pelo Decreto nº 6.527/2008; e (ii) determinar à União Federal que, no prazo de sessenta dias, tome as providências administrativas necessárias para a reativação do Fundo Amazônia, dentro e nos limites das suas competências, com o formato de governança estabelecido no Decreto n. 6.527/2008.

16. Ação direta julgada parcialmente procedente.

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A garantia contratual poderá ser realizada de 5% a 10% do valor do contrato, a depender da análise da complexidade técnica e dos riscos envolvidos.

No entanto, como se depreende do artigo subsequente, a garantia poderá alcançar até 30% do valor inicial do contrato quando se tratar de obra ou serviço de grande vulto.

Não confundir com a garantia prestada como atendimento ao requisito de pré-habilitação que trata o art 58:

Art. 58. Poderá ser exigida, no momento da apresentação da proposta, a comprovação do recolhimento de quantia a título de garantia de proposta, como requisito de pré-habilitação.

• § 1º A garantia de proposta não poderá ser superior a 1% (um por cento) do valor estimado para a contratação.

• § 2º A garantia de proposta será devolvida aos licitantes no prazo de 10 (dez) dias úteis, contado da assinatura do contrato ou da data em que for declarada fracassada a licitação.

• § 3º Implicará execução do valor integral da garantia de proposta a recusa em assinar o contrato ou a não apresentação dos documentos para a contratação.

• § 4º A garantia de proposta poderá ser prestada nas modalidades de que trata o § 1º do art. 96 desta Lei.

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Reviewer #1

Evidence, reproducibility and clarity

In their manuscript de las Mercedes Carro et al investigated the role of Ago proteins during spermatogenesis by producing a triple knockout of Ago 1, 3 and 4. They first describe the pattern of expression of each protein and of Ago2 during the differentiation of male germ cells, then they describe the spermatogenesis phenotype of triple knockout males, study gene deregulation by scRNA seq and identify novel interacting proteins by co-IP mass spectrometry, in particular BRG1/SMARCA4, a chromatin remodeling factor and ATF2 a transcription factor. The main message is that Ago3 and 4 are involved in the regulation of XY gene silencing during meiosis, and also in the control of autosomal gene expression during meiosis. Overall the manuscript is well written, the topic, very interesting and the experiments, well-executed. However, there are some parts of the methodology and data interpretation that are unclear (see below).

Major comments

1= Please clarify how the triple KO was obtained, and if it is constitutive or specific to the male germline. In the result section a Cre (which cre?) is mentioned but it is not mentioned in the M&M. On Figure S1, a MICER VECTOR is shown instead of a deletion, but nothing is explained in the text nor legend. Could the authors provide more details in the results section as well as in the M&M ? This is essential to fully interpret the results obtained for this KO line, and to compare its phenotype to other lines (such as lines 184-9 Comparison of triple KO phenotype with that of Ago4 KO). Also, if it is a constitutive KO, the authors should mention if they observed other phenotypes in triple KO mice since AGO proteins are not only expressed in the male germline.

Response: We apologize for omitting this vital information. We have now incorporated a more detailed description of how the Ago413 mutant was created in the results and M&M sections (line 120 and 686 respectively).

As mentioned in the manuscript, Ago4, Ago1 and Ago3 are widely expressed in mammalian somatic tissues. Mutations or deletions of these genes does not disrupt development; however, there is limited research on the impact of these mutations in mammalian models in vivo. In humans, mutations in Ago1 and Ago3 genes are associated with neurological disorders, autism and intellectual disability (Tokita, M.J.,et al. 2015- doi: 10.1038/ejhg.2014.202., Sakaguchi et al. 2019- doi: 10.1016/j.ejmg.2018.09.004, Schalk et al 2021- doi: 10.1136/jmedgenet-2021-107751). In mouse, global deletion of Ago1 and Ago3 simultaneously was shown to increase mice susceptibility to influenza virus through impaired inflammation responses (Van Stry et al 2012- doi.org/10.1128/jvi.05303-11). Studies performed in female Ago413 mutants (the same mutant line used herein) have shown that knockout mice present postnatal growth retardation with elevated circulating leukocytes (Guidi et al 2023- doi: 10.1016/j.celrep.2023.113515). Other studies of double conditional knockout of Ago1 and Ago3 in the skin associated the loss of these Argonautes with decreased weight of the offspring and severe skin morphogenesis defects (Wang et al 2012- doi: 10.1101/gad.182758.111). In our study, we did not observe major somatic or overt behavioral phenotypes, and we did not observe statistical differences in body weights of null males compared to WT as shown in figure below.

2= The paragraph corresponding to G2/M analysis is unclear to me. Why was this analysis performed? What does the heatmap show in Figure S4? What is G2/M score? (Fig 2D). Lines 219-220, do the authors mean that Pachytene cells are in a cell phase equivalent to G2/M? All this paragraph and associated figures require more explanation to clarify the method and interpretation.

__Response: __We have modified the methods to include more information about how the cell cycle scoring used in Figures 2D and S4 were calculated and will add more information regarding the interpretation of these figures.

3= I have concerns regarding Fig2G: to be convincing the analysis needs to be performed on several replicates, and, it is essential to compare tubules of the same stage - which does not seem to be the case. This does not appear to be the case. Besides, co (immunofluorescent) staining with markers of different cell types should be shown to demonstrate the earlier expression of some markers and their colocalization with markers of the earlier stages.

__Response: __We agree with the Reviewer. New images with staged tubules will be added to the analysis of Figure 2G.

4= one important question that I think the authors should discuss regarding their scRNAseq: clusters are defined using well characterized markers. But Ago triple KO appears to alter the timing of expression of genes... could this deregulation affects the interperetation of scRNAseq clusters and results?

__Response: __We thank the reviewer for this suggestion and agree that including this information is important. We expect that, at most, this dysregulation impacts the edges of these clusters slightly. Given that marker genes that have been used to define cell types in these data are consistently expressed between the knockout and wildtype mice (see Figure S4A), we do not think that the cells in these clusters have different identities, just dysregulated expression programs. We have added the relevant sentence to the discussion, and will include additional supplemental figure panels to document this point more comprehensively.

5= XY gene deregulation is mentioned throughout the result section but only X chromosome genes seem to have been investigated.... Even the gene content of the Y is highly repetitive, it would be very interesting to show the level of expression of Y single copy and Y multicopy genes in a figure 3 panel.

__Response: __We agree with the reviewer that including analysis of Y-linked genes is important. We will add a supplemental figure which includes the Y:Autosome ratio and differential expression analysis.

6= Can the authors elaborate on the observation that X gene upregulation is visible in the KO before MSCI; that is in lept/zygotene clusters (and in spermatogonia, if the difference visible in 3A is significant?)

Response: We do see that X gene expression is upregulated before pachynema. Previous scRNA-seq studies that have looked at MCSI have seen that silencing of genes on the X and Y chromosomes starts before the cell clusters that are defined as pachynema, though silencing is not fully completed until pachynema. We have clarified this point in the manuscript.

7 = miRNA analysis: could the authors indicate if X encoded miRNA were identified and found deregulated? Because Ago4 has been shown to lead to a downregulation of miRNA, among which many X encoded. It is therefore puzzling to see that the triple KO does not recapitulate this observation. Were the analyses performed differently in the present study and in Ago4 KO study?

__Response: __The analysis identifying downregulation of miRNA in the original Ago4 mutant analysis was conducted relative to total small RNA expression. Amongst those altered miRNA families in the Ago4 mutants, we demonstrated both upregulation and downregulation of miRNA. We agree that confirming a similar global downregulation of miRNA counts compared to other small RNAs is important. Therefore, in a revised manuscript, we will add this information to the miRNA analysis section, especially highlighting the X chromosome-associated miRNAs, as well as whether the ratios between other small RNA classes change.

8 = The last results paragraph would also benefit from some additional information. It is not clear why the authors focused on enhancers and did not investigate promoters (or maybe they were but it's unclear). Which regions (size and location from TSS) were investigated for motif enrichment analyses? To what correspond the "transcriptional regulatory regions previously identified using dREG" mentioned in the M&M? I understand it's based on a previous article, but more info in the present manuscript would be useful.

Response: We thank the reviewer for this suggestion. The regions that were used for motif enrichment will be included as a supplementary information in the fully revised manuscript. We have also clarified in the methods that these transcriptional regulatory regions were downloaded from GEO and obtained from previous ChRO-seq data (from GEO) analysis. These data are run through the dREG pipeline that identifies regions predicted to contain transcription start sites, which include promoters and enhancers.

Minor comments

1) In the introduction: The sentence "Ago1 is not expressed in the germline from the spermatogonia stage onwards allowing us to use this model to study the roles of Ago4 and Ago3 in spermatogenesis." is misleading because Ago1 is expressed at least in spermatogonia; It would be more precise to write "after spermatogonia stage" and rephrase the sentence. Otherwise it is surprising to see AGO1 protein in testis lysate and it is not in line with the scRNA seq shown in figure 2.

__Response: __We agree with the Reviewers suggestion and have edited the sentence on line 100. This sentence now reads "Ago1 is not expressed in the germline after the spermatogonia stage allowing us to use this model to study the roles of Ago4 and Ago3 in spermatogenesis".

2) Could the authors precise if AGO proteins are expressed in other tissues? In somatic testicular cells?

__Response: __Expression patterns of mammalian AGOs have been described in somatic and testicular tissues for the mouse by Gonzales-Gonzales et al (2008) by qPCR. They found that Ago2 is expressed in all the somatic tissues analyzed (brain, spleen, heart, muscle and lung) as well as the testis, with the highest expression in brain and lowest in heart. Ago1 is highly expressed in spleen compared to all the tissues analyzed, while Ago3 and Ago4 showed highest expression in testis and brain. Within somatic tissues of the testis, the four argonautes are expressed in Sertoli cells, however, Ago1,3 and 4 expression is very low compared to Ago2, with the latter showing a 10-fold higher transcript level. We have included a sentence with this information in the introduction in line 89.

3) Pattern of expression: How do the authors explain that AGO3 disappears at the diplotene stage and reappears in spermatids?

__Response: __ Single cell RNAseq data in the germline shows reduced transcript for Ago3 from the Pachytene stage onwards, suggesting minimal if any new transcription in round spermatids. We hypothesize that the AGO3 protein present in the round spermatid stage is cytoplasmic, presumably coming from the pool of AGO3 in the chromatoid body, a cytoplasmic structure with functional association with the nucleus in round spermatids (Kotaja et al, 2003 doi: 10.1073/pnas.05093331).

4) It would be useful to show the timing of expression of AGO 1 to 4 throughout spermatogenesis in the first paragraph of the article. Maybe the authors could present data from fig2B earlier?

Response: We understand the Reviewers concern, however, given that Ago expression throughout spermatogenesis was obtained from scRNA seq, we consider that this data should be presented after introducing the Ago413 knockout and the scRNA seq experiment. As Ago1-4 expression was also described in an earlier manuscript by Gonzales-Gonzales et al in the mouse male germline, and our data aligns with this report, we included a sentence about these previous findings in the earlier results section.

5) Line 190: please modify the sentence "reveal no differences in cellular architecture of the seminiferous tubules when compared to wild-type males" to " reveal no gross differences..." since even without quantification of the different cell types it is visible that KO seminiferous tubules are different from WT tubules.

__Response: __We agree with the reviewer, and we modified line 190 (now 173) as suggested. Grossly, seminiferous tubules from Ago413 null males contain the same cell types as in wild type tubules, including spermatozoa. However, our studies show that the number and quality of germ cells is compromised in knockouts, as shown by sperm counts and TUNEL staining.

6) TUNEL analysis: please stage the tubules to determine the stage(s) at which apoptosis is the most predominant.

__Response: __We have complied with the reviewer suggestion. Figure 1G now shows staged seminiferous tubules, and we have replaced the wild type image for one where the staged tubules match the knockout image.

7) Figure S4B does not show an increase of cells at Pachytene stage but at Lepto/zygotene stage (as well as an increase of spermatogonia). Please comment this discrepancy with results shown in Fig2.

__Response: __Figures 2 and S4 show distribution of cells in different substages of spermatogenesis and prophase I measured with very different methods: a cytological approach using chromosome spreads cells vs a transcriptomic approach that involves clustering of cells. We attribute the differences in cell type distribution to differences in the sensitivity of the methods to identify each cell type and therefore identify differences between the number of cells for each group. Moreover, our scRNA-seq data groups the leptotene and zygotene stages together, while the cytological approach allows for separation of these two sub-stages. Importantly, both results show that Ago413 spermatocytes are progressing slower from pachynema into diplonema and/or are dying after pachynema, as stated in line 194 in our manuscript.

8) Fig5H and 5I are not mentioned in the result section. Also, it would be useful to label them with "all chromosomes" and "XY" to differentiate them easily

__Response: __We apologize for the omission and have now cited Figures 5H and 5I in the manuscript (line 453). We have added the suggested labels.

9) Line 530 "data provide further evidence for a functional association between AGO-dependent small RNAs and heterochromatin formation, maintenance and/or silencing." Please rephrase, the present article does not really show that AGO nuclear role depends on small RNAs.

__Response____: __We agree with the reviewer that these data do not directly show a dependence on small RNAs. As our identified localization of AGO proteins to the pericentric heterochromatin coincides with localization of DICER shown previously by Yadav and collaborators (2020, doi: 10.1093/nar/gkaa460), we do believe that our data further implicates small RNAs in the silencing of heterochromatin. Yadav et al shows that DICER localizes to pericentromeric heterochromatin and processes major satellite transcripts into small RNAs in mouse spermatocytes, and cKO germ cells have reduced localization of SUV39H2 and H3K9me3 to the pericentromeric heterochromatin. Given the colocalization of both small RNA producing machinery and AGOs at pericentromeric heterochromatin, the AGOs may bind these small RNAs, and the statement in line 530 refers to how our results provide evidence for the involvement of other RNAi machinery in the silencing of pericentromeric heterochromatin investigated by Yadav et al which likely includes small RNAs.

To clarify this point, we have modified the text accordingly.

10) Line 1256: replace "cite here " by appropriate reference

__Response: __The reference was added to line 1256.

11) Please use SMARCA4 instead of BRG1 name as it is its official name.

__Response: __We have replaced BRG1 with SMARCA4 in the text and figures.

Figures:

Figure 1: Are the pictures shown for Ago3-tagged and floxed from the same stages ? The leptotene stage in 1A looks like a zygotene, while some pachytene/diplotene stage pictures do not look alike.

__Response: __New representative images have been added to figure 1 to match the same substages across the figure.

Figure 1D, please label the Y scale properly (testis weight related to body weight)

__Response: __We have fixed this.

FigS1: Please comment the presence of non-specific bands in the figure legend

__Response: __We have added a sentence in Figure S1 Legend.

Fig 2E and F, please indicate on the figure (in addition to its legend), what are the X and Y axes respectively to facilitate its reading.

__Response: __X and Y axes are now labelled in Figure 2E and F.

2F: please use an easier abbreviation for Spermatocyte than Sp (which could spermatogonia, sperm etc..) such as Scyte I ? (same comment for Fig 3C)

Response: The abbreviation for spermatocyte was changed from Sp to Scyte I in Figures 2 and 3.

Overall, for all figures showing GSEA analyses, could the authors explain what a High positive NES and a High negative NES mean in the results section?

Response: Thank you for this suggestion. We have added this information where the GSEA score of the cell markers is initially introduced.

Significance

Ago proteins are known for their roles in post transcriptional gene regulation via small RNA mediated cleavage of mRNA, which takes places in the cytoplasm. Some Ago proteins have been shown to be also located in the nucleus suggesting other non-canonical roles. It is the case of Ago4 which has been shown to localize to the transcriptionally silenced sex chromosomes (called sex body) of the spermatocyte nucleus, where it contributes to regulate their silencing (Modzelewski et al 2012). Interestingly, Ago4 knockout leads to Ago3 upregulation, including on the sex body indicating that Ago3 and Ago4 are involved in the same nuclear process. In their manuscript, de las Mercedes Carro et al., investigate the consequences of loss of both Ago3 and Ago4 in the male germline by the production of a triple knockout of Ago1, 3 and 4 in the mouse. With this model, the authors describe the role of Ago3 and Ago4 during spermatogenesis and show that they are involved in sex chromosome gene repression in spermatocytes and in round spermatids, as well as in the control of autosomal meiotic gene expression. Triple KO males have impaired meiosis and spermiogenesis, with fewer and abnormal spermatozoa resulting in reduced fertility. Since Ago1 male germline expression is restricted to pre-meiotic germ cells, it is not expected to contribute to the meiotic and postmeiotic phenotypes observed in the triple KO. The strengths of the study are i) the thorough analyses of mRNA expression at the single cell level, and in purified spermatocytes and spermatids (bulk RNAseq), ii) the identification of novel nuclear partners of AGO3/4 relevant for their described nuclear role: ATF2, which they show to also co-localize with the sex body, and BRG1/SMARCA4, a SWI/SNF chromatin remodeler. The main limitation of the study is the lack of information in the method regarding the production of the triple KO, as well as some aspects of the transcriptome and motif analyses. It is also surprising to see that the triple KO does not recapitulate the miRNA deregulation observed in Ago4 KO. The characterization of a non-canonical role of AGO3/4 in male germ cells will certainly influence researchers of the field, and also interest a broader audience studying Argonaute proteins and gene regulation at transcriptional and posttranscriptional levels.

Reviewer #2

Evidence, reproducibility and clarity

In the manuscript titled "Argonaute proteins regulate the timing of the spermatogenic transcriptional program" by Carro et al., the authors present their findings on how Argonaute proteins regulate spermatogenic development. They utilize a mouse model featuring a deletion of the gene cluster on chromosome 4 that contains Ago1, Ago3, and Ago4 to investigate the cumulative roles of AGO3 and AGO4 in spermatogenic cells. The authors characterize the distribution of AGO proteins and their effects on key meiotic milestones such as synapsis, recombination, meiotic transcriptional regulation, and meiotic sex chromosome inactivation (MSCI). They analyze stage-specific transcriptomes in spermatogenic cells using single-cell and bulk RNA sequencing and determine the interactome of AGO3 and AGO4 through mass spectrometry to examine how AGO proteins may regulate gene expression in these cells during meiotic and post-meiotic development. The authors conclude that both AGO3 and AGO4 are essential for regulating the overall gene expression program in spermatogenic cells and specifically modulate MSCI to repress sex-linked genes in pachytene spermatocytes, which may be partially mediated by the proper distribution of DNA damage repair factors. Additionally, AGO3 is suggested to interact with the chromatin remodeler SWI/SNF factor BRG1, facilitating its removal from the sex-chromatin to enable the repression of sex-linked genes during MSCI.

Major Comments: 1. The study utilized a triple knockout mouse model to determine the effect of AGO3 on spermatogenesis, following up on their previous report about the role of AGO4 in spermatogenesis, which resulted from an upregulation of AGO3 in Ago4-/- spermatocytes. However, the results are more difficult to interpret and ascertain the role of AGO3 in these cells, given the absence of any observable phenotype from Ago3 interruption. AGO4 regulates sex body formation, meiotic sex chromosome inactivation (MSCI), and miRNA production in spermatocytes, all of which were noted in the absence of both AGO3 and AGO4, with only an increased incidence of cells containing abnormal RNAPII at the sex chromosomes. It will be necessary to characterize how AGO3 regulates spermatogenic development, including meiotic progression and the regulation of the meiotic transcriptome, and compare these findings with the current observations to determine if the proposed mechanism involving AGO3, BRG1, and possibly AP2 is relevant in this context.

__Response: __While we agree with Reviewer that a single Ago3 knockout will help understand distinct roles of AGO3 and AGO4 in spermatogenesis, the time and resources required to generate a new mouse model are substantial. The analysis included in this current manuscript has already taken over seven years, and with the lengthy production of a new single mutant mouse, validation of the new mouse, and then final analysis, we would be looking at another 3-5 years of analysis. In the current funding climate, and with strong concerns over ensuring reduction in utilization of laboratory mice, we consider this request to be far in excess of what is required to move this important story forward.

The Ago413-/- mouse model has allowed us to associate a nuclear role of Argonaute proteins with a strong reproductive phenotype in the mouse germline. Given the redundancy between Ago3 and Ago4, it is likely that a single Ago3 knockout would have a mild phenotype just like the Ago4 KO. All this said, we agree with the reviewer that analysis of an Ago3 knockout mouse is a valuable next step, just not within this chapter of the story.

1. Does Ago413-/- mice recapitulate the early meiotic entry phenotype observed in Ago4-/- mice? If not, could it be possible that AGO3 promotes meiotic entry, given its strong mRNA expression in spermatogonia according to the scRNAseq data (Fig. 2B)

Response: Our scRNA-seq data shows strong expression of Ago3 in spermatogonia, as mentioned by the Reviewer. Analysis of cell cycle marker expression also shows that the transcriptomic profile of spermatogonia is altered, with higher levels of transcripts corresponding to the later G2/M stages (Figure 2D). Moreover, Ago413 knockouts present an increase in the number of spermatogonial stem cells (Supplementary Figure S4B). However, this cluster represents a pool of quiescent and mitotically active cells entering meiosis, therefore interpretation of these data might be challenging. While specific experiments could be conducted to answer this question, this is outside of the scope of our manuscript. The manuscript as it stands is already rather large, and a full analysis of meiotic entry dynamics would dilute the core message relating to chromatin regulation in the sex body.

1. The authors suggested that the removal of BRG1 by AGO3 is necessary during sex body formation and the eventual establishment of MSCI. However, the BAF complex subunit ARID1A has been shown to facilitate MSCI by regulating promoter accessibility. It will be interesting to determine how BRG1 distribution changes across the genome in the absence of AGO proteins and how that correlates with alterations in sex-linked gene expression.

__Response: __We agree that changes in BRG1 distribution across the genome would be very interesting to identify. However, in this work we show that BRG1/SMARCA4 protein changes its localization in the sex body very rapidly between early to late pachynema. These two substages are only discernable by immunofluorescence using synaptonemal complex markers, as there are currently no available techniques to enrich for these subfractions. Therefore, study of genome occupancy of BRG1 in these specific substages by techniques such as CUT&Tag are not currently possible. However, we are currently working on new methods to distinguish these cell populations and hope eventually to use these purification strategies to perform the studies suggested by this reviewer. Alternatively, the hope is that single cell CUT&Tag methods will become more reliable, and will enable us to address these questions. Both of these options are not currently available to us. The studies by Menon et al (2024-doi:10.7554/eLife.88024.5) provide strong evidence to support that ARID1A is needed to reduce promoter accessibility of XY silenced genes in prophase I through modulation of H3.3 distribution. However, this mechanism and our identification of the removal of BRG1 between early and late pachytema are not inconsistent with one another, as either SMARCA4 or SMARCA2 can associate with ARID1A as part of the cBAF complex, and ARID1A is also not in all forms of the BAF complex which BRG1 are in. The difference between our results and those seen in Menon et al likely indicate that there are multiple forms of the BAF complex which are differentially regulated during MSCI and play different roles in silencing transcription. Further studies of specific BAF subunits are needed to elucidate how different flavors of the BAF complex act at specific genomic locations and meiotic time points.

1. The observations presented in this manuscript (Fig. 1D, 2C, 3D, and 4) suggest a haploinsufficiency of the deleted locus in spermatogenic development. How does this compare with the ablation of either Ago3 or Ago4? Please explain.

Response: Our previous studies in single Ago4 knockouts did not present a heterozygous phenotype (Modzelewski et al 2012, doi: 10.1016/j.devcel.2012.07.003, data not shown). Triple Ago413 knockouts show a much stronger fertility phenotype than single Ago4 knockout. Testis weight of Ago413 homozygous null present a 30% reduction while heterozygous mice show a 15% reduction (Figure 1D), comparable to the 13% reduction previously observed in Ago4-/- males. Sperm counts of Ago413 null and heterozygous males are reduced by 60% and 39% compared to wild type (Figure 1E), respectively, whereas Ago4 null mice have a milder phenotype, with only a 22% reduction in sperm counts. At the MSCI level, both homozygous and heterozygous Ago413 mutant spermatocytes show a similar increase in pachytene spermatocytes with increased RNA pol II ingression into the sex body with respect to wild-type of 35% and 30%, respectively. Ago4 single knockouts show an almost 18% increase in Pol II ingression when compared to wild type. These comparisons are now included in our manuscript in lines 170, 172 and 288. A milder phenotype of the Ago4 knockout and haploinsufficiency in triple Ago413 knockouts but not in Ago4 single knockouts is likely a consequence of the overlapping functions of Ago3 and Ago4 in mammals (and/or overexpression of Ago3 in Ago4 knockouts). In the context of their role in RISC, Wang et al (doi: 10.1101/gad.182758.111) studied the effects of single and double conditional knockouts for Ago1 and Ago2 in miRNA-mediated silencing. They discovered that the interaction between miRNAs and AGOs is highly correlated with the abundance of each AGO protein, and only double knockouts presented an observable phenotype.

Minor Comments: Based on the interactome analysis, it was argued that AGO3 and AGO4 may function separately. Please discuss how AGO3 might compensate for AGO4 (Line 109).

Response: We hypothesize that the combined function of AGO3 and AGO4 is needed for proper sex chromosome inactivation during meiosis. We base this hypothesis on the facts that (i) both proteins localize to the sex body in pachytene spermatocytes, (ii) loss of Ago4 leads to upregulation of Ago3, and (iii) the MSCI phenotype of Ago413 knockout mice is much stronger than the single Ago4 knockout (see above). However, AGO3 and AGO4 might not induce silencing through the same mechanism or pathway. In this work, we observed that their temporal expression in prophase I is different; while AGO3 protein seems to disappear by the diplotene stage, AGO4 is present in the sex body of these cells. Moreover, the proteomic analysis revealed a very low number of common interactors, an observation which could support the idea of AGO3 and AGO4 acting by different (albeit perhaps related) mechanisms to achieve MSCI. It is also possible that common interactors were not identified in our proteomic analysis due to the low abundance of AGO3 and AGO4 in the germ cells, limiting the resolution of the proteomics analysis (note that in order to visualize AGO proteins in WB experiments, at least 60 μg of enriched germ cell lysate must be loaded per lane). Moreover, given the difficulty in obtaining enough isolated pachytene and diplotene spermatocytes to perform immunoprecipitation experiments, we performed IP experiments in whole germ cell lysates, which limits the interpretation of our analysis. If AGO3 and AGO4 protein interactors overlap, then AGO3 would directly substitute for AGO4 leading to silencing in single Ago4 knockouts. However, if AGO3 and AGO4 work together through different, complementary mechanisms, then Ago4 mutant mice likely compensates loss of Ago4 by upregulation of Ago3along with specific interactors of the given pathway. We have added a sentence addressing this matter in line 411 of the results section and lines 506 and 513 of the discussion in the revised manuscript.

In Line 221, it is unclear what is meant by 'cell cycle transcripts'. Does this refer to meiotic transcripts? It is also important to discuss the relevance of the G2/M cell cycle marker genes at later stages of meiotic prophase.

Response: Thank you for this suggestion. We have changed the relevant text to remove redundancies and include more information. We agree that considering the importance of these genes across meiotic prophase is needed, as cells which are in the dividing stage will already have produced the proteins necessary for division. These cells likely correspond to the diplotene/M cluster cells that have a lower G2/M score, potentially causing the bimodal distribution seen in Figure 2D. We have added a sentence addressing this to the manuscript.

While identified as a common interactor of both AGO3 and AGO4 in lines 440-445, HNRNPD is not listed among AGO4 interactors in Table S6. Please correct or explain this discrepancy.

Response: HNRPD was originally identified as an AGO4 interactor using a less strict criteria than the one used in our manuscript: we required consistent enrichment in at least two rounds of IP MS experiments. This reference to HNRNPD was a mistake, given that HNRPD was only enriched in one of our three replicates. Thus, we apologize and have removed the sentence in lines 440-445.

It is unclear whether wild-type cell lysate or lysate containing FLAG-tagged AGO3 was used for BRG1 immunoprecipitation, and which antibody was used to detect AGO3 in the BRG1 IP sample. A co-IP experiment demonstrating interaction between BRG1 and wild-type AGO3 would be ideal in this context. Furthermore, co-localization by IF would be beneficial to determine the subcellular localization and the cell stages the interaction may be occurring. Additionally, co-IP and Western blot methodologies should be included in the methods section.

__Response: __MYC-FLAG tagged AGO3 protein lysates were used for BRG1 Co-Immunoprecipitation, along with an anti MYC antibody to detect AGO3. This is now detailed in the Methods section of our revised manuscript (line 1133).

Regarding BRG1 and AGO3 colocalization by IF, we can confidently show that both AGO3 and BRG1 localize to the sex chromosomes in early pachynema by comparing BRG1/SYCP3 and FLAG-AGO3/SYCP3 stained spreads. We were not able to show colocalization simultaneously on the same cells, given the lack of appropriate antibodies. Our anti FLAG antibody is raised in mouse, while anti BRG1 is raised in rabbit, therefore a non-rabbit, non-mouse anti SYCP3 would be needed to identify prophase I substages, and our lab does not possess such a validated antibody. However, we now have access to a multiplexing kit that allows to use same-species antibodies for immunofluorescence and we can perform these experiments for a revised manuscript.

__Response: __The methods section now includes description of co-IP methodologies (line 1132). Western Blot methodologies are explained in lane 718, under the "Immunoblotting" title.

In line 599, it is unclear what is meant by 'persistence of sex chromosome de-repression'. Please correct or clarify this.

Response: This sentence has been changed and reads: "The persistence of sex chromosome gene expression".

If possible, please add an illustration to summarize the findings together.

Response: We thank the reviewer for this suggestion, and have now added this in Figure 6

Significance

Overall, this study enhances the understanding of gene expression regulation by AGO proteins during spermatogenesis. Several approaches, including functional, histological, and molecular characterization of the triple knockout phenotype, were instrumental in elucidating the role of AGO proteins in MSCI and meiotic as well as postmeiotic gene regulation. The main limitation of the study is that it is challenging to appreciate the role of AGO3 in addition to the previously published role of AGO4 without the inclusion of necessary control groups. Furthermore, the mechanism of action for AGO proteins in meiotic gene regulation was left relatively unexplored. This study presents new findings that will be significant for the research community interested in gene regulation, chromatin biology, and reproductive biology with the above suggestions considered.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

The authors characterize a CRISPR-Cas9 mouse mutant that targets 3 genes that encode AGO family proteins, 2 of which are expressed during spermatogenesis (AGO3 and AGO4) and one that is said is not expressed, AGO1. This mouse mutant showed that AGO3 and AGO4 both contribute to spermatogenesis success as the "Ago413" mutation gave rise to an additive reduction in testis weight, due to spermatocyte apoptosis, and reduction in sperm count. Furthermore, they use insertion mouse mutants for Ago3 and Ago2 that express tagged versions of their corresponding proteins, which they use in combination with pan-AGO antibodies and Ago mutants to show differential expression and localization properties of AGO2, AGO3, and AGO4 (and the absence of AGO1) during spermatogenesis with a particular focus on meiotic prophase. They perform single-cell RNAseq and intricate analyses to demonstrate a change in distribution of meiotic stages in Ago413 mutants, and the overall cell cycle in spermatogonia and spermatocytes is altered. This analysis shows that the mutation leads to an inability to downregulate prior spermatogonia/spermatocyte stage transcripts in a timely manner. On the other hand, later-stage spermatocytes are abnormally expressing spermiogenesis genes. Similar to the Ago4 mutant previously characterized MSCI is disrupted. The authors also show that AGO3 has different interaction partners compared to AGO4 and focus their final assessment on a novel interaction partner of AGO3, BRG1. They show that this factor, which is involved in chromatin remodeling, is aberrantly localized to the sex body during meiotic prophase and diplonema. As BRG1 is involved in open chromatin, it is proposed that AGO3 restricts BRG1 (and related proteins) from the XY chromosome to ensure MSCI. Overall, this paper is very well constructed with mechanistic insights that make this a very impactful contribution to the research community. Major Comments:

1. The abstract contains "Ago413-/- mouse" without any explanation of what that is. The abstract needs to be a stand-alone document that does not require any referencing for context.

Response: We have included a sentence describing Ago413 in line 27

Figure 2C. - The significance bars are confusing as they appear to overlap strangely.

Response: We have modified this figure and now present the significance bars are on top of the data points.

On line 235, the authors state that "we first identified the top non-overlapping upregulated genes for Ago413+/+ germ cells in each cluster. Why did the authors not also select down-regulated genes in each cluster to perform a similar analysis?

__Response: __Thank you for this question. As our goal was to identify genes that are markers of the transcriptional program in each cell type, we used only uniquely upregulated genes for each cluster. Genes that are downregulated for a cluster may be indicative of the transcription in several other cell types, which is not easily interpretable. For a revised manuscript, we will perform this analysis to determine if there is any specific alterations in these downregulated genes.

Their Ago413 mutant characterization does a good job of assessing meiotic prophase and spermatozoa. However, their assessment of the stages in between these is lacking (meiotic divisions and spermiogenesis).

Response: We understand the reviewer's concern, however, it is not usual to study stages between the first meiotic division and spermiogenesis because meiosis II is so rapid and thus we lack tools to dissect it. In general, any defect that impacts meiosis I (and particularly prophase I) leads to cell death during prophase I or at metaphase I due to strictly adhered checkpoints that eradicate defective cells. Thus, the increased TUNEL staining in prophase I indicates to us that defective cells are cleared before exit from meiosis I, and those cells progressing to the spermatid stage are "normal" for meiosis II progression. For these cells that did complete meiosis I and progressed normally through meiosis II, we analyzed their spermiogenic outcome extensively (see section entitled "Post-meiotic spermatids from Ago413-/- males exhibit defective spermiogenesis and poor spermatozoa function"). This section included extensive sperm morphology, sperm motility and sperm fertility through in vitro fertilization assays. That said, we have added a sentence on line 268 to explain the transit through meiosis II.

The discovery of the interaction between BRG1 and AGO3 is exciting. They should assess BRG1 localization in later sub-stages, including late diplonema and diakinesis.

__Response: __BRG1(SMARCA4) was analyzed throughout prophase I, as shown in image 5G, including quantification of fluorescence intensity included the analysis of diplonema (5H-I). However, diakinesis was not included here since there was no observable signal of BRG1 in these cells. We have explained this in lines 459.

ATF2 should have been assessed in more detail, as was done for BRG1 in Figure 5.

__Response: __We agree with the Reviewer, however, staining of chromosome spreads with the anti ATF2 antibody was not possible in our hands after several attempts and changes in staining conditions. However, as staining of sections was successful, we showed localization of ATF2 on spermatocytes by co staining sections with SYCP3 and ATF2.

Reviewer #3 (Significance (Required)): Overall, this paper is very well constructed with mechanistic insights, as described in my reviewer comments, that make this a very impactful contribution to the research community.

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Referee #1

Evidence, reproducibility and clarity

In their manuscript de las Mercedes Carro et al investigated the role of Ago proteins during spermatogenesis by producing a triple knockout of Ago 1, 3 and 4. They first describe the pattern of expression of each protein and of Ago2 during the differentiation of male germ cells, then they describe the spermatogenesis phenotype of triple knockout males, study gene deregulation by scRNA seq and identify novel interacting proteins by co-IP mass spectrometry, in particular BRG1/SMARCA4, a chromatin remodeling factor and ATF2 a transcription factor. The main message is that Ago3 and 4 are involved in the regulation of XY gene silencing during meiosis, and also in the control of autosomal gene expression during meiosis. Overall the manuscript is well written, the topic, very interesting and the experiments, well-executed. However, there are some parts of the methodology and data interpretation that are unclear (see below).

Major comments

1. Please clarify how the triple KO was obtained, and if it is constitutive or specific to the male germline. In the result section a Cre (which cre?) is mentioned but it is not mentioned in the M&M. On Figure S1, a MICER VECTOR is shown instead of a deletion, but nothing is explained in the text nor legend. Could the authors provide more details in the results section as well as in the M&M ? This is essential to fully interpret the results obtained for this KO line, and to compare its phenotype to other lines (such as lines 184-9 Comparison of triple KO phenotype with that of Ago4 KO). Also, if it is a constitutive KO, the authors should mention if they observed other phenotypes in triple KO mice since AGO proteins are not only expressed in the male germline.
2. The paragraph corresponding to G2/M analysis is unclear to me. Why was this analysis performed? What does the heatmap show in Figure S4? What is G2/M score? (Fig 2D). Lines 219-220, do the authors mean that Pachytene cells are in a cell phase equivalent to G2/M? All this paragraph and associated figures require more explanation to clarify the method and interpretation.
3. I have concerns regarding Fig2G: to be convincing the analysis needs to be performed on several replicates, and, it is essential to compare tubules of the same stage - which does not seem to be the case. This does not appear to be the case. Besides, co (immunofluorescent) staining with markers of different cell types should be shown to demonstrate the earlier expression of some markers and their colocalization with markers of the earlier stages.
4. one important question that I think the authors should discuss regarding their scRNAseq: clusters are defined using well characterized markers. But Ago triple KO appears to alter the timing of expression of genes... could this deregulation affects the interperetation of scRNAseq clusters and results?
5. XY gene deregulation is mentioned throughout the result section but only X chromosome genes seem to have been investigated.... Even the gene content of the Y is highly repetitive, it would be very interesting to show the level of expression of Y single copy and Y multicopy genes in a figure 3 panel.
6. Can the authors elaborate on the observation that X gene upregulation is visible in the KO before MSCI; that is in lept/zygotene clusters (and in spermatogonia, if the difference visible in 3A is significant?)
7. miRNA analysis: could the authors indicate if X encoded miRNA were identified and found deregulated? Because Ago4 has been shown to lead to a downregulation of miRNA, among which many X encoded. It is therefore puzzling to see that the triple KO does not recapitulate this observation. Were the analyses performed differently in the present study and in Ago4 KO study?
8. The last results paragraph would also benefit from some additional information. It is not clear why the authors focused on enhancers and did not investigate promoters (or maybe they were but it's unclear). Which regions (size and location from TSS) were investigated for motif enrichment analyses? To what correspond the "transcriptional regulatory regions previously identified using dREG" mentioned in the M&M? I understand it's based on a previous article, but more info in the present manuscript would be useful.

Minor comments

1. In the introduction: The sentence "Ago1 is not expressed in the germline from the spermatogonia stage onwards allowing us to use this model to study the roles of Ago4 and Ago3 in spermatogenesis." is misleading because Ago1 is expressed at least in spermatogonia; It would be more precise to write "after spermatogonia stage" and rephrase the sentence. Otherwise it is surprising to see AGO1 protein in testis lysate and it is not in line with the scRNA seq shown in figure 2.
2. Could the authors precise if AGO proteins are expressed in other tissues? In somatic testicular cells?
3. Pattern of expression: How do the authors explain that AGO3 disappears at the diplotene stage and reappears in spermatids?
4. It would be useful to show the timing of expression of AGO 1 to 4 throughout spermatogenesis in the first paragraph of the article. Maybe the authors could present data from fig2B earlier?
5. Line 190: please modify the sentence "reveal no differences in cellular architecture of the seminiferous tubules when compared to wild-type males" to " reveal no gross differences..." since even without quantification of the different cell types it is visible that KO seminiferous tubules are different from WT tubules.
6. TUNEL analysis: please stage the tubules to determine the stage(s) at which apoptosis is the most predominant.
7. Figure S4B does not show an increase of cells at Pachytene stage but at Lepto/zygotene stage (as well as an increase of spermatogonia). Please comment this discrepancy with results shown in Fig2.
8. Fig5H and 5I are not mentioned in the result section. Also, it would be useful to label them with "all chromosomes" and "XY" to differentiate them easily
9. Line 530 "data provide further evidence for a functional association between AGO-dependent small RNAs and heterochromatin formation, maintenance and/or silencing." Please rephrase, the present article does not really show that AGO nuclear role depends on small RNAs.
10. Line 1256: replace "cite here " by appropriate reference
11. Please use SMARCA4 instead of BRG1 name as it is its official name.

Figures:

Figure 1: Are the pictures shown for Ago3-tagged and floxed from the same stages ? The leptotene stage in 1A looks like a zygotene, while some pachytene/diplotene stage pictures do not look alike.

Figure 1D, please label the Y scale properly (testis weight related to body weight)

FigS1: Please comment the presence of non-specific bands in the figure legend

Fig 2E and F, please indicate on the figure (in addition to its legend), what are the X and Y axes respectively to facilitate its reading.

2F: please use an easier abbreviation for Spermatocyte than Sp (which could spermatogonia, sperm etc..) such as Scyte I ? (same comment for Fig 3C)

Overall, for all figures showing GSEA analyses, could the authors explain what a High positive NES and a High negative NES mean in the results section?

Significance

Ago proteins are known for their roles in post transcriptional gene regulation via small RNA mediated cleavage of mRNA, which takes places in the cytoplasm. Some Ago proteins have been shown to be also located in the nucleus suggesting other non-canonical roles. It is the case of Ago4 which has been shown to localize to the transcriptionally silenced sex chromosomes (called sex body) of the spermatocyte nucleus, where it contributes to regulate their silencing (Modzelewski et al 2012). Interestingly, Ago4 knockout leads to Ago3 upregulation, including on the sex body indicating that Ago3 and Ago4 are involved in the same nuclear process. In their manuscript, de las Mercedes Carro et al., investigate the consequences of loss of both Ago3 and Ago4 in the male germline by the production of a triple knockout of Ago1, 3 and 4 in the mouse. With this model, the authors describe the role of Ago3 and Ago4 during spermatogenesis and show that they are involved in sex chromosome gene repression in spermatocytes and in round spermatids, as well as in the control of autosomal meiotic gene expression. Triple KO males have impaired meiosis and spermiogenesis, with fewer and abnormal spermatozoa resulting in reduced fertility. Since Ago1 male germline expression is restricted to pre-meiotic germ cells, it is not expected to contribute to the meiotic and postmeiotic phenotypes observed in the triple KO. The strengths of the study are i) the thorough analyses of mRNA expression at the single cell level, and in purified spermatocytes and spermatids (bulk RNAseq), ii) the identification of novel nuclear partners of AGO3/4 relevant for their described nuclear role: ATF2, which they show to also co-localize with the sex body, and BRG1/SMARCA4, a SWI/SNF chromatin remodeler. The main limitation of the study is the lack of information in the method regarding the production of the triple KO, as well as some aspects of the transcriptome and motif analyses. It is also surprising to see that the triple KO does not recapitulate the miRNA deregulation observed in Ago4 KO. The characterization of a non-canonical role of AGO3/4 in male germ cells will certainly influence researchers of the field, and also interest a broader audience studying Argonaute proteins and gene regulation at transcriptional and posttranscriptional levels.

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Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

In this work Jeong and colleagues focus on exploring the role of the acyltransferase ZDHHC9 in myelinating OLs in particular in the palmitoylation of several myelin proteins. After confirming the specific enrichment of the Zdhhc9 transcript in mouse and human OLs, the authors examine the subcellular localization of the protein in vitro and observed that in comparison with other isoforms, ZDHHC9 localizes at OLs cell bodies and at discrete puncta in the processes. These observations (Figures 1 and 2) led the authors to hypothesize that ZDHHC9 plays an important role in myelination. No gross changes were detected in OL development in Zdhhc9 KO mice and analyses from P28 Zdhhc9 KO mice crossed with Mobp-EGFP reporter mice did not show changes in EGFP+ OL differentiation (Figure 3).

However, and given the observed subcellular localization of ZDHHC9 in OL processes (Figure 2) and the observation that the percentage of unmyelinated axons is increased in Zdhhc9 KO (Figure 6), early time points to examine the differentiated pools of OLs and their capacity to extend processes/contact axons need to be considered.

We appreciate this point, but due to the order in which experiments were performed, the ZDHHC9 KO mouse colony that we maintained after initial submission of this work contains homozygous MOBP-EGFP, but not the mT/mG transgene that would be most optimal for the proposed experiment. We hope the reviewer appreciates that it would take considerable time and effort regarding mouse breeding to cross out the MOBP and add back the mT/mG. We nonetheless appreciate the importance of the point raised and therefore examined an earlier developmental time point (P21, 3 weeks) to quantify OLs and NG2+ OPCs. In our updated Fig 3C1-C3, we use Mobp-EGFP mice to show that Zdhhc9 KO does not significantly affect the number of EGFP+ OLs at this time point in the cortex, corpus callosum and spinal cord. We also show that in corpus callosum, Zdhhc9 KO does not significantly affect the number of NG2+ OPCs at this earlier time point (Fig 3D, E). Furthermore, immunostaining to detect BCAS1, a marker of pre-mature OLs, also revealed no qualitative difference with ZDHHC9 loss at P21. We show representative images from these BCAS1 experiments in an updated Fig S3. While these new experiments do not address the morphology of OLs in Zdhhc9 KO, they do provide further evidence that deficits in myelination in young Zdhhc9 KO mice (Figure 6) are not likely due to gross differences in OPC or OL numbers during development.

Maturation of OL in Zdhhc9 KO was examined by crossing Zdhhc9 KO with Pdgfra-CreER;R26- EGFP and following the newly EGFP-labelled OPCs following tamoxifen administration. No changes in the numbers of EGFP+ OL were detected. The authors concluded that the loss of ZDHHC9 does not alter oligodendrogenesis in either the young or mature CNS. The authors observed defects in Zdhhc9 KO OL protrusions that they attributed to abnormal OL membrane expansion (Fig 4 and 5). Can they show evidence for this?

This is an important point, and we appreciate the opportunity to explain the reasoning behind our initial statement more fully, while noting that other explanations are possible. Fig 5B (an Imaris-assisted reconstruction using the EGFP cell fill/morphology marker) highlights large spheroid-like distensions along OL processes. We reason that these spheroids are enclosed by the OL lipid membrane because if the membrane were ruptured, the EGFP signal would likely diffuse. This in turn suggests that the caliber of the OL process at the position of the spheroid is grossly abnormal i.e. the membrane has hyper-expanded. Given that OL membrane growth during myelination extends in two directions, i.e., spiral growth to the axonal surface and longitudinal growth along the axon, it is possible that spheroid-like structures are formed by uneven myelin growth. We recognize that we cannot yet conclude whether and how spheroid formation might be linked to the myelination deficit that we observe in Zdhhc9 KO mice. However, defining the subcellular mechanism for spheroid formation may provide further insights into this issue. We have therefore largely retained the original statement but have added the reasoning above to our revised Discussion.

The authors report that Zdhhc9 KO primary and secondary branches in OL were longer, some contained spheroid-like swellings and the OL protrusion complexity was higher. However, these data is partially contradictory to what they show in OL differentiation experiments in vitro (Fig 7). There is also no evidence for increased membrane expansion in Zdhhc9 knockdown myelin forming cells in culture. How to reconcile this? 

We appreciate the reviewer’s interest in this issue. Several non-mutually exclusive factors could account for the differences in OL morphology in vitro versus in vivo caused by Zdhhc9 loss. First, morphology in vivo may well be influenced by the axons and/or other extrinsic components around each OL that are not present in our primary cultures. Second, OL growth in vivo is highly 3-dimensional, whereas growth in culture is largely 2-dimensional – it may be difficult to support formation of spheroids (by definition, a 3-dimensional structure) in the latter situation. Finally, Zdhhc9 is absent in vivo from the beginning of development until the time points examined, whereas in our cultured OL experiments, Zdhhc9 shRNA is virally delivered to OPC cultures at DIV2 and likely acutely affects Zdhhc9 expression predominantly in committed OLs (following the switch to differentiation medium at DIV3). These differences may also affect the ability of other PATs or, potentially, palmitoylation-independent subcellular processes, to compensate for Zdhhc9 loss. We have more fully explained these points in our revised Discussion. 

Reviewer #2 (Public Review):

This study provides an in-depth exploration of the impact of X-linked ZDHHC9 gene mutations on cognitive deficits and epilepsy, with a particular focus on the expression and function of ZDHHC9 in myelin-forming oligodendrocytes (OLs). These findings offer crucial insights into understanding ZDHHC9-related X-linked intellectual disability (XLID) and shed light on the regulatory mechanisms of palmitoylation in myelination. The experimental design and analysis of results are convincing, providing a valuable reference for further research in this field. However, upon careful review, I believe the article still needs further improvement and supplementation in the following aspects:

(1) Regarding the subcellular localization experiment of ZDHHC9 mutants in OL, it is currently limited to in vitro cultured OL, lacking validation in vivo OL or myelin sheath. Additionally, it is necessary to investigate whether the abnormal subcellular localization of ZDHHC9 mutants affects their enzyme activity and palmitoylation modification of substrate proteins.

This is an important point but is technically challenging to address in vivo as it would likely require delivery of AAV to express ZDHHC9wt and XLID mutants specifically in OLs, preferably in the absence of endogenous ZDHHC9. We hope the reviewers would agree that this experiment is beyond the scope of the current study. However, we did compare the ability of ZDHHC9wt and XLID mutants to palmitoylate MBP, and to autopalmitoylate (sometimes used as a surrogate measure of PAT activity) in transfected heterologous cells. Although we recognize that this over-expression system is less physiological than a native OL, it has the benefit of being able to readily compare transfected wt vs mutant forms of ZDHHC9 with minimal contribution from endogenous ZDHHC9. Intriguingly, using this system, we found that autopalmitoylation activity of the XLID ZDHHC9-P150S mutant does not differ significantly from that of ZDHHC9wt, and that this mutant is still capable of palmitoylating MBP. Moreover, the R96W mutant, while impaired in autopalmitoylation, still palmitoylated MBP approximately 50% as effectively as ZDHHC9wt in our cell-based assay. These findings suggest that ZDHHC9-P150S and, probably, ZDHHC9-R96W mutants might still be able to palmitoylate substrates in OLs if they were properly localized. This possibility in turn suggests that impaired subcellular targeting in addition to, or instead of, impaired catalytic activity, may be a key factor in certain cases of ZDHHC9-associated XLID. We have expanded our Figure 8 (new panels 8E-G) to show these additional experiments and have summarized the conclusions above in our revised Discussion. We thank the reviewer for suggesting that we further investigate this issue.

(2) The experimental period (P21+21 days) using genetic labeling to track the development of myelinating cells may not be long enough. It is recommended to extend the observation time and analyze at more time points to more comprehensively reflect the impact of Zdhhc9 KO.

We appreciate this point from the reviewer but, regrettably, we did not maintain the PdgfraCreER; R26-EGFP; Zdhhc9 KO mouse line and hope the reviewer appreciates that it would take considerable time and effort to rederive this line and then perform the suggested extended time course experiments. However, we note for the reviewer that our preliminary studies did not reveal any effect of Zdhhc9 KO on the number of MOBP-EGFP+ OLs in 6-month-old mice (not shown), consistent with a model in which Zdhhc9 loss does not affect OPC-OL commitment per se.

(3) The author speculates that Zdhhc9 may regulate myelination by affecting the membrane localization of specific myelin proteins, but lacks direct experimental evidence to support this. It is suggested to detect the expression and distribution of relevant proteins in the myelin of Zdhhc9 KO mice.

We share the reviewer’s interest in this point but realized that it is more technically challenging to address than might be initially thought. The main protein we would implicate and seek to test is MBP, but we already found that there is no gross change in MBP distribution in vivo in Zdhhc9 KO mice (Fig 3A). However, an anti-MBP antibody recognizes all forms of MBP, not just the specific splice variants whose palmitoylation is affected by ZDHHC9 loss. Specifically assessing nanoscale distribution of these splice variants would require a way (e.g. anti-MBP splice form-specific antibodies that are compatible with immuno-EM) to distinguish these variants from other, non-palmitoylated forms of MBP. Although such an antibody could be an important tool, we hope the reviewers would agree that developing and characterizing such a reagent is beyond the scope of the current study.

We do, however, note that the lack of gross change in MBP distribution and levels in Zdhhc9 KO mice is consistent with the relatively mild phenotype of these mice, compared with shiverer (shi/shi) mice, in which MBP is completely lost. In shiverer, CNS compact myelin is almost absent (PMID: 671037; PMID: 88695; PMID: 460693) and, as the name suggests, mice display a shivering gait, and exhibit seizures and early death. In contrast, Zdhhc9 mice show only subtle behavioral deficits (PMID: 29944857). These differences are all consistent with a model in which Zdhhc9 KO mice, despite their significantly reduced MBP palmitoylation (Fig 8) have grossly normal distribution and levels of MBP when all splice variants are assessed (Fig 3, Fig 8). It is not inconceivable that Zdhhc9 KO mice have a nanoscale change in the distribution of MBP, particularly of specific palmitoylated splice variants, within myelin that profoundly affects myelin ultrastructure, without grossly altering MBP distribution. However, an alternative and not mutually exclusive possibility is that aberrant palmitoylation of other Zdhhc9 substrates accounts for, or contributes to, the abnormalities in myelin at the ultrastructural level. Addressing this issue would require a multi-pronged approach, not just to assess palmitoylation and distribution of such proteins in Zdhhc9 KO, but also to test whether they are direct Zdhhc9 substrates, in order to rule out indirect effects. We hope reviewers would agree that this is best left to a separate study. However, in our revised Discussion we now summarize what can be inferred regarding Zdhhc9-dependent effects on total and splicevariant specific distribution and levels of MBP.  

(4) Although the article mentions the association of Zdhhc9 with intellectual disabilities, it does not involve behavioral analysis of Zdhhc9 KO mice. It is recommended to supplement some behavioral experimental data to support the important role of Zdhhc9 in maintaining normal cognitive function, enhancing the clinical relevance of the article.

We appreciate this point from the reviewer. The behavior of the same ZDHHC9 KO mouse line that we used was reported in PMID: 31747610 and in PMID: 29944857. In the former study, Zdhhc9 KO mice were reported to display seizures reminiscent of phenotypes in human patients with ZDHHC9 mutation. The latter study assessed performance of Zddhc9 KO mice in several tasks that test cognitive function. Specifically the KO mice were reported to display “altered behaviour in the open-field test, elevated plus maze and acoustic startle test that is consistent with a reduced anxiety level; a reduced hang time in the hanging wire test that suggests underlying hypotonia but which may also be linked to reduced anxiety [and] deficits in the Morris water maze test of hippocampal-dependent spatial learning and memory.”. We have incorporate these findings in our revised Discussion, where we summarize how these phenotypes are common, not just to human patients with ZDHHC9 mutation, but also to other human neurodevelopmental conditions and mouse models in which ID is a common feature.

(5) For the abnormal myelination observed in Zdhhc9 KO mice, including unmyelinated large-diameter axons and excessively myelinated small-diameter axons, the article lacks indepth research and explanation on the exact mechanism and mode of action of ZDHHC9 in regulating myelination.

We share the reviewer’s interest in this point but again note that gaining definitive insights into this issue is far from trivial. Convincing evidence of a causative mechanism would require an exhaustive identification of ZDHHC9 in vivo substrates, followed by point mutation of substrate palmitoylation site(s) to determine the extent to which palmitoylation of such protein(s) phenocopies ZDHHC9 loss. Nonetheless, it is possible to break this question down and to summarize what we do and do not know. For example, our experiments in cultured OLs show that ZDHHC9 loss causes call-autonomous deficits in morphological maturation of these cells. We also know that ZDHHC9 loss results in impaired palmitoylation of MBP, a direct substrate for ZDHHC9. Moreover, loss of ZDHHC9 at Golgi outposts in OLs (a phenotype observed with several XLID-associated mutant forms of ZDHHC9, even those with no significant loss of catalytic activity) correlates with intellectual disability. Together, these findings are consistent with a model in which ZDHHC9 action at OL Golgi outposts is critical for normal myelination. However, it is yet to be determined whether the key substrates of ZDHHC9 include MBP, other palmitoyl-proteins that are key constituents of CNS myelin, or proteins whose palmitoylation is important for myelin protein trafficking and targeting. Another non-mutually exclusive possibility is that ZDHHC9 acts at Golgi outposts but indirectly, for example to drive the expression of myelin protein genes. Future experiments, including but not limited to palmitoyl-proteomics in ZDHHC9 (OL-specific) KO mice, will be needed to provide more definitive insights into this issue. We have expanded our Discussion of links between ZDHHC9 mutation and impaired myelination to summarize the above points.

(6) The function of ZDHHC9 in OL may be related to the Golgi apparatus, but its exact role in these structures is still unclear. It is suggested to discuss in more detail the role of ZDHHC9 in the Golgi apparatus in the discussion section.

We appreciate this point, which we considered as related to point (5) above. In our revised Discussion we highlight how ZDHHC9 action at Golgi outposts may involve direct palmitoylation of myelin proteins, palmitoylation of proteins that direct myelin proteins to the myelin membrane and/or activation of gene expression programs that serve to drive myelination. We further note that these possibilities are not mutually exclusive.

(7) More experimental support and in-depth research are needed on the detailed mechanism of how ZDHHC9 and Golga7 cooperatively regulate MBP palmitoylation, and how this decrease in palmitoylation level leads to myelination defects.

This is another important point – our new experiments suggest that, although some XLID mutations markedly affect ZDHHC9’s ability to palmitoylate MBP, others do not, yet all of the mutant forms fail to localize to Golgi outposts. These findings are consistent with a model in which the subcellular location at which ZDHHC9 palmitoylates MBP, and potentially other substrates, is critical for normal myelination. Interestingly, despite their marked differences in basal catalytic activity (as assessed by autopalmitoylation), wt and all XLID forms of ZDHHC9 appear to show enhanced activity (measured by both auto- and MBP palmitoylation) in the presence of ZDHHC9, suggesting that the association with Golga7 (which also localizes to Golgi outposts) is central to ZDHHC9 activity. This model is also highly consistent with the biased expression of Golga7 in OLs, compared to other CNS cell types (Fig 1E, 1F). Moreover, XLID-associated mutant forms of ZDHHC9 also show reduced protein stability and are impaired in their ability to form complexes with Golga7 (also known as Golgi Complex Protein 16kDa; GCP16; PMID: 37035671). Failure of ZDHHC9 XLID mutants to localize to Golgi outposts may thus be due to aberrant trafficking of mutant ZDHHC9 per se, but may also involve impaired association/stabilization of ZDHHC9/Golga7 complexes at these locations. Again, it is possible that either or both of these mechanisms, which are not mutually exclusive, contribute to impaired MBP palmitoylation and/or myelination deficits. We summarize these points in our revised Discussion.

In summary, it is recommended that the authors address the above issues through additional experiments and improved discussions to further strengthen the credibility and clinical relevance of the article.

Recommendations for the authors:  

Reviewer #1 (Recommendations For The Authors):

No gross changes were detected in OL development in Zdhhc9 KO mice and analyses from P28 Zdhhc9 KO mice crossed with Mobp-EGFP reporter mice did not show changes in EGFP+ OL differentiation (Figure 3). However, and given the observed subcellular localization of ZDHHC9 in OL processes (Figure 2) and the observation that the percentage of unmyelinated axons is increased in Zdhhc9 KO (Figure 6), ***early time points to examine the differentiated pools of OLs and their capacity to extend processes/contact axons need to be considered***.

We appreciate this point, but due to the order in which experiments were performed, the ZDHHC9 KO mouse colony that we maintained after initial submission of this work contains homozygous MOBP-EGFP, but not the mT/mG transgene that would be most optimal for the proposed experiment. We hope the reviewer appreciates that it would take considerable time and effort regarding mouse breeding to cross out the MOBP and add back the mT/mG. We nonetheless appreciate the importance of the point raised and therefore examined an earlier developmental time point (P21, 3 weeks) to quantify OLs and NG2+ OPCs. In our updated Fig 3C1-C3, we use Mobp-EGFP mice to show that Zdhhc9 KO does not significantly affect the number of EGFP+ OLs at this time point in the cortex, corpus callosum and spinal cord. We also show that in corpus callosum, Zdhhc9 KO does not significantly affect the number of NG2+ OPCs at this earlier time point (Fig 3D, E). Furthermore, immunostaining to detect BCAS1, a marker of pre-mature OLs, also revealed no qualitative difference with ZDHHC9 loss at P21. We show representative images from these BCAS1 experiments in an updated Fig S3. While these new experiments do not address the morphology of OLs in Zdhhc9 KO, they do provide further evidence that deficits in myelination in young Zdhhc9 KO mice (Figure 6) are not likely due to gross differences in OPC or OL numbers during development.

The authors observed defects in Zdhhc9 KO OL protrusions that they attributed to abnormal OL membrane expansion (Fig 4 and 5). Can they show evidence for this?

This is an important point, and we appreciate the opportunity to explain the reasoning behind our initial statement more fully, while noting that other explanations are possible. Fig 5B (an Imaris-assisted reconstruction using the EGFP cell fill/morphology marker) highlights large spheroid-like distensions along OL processes. We reason that these spheroids are enclosed by the OL lipid membrane because if the membrane were ruptured, the EGFP signal would likely diffuse. This in turn suggests that the caliber of the OL process at the position of the spheroid is grossly abnormal i.e. the membrane has hyper-expanded. Given that OL membrane growth during myelination extends in two directions, i.e., spiral growth to the axonal surface and longitudinal growth along the axon, it is possible that spheroid-like structures are formed by uneven myelin growth. We recognize that we cannot yet conclude whether and how spheroid formation might be linked to the myelination deficit that we observe in Zdhhc9 KO mice.

However, defining the subcellular mechanism for spheroid formation may provide further insights into this issue. We have therefore largely retained the original statement but have added the reasoning above to our revised Discussion.

The authors report that Zdhhc9 KO primary and secondary branches in OL were longer, some contained spheroid-like swellings and the OL protrusion complexity was higher. However, these data is partially contradictory to what they show in OL differentiation experiments in vitro (Fig 7). There is also no evidence for increased membrane expansion in Zdhhc9 knockdown myelin forming cells in culture. How do they reconcile these different findings?

We appreciate the reviewer’s interest in this issue. Several non-mutually exclusive factors could account for the differences in OL morphology in vitro versus in vivo caused by Zdhhc9 loss. First, morphology in vivo may well be influenced by the axons and/or other extrinsic components around each OL that are not present in our primary cultures. Second, OL growth in vivo is highly 3-dimensional, whereas growth in culture is largely 2-dimensional – it may be difficult to support formation of spheroids (by definition, a 3-dimensional structure) in the latter situation. Finally, Zdhhc9 is absent in vivo from the beginning of development until the time points examined, whereas in our cultured OL experiments, Zdhhc9 shRNA is virally delivered to OPC cultures at DIV2 and likely acutely affects Zdhhc9 expression predominantly in committed OLs (following the switch to differentiation medium at DIV3). These differences may also affect the ability of other PATs or, potentially, palmitoylation-independent subcellular processes, to compensate for Zdhhc9 loss. We have more fully explained these points in our revised Discussion. 

Page 7: "The OL processes in this culture condition correspond to large lipid-rich membranous sheets that form spiral membrane expansion on axons in vivo (49)." At which stage are authors referring to? OL processes are extended in culture before membrane formation and this is not clear here. In a 3-days differentiation culture, most OLs have not yet formed a myelin sheath (eg., Figure 2 in Zuchero et al., 2015, Dev Cell).

We appreciate the reviewer highlighting this point. We first note that our oligodendrocyte (OL) culture conditions differ from the immunopanning method used by Zuchero et al., 2015 (original reference (Emery and Dugas, 2013)), which may affect the time course and progression of OL process elaboration and/or myelin sheath formation. We further note that in our cultures most EGFP+ processes are also MBP+ at the time point examined (strictly 3 days plus 9 hours post-differentiation). It thus seems likely that these MBP+ structures largely correspond to the MBP+ wrapping sheaths that occur in vivo, so we have therefore retained our original statement but have added this further explanation.

Minor: Figure 6 (Legend): Time points should be indicated throughout the panels.

We have added this information as requested

Reviewer 2 Recommendations for the Authors:

(1) Regarding the subcellular localization experiment of ZDHHC9 mutants in OL, it is currently limited to in vitro cultured OL, lacking validation in vivo OL or myelin sheath. Additionally, it is necessary to investigate whether the abnormal subcellular localization of ZDHHC9 mutants affects their enzyme activity and palmitoylation modification of substrate proteins.

We thank the reviewer for raising this point. New data in our revised Figure 8 compares autopalmitoylation (sometimes used as a surrogate measure of PAT activity) of ZDHHC9wt and XLID mutants, and their ability to palmitoylate MBP in transfected cells. Intriguingly, we found that autopalmitoylation activity of the ZDHHC9-P150S mutant does not differ significantly from that of ZDHHC9wt, and that this mutant is still capable of palmitoylating MBP. Moreover, the R96W mutant, while impaired in autopalmitoylation, still palmitoylated MBP approximately 50% as effectively as ZDHHC9wt in our cell-based assay. These findings suggest that ZDHHC9-P150S and, probably, ZDHHC9-R96W mutants might still be able to palmitoylate substrates in OLs if they were properly localized. This possibility in turn suggests that impaired subcellular targeting in addition to, or instead of, impaired catalytic activity, may be a key factor in certain cases of ZDHHC9-associated XLID. We have expanded our Figure 8 to show these new experiments and have summarized the conclusions above in our revised Discussion. We thank the reviewer for suggesting that we further investigate this issue.

(2) The experimental period (P21+21 days) using genetic labeling to track the development of myelinating cells may not be long enough. It is recommended to extend the observation time and analyze at more time points to more comprehensively reflect the impact of Zdhhc9 KO.

We appreciate this point from the reviewer but, regrettably, we did not maintain the PdgfraCreER; R26-EGFP; Zdhhc9 KO mouse line and hope the reviewer appreciates that it would take considerable time and effort to rederive this line and then perform the suggested extended time course experiments. However, we note for the reviewer that our preliminary studies did not reveal any effect of Zdhhc9 KO on the number of MOBP-EGFP+ OLs in 6-month-old mice (not shown), consistent with a model in which Zdhhc9 loss does not affect OPC-OL commitment per se.

(3) The author speculates that Zdhhc9 may regulate myelination by affecting the membrane localization of specific myelin proteins, but lacks direct experimental evidence to support this. It is suggested to detect the expression and distribution of relevant proteins in the myelin of Zdhhc9 KO mice.

We share the reviewer’s interest in this point but realized that it is more technically challenging to address than might be initially thought. The main protein we would implicate and seek to test is MBP, but we already found that there is no gross change in MBP distribution in vivo in Zdhhc9 KO mice (Fig 3A). However, an anti-MBP antibody recognizes all forms of MBP, not just the specific splice variants whose palmitoylation is affected by ZDHHC9 loss. Specifically assessing nanoscale distribution of these splice variants would require a way (e.g. am anti-MBP splice form-specific antibody that is compatible with immuno-EM) to distinguish these variants from other, non-palmitoylated forms of MBP. Although such an antibody could be an important tool we hope the reviewers would agree that developing and characterizing such a reagent is beyond the scope of the current study.

We do, however, note that the lack of gross change in MBP distribution and levels in Zdhhc9 KO mice is consistent with the relatively mild phenotype of these mice, compared with shiverer (shi/shi) mice, in which MBP is completely lost. In shiverer, CNS compact myelin is almost absent (PMID: 671037; PMID: 88695; PMID: 460693) and, as the name suggests, mice display a shivering gait, and exhibit seizures and early death. In contrast, Zdhhc9 mice show only subtle behavioral deficits (PMID: 29944857). These differences are all consistent with a model in which Zdhhc9 KO mice, despite their significantly reduced MBP palmitoylation (Fig 8) have grossly normal distribution and levels of MBP when all splice variants are assessed (Fig 3, Fig 8). It is not inconceivable that Zdhhc9 KO mice have a nanoscale change in the distribution of MBP, particularly of specific palmitoylated splice variants, within myelin that profoundly affects myelin ultrastructure, without grossly altering MBP distribution. However, an alternative and not mutually exclusive possibility is that aberrant palmitoylation of other

Zdhhc9 substrates accounts for, or contributes to, the abnormalities in myelin at the ultrastructural level. Addressing this issue would require a multi-pronged approach, not just to assess palmitoylation and distribution of such proteins in Zdhhc9 KO, but also to test whether they are direct Zdhhc9 substrates, in order to rule out indirect effects. We hope reviewers would agree that this is best left to a separate study. However, in our revised Discussion we now summarize what can be inferred regarding Zdhhc9-dependent effects on total and splicevariant specific distribution and levels of MBP.  

(4) Although the article mentions the association of Zdhhc9 with intellectual disabilities, it does not involve behavioral analysis of Zdhhc9 KO mice. It is recommended to supplement some behavioral experimental data to support the important role of Zdhhc9 in maintaining normal cognitive function, enhancing the clinical relevance of the article.

We appreciate this point from the reviewer. The behavior of the same ZDHHC9 KO mouse line that we used was reported in PMID: 31747610 and in PMID: 29944857. In the former study, Zdhhc9 KO mice were reported to display seizures reminiscent of phenotypes in human patients with ZDHHC9 mutation. The latter study assessed performance of Zddhc9 KO mice in several tasks that test cognitive function. Specifically the KO mice were reported to display “altered behaviour in the open-field test, elevated plus maze and acoustic startle test that is consistent with a reduced anxiety level; a reduced hang time in the hanging wire test that suggests underlying hypotonia but which may also be linked to reduced anxiety [and] deficits in the Morris water maze test of hippocampal-dependent spatial learning and memory.”. We have incorporate these findings in our revised Discussion, where we summarize how these phenotypes are common, not just to human patients with ZDHHC9 mutation, but also to other human neurodevelopmental conditions and mouse models in which ID is a common feature.

(5) For the abnormal myelination observed in Zdhhc9 KO mice, including unmyelinated large-diameter axons and excessively myelinated small-diameter axons, the article lacks indepth research and explanation on the exact mechanism and mode of action of ZDHHC9 in regulating myelination.

We share the reviewer’s interest in this point but again note that gaining definitive insights into this issue is far from trivial. Convincing evidence of a causative mechanism would require an exhaustive identification of ZDHHC9 in vivo substrates, followed by point mutation of substrate palmitoylation site(s) to determine the extent to which palmitoylation of such protein(s) phenocopies ZDHHC9 loss. Nonetheless, it is possible to break this question down and to summarize what we do and do not know. For example, our experiments in cultured OLs show that ZDHHC9 loss causes call-autonomous deficits in morphological maturation of these cells. We also know that ZDHHC9 loss results in impaired palmitoylation of MBP, a direct substrate for ZDHHC9. Moreover, loss of ZDHHC9 at Golgi outposts in OLs (a phenotype observed with several XLID-associated mutant forms of ZDHHC9, even those with no significant loss of catalytic activity) correlates with intellectual disability. Together, these findings are consistent with a model in which ZDHHC9 action at OL Golgi outposts is critical for normal myelination. However, it is yet to be determined whether the key substrates of ZDHHC9 include MBP, other palmitoyl-proteins that are key constituents of CNS myelin, or proteins whose palmitoylation is important for myelin protein trafficking and targeting. Another non-mutually exclusive possibility is that ZDHHC9 acts at Golgi outposts but indirectly, for example to drive the expression of myelin protein genes. Future experiments, including but not limited to palmitoyl-proteomics in ZDHHC9 (OL-specific) KO mice, will be needed to provide more definitive insights into this issue. We have expanded our Discussion of links between ZDHHC9 mutation and impaired myelination to summarize the above points.

(6) The function of ZDHHC9 in OL may be related to the Golgi apparatus, but its exact role in these structures is still unclear. It is suggested to discuss in more detail the role of ZDHHC9 in the Golgi apparatus in the discussion section.

We appreciate this point, which we considered as related to point (5) above. In our revised Discussion we highlight how ZDHHC9 action at Golgi outposts may involve direct palmitoylation of myelin proteins, palmitoylation of proteins that direct myelin proteins to the myelin membrane and/or activation of gene expression programs that serve to drive myelination. We further note that these possibilities are not mutually exclusive.

(7) More experimental support and in-depth research are needed on the detailed mechanism of how ZDHHC9 and Golga7 cooperatively regulate MBP palmitoylation, and how this decrease in palmitoylation level leads to myelination defects.

This is another important point – our new experiments suggest that, although some XLID mutations markedly affect ZDHHC9’s ability to palmitoylate MBP, others do not, yet all of the mutant forms fail to localize to Golgi outposts. These findings are consistent with a model in which the subcellular location at which ZDHHC9 palmitoylates MBP, and potentially other substrates, is critical for normal myelination. Interestingly, despite their marked differences in basal catalytic activity (as assessed by autopalmitoylation), wt and all XLID forms of ZDHHC9 appear to show enhanced activity (measured by both auto- and MBP palmitoylation) in the presence of ZDHHC9, suggesting that the association with Golga7 (which also localizes to Golgi outposts) is central to ZDHHC9 activity. This model is also highly consistent with the biased expression of Golga7 in OLs, compared to other CNS cell types (Fig 1E, 1F). Moreover, XLID-associated mutant forms of ZDHHC9 also show reduced protein stability and are impaired in their ability to form complexes with Golga7 (also known as Golgi Complex Protein 16kDa; GCP16; PMID: 37035671). Failure of ZDHHC9 XLID mutants to localize to Golgi outposts may thus be due to aberrant trafficking of mutant ZDHHC9 per se, but may also involve impaired association/stabilization of ZDHHC9/Golga7 complexes at these locations. Again, it is possible that either or both of these mechanisms, which are not mutually exclusive, contribute to impaired MBP palmitoylation and/or myelination deficits. We summarize these points in our revised Discussion.

➀ Between 70 and 90% of people have recurrent episodes of pain, ⒈ İnsanların %70 ila %90’ı tekrarlayan ağrı atakları yaşamaktadır.

➁ and one-third of patients continue to have persistent, recurrent or intermittent pain after their first episode. ⒉ Ve hastaların üçte biri ilk atağından sonra kalıcı, tekrarlayan ya da aralıklı ağrılar yaşamaya devam eder.

➂ In addition to the difficulty with healing, the degenerative process is ongoing with age, and many patients do not minimize potential risk factors. ⒊ İyileşme zorluğuna ek olarak, dejeneratif süreç yaşla birlikte devam eder ve birçok hasta potansiyel risk faktörlerini en aza indirmez.

➃ All of this can contribute to continue episodes of low back pain (LBP). ⒋ Tüm bunlar, bel ağrısının (LBP) devam eden ataklarına katkıda bulunabilir.

➄ The cause of LBP is often multifactorial but combined motions of lumbar flexion with rotation increase risk to the lumbar disk. ⒌ Bel ağrısının nedeni genellikle çok faktörlüdür; ancak belin öne eğilme (fleksiyon) ve dönme hareketlerinin birlikte yapılması, lomber diske yönelik riski artırır.

➅ This is further exacerbated by inflexibilities. ⒍ Bu durum, esneklik eksiklikleriyle daha da kötüleşir.

Reviewer #2 (Public review):

Summary:<br /> This study investigates the impact of mother-child neural synchronization and the quality of parent-child relationships on the development of Theory of Mind (ToM) and social cognition. Utilizing a naturalistic fMRI movie-viewing paradigm, the authors analyzed inter-subject neural synchronization in mother-child dyads and explored the connections between neural maturity, parental caregiving, and social cognitive outcomes. The findings indicate age-related maturation in ToM and social pain networks, emphasizing the importance of dyadic interactions in shaping ToM performance and social skills, thereby enhancing our understanding of the environmental and intrinsic influences on social cognition.

Strengths:<br /> This research addresses a significant question in developmental neuroscience, by linking social brain development with children's behaviors and parenting. It also uses a robust methodology by incorporating neural synchrony measures, naturalistic stimuli, and a substantial sample of mother-child dyads to enhance its ecological validity. Furthermore, the SEM approach provides a nuanced understanding of the developmental pathways associated with Theory of Mind (ToM). The manuscript also addressed many concerns raised in the initial review. The adoption of the neuroconstructivist framework effectively frames neural and cognitive development as reciprocal, addressing prior concerns about causality. The justification for methodological choices, such as omitting resting-state baselines due to scanning challenges in children and using unit-weighted scoring for ToM tasks, further strengthens the study's credibility.

Weaknesses:<br /> (1) The revised introduction has improved, particularly in framing the first goal-developmental changes in ToM and SPM networks-as a "developmental anchor" for goals 2 and 3. However, given prior research on age-related changes in these networks (e.g., Richardson et al., 2018), the authors should clarify whether this goal seeks to replicate prior findings or to extend them under new contexts. Specifying how this part differs from existing work and articulating specific hypotheses would enhance the focus.<br /> (2) I still have some reservations about retaining the slightly causal term "shape" in the title. While the manuscript now carefully avoids causal claims, the title may still be interpreted as implying directionality, especially by non-specialist audiences.<br /> (3) One more question about Figure 2A and 2B: adults and children showed highly similar response curves for video frames, yet some peaks (e.g., T02, T05, T06) are identified as ToM or SPM events only in adults. Whether statistical methods account for the differences? Or whether the corresponding video frames contain subtle social cues that only adults can process?

Example of an annotation on AA Communication Studio text

Reviewer #1 (Public review):

Summary:

The authors state the study's goal clearly: "The goal of our study was to understand to what extent animal individuality is influenced by situational changes in the environment, i.e., how much of an animal's individuality remains after one or more environmental features change." They use visually guided behavioral features to examine the extent of correlation over time and in a variety of contexts. They develop new behavioral instrumentation and software to measure behavior in Buridan's paradigm (and variations thereof), the Y-maze, and a flight simulator. Using these assays, they examine the correlations between conditions for a panel of locomotion parameters. They propose that inter-assay correlations will determine the persistence of locomotion individuality.

Strengths:

The OED defines individuality as "the sum of the attributes which distinguish a person or thing from others of the same kind," a definition mirrored by other dictionaries and the scientific literature on the topic. The concept of behavioral individuality can be characterized as: (1) a large set of behavioral attributes, (2) with inter-individual variability, that are (3) stable over time. A previous study examined walking parameters in Buridan's paradigm, finding that several parameters were variable between individuals, and that these showed stability over separate days and up to 4 weeks (DOI: 10.1126/science.aaw718). The present study replicates some of those findings and extends the experiments from temporal stability to examining correlation of locomotion features between different contexts.

The major strength of the study is using a range of different behavioral assays to examine the correlations of several different behavior parameters. It shows clearly that the inter-individual variability of some parameters is at least partially preserved between some contexts, and not preserved between others. The development of high-throughput behavior assays and sharing the information on how to make the assays is a commendable contribution.

Weaknesses:

The definition of individuality considers a comprehensive or large set of attributes, but the authors consider only a handful. In Supplemental Fig. S8, the authors show a large correlation matrix of many behavioral parameters, but these are illegible and are only mentioned briefly in Results. Why were five or so parameters selected from the full set? How were these selected? Do the correlation trends hold true across all parameters? For assays in which only a subset of parameters can be directly compared, were all of these included in the analysis, or only a subset?

The correlation analysis is used to establish stability between assays. For temporal re-testing, "stability" is certainly the appropriate word, but between contexts it implies that there could be 'instability'. Rather, instead of the 'instability' of a single brain process, a different behavior in a different context could arise from engaging largely (or entirely?) distinct context-dependent internal processes, and have nothing to do with process stability per se. For inter-context similarities, perhaps a better word would be "consistency".

The parameters are considered one-by-one, not in aggregate. This focuses on the stability/consistency of the variability of a single parameter at a time, rather than holistic individuality. It would appear that an appropriate measure of individuality stability (or individuality consistency) that accounts for the high-dimensional nature of individuality would somehow summarize correlations across all parameters. Why was a multivariate approach (e.g. multiple regression/correlation) not used? Treating the data with a multivariate or averaged approach would allow the authors to directly address 'individuality stability', along with the analyses of single-parameter variability stability.

The correlation coefficients are sometimes quite low, though highly significant, and are deemed to indicate stability. For example, in Figure 4C top left, the % of time walked at 23{degree sign}C and 32{degree sign}C are correlated by 0.263, which corresponds to an R2 of 0.069 i.e. just 7% of the 32{degree sign}C variance is predictable by the 23{degree sign}C variance. Is it fair to say that 7% determination indicates parameter stability? Another example: "Vector strength was the most correlated attention parameter... correlations ranged... to -0.197," which implies that 96% (1 - R2) of Y-maze variance is not predicted by Buridan variance. At what level does an r value not represent stability?

The authors describe a dissociation between inter-group differences and inter-individual variation stability, i.e. sometimes large mean differences between contexts, but significant correlation between individual test and retest data. Given that correlation is sensitive to slope, this might be expected to underestimate the variability stability (or consistency). Is there a way to adjust for the group differences before examining correlation? For example, would it be possible to transform the values to in-group ranks prior to correlation analysis?

What is gained by classifying the five parameters into exploration, attention, and anxiety? To what extent have these classifications been validated, both in general, and with regard to these specific parameters? Is increased walking speed at higher temperature necessarily due to increased 'explorative' nature, or could it be attributed to increased metabolism, dehydration stress, or a heat-pain response? To what extent are these categories subjective?

The legends are quite brief and do not link to descriptions of specific experiments. For example, Figure 4a depicts a graphical overview of the procedure, but I could not find a detailed description of this experiment's protocol.

Using the current single-correlation analysis approach, the aims would benefit from re-wording to appropriately address single-parameter variability stability/consistency (as distinct from holistic individuality). Alternatively, the analysis could be adjusted to address the multivariate nature of individuality, so that the claims and the analysis are in concordance with each other.

The study presents a bounty of new technology to study visually guided behaviors. The Github link to the software was not available. To verify successful transfer or open-hardware and open-software, a report would demonstrate transfer by collaboration with one or more other laboratories, which the present manuscript does not appear to do. Nevertheless, making the technology available to readers is commendable.<br /> The study discusses a number of interesting, stimulating ideas about inter-individual variability and presents intriguing data that speaks to those ideas, albeit with the issues outlined above.

While the current work does not present any mechanistic analysis of inter-individual variability, the implementation of high-throughput assays sets up the field to more systematically investigate fly visual behaviors, their variability, and their underlying mechanisms.

Comments on revisions:

I want to express my appreciation for the authors' responsiveness to the reviewer feedback. They appear to have addressed my previous concerns through various modifications including GLM analysis, however, some areas still require clarification for the benefit of an audience that includes geneticists.

(1) GLM Analysis Explanation (Figure 9)<br /> While the authors state that their new GLM results support their original conclusions, the explanation of these results in the text is insufficient. Specifically:

- The interpretation of coefficients and their statistical significance needs more detailed explanation. The audience includes geneticists and other non-statistical people, so the GLM should be explained in terms of the criteria or quantities used to assess how well the results conform with the hypothesis, and to what extent they diverge.<br /> - The criteria used to judge how well the GLM results support their hypothesis are not clearly stated.<br /> - The relationship between the GLM findings and their original correlation-based conclusions needs better integration and connection, leading the reader through your reasoning.

(2) Documentation of Changes<br /> One struggle with the revised manuscript is that no "tracked changes" version was included, so it is hard to know exactly what was done. Without access to the previous version of the manuscript, it is difficult to fully assess the extent of revisions made. The authors should provide a more comprehensive summary of the specific changes implemented, particularly regarding:

(3) Statistical Method Selection<br /> The authors mention using "ridge regression to mitigate collinearity among predictors" but do not adequately justify this choice over other approaches. They should explain:

- Why ridge regression was selected as the optimal method<br /> - How the regularization parameter (λ) was determined<br /> - How this choice affects the interpretation of environmental parameters' influence on individuality

Author response:

The following is the authors’ response to the original reviews

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

The authors state the study's goal clearly: "The goal of our study was to understand to what extent animal individuality is influenced by situational changes in the environment, i.e., how much of an animal's individuality remains after one or more environmental features change." They use visually guided behavioral features to examine the extent of correlation over time and in a variety of contexts. They develop new behavioral instrumentation and software to measure behavior in Buridan's paradigm (and variations thereof), the Y-maze, and a flight simulator. Using these assays, they examine the correlations between conditions for a panel of locomotion parameters. They propose that inter-assay correlations will determine the persistence of locomotion individuality.

Strengths: 

The OED defines individuality as "the sum of the attributes which distinguish a person or thing from others of the same kind," a definition mirrored by other dictionaries and the scientific literature on the topic. The concept of behavioral individuality can be characterized as: 

(1) a large set of behavioral attributes, 

(2) with inter-individual variability, that are 

(3) stable over time. 

A previous study examined walking parameters in Buridan's paradigm, finding that several parameters were variable between individuals, and that these showed stability over separate days and up to 4 weeks (DOI: 10.1126/science.aaw718). The present study replicates some of those findings and extends the experiments from temporal stability to examining the correlation of locomotion features between different contexts.

The major strength of the study is using a range of different behavioral assays to examine the correlations of several different behavior parameters. It shows clearly that the inter-individual variability of some parameters is at least partially preserved between some contexts, and not preserved between others. The development of highthroughput behavior assays and sharing the information on how to make the assays is a commendable contribution.

We thank the reviewer for his exceptionally kind assessment of our work!

Weaknesses: 

The definition of individuality considers a comprehensive or large set of attributes, but the authors consider only a handful. In Supplemental Fig. S8, the authors show a large correlation matrix of many behavioral parameters, but these are illegible and are only mentioned briefly in Results. 

We have now uploaded a high-resolution PDF to the Github Address: https://github.com/LinneweberLab/Mathejczyk_2024_eLife_Individuality/blob/main/S8.pdf, and this is also mentioned in the figure legend for Fig. S8

Why were five or so parameters selected from the full set? How were these selected? 

The five parameters (% of time walked, walking speed, vector strength, angular velocity, and centrophobicity) were selected because they describe key aspects of the investigated behaviors that can be compared directly across assays. Importantly, several parameters we typically use (e.g., Linneweber et al., 2020) cannot be applied under certain conditions, such as darkness or the absence of visual cues. Furthermore, these five parameters encompass three critical aspects of navigation across standard visual behavioral arenas: (1) The “exploration” category is characterized by parameters describing the fly’s activity. (2) Parameters related to “attention” reflect heightened responses to visual cues, but unlike commonly used metrics such as angle or stripe deviations (e.g., Coulomb, 2012; Linneweber et al., 2020), they can also be measured in absence of visual cues and are therefore suitable for cross-assay comparisons. (3) The parameter “centrophobicity,” used as a potential indicator of anxiety, is conceptually linked to the open-field test in mice, where the ratio of wall-to-open-field activity is frequently calculated as a measurement of anxiety (see for example Carter, Sheh, 2015, chapter 2. https://www.sciencedirect.com/book/9780128005118/guide-to-researchtechniques-in-neuroscience). Admittedly, this view is frequently challenged in mice, but it has a long history which is why we use it.

Do the correlation trends hold true across all parameters? For assays in which only a subset of parameters can be directly compared, were all of these included in the analysis, or only a subset? 

As noted above, we only included a subset of parameters in our final analysis, as many were unsuitable for comparison across assays while still providing valuable assayspecific information which are important to relate these results to previous publications.

The correlation analysis is used to establish stability between assays. For temporal retesting, "stability" is certainly the appropriate word, but between contexts, it implies that there could be 'instability'. Rather, instead of the 'instability' of a single brain process, a different behavior in a different context could arise from engaging largely (or entirely?) distinct context-dependent internal processes, and have nothing to do with process stability per se. For inter-context similarities, perhaps a better word would be "consistency". 

Thank you for this suggestion. During the preparation of the manuscript, we indeed frequently alternated between the terms “stability” and “consistency.” And decided to go with “stability” as the only descriptor, to keep it simple. We now fully agree with the reviewer’s argument and have replaced “stability” by “consistency” throughout the current version of the manuscript in order to increase clarity and coherence.

The parameters are considered one by one, not in aggregate. This focuses on the stability/consistency of the variability of a single parameter at a time, rather than holistic individuality. It would appear that an appropriate measure of individuality stability (or individuality consistency) that accounts for the high-dimensional nature of individuality would somehow summarize correlations across all parameters. Why was a multivariate approach (e.g. multiple regression/correlation) not used? Treating the data with a multivariate or averaged approach would allow the authors to directly address 'individuality stability' and analyses of single-parameter variability stability.

We agree with the reviewer that a multivariate analysis adds clear advantages in terms of statistical power, in addition to our chosen approach. On one hand, we believe that the simplicity of our initial analysis, both for correlational and mean data, makes easy for readers to understand and reproduce our data. While preparing the previous version of the manuscript we were skeptical since more complex analyses often involve numerous choices, which can complicate reproducibility. For instance, a recent study in personality psychology (Paul et al., 2024) highlighted the risks of “forking paths” in statistical analysis, showing that certain choices of statistical methods could even reverse findings—a concern mitigated by our simplistic straightforward approach. Still, in preparation of this revised version of the manuscript, we accepted the reviewer’s advice and reanalyzed the data using a generalized linear model. This analysis nicely recapitulates our initial findings and is now summarized in a single figure (Fig. 9).

The correlation coefficients are sometimes quite low, though highly significant, and are deemed to indicate stability. For example, in Figure 4C top left, the % of time walked at 23{degree sign}C and 32{degree sign}C are correlated by 0.263, which corresponds to an R2 of 0.069 i.e. just 7% of the 32{degree sign}C variance is predictable by the 23{degree sign}C variance. Is it fair to say that a 7% determination indicates parameter stability? Another example: "Vector strength was the most correlated attention parameter... correlations ranged... to -0.197," which implies that 96% (1 - R2) of Y-maze variance is not predicted by Buridan variance. At what level does an r value not represent stability?

We agree that this is an important question. Our paper clearly demonstrates that individuality always plays a role in decision-making (and, in this context, any behavioral output can be considered a decision). However, the non-linear relationship between certain situations and the individual’s behavior often reduces the predictive value (or correlation) across contexts, sometimes quite drastically.

For instance, temperature has a relatively linear effect on certain behavioral parameters, leading to predictable changes across individuals. As a result, correlations across temperature conditions are often similar to those observed across time within the same situation. In contrast, this predictability diminishes when comparing conditions like the presence or absence of visual stimuli, the use of different arenas, or different modalities.

For this reason, we believe that significance remains the best indicator for describing how measurable individuality persists, even across vastly different situations.

The authors describe a dissociation between inter-group differences and interindividual variation stability, i.e. sometimes large mean differences between contexts, but significant correlation between individual test and retest data. Given that correlation is sensitive to slope, this might be expected to underestimate the variability stability (or consistency). Is there a way to adjust for the group differences before examining the correlation? For example, would it be possible to transform the values to in-group ranks prior to correlation analysis?  

We thank the reviewer for this suggestion, and we have now addressed this point. To account for slope effects, we have now introduced in-group ranks for our linear model computation (see Fig. 9). 

What is gained by classifying the five parameters into exploration, attention, and anxiety? To what extent have these classifications been validated, both in general and with regard to these specific parameters? Is the increased walking speed at higher temperatures necessarily due to an increased 'explorative' nature, or could it be attributed to increased metabolism, dehydration stress, or a heat-pain response? To what extent are these categories subjective?

We agree that grouping our parameters into traits like exploration, attention, and anxiety always includes subjective decisions. The classification into these three categories is even considered partially controversial in the mouse specific literature, which uses the term “anxiety” in similar experiments (see for exampler Carter, Sheh, 2015, chapter 2 . https://www.sciencedirect.com/book/9780128005118/guide-to-research-techniquesin-neuroscience). Nevertheless, we believe that readers greatly benefit from these categories, since they make it easier to understand (beyond mathematical correlations) which aspects of the flies’ individuality can be considered consistent across situations. Furthermore, these categories serve as a bridge to compare insight from very distinct models.

The legends are quite brief and do not link to descriptions of specific experiments. For example, Figure 4a depicts a graphical overview of the procedure, but I could not find a detailed description of this experiment's protocol.

We assume the reviewer is referring to Figure 3a. The detailed experimental protocol can be found in the Materials and Methods section under Setup 2: IndyTrax Multi-Arena Platform. We have now clarified this in the mentioned figure legend.

Using the current single-correlation analysis approach, the aims would benefit from rewording to appropriately address single-parameter variability stability/consistency (as distinct from holistic individuality). Alternatively, the analysis could be adjusted to address the multivariate nature of individuality, so that the claims and the analysis are in concordance with each other.

The reviewer raises an important point about hierarchies within the concept of animal individuality or personality. We agree that this is best addressed by first focusing on single behavioral traits/parameters and then integrating several trait properties into a cohesive concept of animal personality (holistic individuality). To ensure consistency throughout the text, we have now thoroughly reviewed the entire manuscript clearly distinguish between single-parameter variability stability/consistency and holistic individuality/personality.

The study presents a bounty of new technology to study visually guided behaviors. The GitHub link to the software was not available. To verify the successful transfer of open hardware and open-software, a report would demonstrate transfer by collaboration with one or more other laboratories, which the present manuscript does not appear to do. Nevertheless, making the technology available to readers is commendable.

We have now uploaded all codes and materials to GitHub and made them available as soon as we received the reviewers’ comments. All files and materials can be accessed at https://github.com/LinneweberLab/Mathejczyk_2024_eLife_Individuality, which is now frequently mentioned throughout the revised manuscript.

The study discusses a number of interesting, stimulating ideas about inter-individual variability, and presents intriguing data that speaks to those ideas, albeit with the issues outlined above.

While the current work does not present any mechanistic analysis of inter-individual variability, the implementation of high-throughput assays sets up the field to more systematically investigate fly visual behaviors, their variability, and their underlying mechanisms. 

We thank the reviewer again for the extensive and constructive feedback.

Reviewer #2 (Public Review): 

Summary: 

The authors repeatedly measured the behavior of individual flies across several environmental situations in custom-made behavioral phenotyping rigs.

Strengths: 

The study uses several different behavioral phenotyping devices to quantify individual behavior in a number of different situations and over time. It seems to be a very impressive amount of data. The authors also make all their behavioral phenotyping rig design and tracking software available, which I think is great and I'm sure other folks will be interested in using and adapting it to their own needs.

We thank the reviewer for highlighting the strengths of our study.

Weaknesses/Limitations: 

I think an important limitation is that while the authors measured the flies under different environmental scenarios (i.e. with different lighting and temperature) they didn't really alter the "context" of the environment. At least within behavioral ecology, context would refer to the potential functionality of the expressed behaviors so for example, an anti-predator context, a mating context, or foraging. Here, the authors seem to really just be measuring aspects of locomotion under benign (relatively low-risk perception) contexts. This is not a flaw of the study, but rather a limitation to how strongly the authors can really say that this demonstrates that individuality is generalized across many different contexts. It's quite possible that rank order of locomotor (or other) behaviors may shift when the flies are in a mating or risky context. 

We agree with the reviewer that the definition of environmental context can differ between fields and that behavioral context is differently defined, particularly in ecology. Nevertheless, we highlight that our alternations of environmental context are highly stereotypic, well-defined, and unbiased from any interpretation (we only modified what we stated in the experimental description without designing a specific situation that might be again perceived individually differently. E.g., comparing a context with a predator and one without might result in a binary response because one fraction of the tested individuals might perceive the predator in the predator situation, and the other half does not. 

The analytical framework in terms of statistical methods is lacking. It appears as though the authors used correlations across time/situations to estimate individual variation; however, far more sophisticated and elegant methods exist. The paper would be a lot stronger, and my guess is, much more streamlined if the authors employ hierarchical mixed models to analyse these data these models could capture and estimate differences in individual behavior across time and situations simultaneously. Along with this, it's currently unclear whether and how any statistical inference was performed. Right now, it appears as though any results describing how individuality changes across situations are largely descriptive (i.e. a visual comparison of the strengths of the correlation coefficients?). 

The reviewer raises an important point, also raised by reviewer #1. On one hand, we agree with both reviewers that a more aggregated analysis has clear advantages like more statistical power and has the potential to streamline our manuscript, which is why we added such an analysis (see below). On the other hand, we would also like to defend the initial approach we took, since we think that the simplicity of the analysis for both correlational and mean data is easy to understand and reproduce. More complex analyses necessarily include the selection of a specific statistical toolbox by the experimenters and based on these decisions, different analyses become less comparable and more and more complicated to reproduce, unless the entire decision tree is flawlessly documented. For instance, a recent personality psychology paper investigated the relationship between statistical paths within the decision tree (forking analysis) and their results, leading to very surprising results (Paul et al., 2024), since some paths even reversed their findings. Such a variance in conclusions is hardly possible with the rather simplistic and easily reproducible analysis we performed. One of the major strengths of our study is the simple experimental design, allowing for rather simple and easy to understand analyses.

We nevertheless took the reviewer’s advice very seriously and reanalyzed the data using a generalized linear model, which largely recapitulated the findings of our previously performed “low-tech” analysis in a single figure (Fig. 9).

Another pretty major weakness is that right now, I can't find any explicit mention of how many flies were used and whether they were re-used across situations. Some sort of overall schematic showing exactly how many measurements were made in which rigs and with which flies would be very beneficial. 

We apologize for this inconvenience. A detailed overview of male and female sample sizes has been listed in the supplemental boxplots next to the plots (e.g, Fig S6). Apparently, this was not visible enough. Therefore, we have now also uniformly added the sample sizes to the main figure legends.

I don't necessarily doubt the robustness of the results and my guess is that the author's interpretations would remain the same, but a more appropriate modeling framework could certainly improve their statistical inference and likely highlight some other cool patterns as these methods could better estimate stability and covariance in individual intercepts (and potentially slopes) across time and situation.

As described above, we have now added the suggested analyses. We hope that the reviewer will appreciate the new Fig. 9, which, in our opinion, largely confirms our previous findings using a more appropriate generalized linear modelling framework.

Reviewer #3 (Public Review): 

This manuscript is a continuation of past work by the last author where they looked at stochasticity in developmental processes leading to inter-individual behavioural differences. In that work, the focus was on a specific behaviour under specific conditions while probing the neural basis of the variability. In this work, the authors set out to describe in detail how stable the individuality of animal behaviours is in the context of various external and internal influences. They identify a few behaviours to monitor (read outs of attention, exploration, and 'anxiety'); some external stimuli (temperature, contrast, nature of visual cues, and spatial environment); and two internal states (walking and flying).

They then use high-throughput behavioural arenas - most of which they have built and made plans available for others to replicate - to quantify and compare combinations of these behaviours, stimuli, and internal states. This detailed analysis reveals that:

(1) Many individualistic behaviours remain stable over the course of many days. 

(2) That some of these (walking speed) remain stable over changing visual cues. Others (walking speed and centrophobicity) remain stable at different temperatures.

(3) All the behaviours they tested failed to remain stable over the spatially varying environment (arena shape).

(4) Only angular velocity (a readout of attention) remains stable across varying internal states (walking and flying).

Thus, the authors conclude that there is a hierarchy in the influence of external stimuli and internal states on the stability of individual behaviours.

The manuscript is a technical feat with the authors having built many new highthroughput assays. The number of animals is large and many variables have been tested - different types of behavioural paradigms, flying vs walking, varying visual stimuli, and different temperatures among others. 

We thank the reviewer for this extraordinary kind assessment of our work!

Recommendations for the authors:  

Reviewing Editor (Recommendations For The Authors): 

While appreciating the effort and quality of the work that went into this manuscript, the reviewers identified a few key points that would greatly benefit this work.

(1) Statistical methods adopted. The dataset produced through this work is large, with multiple conditions and comparisons that can be made to infer parameters that both define and affect the individualistic behaviour of an animal. Hierarchical mixed models would be a more appropriate approach to handle such datasets and infer statistically the influence of different parameters on behaviours. We recommend that the authors take this approach in the analyses of their data.

(2) Brevity in the text. We urge the authors to take advantage of eLife's flexible template and take care to elaborate on the text in the results section, the methods adopted, the legends, and the guides to the legends embedded in the main text. The findings are likely to be of interest to a broad audience, and the writing currently targets the specialist.

Reviewer #2 (Recommendations For The Authors): 

I want to start by saying this seems like a really cool study! It's an impressive amount of work and addressing a pretty basic question that is interesting (at least I think so!)

We thank the reviewer again for this assessment!

That said, I would really strongly recommend the authors embrace using mixed/hierarchical models to analyze their data. They're producing some really impressive data and just doing Pearson correlation coefficients across time points and situations is very clunky and actually losing out on a lot of information. The most up-todate, state-of-the-art are mixed models - these models can handle very complex (or not so complex) random structures which can estimate variance and importantly, covariance, in individual intercepts both over time and across situations. I actually think this could add some really cool insights into the data and allow you to characterize the patterns you're seeing in far more detail. It's datasets exactly like this that are tailormade for these complex variance partitioning models! 

As mentioned before, we have now adopted a more appropriate GLM-based data analysis (see above).

Regardless of which statistical methods you decide to use, please explicitly state in your methods exactly what analyses you did. That is completely lacking now and was a bit frustrating. As such, it's completely unclear whether or how statistical inference was performed. How did you do the behavioral clustering? 

We apologize that these points were not clearly represented in the previous version of the manuscript. We have now significantly extended the methods section to include a separate paragraph on the statistical methods used, in order to address this critique and hope that the revised version is clear now.

Also, I could not for the life of me figure out how many flies had been measured. Were they reused across the situation? Or not?

We reused the same flies across situations whenever possible. However, having one fly experience all assays consecutively was not feasible due to their fragility. Instead, individual flies were exposed to at least 2 of the 3 groups of assays used here: in the Indytrax setup ,  the Buridan arenas and variants thereof, and the virtual arenas Hence, we have compared flies across entirely different setups, but the number of times flies can be retested is limited (as otherwise, sample sizes will drop over time, and the flies will have gone through too many experimental alternations). To make this more clear, we have elaborated on this point in the main text, and we added group sample sizes to figure legends r.

What are these "groups" and "populations" that are referred to in the results (e.g. lines 384, 391, 409)?

We apologize for using these two terms somewhat interchangeably without proper introduction/distinction. We have now made this more clear in at the beginning of the results in the main text, by focusing on the term ‘group’. By ‘group’ we refer to the average of all individuals tested in the same situation. Sample sizes in the figure legends now indicate group/population sizes to make this clearer.

Some of the rationale for the development of the behavioral rigs would have actually been nice to include in the intro, rather than in the results.

This rationale is introduced at the beginning of the last paragraph of the introduction. We hope that this now becomes clear in the revised version of the manuscript.

Reviewer #3 (Recommendations For The Authors): 

This manuscript would do well to take advantage of eLife's flexible word limit. I sense that it has been written in brevity for a different journal but I would urge the authors to revisit this and unpack the language here - in the text, in the figure legends, in references to the figures within the text. The way it's currently written, though not misleading, will only speak to the super-specialist or the super-invested :). But the findings are nice, and it would be nice to tailor it to a broader audience.

We appreciate this suggestion. Initially, we were hoping that we had described our results as clearly and brief as possible. We apologize if that was not always the case. The comments and requests of all three reviewers now led to a series of additions to both main text and methods, leading to a significantly expanded manuscript. We hope that these additons are helpful for the general, non-expert audience.

Caths can measure PCWP (LA) pressure and SvO2, and CO.

Reviewer #1 (Public review):

Thank you for allowing me to review the paper "Evidence for deliberate burial of the dead by Homo naledi". This remains a very important site for paleoanthropology. I appreciate the work that the crew, especially the junior members of the team, put into this massive project. I appreciate that the authors did revise the paper since that is not a requirement of eLife. Extensive reviews by peer-reviewers have been provided for this paper, as well as professionally published replies (Martinón-Torres et al., 2023; Foecke et al., 2023). The composition, and citations of this version are much improved, though important information, some requested by reviewers, are buried in the supplementary section. It seems important that the authors make these sections more easily accessible to the general reader. The length of the paper is also unnecessary and impedes the readability of the work. Concise clarity is an expectation of most journals. The Netflix documentary was made to appeal to a mass audience, I would hope that the goal of the accompanying publication would be to enable readers to fully comprehend the work behind the claims.

This version of the paper considers at great length many possibilities for how the H. naledi skeletal material came to rest in the cave system with some additional figures and data provided. However, quite a lot is still unclear. In my original review I stated, "The authors have repeatedly described how incredibly challenging it is to get into and out of this cave system and all of its chambers." This was a point emphasized in the Netflix documentary. In this version of the paper the authors have included within the supplementary section a brief discussion of other entrances. The work by Robbins et al. 2021 (a peer-reviewed paper in the impact factor rated journal Chemical Geology) is extremely relevant here. In this revision it is noted in the supplementary section that if the Postbox chamber was used as an opening, it would have reduced the length of the access to the system by 80 m. This fact seems important. This section should be moved out of the supplementary material and expanded because the conclusions published by Robbins et al. (2021) indicate a completely different route by which H. naledi accessed the cave, but this is hardly mentioned in the revision and deserves attention. To quote the Robbins et al.'s (2021) discussion section 6.3:

"We acknowledge that additional data is required in order to confidently assess the relative timing of the Dragon's Back collapse and entry of H. naledi. Nonetheless, the stratigraphic and geochronologic observations presented here, together with those previously published (Dirks et al., 2017) are consistent with the following scenario. Prior to the collapse of the Dragon's Back, sometime before 241 ka (new minimum age for H. naledi from RS68), the cave could be entered by H. naledi via a shaft in the roof of the Postbox Chamber. From there H. naledi could walk along a straight passage that follows a gently descending, SW trending fracture into the Dragon's Back Chamber and, with the Dragon's Back block still attached to the roof, would have only needed to climb over a ~5 m high sill to access the Dinaledi Subsystem behind it. This sill and narrow fracture system behind the Dragon's Back block would have been a major impediment to any flood waters and most other fauna into the Dinaledi Subsystem, but it would have been a more accessible route than that today."

The paper's conclusion continues, "The new dates further constrain the minimum age of H. naledi to 241 ka. Thus, H. naledi entered the subsystem between 241 ka and 335 ka, during a glacial period, when clastic sediment along the access route into the Dinaledi Subsystem experienced erosion. H. naledi would have probably entered the cave in the same way as the clastic sediments did, through an opening in the roof of the Postbox Chamber and may have entered via the Dragon's Back Chamber by climbing a 5 m high sill and passing below the Dragon's Back Block that was then still attached to the roof, to enter the Dinaledi Subsystem. In this context it is important to emphasize that it was not the Dragon's Back Block that prevented high-energy transport of coarse siliciclastic sediment from the Dragon's Back Chamber into the Dinaledi Subsystem, but rather the in situ floor block in the back wall of the Dragon's Back Chamber, against which the Dragon's Back Block slumped after it fell." This conclusion is very different from the complex pathway suggested by Berger et al. Martinón-Torres et al., 2023 also requested elaboration on this point in their reply by stating, "Moreover, recent studies by the Rising Star Cave team also point to a possible different and easier accesses for H. naledi into the fossil-bearing cave chambers than the current restricted access chute used by the research team, making clear that the degree of accessibility remains an open question (Robbins et al., 2021). Based on extensive dating studies of speleothem, this research (Robbins et al., 2021) implies that prior to 241 ka and the collapse of the Dragon's Back block hominins and other species could have more easily entered the cave via the Post Box Chamber and beneath the Dragon's Back Block before it fell. This gives access to a series of rifts that allow easier entry to the Dinaledi and other chambers beyond the present-day chute."

Because this paper introduces very different sets of possibilities, it seems impossible to derive an understanding of the processes that occurred 335-241 ka throughout the cave system without going into detail on these other openings, especially openings that are hypothesized to have been used by the hominins in question.

The world cares deeply about the H. naledi hominins and their story. I hope that in the coming years these issues are addressed, and perhaps other independent teams are allowed to do a full analysis since science is about replication. In any case, the excavation team has contributed important fossils to paleoanthropology.

Literature cited:

• Foecke, Kimberly K., Queffelec, Alain, & Pickering, Robyn. (2023). No Sedimentological Evidence for Deliberate Burial by Homo naledi - A Case Study Highlighting the Need for Best Practices in Geochemical Studies Within Archaeology and Paleoanthropology. PaleoAnthropology, 2024.

• Martinón-Torres, M., Garate, D., Herries, A. I. R., & Petraglia, M. D. (2023). No scientific evidence that Homo naledi buried their dead and produced rock art. Journal of Human Evolution, 103464. https://doi.org/10.1016/j.jhevol.2023.103464

• Robbins, J. L., Dirks, P. H. G. M., Roberts, E. M., Kramers, J. D., Makhubela, T. V., HilbertWolf, H. L., Elliott, M., Wiersma, J. P., Placzek, C. J., Evans, M., & Berger, L. R. (2021). Providing context to the Homo naledi fossils: Constraints from flowstones on the age of sediment deposits in Rising Star Cave, South Africa. Chemical Geology, 567, 120108. https://doi.org/10.1016/j.chemgeo.2021.120108

Reviewer #2 (Public review):

Before providing my review of the revised version of this study by Berger et al., which explores potential deliberate burials of Homo naledi within the Rising Star Cave System, I would like to briefly summarize the key points from my previous review of the earlier version (in 2023). Summarizing my previous review will provide context for assessing how effectively the revised study addresses the concerns I raised previously (in 2023).

In my earlier comments, I highlighted significant methodological and analytical shortcomings that, in my view, undermined the authors' claim of intentional burials by Homo naledi. While the study presented detailed geological and fossil data, I found the evidence for intentional burials unconvincing due to insufficient application of archaeothanatological principles and other methodological gaps.

My key concerns included:

(1) The absence of a comprehensive archaeothanatological analysis, particularly with respect to taphonomic changes, bone articulations, and displacement patterns such as the collapse of sediments and bone remains into voids created by decomposition.

(2) Missing or unclear illustrations of bone arrangements, which are critical for interpreting burial positions and processes.

(3) A lack of detailed discussion on the sequence of decomposition, joint disarticulation, sediment infill, and secondary bone displacement.

To convincingly support claims of deliberate burial, I argued that the study must reconstruct the timeline and processes surrounding death and deposition while clearly distinguishing natural taphonomic changes from intentional human actions. I emphasized the importance of integrating established archaeothanatological frameworks, such as those outlined by Duday et al. or Boulestin et al., to provide the necessary analytical rigor.

I will now explain how the revised version of this study has successfully addressed all the concerns raised in my previous review and why I now think that the authors provide sufficient evidence for the presence of "repeated and patterned" deliberate burials (referred to as "cultural burials" by the authors) by Homo naledi within the Rising Star Cave System.

In their revised manuscript, the authors have implemented substantial improvements in methodology, analytical depth, and overall presentation, which have effectively resolved the critical issues I previously highlighted. These revisions greatly strengthen their argument for intentional funerary practices. Importantly, the authors remain cautious in their interpretation of the evidence, explicitly refraining from inferring "symbolic" behavior or complex cognitive motivations behind these burials. Instead, they focus on presenting clear evidence for deliberate, patterned practices while leaving the broader implications for Homo naledi's cultural and cognitive capacities open for further investigation. This cautious approach adds to the credibility of their conclusions and avoids overextending the interpretation of the data.

The authors' enhanced application of archaeothanatological principles now offers a more comprehensive and convincing interpretation of the burial features. Key gaps in the earlier version, such as the absence of detailed reconstructions of taphonomic processes, bone articulations, and displacement patterns, have been addressed with thorough analyses and clearer illustrations. The study also now includes a well-structured timeline of events surrounding death and deposition, demonstrating an improved ability to differentiate between natural processes and deliberate human actions. These additions lend greater clarity and rigor to the evidence, making the argument for intentional burials both robust and persuasive.

Furthermore, the revised study presents detailed data on skeletal arrangements, decomposition sequences, and spatial patterns. This information is now relatively well illustrated and contextualized, enabling readers to better understand the complex processes involved in these burial practices. Importantly, the authors provide a stronger theoretical framework, integrating established archaeothanatological methodologies and taphonomic studies that situate their findings within broader archaeological and anthropological discussions of funerary behavior.

That being said, there remain relatively minor issues that could be refined further. Addressing these would help ensure the study is as clear and accessible as possible to the reader. Such adjustments would enhance the overall readability and reinforce the study's impact within the scientific community.

A - Suggested changes:

While the revised version of this study marks a significant improvement, successfully addresses my previous major concerns and provides a convincing argument for deliberate burials by Homo naledi, I believe that including both one summary table + one summary figure for each of the three main locations and the-Hill Antechamber, and Dinaledi Chamber (Feature 1 and Puzzle Box)-would further enhance the clarity and accessibility of the findings. Such tables and figures would serve as a valuable reference, allowing readers to more easily follow how the detailed patterns observed at each site fit the criteria for distinguishing intentional from natural processes.

The summary tables should consolidate key information for each location, such as:

(1) Bone articulations: A comprehensive list of articulated skeletal elements, categorized by their anatomical relationships (e.g., labile vs. stable articulations).

(2) Displacement patterns: Documentation of any spatial shifts in bone positions, noting directions and extents of disarticulation.

(3) Sequence of decomposition: Observations regarding the sequence of decomposition, joint disarticulation and associated changes in bone arrangements.

(4) Sediment interaction: Notes on sediment infill and its timing relative to decomposition, including evidence of secondary voids or delayed sediment deposition.

(5) Distinguishing criteria: Clear indications of how each observed pattern supports intentional burial (e.g., structured placement, lack of natural transport mechanisms) versus natural processes (e.g., random dispersal, sediment-driven bone displacement).<br /> Including such tables would not only summarize the complex taphonomic and archaeothanatological data but also allow readers to quickly assess how the evidence supports the authors' conclusions. This approach would bridge the gap between the detailed narrative descriptions and the criteria necessary to differentiate deliberate funerary practices from natural occurrences.

To streamline the main text further, many of the detailed descriptions of individual bones, specific displacement measurements, and other intricate observations could be moved to the supplementary data. This reorganization would maintain the richness of the data for those who wish to explore it in depth, while the summary tables would present the key findings concisely in the main text. This balance between accessibility and detail would ensure that the study appeals to both specialists requiring comprehensive data and readers looking for an overarching understanding of the findings.

In addition to these structural changes, it is crucial to ensure that evidence is consistently illustrated throughout the text.

Importantly the skeletal part representation is provided for Dinaledi Feature 1 in Figure 14, but similar data is not presented for the other burial features, such as those in the Hill Antechamber or Puzzle Box. This inconsistency could make it more challenging for readers to compare the features and fully appreciate the patterns of burial behavior across the different locations. Ensuring that similar types of evidence and analyses are presented uniformly for all features would strengthen the study and make its conclusions more cohesive and compelling.

Adding supplementary figures to represent the skeletal part distribution (as in Figure 14) within each excavated area (i.e., not only for Dinaledi Feature 1 but also for Hill Antechamber and Puzzle Box) would significantly enhance the study's clarity and accessibility. These figures could provide a visual summary of skeletal part representation, allowing readers to easily understand the nature of human remains within each burial context.

Specifically, such figures could:

(1) Illustrate Skeletal Part Representation: By visually mapping the presence and location of various skeletal elements, the figures would make it easier for readers to assess the completeness and arrangement of remains in each feature. This is particularly important for interpreting patterns of bone articulation and disarticulation.<br /> For example, it is quite challenging to determine the exact number and characteristics of the human skeletal remains identified within the Puzzle Box and those recovered through the "subsurface collection" in its surrounding area. The authors state that "at least six individuals" were identified in this area (during "subsurface collection") but provide no further clarification. They simply mention that "most elements" were described previously, without specifying which elements or where this prior description can be found.

(2) Highlight Articulations and Displacements: Figures could indicate which bones are articulated and their relative positions, as well as the spatial distribution of disarticulated elements. This would provide a clear visual context to support interpretations of taphonomic processes.

(3) Facilitate Comparisons Across Locations: By presenting skeletal part representation consistently for each location, the figures would enable readers to directly compare features, reinforcing the argument for "repeated and patterned" behavior.

(4) Simplify Complex Data: Instead of relying solely on textual descriptions, the visual format would allow readers to quickly grasp the key findings, making the study more accessible to a broader audience

By including such figures alongside the proposed summary tables in the main text, the study would achieve a balance between detailed narrative descriptions and concise, visual representation of the data. This approach would strengthen the overall presentation and support the authors' conclusions effectively.

Again, by presenting the data in a structured and comparative format, the new tables + figures could also highlight the differences and similarities between the three locations. This would reinforce the argument for "repeated and patterned" behavior, as the tables would make it easier to observe consistent burial practices across different contexts within the Rising Star Cave System.

Adding these summary tables + figures, ensuring consistent presentation of evidence, and reallocating detailed descriptions to supplementary materials would not require significant new analysis. However, these organizational adjustments would greatly enhance the study's clarity, readability, and overall impact.

B - A few additional changes are needed:

Figure 8: This figure is critical but lacks clarity. Specifically:

Panels 8a-c suffer from low contrast, making details difficult to discern.<br /> Panel 8d (sediment profile) is too small and lacks annotations that would aid interpretation.<br /> Figure S7: While this figure has significantly better contrast than Figures 8a-c, I am unable to identify the "articulated foot ... at right of frame," as mentioned in the caption. Please clarify this by adding annotations directly to the figure.

Page 4, 2nd paragraph: In the sentence "Researchers thus have diverse opinions about how to test whether ...," the word "opinions" should be replaced with a more precise term, such as "approaches."

C - In conclusion, I am impressed by the significant effort and meticulous work that has gone into this revised version of the study. The quality of the new evidence presented is commendable, and the findings now convincingly demonstrate not only clear evidence of intentional burial practices by Homo naledi but also compelling indications of post-depositional reworking. These advancements reflect a major improvement in the study's analytical rigor and the robustness of its conclusions, making it a valuable contribution to the understanding of early hominin funerary behavior.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This work tried to map the synaptic connectivity between the inputs and outputs of the song premotor nucleus, HVC in zebra finches to understand how sensory (auditory) to motor circuits interact to coordinate song production and learning. The authors optimized the optogenetic technique via AAV to manipulate auditory inputs from a specific auditory area one-by-one and recorded synaptic activity from a neuron with whole-cell recording from slice preparation with identification of the projection area by retrograde neuronal tracing. This thorough and detailed analysis provides compelling evidence of synaptic connections between 4 major auditory inputs (3 forebrain and 1 thalamic region) within three projection neurons in the HVC; all areas give monosynaptic excitatory inputs and polysynaptic inhibitory inputs, but proportions of projection to each projection neuron varied. They also find specific reciprocal connections between mMAN and Av. Taken together the authors provide the map of the synaptic connection between intercortical sensory to motor areas which is suggested to be involved in zebra finch song production and learning.

Strengths:

The authors optimized optogenetic tools with eGtACR1 by using AAV which allow them to manipulate synaptic inputs in a projection-specific manner in zebra finches. They also identify HVC cell types based on projection area. With their technical advance and thorough experiments, they provided detailed map synaptic connections.

Weaknesses:

As it is the study in brain slice, the functional implication of synaptic connectivity is limited. Especially as all the experiments were done in the adult preparation, there could be a gap in discussing the functions of developmental song learning.

We thank the reviewer for their appreciation of our work. Although we agree that there can be limitations to brain slice preparations, the approaches used here for synaptic connectivity mapping are well-designed to identify long-range synaptic connectivity patterns. Optogenetic stimulation of axon terminals in brain slices does not require intact axons and works well when axons are cut, allowing identification of all inputs expressing optogenetic channels from aXerent regions. Terminal stimulation in slices yields stable post-synaptic responses for hours without rundown, assuring that polysynaptic and monosynaptic connections can be reliably identified in our brain slices.  Additionally, conducting similar types of experiments in vivo can run into important limitations. First, the extent of TTX and 4-AP diXusion, which is necessary for identification of long-range monosynaptic connections, can be diXicult to verify in vivo - potentially confounding identification of monosynaptic connectivity.  Second, conducting whole-cell patch-clamp experiments in vivo, particularly in deeper brain regions, is technically challenging, and would limit the number of cells that can be patched and increase the number of animals needed. 

We agree that there may well be important diXerences between adult connectivity and connectivity patterns in the juvenile brain. Indeed, learning and experience during development almost certainly shape connectivity patterns and these patterns of connectivity may change incrementally and/or dynamically during development. Ultimately, adult connectivity patterns are the result of changes in the brain that accrue over development. Given that this is the first study mapping long-range connectivity of HVC input-output pathways, we reasoned that the adult connectivity would provide a critical reference allowing future studies to map diXerent stages of juvenile connectivity and the changes in connectivity driven by milestones like forming a tutor song memory, sensorimotor learning, and song crystallization.

In this revision we worked to better highlight the points raised above and thank the reviewer for their comments.

Reviewer #2 (Public review):

Summary:

The manuscript describes synaptic connectivity in the Songbird cortex's four main classes of sensory neuron aXerents onto three known classes of projection neurons of the pre-motor cortical region HVC. HVC is a region associated with the generation of learned bird songs. Investigators here use all male zebra finches to examine the functional anatomy of this region using patch clamp methods combined with optogenetic activation of select neuronal groups.

Strengths:

The quality of the recordings is extremely high and the quantity of data is on a very significant scale, this will certainly aid the field.

Weaknesses:

The authors could make the figures a little easier to navigate. Most of the figures use actual anatomical images but it would be nice to have this linked with a zebra finch atlas in more of a cartoon format that accompanied each fluro image. Additionally, for the most part, figures showing the labeling lack scale bar values (in um). These should be added not just shown in the legends.

The authors could make it clear in the abstract that this is all male zebra finches - perhaps this is obvious given the bird song focus, but it should be stated. The number of recordings from each neuron class and the overall number of birds employed should be clearly stated in the methods (this is in the figures, but it should say n=birds or cells as appropriate).

The authors should consider sharing the actual electrophysiology records as data.

We thank the reviewer for their assessment of our research and suggestions. We have implemented many of these suggestions and provide details in our response to their specific Recommendations. Additionally, we are organizing our data and will make it publicly available with the version of record.

Reviewer #3 (Public review):

Nucleus HVC is critical both for song production as well as learning and arguably, sitting at the top of the song control system, is the most critical node in this circuit receiving a multitude of inputs and sending precisely timed commands that determine the temporal structure of song. The complexity of this structure and its underlying organization seem to become more apparent with each experimental manipulation, and yet our understanding of the underlying circuit organization remains relatively poorly understood. In this study, Trusel and Roberts use classic whole-cell patch clamp techniques in brain slices coupled with optogenetic stimulation of select inputs to provide a careful characterization and quantification of synaptic inputs into HVC. By identifying individual projection neurons using retrograde tracer injections combined with pharmacological manipulations, they classify monosynaptic inputs onto each of the three main classes of glutamatergic projection neurons in HVC (RA-, Area X- and Av-projecting neurons). This study is remarkable in the amount of information that it generates, and the tremendous labor involved for each experiment, from the expression of opsins in each of the target inputs (Uva, NIf, mMAN, and Av), the retrograde labelling of each type of projection neuron, and ultimately the optical stimulation of infected axons while recording from identified projection neurons. Taken together, this study makes an important contribution to increasing our identification, and ultimately understanding, of the basic synaptic elements that make up the circuit organization of HVC, and how external inputs, which we know to be critical for song production and learning, contribute to the intrinsic computations within this critic circuit.

This study is impressive in its scope, rigorous in its implementation, and thoughtful regarding its limitations. The manuscript is well-written, and I appreciate the clarity with which the authors use our latest understanding of the evolutionary origins of this circuit to place these studies within a larger context and their relevance to the study of vocal control, including human speech. My comments are minor and primarily about legibility, clarification of certain manipulations, and organization of some of the summary figures.

We thank the reviewer for their thoughtful assessment of our research.

Recommendations for the authors:

The following recommendations were considered by all reviewers to be important to incorporate for improving this paper:

(1) Clarify the site of viral injection and the possibility of labeling other structures a) Show images of viral injection sites.

We provide a representative image of viral expression for each pathway studied in this manuscript. Please see panel A in Figures 2-3 and 5-6 showing our viral expression in Uva, NIf, mMAN, and Av respectively.  

b) Include in discussion caveats that the virus may spread beyond the boundaries of structures (e.g. especially injections into NIF could spread into Field L).

For each HVC aXerent nucleus we have now included a sentence describing the possible spread of viral infection in surrounding structures in the Results. We also now expanded the image from the Av section to include NIf, to showcase lack of viral expression in NIf (see Fig. 6A).

(2) Clarify the logic and precise methods of the TTX and 4-AP experiments

a) Please see the detailed issue raised by Reviewer 3, Major Point 1 below.

The TTX and 4AP application is the gold-standard of opsin-assisted synaptic circuit interrogation, pioneered by the Svoboda lab in 2009 (Petreanu, Mao et al. 2009) and widely used to assess monosynaptic connectivity in multiple brain circuits, as summarized in a recent review(Linders, Supiot et al. 2022). We now better describe the logic of this approach in the second paragraph of the Results section and cite the first description of this method from the Svoboda lab and a recent review weighing this method with other optogenetic methods for tracing synaptic connections in the brain.

(3) Include caveats in discussion

a) Note that there may be other inputs to HVC that were not examined in this study (e.g. CMM, Field L)

In our original manuscript we did state “Although a complete description of HVC circuitry will require the examination of other potential inputs (i.e. RA_HVC PNs, A11 glutamatergic neurons(Roberts, Klein et al. 2008, Ben-Tov, Duarte et al. 2023)) and a characterization of interneuron synaptic connectivity, here we provide a map of the synaptic connections between the 4 best described aPerents to HVC and its 3 populations of projection neurons” in the last paragraph of the Discussion. We have now edited this sentence to include the projection from NCM to HVC and cited Louder et al., 2024.

We have extensively mapped input pathways to HVC, and consistent with Vates (Vates, Broome et al. 1996) we have not found evidence that Field L projects to HVC. Rather that it projects to the shelf region outside of HVC. Consistent with this, we do not see retrogradely labeled neurons in Field L following tracer injections confined to HVC (see Fig. 3G). Additionally, we find that CM projections to HVC arise from the nucleus Avalanche (Roberts, Hisey et al. 2017) which we specifically examine in this study. We do not dispute that there may be other pathways projecting to HVC that will need to be examined in the future, including known projections from neuromodulatory regions and RA, from developmentally restricted pathway(s) like NCM (Louder, Kuroda et al. 2024), and from yet unidentified pathways.

b) Also note that birds in this study were adults and that some inputs to HVC likely to be important for learning may recede during development (e.g. Louder et al, 2024).

In the second to last paragraph of the Discussion we now state: While our opsin-assisted circuit mapping provides us with a new level of insight into HVC synaptic circuitry, there are limitations to this research that should be considered. All circuit mapping in this study was carried out in brain slices from adult male zebra finches. Future studies will be needed to examine how this adult connectivity pattern relates to patterns of connectivity in juveniles during sensory or sensorimotor phases of vocal learning and connectivity patterns in female birds.   

(4) Consider cosmetic changes to figures as suggested by Reviewers 2-3 below.

We thank the reviewers for their suggestions and have implemented the changes as best we can.

(5) Address all minor issues raised below.

Reviewer #1 (Recommendations for the authors):

I see this study is well designed to answer the author's specific question, mapping synaptic auditorymotor connections within HVC. Their experiments with advanced techniques of projection-specific optogenetic manipulation of synaptic inputs and retrograde identification of projection areas revealed input-output combination selective synaptic mapping.

As I found this study advanced our knowledge with the compelling dataset, I have only some minor comments here.

(1) One technical concern is we don't see how much the virus infection was focused on the target area and if we can ignore the eXect of synaptic connectivity from surrounding areas. As the amount of virus they injected is large (1.5ul) and target areas are small, we assume the virus might spread to the surrounding area, such as field L which also projects to HVC when targeting Nif. While I think the majority of the projections were from their target areas, it would be better to mention (also the images with larger view areas) the possibilities of projections of surrounding areas.

We agree with the reviewer about the concern about specificity of viral expression. For this reason, we included sample images of the viral expression in each target area (panel A in Fig. 2,3,5,6). We have now also included a sentence at the beginning of each subsection of our Result to describe how we have ensured interpretability of the results. Uva and mMAN’s surrounding areas are not known to project to HVC. Possible cross-infection is an issue for Av and NIf, and we checked each bird’s injection site to ensure that eGtACR1+ cells were not visible in the unintended HVC-projecting areas.

As mentioned in our response the public comment, consistent with Vates (Vates, Broome et al. 1996) we do not see evidence that Field L projects directly to HVC (see Fig. 3G).

(2) Another concern about the technical issue is the damage to axonal projections. While I understand the authors stimulated axonal terminals axonal projections were assumed to be cut and their ability to release neurotransmitters would be reduced especially after long-term survival or repeated stimulation. Mentioning whether projection pathways were within their 230um-thick slice (probably depends on input sites) or not and the eXect of axonal cut would be helpful.

We agree that slice electrophysiology has limitations. However, we disagree with the claim of reduced reliability or stability of the evoked response. We and others find that electrical and optogenetic repeated terminal stimulation in slices can yield stable post-synaptic responses for tens of minutes and even hours (Bliss and Gardner-Medwin 1973, Bliss and Lomo 1973, Liu, Kurotani et al. 2004, Pastalkova, Serrano et al. 2006, Xu, Yu et al. 2009, Trusel, Cavaccini et al. 2015, Trusel, Nuno-Perez et al. 2019). Indeed, long-term synaptic plasticity experiments in most preparations and across brain areas rely on such stability of the presynaptic machinery for synaptic release, despite axons being severed from their parent soma. Our assumption is the vast majority, if not all, connections between axon terminals and their cell body in the aXerent regions have been cut in our preparations. Nonetheless, the diversity of outcomes we report (currents returning after TTX+4AP or not, depending on the specific combination of input and HVCPN class) is consistent with the robustness of the synaptic interrogation method. 

(3) While I understand this study focused on 4 major input areas and the authors provide good pictures of synaptic HVC connections from those areas, HVC has been reported to receive auditory inputs from other areas as well (CMM, FieldL, etc.). It is worth mentioning that there are other auditory inputs and would be interesting to discuss coordination with the inputs from other areas.

We have extensively mapped input pathways to HVC, and consistent with Vates (Vates, Broome et al. 1996) we have not found evidence that Field L projects to HVC. Rather that it projects to the shelf region outside of HVC. Consistent with this, we do not see retrogradely labeled neurons in Field L following tracer injections confined to HVC (see Fig. 3G). Additionally, we find that CM projections to HVC arise from the nucleus Avalanche (Roberts, Hisey et al. 2017) which we specifically examine in this study. We do not dispute that there may be other pathways projecting to HVC that will need to be examined in the future, including known projections from neuromodulatory regions and RA, from developmentally restricted pathway(s) like NCM (Louder, Kuroda et al. 2024), and from yet unidentified pathways.

(4) The HVC local neuronal connections have been reported to be modified and a recent study revealed the transient auditory inputs into HVC during song learning period. The author discusses the functions of HVC synaptic connections on song learning (also title says synaptic connection for song learning), however, the experiments were done in adults and dp not discuss the possibility of diXerent synaptic connection mapping in juveniles in the song learning period. Mentioning the neuronal activities and connectivity changes during song learning is important. Also, it would be helpful for the readers to discuss the potential diXerences between juveniles/adults if they want to discuss the functions of song learning.

We now mention in the Discussion that this is an important caveat of our research and that future studies will be needed to examine how these adult connectivity patterns relate to connectivity patterns in juveniles during sensory or sensorimotor phases of vocal learning and connectivity patterns in female birds. Nonetheless, the title and abstract cite song learning because it is important for the broader public to understand that at least some of these aXerent brain regions carry an essential role in song learning (Foster and Bottjer 2001, Roberts, Gobes et al. 2012, Roberts, Hisey et al. 2017, Zhao, Garcia-Oscos et al. 2019, Koparkar, Warren et al. 2024).

Reviewer #2 (Recommendations for the authors):

The work is very detailed and will be an important resource to those working in the field. The recordings are of a high quality and lots of information is included such as measures of response kinetics amplitude and pharmacological confirmation of excitatory and inhibitory synaptic responses. In general, I feel the quality is extremely high and the quantity of data is on a very significant exhaustive scale that will certainly aid the field. I have come at this conclusion as a non zebra finch person but I feel the connection information shown will be of benefit given its high quality.

Figure 7 is a nice way of showing the overall organization. Optional suggestion, consider highlighting anything in Figure 7 that results in a new understanding of the song system as compared to previous work on anatomy and function.

We thank the reviewer for the kind comments about our research. We have highlighted our newly found connection between mMAN and Av and all the connections onto the HVC PNs in Panel B are newly identified in this study.

Reviewer #3 (Recommendations for the authors):

Major points

(1) Clarification regarding methods for determining monosynaptic events:

One of the manipulations that I struggled the most with was those describing the use of TTX + 4AP to isolate monosynaptic events. Initially, not being as familiar with the use of optically based photostimulation of axons to release transmitter locally, I was initially confused by statements such as "we found that oEPSC returned after application of TTX+4AP". This might be clear to someone performing these manipulations, but a bit more clarification would be helpful. Should I assume that an existing monosynaptic EPSC would be masked by co-occurring polysynaptic IPSCs which disappear following application of TTX + 4AP, thereby unmasking the monosynaptic EPSC, thereby causing the EPSC to "return"? A word that I am not sure works. Continuing my confusion with these experiments, I am unsure how this cocktail of drugs is added, if it is even added as a cocktail, which is what I initially assumed. The methods and the results are not so clear if they are added in sequence and why and if traces are recorded after the addition of both drugs or if they are recorded for TTX and then again for TTX + 4AP. Finally, looking at the traces in the experimental figures (e.g. Figures 2F, 3F, 5F, and 6F), it is diXicult to see what is being shown, at least for me. First, the authors need to describe better in the results why they stimulate twice in short succession and why they seem to use the response to the second pulse (unless I am mistaken) to measure the monosynaptic event. Second, I was confused by the traces (which are very small) in the presence of TTX. I would have expected to see a response if there was a monosynaptic EPSC but I only seem to see a flat line.  

The confusion that I list above might be due in part to my ignorance, but it is important in these types of papers not to assume too much expertise if you want readers with a less sophisticated understanding of synaptic physiology to understand the data. In other words, a little bit more clarity and hand-holding would be welcome.

We understand the reviewer’s confusion about the methodology.  In Voltage clamp, the amplifier injects current through the electrode maintaining the membrane voltage to -70mV, where the equilibrium potential for Cl- is near equilibrium, and therefore the only synaptic current evoked by light stimulation is due to cation influx, mainly through AMPA receptors (see Fig. 1).  Therefore, cooccurring polysynaptic IPSCs wouldn’t be visible. We examine those holding the membrane voltage at +10mV, see Fig. 1. TTX application suppresses V-dependent Na+ channels and therefore stops all neurotransmission. We show the traces upon TTX to show that currents we were recording prior to TTX application were of synaptic origin, and not due to accidental expression of opsin in the patched cell. Also, this ensures that any current visible after 4AP application is due to monosynaptic transmission and not to a failure of TTX application.

After recording and light stimulation with TTX, we then add 4AP, which is a blocker of presynaptic K+ channels. This prevents the repolarization of the terminals that would occur in response to opsinmediated local depolarization. 4AP application, therefore, allows local opsin-driven depolarizations to reach the threshold for Ca2+-dependent vesicle docking and release. This procedure selectively reveals or unmasks the monosynaptic currents because any non-monosynaptically connected neuron would still need V-dependent Na+ channels to eXectively produce indirect neurotransmission onto the patched cell. The TTX and 4AP application is the gold-standard of opsinassisted synaptic circuit interrogation, pioneered by the Svoboda lab in 2009 and widely used to assess monosynaptic connectivity in multiple brain circuits, as summarized in a recent review (Linders et al., 2022). We now include 2 more sentences near the beginning of the Results to clarify this process and directly point to the Linders review for researchers wanting a deeper explanation of this technique. 

The double stimulation is unrelated to our testing of monosynaptic connections. We originally conducted the experiments by delivering 2 pulses of light separated by 50ms, a common way to examine the pair-pulse ratio (PPR) – a physiological measure which is used to probe synapses for short-term plasticity and release probability. However, through discussions with colleagues we realized that the slow decay time of eGtACR1 may complicate interpretation of the response to the second light pulse. Thus, we elected to not report these results and indicated this in the Methods section:  “We calculated the paired-pulse ratio (PPR) as the amplitude of the second peak divided by the amplitude of the first peak elicited by the twin stimuli, however due to slow kinetics of eGtACR1 the results would be diPicult to interpret, and therefore we are not currently reporting them.” 

(2) Suggestions for improving summary figures:

Summary Figure 1a: The circuit diagram (schematic to the right of 1a) is OK but I initially found it a bit diXicult to interpret. For example, it is not clear why pink RA projecting neurons don't reach as far to the right as X or Av projecting neurons, suggesting that they are not really projection neurons. Also, the big question marks in the intermediate zone are not entirely intuitive. It seems there might be a better way of representing this. It might also be worth stating in the figure legend that the interconnectivity patterns shown in the figure between PNs in HVC are based on specific prior studies.

We thank the reviewer for the constructive criticism. We have modified the figure to extend the RA projection line and mentioned in the figure legend that connectivity between PNs is based on prior studies.

Summary Figure 1a: I am not sure I love this figure. There are a few minor issues. First, there are too many browns [Nif/AV and mMAN] which makes it more challenging to clearly disambiguate the diXerent projections. Second, it is unclear why this figure does not represent projections from RA to HVC. My biggest concern with this figure is that it oversimplifies some of the findings. From the figure, one gets the impression that Uva only projects to RA-PNs and that Av only projects to X-PNs even though the authors show connections to other PNs. With the small sample size in this current study for each projection and each PN type, one really cannot rule out that these "minority" projections are not important. I, therefore, suggest that the authors qualitatively represent the strength/probability of connections by weighting with thickness of aXerent connections.

We assume the reviewer is commenting on our summary figure panel 7B. We agree with the referee that this is a simplified representation of our findings. We had indeed indicated in the legend that this was just a “Schematic of the HVC aXerent connectivity map resulting from the present work” and that “For conceptualization purposes, aXerent connectivity to HVC-PNs is shown only when the rate of monosynaptic connectivity reaches 50% of neurons examined”. We have added a title to highlight that this is but a simplification. We have now adjusted the colors to make the figure easier to follow. Based on the reviewers critique we searched for a better method for summarizing the complex connectivity patterns described in this research. We settled on a Sankey diagram of connectivity. This is now Figure 7C. In this diagram, we are able to show the proportion of connections from each input pathway onto each class of neuron and if these connections are poly or monosynaptic. We find this to a straightforward way of displaying all of the connectivity patterns identified in our figure 2-3 and 4-5 look forward to understanding if the reviewers find this a useful way of illustrating our findings.

Minor points:

(1) Line 50 - typo - song circuits.

Thank you for catching this.

(2) Line 106 - 111 - The findings suggest that 100% of Uva projections onto HVCRA neurons are monosynaptic. However, because the authors only tested 6 neurons their statements that their findings are so diXerent from other studies, should be somewhat tempered since these other studies (e.g. Moll et al.) looked at 251 neurons in HVC and sampling bias could still somewhat explain the diXerence.

We observed oEPSCs in 43 of 51 (84.3%) HVC-RA neurons recorded (mean rise time = 2.4 ms) and monosynaptic connections onto 100% of the HVC-RA neurons tested (n = 6). Moll et al. combined electrical stimulation of Uva with two-photon calcium imaging (GCaMP6s) of putative HVC-RA neurons (n = 251 neurons). We should note that these are putative HVC-RA neurons because they were not visually identified using retrograde tracing or using some other molecular handle. They report that only ~16% of HVC-RA neurons showed reliable calcium responses following Uva stimulation. Although the experiments by Moll et al are technically impressive, calcium imaging is an insensitive technique for measuring post-synaptic responses, particularly subthreshold responses, when compared to whole-cell patch-clamp recordings. This approach cannot identify monosynaptic connections and is likely limited to only be sensitive suprathreshold activity that likely relies on recruitment of other polysynaptic inputs onto the neurons in HVC. Furthermore, as indicated in the Discussion, our opsin-mediated synaptic interrogation recruits any eGtACR1+ Uva terminal in the slice and therefore will have great likelihood of revealing any existing connections. 

A limitation of whole-cell patch-clamp recordings is that it is a laborious low throughput technique. Future experiments using better imaging approaches, like voltage imaging, may be able to weigh in on diXerences between what we report here using whole-cell patch-clamp recordings from visually identified HVC-RA neurons combined with optogenetic manipulations of Uva terminals and the calcium imaging results reported by Moll. Nonetheless, whole-cell patch-clamp recordings combined with optogenetic manipulations is likely to remain the most sensitive method for identifying synaptic connectivity.

(3) Figure 2G - the significance of white circles is not clear.

The figure legend indicates that those highlight and mark the position of “retrogradely labeled HVCprojecting neurons in Uva (cyan, white circles)” to facilitate identification of colocalization with the in-situ markers.

(4) Line 135 - Cardin et al. (J. Neurophys. 2004) is the first to show that song production does not require Nif.

We thank the reviewer pointing this out and we have cited this important study. 

(5) Line 183 - This is a confusing sentence because I initially thought that mMAN-mMANHVC PNs was a category!

We switched the dash with a colon.

(6) Figure 4d could use some arrows to identify what is shown. It is assumed that the box represents mMAN. Should it be assumed that Av is not in the plane of this section? If not, this should be stated in the legend. It is also unclear where the anterograde projections are. Is this the dork highway that goes from the box to the dorsal surface? If yes this should be indicated but it should also be made clear why the projections go both in the dorsal as well as the ventral directions.

The inset, as indicated by the lines around it, is a magnification of the terminal fields in Av. We added an explanation of the inset.

(7) Discussion. In the introduction, the authors mention projections from RA to HVC but never end up studying them in the current manuscript which seems like a missed opportunity and perhaps even a weakness of the study. In the discussion, it would certainly be good for the authors to at least discuss the possible significance of these projections and perhaps why they decided not to study them.

We thank the reviewer for the comment. Unfortunately, we couldn’t reliably evoke interpretable currents from RA, and we elected to publish the current version of the paper with these 4 major inputs. Nonetheless, we have indicated in the Introduction and in the Discussion that more inputs (e.g. RA, A11, NCM) remain to be evaluated. 

(8) Line 622 - Is this reference incomplete?

We thank the reviewer. We have corrected the reference.

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Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Wang et al., recorded concurrent EEG-fMRI in 107 participants during nocturnal NREM sleep to investigate brain activity and connectivity related to slow oscillations (SO), sleep spindles, and in particular their co-occurrence. The authors found SO-spindle coupling to be correlated with increased thalamic and hippocampal activity, and with increased functional connectivity from the hippocampus to the thalamus and from the thalamus to the neocortex, especially the medial prefrontal cortex (mPFC). They concluded the brain-wide activation pattern to resemble episodic memory processing, but to be dissociated from task-related processing and suggest that the thalamus plays a crucial role in coordinating the hippocampal-cortical dialogue during sleep.

The paper offers an impressively large and highly valuable dataset that provides the opportunity for gaining important new insights into the network substrate involved in SOs, spindles, and their coupling. However, the paper does unfortunately not exploit the full potential of this dataset with the analyses currently provided, and the interpretation of the results is often not backed up by the results presented. I have the following specific comments.

Thank you for your thoughtful and constructive feedback. We greatly appreciate your recognition of the strengths of our dataset and findings Below, we address your specific comments and provide responses to each point you raised to ensure our methods and results are as transparent and comprehensible as possible. We hope these revisions address your comments and further strengthen our manuscript. Thank you again for the constructive feedback.

(1) The introduction is lacking sufficient review of the already existing literature on EEG-fMRI during sleep and the BOLD-correlates of slow oscillations and spindles in particular (Laufs et al., 2007; Schabus et al., 2007; Horovitz et al., 2008; Laufs, 2008; Czisch et al., 2009; Picchioni et al., 2010; Spoormaker et al., 2010; Caporro et al., 2011; Bergmann et al., 2012; Hale et al., 2016; Fogel et al., 2017; Moehlman et al., 2018; Ilhan-Bayrakci et al., 2022). The few studies mentioned are not discussed in terms of the methods used or insights gained.

We acknowledge the need for a more comprehensive review of prior EEG-fMRI studies investigating BOLD correlates of slow oscillations and spindles. However, these articles are not all related to sleep SO or spindle. Articles (Hale et al., 2016; Horovitz et al., 2008; Laufs, 2008; Laufs, Walker, & Lund, 2007; Spoormaker et al., 2010) mainly focus on methodology for EEG-fMRI, sleep stages, or brain networks, which are not the focus of our study. Thank you again for your attention to the comprehensiveness of our literature review, and we will expand the introduction to include a more detailed discussion of the existing literature, ensuring that the contributions of previous EEG-fMRI sleep studies are adequately acknowledged.  

Introduction, Page 4 Lines 62-76

“Investigating these sleep-related neural processes in humans is challenging because it requires tracking transient sleep rhythms while simultaneously assessing their widespread brain activation. Recent advances in simultaneous EEG-fMRI techniques provide a unique opportunity to explore these processes. EEG allows for precise event-based detection of neural signal, while fMRI provides insight into the broader spatial patterns of brain activation and functional connectivity (Horovitz et al., 2008; Huang et al., 2024; Laufs, 2008; Laufs, Walker, & Lund, 2007; Schabus et al., 2007; Spoormaker et al., 2010). Previous EEG-fMRI studies on sleep have focused on classifying sleep stages or examining the neural correlates of specific waves (Bergmann et al., 2012; Caporro et al., 2012; Czisch et al., 2009; Fogel et al., 2017; Hale et al., 2016; Ilhan-Bayrakcı et al., 2022; Moehlman et al., 2019; Picchioni et al., 2011). These studies have generally reported that slow oscillations are associated with widespread cortical and subcortical BOLD changes, whereas spindles elicit activation in the thalamus, as well as in several cortical and paralimbic regions. Although these findings provide valuable insights into the BOLD correlates of sleep rhythms, they often do not employ sophisticated temporal modeling (Huang et al., 2024), to capture the dynamic interactions between different oscillatory events, e.g., the coupling between SOs and spindles.”

(2) The paper falls short in discussing the specific insights gained into the neurobiological substrate of the investigated slow oscillations, spindles, and their interactions. The validity of the inverse inference approach ("Open ended cognitive state decoding"), assuming certain cognitive functions to be related to these oscillations because of the brain regions/networks activated in temporal association with these events, is debatable at best. It is also unclear why eventually only episodic memory processing-like brain-wide activation is discussed further, despite the activity of 16 of 50 feature terms from the NeuroSynth v3 dataset were significant (episodic memory, declarative memory, working memory, task representation, language, learning, faces, visuospatial processing, category recognition, cognitive control, reading, cued attention, inhibition, and action).

Thank you for pointing this out, particularly regarding the use of inverse inference approaches such as “open-ended cognitive state decoding.” Given the concerns about the indirectness of this approach, we decided to remove its related content and results from Figure 3 in the main text and include it in Supplementary Figure 7. We will refocus the main text on direct neurobiological insights gained from our EEG-fMRI analyses, particularly emphasizing the hippocampal-thalamocortical network dynamics underlying SO-spindle coupling, and we will acknowledge the exploratory nature of these findings and highlight their limitations.

Discussion, Page 17-18 Lines 323-332

“To explore functional relevance, we employed an open-ended cognitive state decoding approach using meta-analytic data (NeuroSynth: Yarkoni et al. (2011)). Although this method usefully generates hypotheses about potential cognitive processes, particularly in the absence of a pre- and post-sleep memory task, it is inherently indirect. Many cognitive terms showed significant associations (16 of 50), such as “episodic memory,” “declarative memory,” and “working memory.” We focused on episodic/declarative memory given the known link with hippocampal reactivation (Diekelmann & Born, 2010; Staresina et al., 2015; Staresina et al., 2023). Nonetheless, these inferences regarding memory reactivation should be interpreted cautiously without direct behavioral measures. Future research incorporating explicit tasks before and after sleep would more rigorously validate these potential functional claims.”

(3) Hippocampal activation during SO-spindles is stated as a main hypothesis of the paper - for good reasons - however, other regions (e.g., several cortical as well as thalamic) would be equally expected given the known origin of both oscillations and the existing sleep-EEG-fMRI literature. However, this focus on the hippocampus contrasts with the focus on investigating the key role of the thalamus instead in the Results section.

We appreciate your insight regarding the relative emphasis on hippocampal and thalamic activation in our study. We recognize that the manuscript may currently present an inconsistency between our initial hypothesis and the main focus of the results. To address this concern, we will ensure that our Introduction and Discussion section explicitly discusses both regions, highlighting the complementary roles of the hippocampus (memory processing and reactivation) and the thalamus (spindle generation and cortico-hippocampal coordination) in SO-spindle dynamics.

Introduction, Page 5 Lines 87-103

“To address this gap, our study investigates brain-wide activation and functional connectivity patterns associated with SO-spindle coupling, and employs a cognitive state decoding approach (Margulies et al., 2016; Yarkoni et al., 2011)—albeit indirectly—to infer potential cognitive functions. In the current study, we used simultaneous EEG-fMRI recordings during nocturnal naps (detailed sleep staging results are provided in the Methods and Table S1) in 107 participants. Although directly detecting hippocampal ripples using scalp EEG or fMRI is challenging, we expected that hippocampal activation in fMRI would coincide with SO-spindle coupling detected by EEG, given that SOs, spindles, and ripples frequently co-occur during NREM sleep. We also anticipated a critical role of the thalamus, particularly thalamic spindles, in coordinating hippocampal-cortical communication.

We found significant coupling between SOs and spindles during NREM sleep (N2/3), with spindle peaks occurring slightly before the SO peak. This coupling was associated with increased activation in both the thalamus and hippocampus, with functional connectivity patterns suggesting thalamic coordination of hippocampal-cortical communication. These findings highlight the key role of the thalamus in coordinating hippocampal-cortical interactions during human sleep and provide new insights into the neural mechanisms underlying sleep-dependent brain communication. A deeper understanding of these mechanisms may contribute to future neuromodulation approaches aimed at enhancing sleep-dependent cognitive function and treating sleep-related disorders.”

Discussion, Page 16-17 Lines 292-307

“When modeling the timing of these sleep rhythms in the fMRI, we observed hippocampal activation selectively during SO-spindle events. This suggests the possibility of triple coupling (SOs–spindles–ripples), even though our scalp EEG was not sufficiently sensitive to detect hippocampal ripples—key markers of memory replay (Buzsáki, 2015). Recent iEEG evidence indicates that ripples often co-occur with both spindles (Ngo, Fell, & Staresina, 2020) and SOs (Staresina et al., 2015; Staresina et al., 2023). Therefore, the hippocampal involvement during SO-spindle events in our study may reflect memory replay from the hippocampus, propagated via thalamic spindles to distributed cortical regions.

The thalamus, known to generate spindles (Halassa et al., 2011), plays a key role in producing and coordinating sleep rhythms (Coulon, Budde, & Pape, 2012; Crunelli et al., 2018), while the hippocampus is found essential for memory consolidation (Buzsáki, 2015; Diba & Buzsá ki, 2007; Singh, Norman, & Schapiro, 2022). The increased hippocampal and thalamic activity, along with strengthened connectivity between these regions and the mPFC during SO-spindle events, underscores a hippocampal-thalamic-neocortical information flow. This aligns with recent findings suggesting the thalamus orchestrates neocortical oscillations during sleep (Schreiner et al., 2022). The thalamus and hippocampus thus appear central to memory consolidation during sleep, guiding information transfer to the neocortex, e.g., mPFC.”

(4) The study included an impressive number of 107 subjects. It is surprising though that only 31 subjects had to be excluded under these difficult recording conditions, especially since no adaptation night was performed. Since only subjects were excluded who slept less than 10 min (or had excessive head movements) there are likely several datasets included with comparably short durations and only a small number of SOs and spindles and even less combined SO-spindle events. A comprehensive table should be provided (supplement) including for each subject (included and excluded) the duration of included NREM sleep, number of SOs, spindles, and SO+spindle events. Also, some descriptive statistics (mean/SD/range) would be helpful.

We appreciate your recognition of our sample size and the challenges associated with simultaneous EEG-fMRI sleep recordings. We acknowledge the importance of transparently reporting individual subject data, particularly regarding sleep duration and the number of detected SOs, spindles, and SO-spindle events. To address this, we will provide comprehensive tables in the supplementary materials, contains descriptive information about sleep-related characteristics (Table S1), as well as detailed information about sleep waves at each sleep stage for all 107 subjects(Table S2-S4), listing for each subject:(1)Different sleep stage duration; (2)Number of detected SOs; (3)Number of detected spindles; (4)Number of detected SO-spindle coupling events; (5)Density of detected SOs; (6)Density of detected spindles; (7)Density of detected SO-spindle coupling events.

However, most of the excluded participants were unable to fall asleep or had too short a sleep duration, so they basically had no NREM sleep period, so it was impossible to count the NREM sleep duration, SO, spindle, and coupling numbers.

Supplementary Materials, Page 42-54, Table S1-S4

(5) Was the 20-channel head coil dedicated for EEG-fMRI measurements? How were the electrode cables guided through/out of the head coil? Usually, the 64-channel head coil is used for EEG-fMRI measurements in a Siemens PRISMA 3T scanner, which has a cable duct at the back that allows to guide the cables straight out of the head coil (to minimize MR-related artifacts). The choice for the 20-channel head coil should be motivated. Photos of the recording setup would also be helpful.

Thank you for your comment regarding our choice of the 20-channel head coil for EEG-fMRI measurements. We acknowledge that the 64-channel head coil is commonly used in Siemens PRISMA 3T scanners; however, the 20-channel coil was selected due to specific practical and technical considerations in our study. In particular, the 20-channel head coil was compatible with our EEG system and ensured sufficient signal-to-noise ratio (SNR) for both EEG and fMRI acquisition. The EEG electrode cables were guided through the lateral and posterior openings of the head coil, secured with foam padding to reduce motion and minimize MR-related artifacts. Moreover, given the extended nature of nocturnal sleep recordings, the 20-channel coil allowed us to maintain participant comfort while still achieving high-quality simultaneous EEG-fMRI data.

We have made this clearer in the revised manuscript. 

Methods, Page 20 Lines 385-392

“All MRI data were acquired using a 20-channel head coil on a research-dedicated 3-Tesla Siemens Magnetom Prisma MRI scanner. Earplugs and cushions were provided for noise protection and head motion restriction. We chose the 20-channel head coil because it was compatible with our EEG system and ensured sufficient signal-to-noise ratio (SNR) for both EEG and fMRI acquisition. The EEG electrode cables were guided through the lateral and posterior openings of the head coil, secured with foam padding to reduce motion and minimize MR-related artifacts. Moreover, given the extended nature of nocturnal sleep recordings, the 20-channel coil helped maintain participant comfort while still achieving high-quality simultaneous EEG-fMRI data.”

(6) Was the EEG sampling synchronized to the MR scanner (gradient system) clock (the 10 MHz signal; not referring to the volume TTL triggers here)? This is a requirement for stable gradient artifact shape over time and thus accurate gradient noise removal.

Thank you for raising this important point. We confirm that the EEG sampling was synchronized to the MR scanner’s 10 MHz gradient system clock, ensuring a stable gradient artifact shape over time and enabling accurate artifact removal. This synchronization was achieved using the standard clock synchronization interface of the EEG amplifier, minimizing timing jitter and drift. As a result, the gradient artifact waveform remained stable across volumes, allowing for more effective artifact correction during preprocessing. We appreciate your attention to this critical aspect of EEG-fMRI data acquisition.

We have made this clearer in the revised manuscript. 

Methods, Page 19-20 Lines 371-383

“EEG was recorded simultaneously with fMRI data using an MR-compatible EEG amplifier system (BrainAmps MR-Plus, Brain Products, Germany), along with a specialized electrode cap. The recording was done using 64 channels in the international 10/20 system, with the reference channel positioned at FCz. In order to adhere to polysomnography (PSG) recording standards, six electrodes were removed from the EEG cap: one for electrocardiogram (ECG) recording, two for electrooculogram (EOG) recording, and three for electromyogram (EMG) recording. EEG data was recorded at a sample rate of 5000 Hz, the resistance of the reference and ground channels was kept below 10 kΩ, and the resistance of the other channels was kept below 20 kΩ. To synchronize the EEG and fMRI recordings, the BrainVision recording software (BrainProducts, Germany) was utilized to capture triggers from the MRI scanner. The EEG sampling was synchronized to the MR scanner’s 10 MHz gradient system clock, ensuring a stable gradient artifact shape over time and enabling accurate artifact removal. This was achieved via the standard clock synchronization interface of the EEG amplifier, minimizing timing jitter and drift.”

(7) The TR is quite long and the voxel size is quite large in comparison to state-of-the-art EPI sequences. What was the rationale behind choosing a sequence with relatively low temporal and spatial resolution?

We acknowledge that our chosen TR and voxel size are relatively long and large compared to state-of-the-art EPI sequences. This decision was made to optimize the signal-to-noise ratio (SNR) and reduce susceptibility-related distortions, which are particularly critical in EEG-fMRI sleep studies where head motion and physiological noise can be substantial. A longer TR allowed us to sample whole-brain activity with sufficient coverage, while a larger voxel size helped enhance BOLD sensitivity and minimize partial volume effects in deep brain structures such as the thalamus and hippocampus, which are key regions of interest in our study. We appreciate your concern and hope this clarification provides sufficient rationale for our sequence parameters.

We have made this clearer in the revised manuscript. 

Methods, Page 20-21 Lines 398-408

“Then, the “sleep” session began after the participants were instructed to try and fall asleep. For the functional scans, whole-brain images were acquired using k-space and steady-state T2*-weighted gradient echo-planar imaging (EPI) sequence that is sensitive to the BOLD contrast. This measures local magnetic changes caused by changes in blood oxygenation that accompany neural activity (sequence specification: 33 slices in interleaved ascending order, TR = 2000 ms, TE = 30 ms, voxel size = 3.5 × 3.5 × 4.2 mm3, FA = 90°, matrix = 64 × 64, gap = 0.7 mm). A relatively long TR and larger voxel size were chosen to optimize SNR and reduce susceptibility-related distortions, which are critical in EEG-fMRI sleep studies where head motion and physiological noise can be substantial. The longer TR allowed whole-brain coverage with sufficient temporal resolution, while the larger voxel size helped enhance BOLD sensitivity and minimize partial volume effects in deep brain structures (e.g., the thalamus and hippocampus), which are key regions of interest in this study.”

(8) The anatomically defined ROIs are quite large. It should be elaborated on how this might reduce sensitivity to sleep rhythm-specific activity within sub-regions, especially for the thalamus, which has distinct nuclei involved in sleep functions.

We appreciate your insight regarding the use of anatomically defined ROIs and their potential limitations in detecting sleep rhythm-specific activity within sub-regions, particularly in the thalamus. Given the distinct functional roles of thalamic nuclei in sleep processes, we acknowledge that using a single, large thalamic ROI may reduce sensitivity to localized activity patterns. To address this, we will discuss this limitation in the revised manuscript, acknowledging that our approach prioritizes whole-structure effects but may not fully capture nucleus-specific contributions.

Discussion, Page 18 Lines 333-341

“Despite providing new insights, our study has several limitations. First, our scalp EEG did not directly capture hippocampal ripples, preventing us from conclusively demonstrating triple coupling. Second, the combination of EEG-fMRI and the lack of a memory task limit our ability to parse fine-grained BOLD responses at the DOWN- vs. UP-states of SOs and link observed activations to behavioral outcomes. Third, the use of large anatomical ROIs may mask subregional contributions of specific thalamic nuclei or hippocampal subfields. Finally, without a memory task, we cannot establish a direct behavioral link between sleep-rhythm-locked activation and memory consolidation. Future studies combining techniques such as ultra-high-field fMRI or iEEG with cognitive tasks may refine our understanding of subregional network dynamics and functional significance during sleep.”

(9) The study reports SO & spindle amplitudes & densities, as well as SO+spindle coupling, to be larger during N2/3 sleep compared to N1 and REM sleep, which is trivial but can be seen as a sanity check of the data. However, the amount of SOs and spindles reported for N1 and REM sleep is concerning, as per definition there should be hardly any (if SOs or spindles occur in N1 it becomes by definition N2, and the interval between spindles has to be considerably large in REM to still be scored as such). Thus, on the one hand, the report of these comparisons takes too much space in the main manuscript as it is trivial, but on the other hand, it raises concerns about the validity of the scoring.

We appreciate your concern regarding the reported presence of SOs and spindles in N1 and REM sleep and the potential implications. Our detection method for detecting SO, spindle, and coupling were originally designed only for N2&N3 sleep data based on the characteristics of the data itself, and this method is widely recognized and used in the sleep research (Hahn et al., 2020; Helfrich et al., 2019; Helfrich et al., 2018; Ngo, Fell, & Staresina, 2020; Schreiner et al., 2022; Schreiner et al., 2021; Staresina et al., 2015; Staresina et al., 2023). While, because the detection methods for SO and spindle are based on percentiles, this method will always detect a certain number of events when used for other stages (N1 and REM) sleep data, but the differences between these events and those detected in stage N23 remain unclear. We will acknowledge the reasons for these results in the Methods section and emphasize that they are used only for sanity checks.

Methods, Page 25 Lines 515-524

“We note that the above methods for detecting SOs, spindles, and their couplings were originally developed for N2 and N3 sleep data, based on the specific characteristics of these stages. These methods are widely recognized in sleep research (Hahn et al., 2020; Helfrich et al., 2019; Helfrich et al., 2018; Ngo, Fell, & Staresina, 2020; Schreiner et al., 2022; Schreiner et al., 2021; Staresina et al., 2015; Staresina et al., 2023). However, because this percentile-based detection approach will inherently identify a certain number of events if applied to other stages (e.g., N1 and REM), the nature of these events in those stages remains unclear compared to N2/N3. We nevertheless identified and reported the detailed descriptive statistics of these sleep rhythms in all sleep stages, under the same operational definitions, both for completeness and as a sanity check. Within the same subject, there should be more SOs, spindles, and their couplings in N2/N3 than in N1 or REM (see also Figure S2-S4, Table S1-S4).”

(10) Why was electrode F3 used to quantify the occurrence of SOs and spindles? Why not a midline frontal electrode like Fz (or a number of frontal electrodes for SOs) and Cz (or a number of centroparietal electrodes) for spindles to be closer to their maximum topography?

We appreciate your suggestion regarding electrode selection for SO and spindle quantification. Our choice of F3 was primarily based on previous studies (Massimini et al., 2004; Molle et al., 2011), where bilateral frontal electrodes are commonly used for detecting SOs and spindles. Additionally, we considered the impact of MRI-related noise and, after a comprehensive evaluation, determined that F3 provided an optimal balance between signal quality and artifact minimization. We also acknowledge that alternative electrode choices, such as Fz for SOs and Cz for spindles, could provide additional insights into their topographical distributions.

(11) Functional connectivity (hippocampus -> thalamus -> cortex (mPFC)) is reported to be increased during SO-spindle coupling and interpreted as evidence for coordination of hippocampo-neocortical communication likely by thalamic spindles. However, functional connectivity was only analysed during coupled SO+spindle events, not during isolated SOs or isolated spindles. Without the direct comparison of the connectivity patterns between these three events, it remains unclear whether this is specific for coupled SO+spindle events or rather associated with one or both of the other isolated events. The PPIs need to be conducted for those isolated events as well and compared statistically to the coupled events.

We appreciate your critical perspective on our functional connectivity analysis and the interpretation of hippocampus-thalamus-cortex (mPFC) interactions during SO-spindle coupling. We acknowledge that, in the current analysis, functional connectivity was only examined during coupled SO-spindle events, without direct comparison to isolated SOs or isolated spindles. To address this concern, we have conducted PPI analyses for all three ROIs(Hippocampus, Thalamus, mPFC) and all three event types (SO-spindle couplings, isolated SOs, and isolated spindles). Our results indicate that neither isolated SOs nor isolated Spindles yielded significant connectivity changes in all three ROIs, as all failed to survive multiple comparison corrections. This suggests that the observed connectivity increase is specific to SO-spindle coupling, rather than being independently driven by either SOs or spindles alone.

Results, Page 14 Lines 248-255

“Crucially, the interaction between FC and SO-spindle coupling revealed that only the functional connectivity of hippocampus -> thalamus (ROI analysis, t(106) = 1.86, p = 0.0328) and thalamus -> mPFC (ROI analysis, t(106) = 1.98, p = 0.0251) significantly increased during SO-spindle coupling, with no significant changes in all other pathways (Fig. 4e). We also conducted PPI analyses for the other two events (SOs and spindles), and neither yielded significant connectivity changes in the three ROIs, as all failed to survive whole-brain FWE correction at the cluster level (p < 0.05). Together, these findings suggest that the thalamus, likely via spindles, coordinates hippocampal-cortical communication selectively during SO-spindle coupling, but not isolated SOs or spindle events alone.”

(12) The limited temporal resolution of fMRI does indeed not allow for easily distinguishing between fMRI activation patterns related to SO-up- vs. SO-down-states. For this, one could try to extract the amplitudes of SO-up- and SO-down-states separately for each SO event and model them as two separate parametric modulators (with the risk of collinearity as they are likely correlated).

We appreciate your insightful comment regarding the challenge of distinguishing fMRI activation patterns related to SO-up vs. SO-down states due to the limited temporal resolution of fMRI. While our current analysis does not differentiate between these two phases, we acknowledge that separately modeling SO-up and SO-down states using parametric modulators could provide a more refined understanding of their distinct neural correlates. However, as you notes, this approach carries the risk of collinearity, and there is indeed a high correlation between the two amplitudes across all subjects in our results (r=0.98). Future studies could explore more on leveraging high-temporal-resolution techniques. While implementing this in the current study is beyond our scope, we will acknowledge this limitation in the Discussion section.

Discussion, Page 17 Lines 308-322

“An intriguing aspect of our findings is the reduced DMN activity during SOs when modeled at the SO trough (DOWN-state). This reduced DMN activity may reflect large-scale neural inhibition characteristic of the SO trough. The DMN is typically active during internally oriented cognition (e.g., self-referential processing or mind-wandering) and is suppressed during external stimuli processing (Yeshurun, Nguyen, & Hasson, 2021). It is unlikely, however, that this suppression of DMN during SO events is related to a shift from internal cognition to external responses given it is during deep sleep time. Instead, it could be driven by the inherent rhythmic pattern of SOs, which makes it difficult to separate UP- from DOWN-states (the two temporal regressors were highly correlated, and similar brain activation during SOs events was obtained if modelled at the SO peak instead, Fig. S5). Since the amplitude at the SO trough is consistently larger than that at the SO peak, the neural activation we detected may primarily capture the large-scale inhibition from DOWN-state. Interestingly, no such DMN reduction was found during SO-spindle coupling, implying that coupling may involve distinct neural dynamics that partially re-engage DMN-related processes, possibly reflecting memory-related reactivation. Future research using high-temporal-resolution techniques like iEEG could clarify these possibilities.”

Discussion, Page 18 Lines 333-341

“Despite providing new insights, our study has several limitations. First, our scalp EEG did not directly capture hippocampal ripples, preventing us from conclusively demonstrating triple coupling. Second, the combination of EEG-fMRI and the lack of a memory task limit our ability to parse fine-grained BOLD responses at the DOWN- vs. UP-states of SOs and link observed activations to behavioral outcomes. Third, the use of large anatomical ROIs may mask subregional contributions of specific thalamic nuclei or hippocampal subfields. Finally, without a memory task, we cannot establish a direct behavioral link between sleep-rhythm-locked activation and memory consolidation. Future studies combining techniques such as ultra-high-field fMRI or iEEG with cognitive tasks may refine our understanding of subregional network dynamics and functional significance during sleep.”

(13) L327: "It is likely that our findings of diminished DMN activity reflect brain activity during the SO DOWN-state, as this state consistently shows higher amplitude compared to the UP-state within subjects, which is why we modelled the SO trough as its onset in the fMRI analysis." This conclusion is not justified as the fact that SO down-states are larger in amplitude does not mean their impact on the BOLD response is larger.

We appreciate your concern regarding our interpretation of diminished DMN activity reflecting the SO down-state. We acknowledge that the current expression is somewhat misleading, and our interpretation of it is: it could be driven by the inherent rhythmic pattern of SOs, which makes it difficult to separate UP- from DOWN-states (the two temporal regressors were highly correlated, and similar brain activation during SOs events was obtained if modelled at the SO peak instead). Since the amplitude at the SO trough is consistently larger than that at the SO peak, the neural activation we detected may primarily capture the large-scale inhibition from DOWN-state. And we will make this clear in the Discussion section.

Discussion, Page 17 Lines 308-322

“An intriguing aspect of our findings is the reduced DMN activity during SOs when modeled at the SO trough (DOWN-state). This reduced DMN activity may reflect large-scale neural inhibition characteristic of the SO trough. The DMN is typically active during internally oriented cognition (e.g., self-referential processing or mind-wandering) and is suppressed during external stimuli processing (Yeshurun, Nguyen, & Hasson, 2021). It is unlikely, however, that this suppression of DMN during SO events is related to a shift from internal cognition to external responses given it is during deep sleep time. Instead, it could be driven by the inherent rhythmic pattern of SOs, which makes it difficult to separate UP- from DOWN-states (the two temporal regressors were highly correlated, and similar brain activation during SOs events was obtained if modelled at the SO peak instead, Fig. S5). Since the amplitude at the SO trough is consistently larger than that at the SO peak, the neural activation we detected may primarily capture the large-scale inhibition from DOWN-state. Interestingly, no such DMN reduction was found during SO-spindle coupling, implying that coupling may involve distinct neural dynamics that partially re-engage DMN-related processes, possibly reflecting memory-related reactivation. Future research using high-temporal-resolution techniques like iEEG could clarify these possibilities.”

(14) Line 77: "In the current study, while directly capturing hippocampal ripples with scalp EEG or fMRI is difficult, we expect to observe hippocampal activation in fMRI whenever SOs-spindles coupling is detected by EEG, if SOs- spindles-ripples triple coupling occurs during human NREM sleep". Not all SO-spindle events are associated with ripples (Staresina et al., 2015), but hippocampal activation may also be expected based on the occurrence of spindles alone (Bergmann et al., 2012).

We appreciate your clarification regarding the relationship between SO-spindle coupling and hippocampal ripples. We acknowledge that not all SO-spindle events are necessarily accompanied by ripples (Staresina et al., 2015). However, based on previous research, we found that hippocampal ripples are significantly more likely to occur during SO-spindle coupling events. This suggests that while ripple occurrence is not guaranteed, SO-spindle coupling creates a favorable network state for ripple generation and potential hippocampal activation. To ensure accuracy, we will revise the manuscript to delete this misleading sentence in the Introduction section and acknowledge in the Discussion that our results cannot conclusively directly observe the triple coupling of SO, spindle, and hippocampal ripples.

Discussion, Page 18 Lines 333-341

“Despite providing new insights, our study has several limitations. First, our scalp EEG did not directly capture hippocampal ripples, preventing us from conclusively demonstrating triple coupling. Second, the combination of EEG-fMRI and the lack of a memory task limit our ability to parse fine-grained BOLD responses at the DOWN- vs. UP-states of SOs and link observed activations to behavioral outcomes. Third, the use of large anatomical ROIs may mask subregional contributions of specific thalamic nuclei or hippocampal subfields. Finally, without a memory task, we cannot establish a direct behavioral link between sleep-rhythm-locked activation and memory consolidation. Future studies combining techniques such as ultra-high-field fMRI or iEEG with cognitive tasks may refine our understanding of subregional network dynamics and functional significance during sleep.”

Reviewer #2 (Public review):

In this study, Wang and colleagues aimed to explore brain-wide activation patterns associated with NREM sleep oscillations, including slow oscillations (SOs), spindles, and SO-spindle coupling events. Their findings reveal that SO-spindle events corresponded with increased activation in both the thalamus and hippocampus. Additionally, they observed that SO-spindle coupling was linked to heightened functional connectivity from the hippocampus to the thalamus, and from the thalamus to the medial prefrontal cortex-three key regions involved in memory consolidation and episodic memory processes.

This study's findings are timely and highly relevant to the field. The authors' extensive data collection, involving 107 participants sleeping in an fMRI while undergoing simultaneous EEG recording, deserves special recognition. If shared, this unique dataset could lead to further valuable insights. While the conclusions of the data seem overall well supported by the data, some aspects with regard to the detection of sleep oscillations need clarification.

The authors report that coupled SO-spindle events were most frequent during NREM sleep (2.46 [plus minus] 0.06 events/min), but they also observed a surprisingly high occurrence of these events during N1 and REM sleep (2.23 [plus minus] 0.09 and 2.32 [plus minus] 0.09 events/min, respectively), where SO-spindle coupling would not typically be expected. Combined with the relatively modest SO amplitudes reported (~25 µV, whereas >75 µV would be expected when using mastoids as reference electrodes), this raises the possibility that the parameters used for event detection may not have been conservative enough - or that sleep staging was inaccurately performed. This issue could present a significant challenge, as the fMRI findings are largely dependent on the reliability of these detected events.

Thank you very much for your thorough and encouraging review. We appreciate your recognition of the significance and relevance of our study and dataset, particularly in highlighting how simultaneous EEG-fMRI recordings can provide complementary insights into the temporal dynamics of neural oscillations and their associated spatial activation patterns during sleep. In the sections that follow, we address each of your comments in detail. We have revised the text and conducted additional analyses wherever possible to strengthen our argument, clarify our methodological choices. We believe these revisions improve the clarity and rigor of our work, and we thank you for helping us refine it.

We appreciate your insightful comments regarding the detection of sleep oscillations. Our methods for detecting SOs, spindles, and their couplings were originally developed for N2 and N3 sleep data, based on the specific characteristics of these stages. These methods are widely recognized in sleep research (Hahn et al., 2020; Helfrich et al., 2019; Helfrich et al., 2018; Ngo, Fell, & Staresina, 2020; Schreiner et al., 2022; Schreiner et al., 2021; Staresina et al., 2015; Staresina et al., 2023). However, because this percentile-based detection approach will inherently identify a certain number of events if applied to other stages (e.g., N1 and REM), the nature of these events in those stages remains unclear compared to N2/N3. We nevertheless identified and reported the detailed descriptive statistics of these sleep rhythms in all sleep stages, under the same operational definitions, both for completeness and as a sanity check. Within the same subject, there should be more SOs, spindles, and their couplings in N2/N3 than in N1 or REM. We will acknowledge the reasons for these results in the Methods section and emphasize that they are used only for sanity checks.

Regarding the reported SO amplitudes (~25 µV), during preprocessing, we applied the Signal Space Projection (SSP) method to more effectively remove MRI gradient artifacts and cardiac pulse noise. While this approach enhances data quality, it also reduces overall signal power, leading to systematically lower reported amplitudes. Despite this, our SO detection in NREM sleep (especially N2/N3) remain physiologically meaningful and are consistent with previous fMRI studies using similar artifact removal techniques. We appreciate your careful evaluation and valuable suggestions.

In addition, we will provide comprehensive tables in the supplementary materials, contains descriptive information about sleep-related characteristics (Table S1), as well as detailed information about sleep waves at each sleep stage for all 107 subjects(Table S2-S4), listing for each subject:(1)Different sleep stage duration; (2)Number of detected SOs; (3)Number of detected spindles; (4)Number of detected SO-spindle coupling events; (2)Density of detected SOs; (3)Density of detected spindles; (4)Density of detected SO-spindle coupling events.

Methods, Page 25 Lines 515-524

“We note that the above methods for detecting SOs, spindles, and their couplings were originally developed for N2 and N3 sleep data, based on the specific characteristics of these stages. These methods are widely recognized in sleep research (Hahn et al., 2020; Helfrich et al., 2019; Helfrich et al., 2018; Ngo, Fell, & Staresina, 2020; Schreiner et al., 2022; Schreiner et al., 2021; Staresina et al., 2015; Staresina et al., 2023). However, because this percentile-based detection approach will inherently identify a certain number of events if applied to other stages (e.g., N1 and REM), the nature of these events in those stages remains unclear compared to N2/N3. We nevertheless identified and reported the detailed descriptive statistics of these sleep rhythms in all sleep stages, under the same operational definitions, both for completeness and as a sanity check. Within the same subject, there should be more SOs, spindles, and their couplings in N2/N3 than in N1 or REM (see also Figure S2-S4, Table S1-S4).”

Supplementary Materials, Page 42-54, Table S1-S4

Reviewer #3 (Public review):

Summary:

Wang et al., examined the brain activity patterns during sleep, especially when locked to those canonical sleep rhythms such as SO, spindle, and their coupling. Analyzing data from a large sample, the authors found significant coupling between spindles and SOs, particularly during the upstate of the SO. Moreover, the authors examined the patterns of whole-brain activity locked to these sleep rhythms. To understand the functional significance of these brain activities, the authors further conducted open-ended cognitive state decoding and found a variety of cognitive processing may be involved during SO-spindle coupling and during other sleep events. The authors next investigated the functional connectivity analyses and found enhanced connectivity between the hippocampus, the thalamus, and the medial PFC. These results reinforced the theoretical model of sleep-dependent memory consolidation, such that SO-spindle coupling is conducive to systems-level memory reactivation and consolidation.

Strengths:

There are obvious strengths in this work, including the large sample size, state-of-the-art neuroimaging and neural oscillation analyses, and the richness of results.

Weaknesses:

Despite these strengths and the insights gained, there are weaknesses in the design, the analyses, and inferences.

Thank you for your detailed and thoughtful review of our manuscript. We are delighted that you recognize our advanced analysis methods and rich results of neuroimaging and neural oscillations as well as the large sample size data. In the following sections, we provide detailed responses to each of your comments. And we have revised the text and conducted additional analyses to strengthen our arguments and clarify our methodological choices. We believe these revisions enhance the clarity and rigor of our work, and we sincerely appreciate your thoughtful feedback in helping us refine the manuscript.

(1) A repeating statement in the manuscript is that brain activity could indicate memory reactivation and thus consolidation. This is indeed a highly relevant question that could be informed by the current data/results. However, an inherent weakness of the design is that there is no memory task before and after sleep. Thus, it is difficult (if not impossible) to make a strong argument linking SO/spindle/coupling-locked brain activity with memory reactivation or consolidation.

We appreciate your suggestion regarding the lack of a pre- and post-sleep memory task in our study design. We acknowledge that, in the absence of behavioral measures, it is hard to directly link SO-spindle coupling to memory consolidation in an outcome-driven manner. Our interpretation is instead based on the well-established role of these oscillations in memory processes, as demonstrated in previous studies. We sincerely appreciate this feedback and will adjust our Discussion accordingly to reflect a more precise interpretation of our findings.

Discussion, Page 18 Lines 333-341

“Despite providing new insights, our study has several limitations. First, our scalp EEG did not directly capture hippocampal ripples, preventing us from conclusively demonstrating triple coupling. Second, the combination of EEG-fMRI and the lack of a memory task limit our ability to parse fine-grained BOLD responses at the DOWN- vs. UP-states of SOs and link observed activations to behavioral outcomes. Third, the use of large anatomical ROIs may mask subregional contributions of specific thalamic nuclei or hippocampal subfields. Finally, without a memory task, we cannot establish a direct behavioral link between sleep-rhythm-locked activation and memory consolidation. Future studies combining techniques such as ultra-high-field fMRI or iEEG with cognitive tasks may refine our understanding of subregional network dynamics and functional significance during sleep.”

(2) Relatedly, to understand the functional implications of the sleep rhythm-locked brain activity, the authors employed the "open-ended cognitive state decoding" method. While this method is interesting, it is rather indirect given that there were no behavioral indices in the manuscript. Thus, discussions based on these analyses are speculative at best. Please either tone down the language or find additional evidence to support these claims.

Moreover, the results from this method are difficult to understand. Figure 3e showed that for all three types of sleep events (SO, spindle, SO-spindle), the same mental states (e.g., working memory, episodic memory, declarative memory) showed opposite directions of activation (left and right panels showed negative and positive activation, respectively). How to interpret these conflicting results? This ambiguity is also reflected by the term used: declarative memory and episodic memories are both indexed in the results. Yet these two processes can be largely overlapped. So which specific memory processes do these brain activity patterns reflect? The Discussion shall discuss these results and the limitations of this method.

We appreciate your critical assessment of the open-ended cognitive state decoding method and its interpretational challenges. Given the concerns about the indirectness of this approach, we decided to remove its related content and results from Figure 3 in the main text and include it in Supplementary Figure 7. 

Due to the complexity of memory-related processes, we acknowledge that distinguishing between episodic and declarative memory based solely on this approach is not straightforward. We will revise the Supplementary Materials to explicitly discuss these limitations and clarify that our findings do not isolate specific cognitive processes but rather suggest general associations with memory-related networks.

Discussion, Page 17-18 Lines 323-332

“To explore functional relevance, we employed an open-ended cognitive state decoding approach using meta-analytic data (NeuroSynth: Yarkoni et al. (2011)). Although this method usefully generates hypotheses about potential cognitive processes, particularly in the absence of a pre- and post-sleep memory task, it is inherently indirect. Many cognitive terms showed significant associations (16 of 50), such as “episodic memory,” “declarative memory,” and “working memory.” We focused on episodic/declarative memory given the known link with hippocampal reactivation (Diekelmann & Born, 2010; Staresina et al., 2015; Staresina et al., 2023). Nonetheless, these inferences regarding memory reactivation should be interpreted cautiously without direct behavioral measures. Future research incorporating explicit tasks before and after sleep would more rigorously validate these potenial functional claims.”

(3) The coupling strength is somehow inconsistent with prior results (Hahn et al., 2020, eLife, Helfrich et al., 2018, Neuron). Specifically, Helfrich et al. showed that among young adults, the spindle is coupled to the peak of the SO. Here, the authors reported that the spindles were coupled to down-to-up transitions of SO and before the SO peak. It is possible that participants' age may influence the coupling (see Helfrich et al., 2018). Please discuss the findings in the context of previous research on SO-spindle coupling.

We appreciate your concern regarding the temporal characteristics of SO-spindle coupling. We acknowledge that the SO-spindle coupling phase results in our study are not identical to those reported by Hahn et al. (2020); Helfrich et al. (2018). However, these differences may arise due to slight variations in event detection parameters, which can influence the precise phase estimation of coupling. Notably, Hahn et al. (2020) also reported slight discrepancies in their group-level coupling phase results, highlighting that methodological differences can contribute to variability across studies. Furthermore, our findings are consistent with those of Schreiner et al. (2021), further supporting the robustness of our observations.  

That said, we acknowledge that our original description of SO-spindle coupling as occurring at the "transition from the lower state to the upper state" was not entirely precise. The -π/2 phase represents the true transition point, while our observed coupling phase is actually closer to the SO peak rather than strictly at the transition. We will revise this statement in the manuscript to ensure clarity and accuracy in describing the coupling phase.  

Discussion, Page 16 Lines 283-291

“Our data provide insights into the neurobiological underpinnings of these sleep rhythms. SOs, originating mainly in neocortical areas such as the mPFC, alternate between DOWN- and UP-states. The thalamus generates sleep spindles, which in turn couple with SOs. Our finding that spindle peaks consistently occurred slightly before the UP-state peak of SOs (in 83 out of 107 participants), concurs with prior studies, including Schreiner et al. (2021). Yet it differs from some results suggesting spindles might peak right at the SO UP-state (Hahn et al., 2020; Helfrich et al., 2018). Such discrepancies could arise from differences in detection algorithms, participant age (Helfrich et al., 2018), or subtle variations in cortical-thalamic timing. Nonetheless, these results underscore the importance of coordinated SO-spindle interplay in supporting sleep-dependent processes.”

(4) The discussion is rather superficial with only two pages, without delving into many important arguments regarding the possible functional significance of these results. For example, the author wrote, "This internal processing contrasts with the brain patterns associated with external tasks, such as working memory." Without any references to working memory, and without delineating why WM is considered as an external task even working memory operations can be internal. Similarly, for the interesting results on SO and reduced DMN activity, the authors wrote "The DMN is typically active during wakeful rest and is associated with self-referential processes like mind-wandering, daydreaming, and task representation (Yeshurun, Nguyen, & Hasson, 2021). Its reduced activity during SOs may signal a shift towards endogenous processes such as memory consolidation." This argument is flawed. DMN is active during self-referential processing and mind-wandering, i.e., when the brain shifts from external stimuli processing to internal mental processing. During sleep, endogenous memory reactivation and consolidation are also part of the internal mental processing given the lack of external environmental stimulation. So why during SO or during memory consolidation, the DMN activity would be reduced? Were there differences in DMN activity between SO and SO-spindle coupling events?

We appreciate your concerns regarding the brevity of the discussion and the need for clearer theoretical arguments. We will expand this section to provide more in-depth interpretations of our findings in the context of prior literature. Regarding working memory (WM), we acknowledge that our phrasing was ambiguous. We will modify this statement in the Discussion section.

For the SO-related reduction in DMN activity, we recognize the need for a more precise explanation. This reduced DMN activity may reflect large-scale neural inhibition characteristic of the SO trough. The DMN is typically active during internally oriented cognition (e.g., self-referential processing or mind-wandering) and is suppressed during external stimuli processing (Yeshurun, Nguyen, & Hasson, 2021). It is unlikely, however, that this suppression of DMN during SO events is related to a shift from internal cognition to external responses given it is during deep sleep time. Instead, it could be driven by the inherent rhythmic pattern of SOs, which makes it difficult to separate UP- from DOWN-states (the two temporal regressors were highly correlated, and similar brain activation during SOs events was obtained if modelled at the SO peak instead). Since the amplitude at the SO trough is consistently larger than that at the SO peak, the neural activation we detected may primarily capture the large-scale inhibition from DOWN-state.

To address your final question, we have conducted the additional post hoc comparison of DMN activity between isolated SOs and SO-spindle coupling events. Our results indicate that

DMN activation during SOs was significantly lower than during SO-spindle coupling (t(106) = -4.17, p < 1e-4). This suggests that SO-spindle coupling may involve distinct neural dynamics that partially re-engage DMN-related processes, possibly reflecting memory-related reactivation. We appreciate your constructive feedback and will integrate these expanded analyses and discussions into our revised manuscript.

Results, Page 11 Lines 199-208

“Spindles were correlated with positive activation in the thalamus (ROI analysis, t(106) = 15.39, p < 1e-4), the anterior cingulate cortex (ACC), and the putamen, alongside deactivation in the DMN (Fig. 3c). Notably, SO-spindle coupling was linked to significant activation in both the thalamus (ROI analysis, t(106) \= 3.38, p = 0.0005) and the hippocampus (ROI analysis, t(106) \= 2.50, p = 0.0070, Fig. 3d). However, no decrease in DMN activity was found during SO-spindle coupling, and DMN activity during SO was significantly lower than during coupling (ROI analysis, t(106) \= -4.17, p < 1e-4). For more detailed activation patterns, see Table S5-S7. We also varied the threshold used to detect SO events to assess its effect on hippocampal activation during SO-spindle coupling and observed that hippocampal activation remained significant when the percentile thresholds for SO detection ranged between 71% and 80% (see Fig. S6).”

Discussion, Page 17-18 Lines 308-332

“An intriguing aspect of our findings is the reduced DMN activity during SOs when modeled at the SO trough (DOWN-state). This reduced DMN activity may reflect large-scale neural inhibition characteristic of the SO trough. The DMN is typically active during internally oriented cognition (e.g., self-referential processing or mind-wandering) and is suppressed during external stimuli processing (Yeshurun, Nguyen, & Hasson, 2021). It is unlikely, however, that this suppression of DMN during SO events is related to a shift from internal cognition to external responses given it is during deep sleep time. Instead, it could be driven by the inherent rhythmic pattern of SOs, which makes it difficult to separate UP- from DOWN-states (the two temporal regressors were highly correlated, and similar brain activation during SOs events was obtained if modelled at the SO peak instead, Fig. S5). Since the amplitude at the SO trough is consistently larger than that at the SO peak, the neural activation we detected may primarily capture the large-scale inhibition from DOWN-state. Interestingly, no such DMN reduction was found during SO-spindle coupling, implying that coupling may involve distinct neural dynamics that partially re-engage DMN-related processes, possibly reflecting memory-related reactivation. Future research using high-temporal-resolution techniques like iEEG could clarify these possibilities.

To explore functional relevance, we employed an open-ended cognitive state decoding approach using meta-analytic data (NeuroSynth: Yarkoni et al. (2011)). Although this method usefully generates hypotheses about potential cognitive processes, particularly in the absence of a pre- and post-sleep memory task, it is inherently indirect. Many cognitive terms showed significant associations (16 of 50), such as “episodic memory,” “declarative memory,” and “working memory.” We focused on episodic/declarative memory given the known link with hippocampal reactivation (Diekelmann & Born, 2010; Staresina et al., 2015; Staresina et al., 2023). Nonetheless, these inferences regarding memory reactivation should be interpreted cautiously without direct behavioral measures. Future research incorporating explicit tasks before and after sleep would more rigorously validate these potential functional claims.”

Recommendations for the authors:

Reviewing Editor Comment:

The reviewers think that you are working on a relevant and important topic. They are praising the large sample size used in the study. The reviewers are not all in line regarding the overall significance of the findings, but they all agree the paper would strongly benefit from some extra work, as all reviewers raise various critical points that need serious consideration.

We appreciate your recognition of the relevance and importance of our study, as well as your acknowledgment of the large sample size as a strength of our work. We understand that there are differing perspectives regarding the overall significance of our findings, and we value the constructive critiques provided. We are committed to addressing the key concerns raised by all reviewers, including refining our analyses, clarifying our interpretations, and incorporating additional discussions to strengthen the manuscript. Below, we address your specific recommendations and provide responses to each point you raised to ensure our methods and results are as transparent and comprehensible as possible. We believe that these revisions will significantly enhance the rigor and impact of our study, and we sincerely appreciate your thoughtful feedback in helping us improve our work.

Reviewer #1 (Recommendations for the authors):

(1) The phrase "overnight sleep" suggests an entire night, while these were rather "nocturnal naps". Please rephrase.

Response: Thank you for pointing this out. We have revised the phrasing in our manuscript to "nocturnal naps" instead of "overnight sleep" to more accurately reflect the duration of the sleep recordings.

(2) Sleep staging results (macroscopic sleep architecture) should be provided in more detail (at least min and % of the different sleep stages, sleep onset latency, total sleep duration, total recording duration), at least mean/SD/range.

Thank you for this suggestion. We will provide comprehensive tables in the supplementary materials, contains descriptive information about sleep-related characteristics. This information will help provide a clearer overview of the macroscopic sleep architecture in our dataset.

Reviewer #2 (Recommendations for the authors):

In order to allow for a better estimation of the reliability of the detected sleep events, please:

(1) Provide densities and absolute numbers of all detected SOs and spindles (N1, NREM, and REM sleep).

Thank you for pointing this out. We will provide comprehensive tables in the supplementary materials, contains detailed information about sleep waves at each sleep stage for all 107 subjects (Table S2-S4), listing for each subject:1) Different sleep stage duration; 2) Number of detected SOs; 3) Number of detected spindles; 4) Number of detected SO-spindle coupling events; 5) Density of detected SOs; 6) Density of detected spindles; 7) Density of detected SO-spindle coupling events.

Supplementary Materials, Page 43-54, Table S2-S4

(2) Show ERPs for all detected SOs and spindles (per sleep stage).

Thank you for the suggestion. We will provide ERPs for all detected SOs and spindles, separated by sleep stage (N1, N2&N3, and REM) in supplementary Fig. S2-S4. These ERP waveforms will help illustrate the characteristic temporal profiles of SOs and spindles across different sleep stages.

Methods, Page 25, Line 525-532

“Event-related potentials (ERP) analysis. After completing the detection of each sleep rhythm event, we performed ERP analyses for SOs, spindles, and coupling events in different sleep stages. Specifically, for SO events, we took the trough of the DOWN-state of each SO as the zero-time point, then extracted data in a [-2 s to 2 s] window from the broadband (0.1–30 Hz) EEG and used [-2 s to -0.5 s] for baseline correction; the results were then averaged across 107 subjects (see Fig. S2a). For spindle events, we used the peak of each spindle as the zero-time point and applied the same data extraction window and baseline correction before averaging across 107 subjects (see Fig. S2b). Finally, for SO-spindle coupling events, we followed the same procedure used for SO events (see Fig. 2a, Figs. S3–S4).”

(3) Provide detailed info concerning sleep characteristics (time spent in each sleep stage etc.).

Thank you for this suggestion. Same as the response above, we will provide comprehensive tables in the supplementary materials, contains descriptive information about sleep-related characteristics.

Supplementary Materials, Page 42, Table S1 (same as above)

(4) What would happen if more stringent parameters were used for event detection? Would the authors still observe a significant number of SO spindles during N1 and REM? Would this affect the fMRI-related results?

Thank you for this suggestion. Our methods for detecting SOs, spindles, and their couplings were originally developed for N2 and N3 sleep data, based on the specific characteristics of these stages. These methods are widely recognized in sleep research (Hahn et al., 2020; Helfrich et al., 2019; Helfrich et al., 2018; Ngo, Fell, & Staresina, 2020; Schreiner et al., 2022; Schreiner et al., 2021; Staresina et al., 2015; Staresina et al., 2023). However, because this percentile-based detection approach will inherently identify a certain number of events if applied to other stages (e.g., N1 and REM), the nature of these events in those stages remains unclear compared to N2/N3. We nevertheless identified and reported the detailed descriptive statistics of these sleep rhythms in all sleep stages, under the same operational definitions, both for completeness and as a sanity check. Within the same subject, there should be more SOs, spindles, and their couplings in N2/N3 than in N1 or REM (see also Figure S2-S4, Table S1-S4).

Furthermore, in order to explore the impact of this on our fMRI results, we conducted an additional sensitivity analysis by applying different detection parameters for SOs. Specifically, we adjusted amplitude percentile thresholds for SO detection (the parameter that has the greatest impact on the results). We used the hippocampal activation value during N2&N3 stage SO-spindle coupling as an anchor value and found that when the parameters gradually became stricter, the results were similar to or even better than the current results. However, when we continued to increase the threshold, the results began to gradually decrease until the threshold was increased to 80%, and the results were no longer significant. This indicates that our results are robust within a specific range of parameters, but as the threshold increases, the number of trials decreases, ultimately weakening the statistical power of the fMRI analysis.

Thank you again for your suggestions on sleep rhythm event detection. We will add the results in Supplementary and revise our manuscript accordingly.

Results, Page 11, Line 199-208

“Spindles were correlated with positive activation in the thalamus (ROI analysis, t(106) = 15.39, p < 1e-4), the anterior cingulate cortex (ACC), and the putamen, alongside deactivation in the DMN (Fig. 3c). Notably, SO-spindle coupling was linked to significant activation in both the thalamus (ROI analysis, t(106) \= 3.38, p = 0.0005) and the hippocampus (ROI analysis, t(106) \= 2.50, p = 0.0070, Fig. 3d). However, no decrease in DMN activity was found during SO-spindle coupling, and DMN activity during SO was significantly lower than during coupling (ROI analysis, t(106) \= -4.17, p < 1e-4). For more detailed activation patterns, see Table S5-S7. We also varied the threshold used to detect SO events to assess its effect on hippocampal activation during SO-spindle coupling and observed that hippocampal activation remained significant when the percentile thresholds for SO detection ranged between 71% and 80% (see Fig. S6).”

Finally, we sincerely thank all again for your thoughtful and constructive feedback. Your insights have been invaluable in refining our analyses, strengthening our interpretations, and improving the clarity and rigor of our manuscript. We appreciate the time and effort you have dedicated to reviewing our work, and we are grateful for the opportunity to enhance our study based on your recommendations.  

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add hyphen for transition to next page

Aggiungerei qualche informazione più specifica (metto questo esempio, da adattare a Gorizia):

https://aisp-sis.com/popdays-2025/#venue

Metterei solo la current con le sue travel information e a parte Past Summer Schools (come schools non venues)

Ritengo che potremmo inserire maggiori informazioni sulla sede del corso solamente dopo aver ricevuto -almeno- un ok informale da parte della Regione; all'inizio è meglio lasciare un generico "Gorizia"

Reviewer #3 (Public review):

Summary:

Kim et al. present a study of the neural dynamics underlying reversal learning in monkey PFC and neural networks. Their main finding is that neural activity during fixation resembles a line attractor storing the current belief of the reversal state of the task. This is followed by richer dynamics unfolding throughout the remainder of the trial, which eventually converge to a new point on the line attractor by the start of the next trial. The idea of studying neural dynamics throughout the task (including intervening behaviour) is interesting, and the data provides some insights into the neural dynamics driving reversal learning. The modelling seems to support the analyses, but both the modelling and analyses also leave several open questions.

Strengths:

The paper addresses an interesting topic of the neural dynamics underlying reversal learning in PFC, using a combination of biological and simulated data. Reversal learning has been studied extensively in neuroscience, but this paper takes a step further by analysing neural dynamics throughout the trials instead of focusing on just the evidence integration epoch.

The authors show some close parallels between the experimental data and RNN simulations, both in terms of behaviour and neural dynamics. The analyses of how rewarded and unrewarded trials differentially affect dynamics throughout the trials in RNNs and PFC were particularly interesting. This work has the potential to provide new insights into the neural underpinnings of reversal learning.

Weaknesses:

Data analyses:

While the analyses seem mostly sound, one shortcoming is that they are all aligned to the inferred reversal trial rather than the true experimental reversal trial. For example, the analyses showing that 'x_rev' decays strongly after the reversal trial, irrespective of the reward outcome, seem like they are true essentially by design. The choice to align to the inferred reversal trial also makes this trial seem 'special' (e.g. in Fig 2 & Fig 6A), but it is unclear whether this is a real feature of the data or an artifact of effectively conditioning on a change in behaviour. It would be useful to investigate whether any of these analyses differ when aligned to the true reversal trial. It is also unsurprising that x_rev increases before the reversal and decreases after the reversal (it is hard to imagine a system where this is not the case), yet all of Fig 6 and several other analyses are devoted to this point.

Most of the analyses focus on the dynamics specifically in the x_rev subspace, but a major point of the paper is to say that biological (and artificial) networks may also have to do other things at different times in the trial. If that is the case, it would be interesting to also ask what happens in other subspaces of neural activity, which are not specifically related to evidence integration or choice - are there other subspaces that explain substantial variance? Do they relate to any meaningful features of the experiment?

This is especially important when considering analyses trying to establish the presence (or absence) of attractor dynamics in the circuit. In particular, activity in the x_rev subspace both affects and depends on other subspaces of neural activity, so it is not as meaningful to analyse the dynamics of this subspace in isolation. It would e.g. have been preferable to analyse the early-trial dynamics in the full state space and then possibly projecting onto x_rev, rather than first projecting activity onto x_rev and then fitting a linear autoregressive model.

Modelling:

There are a number of surprising and non-standard modelling choices made in this paper. For example, the choice to only use inhibitory neurons is non-conventional and it is not clear whether and how this impacts the results. The inputs are also provided without any learnable input weights, which makes it harder to interpret the input-driven dynamics during the different phases of a trial.

It is surprising that the RNN is "trained to flip its preferred choice a few trials after the inferred scheduled reversal trial", with the reversal trial inferred by an ideal Bayesian observer. A more natural approach would be to directly train the RNN to solve the task (by predicting the optimal choice) and then investigating the emergent behaviour & dynamics. If the authors prefer their imitation learning approach, it is also surprising that the network is trained to predict the reversal trial inferred using Bayesian smoothing instead of Bayesian filtering.

Finally, it was surprising that the network is trained and tested with different block lengths (24 & 36 trials, respectively), and it is not mentioned whether or how this affects behaviour.

Author response:

The following is the authors’ response to the original reviews

Main revision made to the manuscript

The main revision made to the manuscript is to reconcile our findings with the line attractor model. The revision is based on Reviewer 1’s comment on reinterpreting our results as a superposition of an attractor model with fast timescale dynamics. We expanded our analysis regime to the start of a trial and characterized the overall within-trial dynamics to reinterpret our findings.

We first acknolwedge that our results are not in contradiction with evidence integration on a line attractor. As pointed out by the reviewers, our finding that the integration of reward outcome explains the reversal probability activity x_rev (Figure 3) is compatible with the line attractor model. However, the reward integration equation is an algebraic relation and does not characterize the dynamics of reversal probability activity. So a closer analysis on the neural dynamics is needed to assess the feasibility of line attractor.

In the revised manuscript, we show that x_rev exhibits two different activity modes (Figure 4). First, x_rev has substantial non-stationary dynamics during a trial, and this non-stationary activity is incompatible with the line attractor model, as claimed in the original manuscript. Second, we present new results showing that x_rev is stationary (i.e., constant in time) and stable (i.e., contracting) at the start of a trial. These two properties of x_rev support that it is a point attractor at the start of a trial and is compatible with the line attractor model. 

We further analyze how the two activity modes are linked (Figure 4, Support vector regression). We show that the non-stationary activity is predictable from the stationary activity if the underlying dynamics can be inferred. In other words, the non-stationary activity during a trial is generated by an underlying dynamics with the initial condition provided by the stationary state at the start of trial.

These results suggest an extension of the line attractor model where an attractor state at the start of a trial provides an initial condition from which non-stationary activity is generated during a trial by an underlying dynamics associated with task-related behavior (Figure 4, Augmented model). 

The separability of non-stationary trajectories (Figure 5 and 6) is a property of the non-stationary dynamics that allows separable points in the initial stationary state to remain separable during a trial, thus making it possible to represent distinct probabilistic values in non-stationary activity.

This revised interpretation of our results (1) retains our original claim that the non-stationary dynamics during a trial is incompatible with the line attractor model and (2) introduces attractor state at the start of a trial which is compatible with the line attractor model. Our anlaysis shows that the two activity modes are linked by an underlying dynamics, and the attractor state serves as initial state to launch the non-stationary activity.

Responses to the Public Reviews:

Reviewer # 1:

(1) To provide better explanation of the reversal learning task and network training method, we added detailed description of RNN and monkey task structure (Result Section 1), included a schematic of target outputs (Figure1B), explained the rationale behind using inhibitory network model (Method Section 1) and explained the supervised RNN training scheme (Result Section 1). This information can also be found in the Methods.

(2) Our understanding is that the augmented model discussed in the previous page is aligned with the model suggested by Reviewer 1: “a curved line attractor, with faster timescale dynamics superimposed on this structure”. It is likely that the “fast” non-stationary activity observed during the trial is driven by task-related behavior, thus is transient. For instance, we do not observe such non-stationary activity in the inter-trial-interval when the task-related behavior is absent. For this reason, the non-stationary trajectories were not considered to be part of the attractor. Instead, they are transient activity generated by the underlying neural dynamics associated with task-related behavior. We believe such characterization of faster timescale dynamics is consistent with Reviewer 1’s view and wanted to clarify that there are two different activity modes.

(3) We appreciate the reviewers (Reviewer 1 and Reviewer 2) comment that TDR may be limited in isolating the neural subspace of interest. Our study presents what could be learned from TDR but is by no means the only way to interpret the neural data. It would be of future work to apply other methods for isolating task-related neural activities.

We would appreciate it if the reviewers could share thoughts on what other alternative methods could better isolate the reversal probability activity.

Reviewer # 2:

(1) (i) We respectfully disagree with Reviewer 2’s comment that “no action is required to be performed by neurons in the RNN”. In our network setup, the output of RNN learns to choose a sign (+ or -), as Reviewer 2 pointed out, to make a choice. This is how the RNN takes an action. It is unclear to us what Reviewer 2 has intended by “action” and how reaching a target value (not just taking a sign) would make a significant difference in how the network performs the task. 

(ii)  From Reviewer 2’s comment that “no intervening behavior is thus performed by neurons”, we noticed that the term “intervening behavior” has caused confusion. It refers to task-related behavior, such as making choices or receiving reward, that the subject must perform across trials before reversing its preferred choice. These are the behaviors that intervene the reversal of preferred choice. To clarify its meaning, in the revised manuscript, we changed the term to “task-related behavior” and put them in context. For example, in the Introduction we state that “However, during a trial, task-related behavior, such as making decisions or receiving feedback, produced …”

(iii) As pointed out by Reviewer 2, the lack of fixation period in the RNN could make differences in the neural dynamics of RNN and PFC, especially at the start of a trial. We demonstrate this issue in Result Section 4 where we analyze the stationary activity at the start of a trial. We find that fixating the choice output to zero before making a choice promotes stationary activity and makes the RNN activity more similar to the PFC activity.

Reviewer #3:

(1) (i) In the previous study (Figure 1 in [Bartolo and Averbeck ‘20]), it was shown that neural activity can predict the behavioral reversal trial. This is the reason we examined the neural activity in the trials centered at the behavioral reversal trial. We explained in Result Section 2 that we followed this line of analysis in our study.

(ii) We would like to emphasize that the main point of Figures 4 and 5 is to show the separability of neural trajectories: the entire trajectory shifts without overlapping. It is not obvious that high-dimensional neural population activity from two trials should remain separated when their activities are compressed into a one-dimensional subspace. The onedimensional activities can easily collide since their activities are compressed into a lowdimensional space. We revised the manuscript to bring out these points. We added an opening paragraph that discusses separability of trajectories and revised the main text to bring out the findings on separability. 

(iii) We agree with Reviewer 3 that it would be interesting to look at what happens in other subspace of neural activity that are not related to reversal probability and characterize how different neural subspace interact with each. However, the focus of this paper was the reversal probability activity, and we’d consider these questions out of the scope of current paper. We point out that, using the same dataset, neural activity related to other experimental variables were analyzed in other papers [Bartolo and Averbeck ’20; Tang, Bartolo and Averbeck ‘21] 

(2) (i) In the revised manuscript, we added explanation on the rational behind choosing inhibitory network as a simplified model for the balanced state. In brief, strong inhibitory recurrent connections with strong excitatory external input operates in the balanced state, as in the standard excitatory-inhibitory network. We included references that studied this inhibitory network. We also explained the technical reason (GPU memory) for choosing the inhibitory model.

(ii) We thank the reviewer for pointing out that the original manuscript did not mention how the feedback and cue were initialized. They were random vectors sample from Gaussian distribution. We added this information in the revised manuscript. In our opinion, it is common to use random external inputs for training RNNs, as it is a priori unclear how to choose them. In fact, it is possible to analyze the effects of random feedback on one-dimensional x_rev dynamics by projecting the random feedback vector to the reversal probability vector. This is shown in Figure 4F.

(iii) We agree that it would be more natural to train the RNN to solve the task without using the Bayesian model. We point out this issue in the Discussion in the revised manuscript.

Recommendations for the authors:

Reviewer #1:

(1) My understanding of network training was that a Bayesian ideal observer signaled target output based on previous reward outcomes. However, the authors never mention that networks are trained by supervised learning in the main text until the last paragraph of the discussion. There is no mention that there was an offset in the target based on the behavior of the monkeys in the main text. These are really important things to consider in the context of the network solution after training. I couldn't actually find any figure that presents the target output for the network. Did I miss something key here?

In Result Section 1, we added a paragraph that describes in detail how the RNN is trained. We explained that the network is first simulated and then the choice outputs and reward outcomes are fed into the Bayesian model to infer the scheduled reversal trial. A few trials are added to the inferred reversal trial to obtain the behavioral reversal trial, as found in a previous study [Bartolo and Averbeck ‘20]. Then the network weights are updated by backpropagation-through-time via supervised learning. 

In the original manuscript, the target output for the network was described in Methods Section 2.5, Step 4. To make this information readily accessible, we added a schematic in Figure 1B that shows the scheduled, inferred and behavioral reversal trials. It also shows how the target choice ouputs are defined. They switch abruptly at the behavioral reversal trial.

(2) The role of block structure in the task is an important consideration. What are the statistics of block switches? The authors say on average the reversals are every 36 trials, but also say there are random block switches. The reviewer's notes suggest that both the networks and monkeys may be learning about the typical duration of blocks, which could influence their expectations of reversals. This aspect of the task design should be explained more thoroughly and considered in the context of Figure 1E and 5 results.

We provided more detailed description of the reversal learning task in Result Section 1. We clarified that (1) a task is completed by executing a block of fixed number of trials and (2) reversal of reward schedule occurrs at a random trial around the mid-trial in a block. The differences in the number of trials in a block that the RNNs (36) and the monkeys (80) perform are also explained. We also pointed out the differences in how the reversal trial is randomly sampled.

However, it is unclear what Reviewer 1 meant by random block switches. Our reversal learning task is completed when a block of fixed number of trials is executed. Reversal of reward schedule occurs only once on a randomly selected trial in the block, and the reversed reward schedule is maintained until the end of a block. It is different from other versions of reveral learning where the reward schedule switches multiple times across trials. We clarified this point in Result Section 1.

(3) The relationship between the supervised learning approach used in the RNNs and reinforcement learning was confused in the discussion. "Although RNNs in our study were trained via supervised learning, animals learn a reversal-learning task from reward feedback, making it into a reinforcement learning (RL) problem." This is fundamentally not true. In the case of this work, the outcome of the previous trial updates the target output, rather than the trial and error type learning as is typical in reinforcement learning. Networks are not learning by reinforcement learning and this statement is confusing.

We agree with Reviewer 1’s comment that the statement in the original manuscript is confusing. Our intention was to point out that our study used supervised learning, and this is different from animals learn by reinforcement learning in rea life. We revised the sentence in Discussion as follows:

“The RNNs in our study were trained via supervised learning. However, in real life, animals learn a reversal learning task via reinforcement learning (RL), i.e., learn the task from reward outcomes.”

(4) The distinction between line attractors and the dynamic trajectories described by the authors deserves further investigation. A significant concern arises from the authors' use of targeted dimensionality reduction (TDR), a form of regression, to identify the axis determining reversal probability. While this approach can reveal interesting patterns in the data, it may not necessarily isolate the dimension along which the RNN computes reversal probability. This limitation could lead to misinterpretation of the underlying neural dynamics.

a) This manuscript cites work described in "Prefrontal cortex as a meta-reinforcement learning system," which examined a similar task. In that study, the authors identified a v-shaped curve in the principal component space of network states, representing the probability of choosing left or right.

Importantly, this curve is topologically equivalent to a line and likely represents a line attractor. However, regressing against reversal probability in such a case would show that a single principal component (PC2) directly correlates with reversal probability.

b) The dynamics observed in the current study bear a striking resemblance to this structure, with the addition of intervening loops in the network state corresponding to within-trial state evolution. Crucially, these observations do not preclude the existence of a line attractor. Instead, they may reflect the network's need to produce fast timescale dynamics within each trial, superimposed on the slower dynamics of the line attractor.

c) This alternative interpretation suggests that reward signals could function as inputs that shift the network state along the line attractor, with information being maintained across trials. The fast "intervening behaviors" observed by the authors could represent faster timescale dynamics occurring on top of the underlying line attractor dynamics, without erasing the accumulated evidence for reversals.

d) Given these considerations, the authors' conclusion that their results are better described by separable dynamic trajectories rather than fixed points on a line attractor may be premature. The observed dynamics could potentially be reconciled with a more nuanced understanding of line attractor models, where the attractor itself may be curved and coexist with faster timescale dynamics.

We appreciate the insightful comments on (1) the similarity of the work by Wang et al ’18 with our findings and (2) an alternative interpretation that augments the line attractor with fast timescale dynamics. 

(1) We added a discussion of the work by Wang et al ’18 in Result Section 2 to point out the similarity of their findings in the principal component space with ours in the x_rev and x_choice space. We commented that such network dynamics could emerge when learning to perform the reversal learning the task, regardless of the training schemes. 

We also mention that the RL approach in Wang et al ’18 does not consider within-trial dynamics, therefore lacks the non-stationary activity observed during the trial in the PFC of monkeys and our trained RNNs.

(2) We revised our original manuscript substantially to reconcile the line attractor model with the nonstationary activity observed during a trial. 

Here are the highlights of the revised interpretation of the PFC and the RNN network activity

- The dynamics of x_rev consists of two activity modes, i.e., stationary activity at the start of a trial and non-stationary activity during the trial. Schematic of the augmented model that reconciles two activity modes is shown in Figure 4A. Analysis of the time derivative (dx_reverse / dt) and contractivity of the stationary state are shown in Figure 4B,C to demonstrate two activity modes.

- We discuss in Result Section 4 main text that the stationary activity is consistent with the line attractor model, but the non-stationary activity deviates from the model. 

- The two activity modes are linked dynamically. There is an underlying dynamics that can map the stationary state to the non-stationary trajectory. This is shown by predicting the nonstationary trajectory with the stationary state using a support vector regression model. The prediction results are shown in Figure 4D,E,F.

- We discuss in Result Section 4 an extension of the standard line attractor model: points on the line attractor can serve as initial states that launch non-stationary activity associated with taskrelated behavior.

- The separability of neural trajectories presented in Result Section 5 is framed as a property of the non-stationary dynamics associated with task-related behavior.

To strengthen their claims, the authors should:

(1) Provide a more detailed description of their RNN training paradigm and task structure, including clear illustrations of target outputs.

(2) Discuss how their findings relate to and potentially extend previous work on similar tasks, particularly addressing the similarities and differences with the v-shaped state organization observed in reinforcement learning contexts. (https://www.nature.com/articles/s41593-018-0147-8 Figure1).

(3) Explore whether their results could be consistent with a curved line attractor model, rather than treating line attractors and dynamic trajectories as mutually exclusive alternatives.

Our response to these three comments is described above.

Addressing these points would significantly enhance the impact of the study and provide a more nuanced understanding of how reversal probabilities are represented in neural circuits.

In conclusion, while this study provides interesting insights into the neural representation of reversal probability, there are several areas where the methodology and interpretations could be refined.

Additional Minor Concerns:

(1) Network Training and Reversal Timing: The authors mention that the network was trained to switch after a reversal to match animal behavior, stating "Maximum a Posterior (MAP) of the reversal probability converges a few trials past the MAP estimate." More explanation of how this training strategy relates to actual animal behavior would enhance the reader's understanding of the meaning of the model's similarity to animal behavior in Figure 1.

In Method Section 2.5, we described how our observation that the running estimate of MAP converges a few trials after the actual MAP is analogous to the animal’s reversal behavior.

“This observation can be interpreted as follows. If a subject performing the reversal learning task employs the ideal observer model to detect the trial at which reward schedule is reversed, the subject can infer the reversal of reward schedule a few trials past the actual reversal and then switch its preferred choice. This delay in behavioral reversal, relative to the reversal of reward schedule, is analogous to the monkeys switching their preferred choice a few trials after the reversal of reward schedule.”

In Step 4, we also mentioned that the target choice outputs are defined based on our observation in Step 3.

“We used the observation from Step 3 to define target choice outputs that switch abruptly a few trials after the reversal of reward schedule, denoted as $t^*$ in the following. An example of target outputs are shown in Fig.\,\ref{fig_behavior}B.”

(2) How is the network simulated in step 1 of training? Is it just randomly initialized? What defines this network structure?

The initial state at the start of a block was random. We think the initial state is less relevant as the external inputs (i.e., cue and feedback) are strong and drive the network dynamics. We mentioned these setup and observation in Step 1 of training.

“Step 1. Simulate the network starting from a random initial state, apply the external inputs, i.e., cue and feedback inputs, at each trial and store the network choices and reward outcomes at all the trials in a block. The network dynamics is driven by the external inputs applied periodically over the trials.”

(3) Clarification on Learning Approach: More description of the approach in the main text would be beneficial. The statement "Here, we trained RNNs that learned from a Bayesian inference model to mimic the behavioral strategies of monkeys performing the reversal learning task [2, 4]" is somewhat confusing, as the model isn't directly fit to monkey data. A more detailed explanation of how the Bayesian inference model relates to monkey behavior and how it's used in RNN training would improve clarity.

We described the learning approach in more detail, but also tried to be concise without going into technical details.

We revised the sentence in Introduction as follows:

“We sought to train RNNs to mimic the behavioral strategies of monkeys performing the reversal learning task. Previous studies \cite{costa2015reversal, bartolo2020prefrontal} have shown that a Bayesian inference model can capture a key aspect of the monkey's behavioral strategy, i.e., adhere to the preferred choice until the reversal of reward is detected and then switch abruptly. We trained the RNNs to replicate this behavioral strategy by training them on target behaviors generated from the Bayesian model.”

We also added a paragraph in Result Section 1 that explains in detail how the training approach works.

(4) In Figure 1B, it would be helpful to show the target output.

We added a figure in Fig1B that shows a schematic of how the target output is generated.

(5) An important point to consider is that a line attractor can be curved while still being topologically equivalent to a line. This nuance makes Figure 4A somewhat difficult to interpret. It might be helpful to discuss how the observed dynamics relate to potentially curved line attractors, which could provide a more nuanced understanding of the neural representations.

As discussed above, we interpret the “curved” activity during the trial as non-stationary activity. We do not think this non-stationary activity would be characterized as attractor. Attractor is (1) a minimal set of states that is (2) invariant under the dynamics and (3) attracting when perturbed into its neighborhood [Strogatz, Nonlinear dynamics and chaos]. If we consider the autonomous system without the behavior-related external input as the base system, then the non-stationary states could satisfy (2) and (3) but not (1), so they are not part of the attractor. If we include the behavior-related external input to the autonomous dynamics, then it may be possible that the non-stationary trajectories are part of the attractor. We adopted the former interpretation as the behavior-related inputs are external and transient.

(6) The results of the perturbation experiments seem to follow necessarily from the way x_rev was defined. It would be valuable to clarify if there's more to these results than what appears to be a direct consequence of the definition, or if there are subtleties in the experimental design or analysis that aren't immediately apparent.

The neural activity x_rev is correlated to the reversal probability, but it is unclear if the activity in this neural subspace is causally linked to behavioral variables, such as choice output. We added this explanation at the beginning of Results Section 7 to clarify the reason for performing the perturbation experiments.

“The neural activity $x_{rev}$ is obtained by identifying a neural subspace correlated to reversal probability. However, it remains to be shown if activity within this neural subspace is causally linked to behavioral variables, such as choice output.”

Reviewer #2:

Below is a list of things I have found difficult to understand, and been puzzled/concerned about while reading the manuscript:

(1) It would be nice to say a bit more about the dataset that has been used for PFC analysis, e.g. number of neurons used and in what conditions is Figure 2A obtained (one has to go to supplementary to get the reference).

We added information about the PFC dataset in the opening paragraph of Result Section 2 to provide an overview of what type of neural data we’ve analyzed. It includes information about the number of recorded neurons, recording method and spike binning process.

(2) It would be nice to give more detail about the monkey task and better explain its trial structure.

In Result Section 1 we added a description of the overall task structure (and its difference with other versions of revesal learning task), the RNN / monkey trial structure and differences in RNN and monkey tasks.

(3) In the introduction it is mentioned that during the hold period, the probability of reversal is represented. Where does this statement come from?

The fact that neural activity during a hold period, i.e., fixation period before presenting the target images, encodes the probability of reversal was demonstrated in a previous study (Bartolo and Averbeck ’20). 

We realize that our intention was to state that, during the hold period, the reversal probability activity is stationary as in the line attractor model, instead of focusing on that the probability of reversal is represented during this period. We revised the sentence to convey this message. In addition, we revised the entire paragraph to reinterpret our findings: there are two activity modes where the stationary activity is consistent with the line attractor model but the non-stationary activity deviates from it.

(4) "Around the behavioral reversal trial, reversal probabilities were represented by a family of rankordered trajectories that shifted monotonically". This sentence is confusing and hard to understand.

Thank you for point this out. We rewrote the paragraph to reflect our revised interpretation. This sentence was removed, as it can be considered as part of the result on separable trajectories.

(5) For clarity, in the first section, when it is written that "The reversal behavior of trained RNNs was similar to the monkey's behavior on the same task" it would be nice to be more precise, that this is to be expected given the strategy used to train the network.

We removed this sentence as it makes a blanket statement. Instead, we compared the behavioral outputs of the RNNs and the monkeys one by one.

We added a sentence in Result Section 1 that the RNN’s abrupt behavioral reversal is expected as they are trained to mimic the target choice outputs of the Bayesian model.

“Such abrupt reversal behavior was expected as the RNNs were trained to mimic the target outputs of the Bayesian inference model.”

(6) What is the value of tau used in eq (1), and how does it compare to trial duration?

We described the value of time constant tau in Eq (1) and also discussed in Result Section 1 that tau=20ms is much faster than trial duration 500ms, thus the persistent behavior seen in trained RNNs is due to learning.

(7) It would be nice to expand around the notion of « temporally flexible representation » to help readers grasp what this means.

Instead of stating that the separable dynamic trajectories have “temporally flexible representation”, we break down in what sense it is temporally flexible: separable dynamic trajectories can accommodate the effects that task-related behavior have on generating non-stationary neural dynamics.

“In sum, our results show that, in a probabilistic reversal learning task, recurrent neural networks encode reversal probability by adopting, not only stationary states as in a line attractor, but also separable dynamic trajectories that can represent distinct probabilistic values while accommodating non-stationary dynamics associated with task-related behavior.”

Reviewer #3:

(1) Data:

It would be useful to describe the experimental task, recording setup, and analyses in much more detail - both in the text and in the methods. What part of PFC are the recordings from? How many neurons were recorded over how many sessions? Which other papers have they been used in? All of these things are important for the reader to know, but are not listed anywhere. There are also some inconsistencies, with the main text e.g. listing the 'typical block length' as 36 trials, and the methods listing the block length as 24 trials (if this is a difference between the biological data and RNN, that should be more explicit and motivated).

We provided more detailed description of the monkey experimental task and PFC recordings in Result Section 1. We also added a new section in Methods 2.1 to describe the monkey experiment.

The experimental analyses should be explained in more detail in the methods. There is e.g. no detailed description of the analysis in Figure 6F.

We added a new section in Methods 6 to describe how the residual PFC activity is computed. It also describes the RNN perturbation experiments.

Finally, it would be useful for more analyses of monkey behaviour and performance, either in the main text or supplementary figures.

We did not pursue this comment as it is unclear how additional behavioral analyses would improve the manuscript.

(2) Model:

When fitting the network, 'step 1' of training in 2.3 seems superfluous. The posterior update from getting a reward at A is the same as that from not getting a reward at B (and vice versa), and it is therefore completely independent of the network choice. The reversal trial can therefore be inferred without ever simulating the network, simply by generating a sample of which trials have the 'good' option being rewarded and which trials have the 'bad' option being rewarded.

We respectfully disagree with Reviewer 3’s comment that the reversal trial can be inferred without ever simulating the network. The only way for the network to know about the underlying reward schedule is to perform the task by itself. By simulating the network, it can sample the options and the reward outcomes. 

Our understanding is that Review 3 described a strategy that a human would use to perform this task. Our goal was to train the RNN to perform the task.

Do the blocks always start with choice A being optimal? Is everything similar if the network is trained with a variable initial rewarded option? E.g. in Fig 6, would you see the appropriate swap in the effect of the perturbation on choice probability if choice B was initially optimal?

Thank you for pointing out that the initial high-value option can be random. When setting up the reward schedule, the initial high-value option was chosen randomly from two choice outputs and, at the scheduled reversal, it was switched to the other option. We did not describe this in the original manuscript.

We added a descrption in Training Scheme Step 4 that the the initial high-value option is selected randomly. This is also explained in Result Section 1 when we give an overview of the RNN training procedure.

(3) Content:

It is rarely explained what the error bars represent (e.g. Figures 3B, 4C, ...) - this should be clear in all figures.

We added that the error bars represent the standard error of mean.

Figure 2A: this colour scheme is not great. There are abrupt colour changes both before and after the 'reversal' trial, and both of the extremes are hard to see.

We changed the color scheme to contrast pre- and post-reversal trials without the abrupt color change.

Figure 3E/F: how is prediction accuracy defined?

We added that the prediction accuracy is based on Pearson correlation.

Figure 4B: why focus on the derivative of the dynamics? The subsequent plots looking at the actual trajectories are much easier to understand. Also - what is 'relative trial' relative to?

The derivative was analyzed to demonstrate stationarity or non-stationarity of the neural activity. We think it will be clearer in the revised manuscript that the derivative allows us to characterize those two activity modes.

Relative trial number indicate the trial position relative to the behavioral reversal trial. We added this description to the figures when “relative trial” is used.

Figure 4C: what do these analyses look like if you match the trial numbers for the shift in trajectories? As it is now, there will presumably be more rewarded trials early and late in each block, and more unrewarded trials around the reversal point. Does this introduce biases in the analysis? A related question is (i) why the black lines are different in the top and bottom plots, and (ii) why the ends of the black lines are discontinuous with the beginnings of the red/blue lines.

We could not understand what Reviewer 3 was asking in this comment. It’d help if Review 3 could clarify the following question:

“Figure 4C: what do these analyses look like if you match the trial numbers for the shift in trajectories?”

Question (i): We wanted to look at how the trajectory shifts in the subsequent trial if a reward is or is not received in the current trial. The top panel analyzed all the trials in which the subsquent trial did not receive a reward. The bottom panel analyzed all the trials in which the subsequent trial received a reward. So, the trials analyzed in the top and bottom panels are different, and the black lines (x_rev of “current” trial) in the top and bottom panels are different.

Question (ii): Black line is from the preceding trial of the red/blue lines, so if trials are designed to be continuous with the inter-trial-interval, then black and red/blue should be continuous. However, in the monkey experiment, the inter-trial-intervals were variable, so the end of current trial does not match with the start of next trial. The neural trajectories presented in the manuscript did not include the activity in this inter-trial-interval.

Figure 6C: are the individual dots different RNNs? Claiming that there is a decrease in Delta x_choice for a v_+ stimulation is very misleading.

Yes individual dots are different RNN perturbations. We added explanation about the dots in Figure7C caption. 

We agree with the comment that \Delta x_choice did not decrease. This sentence was removed. Instead, we revised the manuscript to state that x_choice for v_+ stimulation was smaller than the x_choice for v_- stimulation. We performed KS-test to confirm statistical significance.

Discussion: "...exhibited behaviour consistent with an ideal Bayesian observer, as found in our study". The RNN was explicitly trained to reproduce an ideal Bayesian observer, so this can only really be considered an assumption (not a result) in the present study.

We agree that the statement in the original manuscript is inaccurate. It was revised to reflect that, in the other study, behavior outputs similar to a Bayesian observer emerged by simply learning to do the task, intead of directly mimicking the outputs of Bayesian observer as done in our study.

“Authors showed that trained RNNs exhibited behavior outputs consistent with an ideal Bayesian observer without explicitly learning from the Bayesian observer. This finding shows that the behavioral strategies of monkeys could emerge by simply learning to do the task, instead of directly mimicking the outputs of Bayesian observer as done in our study.”

Methods: Would the results differ if your Bayesian observer model used the true prior (i.e. the reversal happens in the middle 10 trials) rather than a uniform prior? Given the extensive literature on prior effects on animal behaviour, it is reasonable to expect that monkeys incorporate some non-uniform prior over the reversal point.

Thank you for pointing out the non-uniform prior. We haven’t conducted this analysis, but would guess that the convergence to the posterior distribution would be faster. We’d have to perform further analysis, which is out of the scope of this paper, to investigate whether the posteior distribution would be different from what we obtained from uniform prior.

Making the code available would make the work more transparent and useful to the community.

The code is available in the following Github repository: https://github.com/chrismkkim/LearnToReverse

Primair = vanaf het begin van seksuele activiteit Secundair = begint later, na een periode zonder klachten.

Será que não vale colocarmos em carrossel? Semelhante ao que fizemos na home e outras páginas.

Tirar essa sessão a incorporar os principais benefícios - que ainda não ficaram evidentes, na sessão abaixo.

Essa sessão não deveria estar posicionada ao final da página e com uma outro disposição dos elementos (Podemos fazer algo parecido com o que fizemos na página de vendas da imersão SOC).

Deixar os tópicos com o mesmo número de linhas no mobile

• Precisamos respeitar as fontes originais;
• Não curti muito a imagem ao lado da headline

Reviewer #2 (Public review):

The manuscript by Lacy et al. is well written, with a clear and compelling introduction that effectively conveys the significance of the study. The methods are appropriate and well-executed, and the results, both in the main text and supplementary materials, are presented in a clear and detailed manner. The authors interpret their findings with appropriate caution.

This work makes a valuable contribution to our understanding of the evolution of complementary sex determination (CSD) in ants. In particular, it provides important evidence for the ancient origin of a non-coding locus implicated in sex determination, and shows that, remarkably, this sex locus is conserved even in an ant species with a non-canonical reproductive system that typically does not produce males. I found this to be an excellent and well-rounded study, carefully analyzed and well contextualized.

That said, I do have a few minor comments, primarily concerning the discussion of the potential 'ghost' CSD locus. While the authors acknowledge (line 367) that they currently have no data to distinguish among the alternative hypotheses, I found the evidence for an additional CSD locus presented in the results (lines 261-302) somewhat limited and at times a bit difficult to follow. I wonder whether further clarification or supporting evidence could already be extracted from the existing data. Specifically:

(1) Line 268: I doubt the relevance of comparing the proportion of diploid males among all males between lines A and B to infer the presence of additional CSD loci. Since the mechanisms producing these two types of males differ, it might be more appropriate to compare the proportion of diploid males among all diploid offspring. This ratio has been used in previous studies on CSD in Hymenoptera to estimate the number of sex loci (see, for example, Cook 1993, de Boer et al. 2008, 2012, Ma et al. 2013, and Chen et al., 2021). The exact method might not be applicable to clonal raider ants, but I think comparing the percentage of diploid males among the total number of (diploid) offspring produced between the two lineages might be a better argument for a difference in CSD loci number.

(2) If line B indeed carries an additional CSD locus, one would expect that some females could be homozygous at the ANTSR locus but still viable, being heterozygous only at the other locus. Do the authors detect any females in line B that are homozygous at the ANTSR locus? If so, this would support the existence of an additional, functionally independent CSD locus.

(3) Line 281: The description of the two tra-containing CSD loci as "conserved" between Vollenhovia and the honey bee may be misleading. It suggests shared ancestry, whereas the honey bee csd gene is known to have arisen via a relatively recent gene duplication from fem/tra (10.1038/nature07052). It would be more accurate to refer to this similarity as a case of convergent evolution rather than conservation.

(4) Finally, since the authors successfully identified multiple alleles of the first CSD locus using previously sequenced haploid males, I wonder whether they also observed comparable allelic diversity at the candidate second CSD locus. This would provide useful supporting evidence for its functional relevance.

Overall, these are relatively minor points in the context of a strong manuscript, but I believe addressing them would improve the clarity and robustness of the authors' conclusions.

Author response:

Reviewer #1 (Public review):

This study investigates the sex determination mechanism in the clonal ant Ooceraea biroi, focusing on a candidate complementary sex determination (CSD) locus-one of the key mechanisms supporting haplodiploid sex determination in hymenopteran insects. Using whole genome sequencing, the authors analyze diploid females and the rarely occurring diploid males of O. biroi, identifying a 46 kb candidate region that is consistently heterozygous in females and predominantly homozygous in diploid males. This region shows elevated genetic diversity, as expected under balancing selection. The study also reports the presence of an lncRNA near this heterozygous region, which, though only distantly related in sequence, resembles the ANTSR lncRNA involved in female development in the Argentine ant, Linepithema humile (Pan et al. 2024). Together, these findings suggest a potentially conserved sex determination mechanism across ant species. However, while the analyses are well conducted and the paper is clearly written, the insights are largely incremental. The central conclusion - that the sex determination locus is conserved in ants - was already proposed and experimentally supported by Pan et al. (2024), who included O. biroi among the studied species and validated the locus's functional role in the Argentine ant. The present study thus largely reiterates existing findings without providing novel conceptual or experimental advances.

Although it is true that Pan et al., 2024 demonstrated (in Figure 4 of their paper) that the synteny of the region flanking ANTSR is conserved across aculeate Hymenoptera (including O. biroi), Reviewer 1’s claim that that paper provides experimental support for the hypothesis that the sex determination locus is conserved in ants is inaccurate. Pan et al., 2024 only performed experimental work in a single ant species (Linepithema humile) and merely compared reference genomes of multiple species to show synteny of the region, rather than functionally mapping or characterizing these regions.

Other comments:

The mapping is based on a very small sample size: 19 females and 16 diploid males, and these all derive from a single clonal line. This implies a rather high probability for false-positive inference. In combination with the fact that only 11 out of the 16 genotyped males are actually homozygous at the candidate locus, I think a more careful interpretation regarding the role of the mapped region in sex determination would be appropriate. The main argument supporting the role of the candidate region in sex determination is based on the putative homology with the lncRNA involved in sex determination in the Argentine ant, but this argument was made in a previous study (as mentioned above).

Our main argument supporting the role of the candidate region in sex determination is not based on putative homology with the lncRNA in L. humile. Instead, our main argument comes from our genetic mapping (in Fig. 2), and the elevated nucleotide diversity within the identified region (Fig. 4). Additionally, we highlight that multiple genes within our mapped region are homologous to those in mapped sex determining regions in both L. humile and Vollenhovia emeryi, possibly including the lncRNA.

In response to the Reviewer’s assertion that the mapping is based on a small sample size from a single clonal line, we want to highlight that we used all diploid males available to us. Although the primary shortcoming of a small sample size is to increase the probability of a false negative, small sample sizes can also produce false positives. We used two approaches to explore the statistical robustness of our conclusions. First, we generated a null distribution by randomly shuffling sex labels within colonies and calculating the probability of observing our CSD index values by chance (shown in Fig. 2). Second, we directly tested the association between homozygosity and sex using Fisher’s Exact Test (shown in Supplementary Fig. S2). In both cases, the association of the candidate locus with sex was statistically significant after multiple-testing correction using the Benjamini-Hochberg False Discovery Rate. These approaches are clearly described in the “CSD Index Mapping” section of the Methods.

We also note that, because complementary sex determination loci are expected to evolve under balancing selection, our finding that the mapped region exhibits a peak of nucleotide diversity lends orthogonal support to the notion that the mapped locus is indeed a complementary sex determination locus.

The fourth paragraph of the results and the sixth paragraph of the discussion are devoted to explaining the possible reasons why only 11/16 genotyped males are homozygous in the mapped region. The revised manuscript will include an additional sentence (in what will be lines 384-388) in this paragraph that includes the possible explanation that this locus is, in fact, a false positive, while also emphasizing that we find this possibility to be unlikely given our multiple lines of evidence.

In response to Reviewer 1’s suggestion that we carefully interpret the role of the mapped region in sex determination, we highlight our careful wording choices, nearly always referring to the mapped locus as a “candidate sex determination locus” in the title and throughout the manuscript. For consistency, the revised manuscript version will change the second results subheading from “The O. biroi CSD locus is homologous to another ant sex determination locus but not to honeybee csd” to “O. biroi’s candidate CSD locus is homologous to another ant sex determination locus but not to honeybee csd,” and will add the word “candidate” in what will be line 320 at the beginning of the Discussion, and will change “putative” to “candidate” in what will be line 426 at the end of the Discussion.

In the abstract, it is stated that CSD loci have been mapped in honeybees and two ant species, but we know little about their evolutionary history. But CSD candidate loci were also mapped in a wasp with multi-locus CSD (study cited in the introduction). This wasp is also parthenogenetic via central fusion automixis and produces diploid males. This is a very similar situation to the present study and should be referenced and discussed accordingly, particularly since the authors make the interesting suggestion that their ant also has multi-locus CSD and neither the wasp nor the ant has tra homologs in the CSD candidate regions. Also, is there any homology to the CSD candidate regions in the wasp species and the studied ant?

In response to Reviewer 1’s suggestion that we reference the (Matthey-Doret et al. 2019) study in the context of diploid males being produced via losses of heterozygosity during asexual reproduction, the revised manuscript will include the following sentence: “Therefore, if O. biroi uses CSD, diploid males might result from losses of heterozygosity at sex determination loci (Fig. 1C), similar to what is thought to occur in other asexual Hymenoptera that produce diploid males (Rabeling and Kronauer 2012; Matthey-Doret et al. 2019).”

We note, however, that in their 2019 study, Matthey-Doret et al. did not directly test the hypothesis that diploid males result from losses of heterozygosity at CSD loci during asexual reproduction, because the diploid males they used for their mapping study came from inbred crosses in a sexual population of that species.

We address this further below, but we want to emphasize that we do not intend to argue that O. biroi has multiple CSD loci. Instead, we suggest that additional, undetected CSD loci is one possible explanation for the absence of diploid males from any clonal line other than clonal line A. In response to Reviewer 1’s suggestion that we reference the (Matthey-Doret et al. 2019) study in the context of multilocus CSD, the revised manuscript version will include the following additional sentence in the fifth paragraph of the discussion: “Multi-locus CSD has been suggested to limit the extent of diploid male production in asexual species under some circumstances (Vorburger 2013; Matthey-Doret et al. 2019).”

Regarding Reviewer 2’s question about homology between the putative CSD loci from the (Matthey-Doret et al. 2019) study and O. biroi, we note that there is no homology. The revised manuscript version will have an additional Supplementary Table (which will be the new Supplementary Table S3) that will report the results of this homology search. The revised manuscript will also include the following additional sentence in the Results: “We found no homology between the genes within the O. biroi CSD index peak and any of the genes within the putative L. fabarum CSD loci (Supplementary Table S3).”

The authors used different clonal lines of O. biroi to investigate whether heterozygosity at the mapped CSD locus is required for female development in all clonal lines of O. biroi (L187-196). However, given the described parthenogenesis mechanism in this species conserves heterozygosity, additional females that are heterozygous are not very informative here. Indeed, one would need diploid males in these other clonal lines as well (but such males have not yet been found) to make any inference regarding this locus in other lines.

We agree that a full mapping study including diploid males from all clonal lines would be preferable, but as stated earlier in that same paragraph, we have only found diploid males from clonal line A. We stand behind our modest claim that “Females from all six clonal lines were heterozygous at the CSD index peak, consistent with its putative role as a CSD locus in all O. biroi.” In the revised manuscript version, this sentence (in what will be lines 199-201) will be changed slightly in response to a reviewer comment below: “All females from all six clonal lines (including 26 diploid females from clonal line B) were heterozygous at the CSD index peak, consistent with its putative role as a CSD locus in all O. biroi.”

Reviewer #2 (Public review):

The manuscript by Lacy et al. is well written, with a clear and compelling introduction that effectively conveys the significance of the study. The methods are appropriate and well-executed, and the results, both in the main text and supplementary materials, are presented in a clear and detailed manner. The authors interpret their findings with appropriate caution.

This work makes a valuable contribution to our understanding of the evolution of complementary sex determination (CSD) in ants. In particular, it provides important evidence for the ancient origin of a non-coding locus implicated in sex determination, and shows that, remarkably, this sex locus is conserved even in an ant species with a non-canonical reproductive system that typically does not produce males. I found this to be an excellent and well-rounded study, carefully analyzed and well contextualized.

That said, I do have a few minor comments, primarily concerning the discussion of the potential 'ghost' CSD locus. While the authors acknowledge (line 367) that they currently have no data to distinguish among the alternative hypotheses, I found the evidence for an additional CSD locus presented in the results (lines 261-302) somewhat limited and at times a bit difficult to follow. I wonder whether further clarification or supporting evidence could already be extracted from the existing data. Specifically:

We agree with Reviewer 2 that the evidence for a second CSD locus is limited. In fact, we do not intend to advocate for there being a second locus, but we suggest that a second CSD locus is one possible explanation for the absence of diploid males outside of clonal line A. In our initial version, we intentionally conveyed this ambiguity by titling this section “O. biroi may have one or multiple sex determination loci.” However, we now see that this leads to undue emphasis on the possibility of a second locus. In the revised manuscript, we will split this into two separate sections: “Diploid male production differs across O. biroi clonal lines” and “O. biroi lacks a tra-containing CSD locus.”

(1) Line 268: I doubt the relevance of comparing the proportion of diploid males among all males between lines A and B to infer the presence of additional CSD loci. Since the mechanisms producing these two types of males differ, it might be more appropriate to compare the proportion of diploid males among all diploid offspring. This ratio has been used in previous studies on CSD in Hymenoptera to estimate the number of sex loci (see, for example, Cook 1993, de Boer et al. 2008, 2012, Ma et al. 2013, and Chen et al., 2021). The exact method might not be applicable to clonal raider ants, but I think comparing the percentage of diploid males among the total number of (diploid) offspring produced between the two lineages might be a better argument for a difference in CSD loci number.

We want to re-emphasize here that we do not wish to advocate for there being two CSD loci in O. biroi. Rather, we want to explain that this is one possible explanation for the apparent absence of diploid males outside of clonal line A. We hope that the modifications to the manuscript described in the previous response help to clarify this.

Reviewer 2 is correct that comparing the number of diploid males to diploid females does not apply to clonal raider ants. This is because males are vanishingly rare among the vast numbers of females produced. We do not count how many females are produced in laboratory stock colonies, and males are sampled opportunistically. Therefore, we cannot report exact numbers. However, we will add the following sentence to the revised manuscript: “Despite the fact that we maintain more colonies of clonal line B than of clonal line A in the lab, all the diploid males we detected came from clonal line A.”

(2) If line B indeed carries an additional CSD locus, one would expect that some females could be homozygous at the ANTSR locus but still viable, being heterozygous only at the other locus. Do the authors detect any females in line B that are homozygous at the ANTSR locus? If so, this would support the existence of an additional, functionally independent CSD locus.

We thank the reviewer for this suggestion, and again we emphasize that we do not want to argue in favor of multiple CSD loci. We just want to introduce it as one possible explanation for the absence of diploid males outside of clonal line A.

The 26 sequenced diploid females from clonal line B are all heterozygous at the mapped locus, and the revised manuscript will clarify this in what will be lines 199-201. Previously, only six of those diploid females were included in Supplementary Table S2, and that will be modified accordingly.

(3) Line 281: The description of the two tra-containing CSD loci as "conserved" between Vollenhovia and the honey bee may be misleading. It suggests shared ancestry, whereas the honey bee csd gene is known to have arisen via a relatively recent gene duplication from fem/tra (10.1038/nature07052). It would be more accurate to refer to this similarity as a case of convergent evolution rather than conservation.

In the sentence that Reviewer 2 refers to, we are representing the assertion made in the (Miyakawa and Mikheyev 2015) paper in which, regarding their mapping of a candidate CSD locus that contains two linked tra homologs, they write in the abstract: “these data support the prediction that the same CSD mechanism has indeed been conserved for over 100 million years.” In that same paper, Miyakawa and Mikheyev write in the discussion section: “As ants and bees diverged more than 100 million years ago, sex determination in honey bees and V. emeryi is probably homologous and has been conserved for at least this long.”

As noted by Reviewer 2, this appears to conflict with a previously advanced hypothesis: that because fem and csd were found in Apis mellifera, Apis cerana, and Apis dorsata, but only fem was found in Mellipona compressipes, Bombus terrestris, and Nasonia vitripennis, that the csd gene evolved after the honeybee (Apis) lineage diverged from other bees (Hasselmann et al. 2008). However, it remains possible that the csd gene evolved after ants and bees diverged from N. vitripennis, but before the divergence of ants and bees, and then was subsequently lost in B. terrestris and M. compressipes. This view was previously put forward based on bioinformatic identification of putative orthologs of csd and fem in bumblebees and in ants [(Schmieder et al. 2012), see also (Privman et al. 2013)]. However, subsequent work disagreed and argued that the duplications of tra found in ants and in bumblebees represented convergent evolution rather than homology (Koch et al. 2014). Distinguishing between these possibilities will be aided by additional sex determination locus mapping studies and functional dissection of the underlying molecular mechanisms in diverse Aculeata.

Distinguishing between these competing hypotheses is beyond the scope of our paper, but the revised manuscript will include additional text to incorporate some of this nuance. We will include these modified lines below:

“A second QTL region identified in V. emeryi (V.emeryiCsdQTL1) contains two closely linked tra homologs, similar to the closely linked honeybee tra homologs, csd and fem (Miyakawa and Mikheyev 2015). This, along with the discovery of duplicated tra homologs that undergo concerted evolution in bumblebees and ants (Schmieder et al. 2012; Privman et al. 2013) has led to the hypothesis that the function of tra homologs as CSD loci is conserved with the csd-containing region of honeybees (Schmieder et al. 2012; Miyakawa and Mikheyev 2015). However, other work has suggested that tra duplications occurred independently in honeybees, bumblebees, and ants (Hasselmann et al. 2008; Koch et al. 2014), and it remains to be demonstrated that either of these tra homologs acts as a primary CSD signal in V. emeryi.”

(4) Finally, since the authors successfully identified multiple alleles of the first CSD locus using previously sequenced haploid males, I wonder whether they also observed comparable allelic diversity at the candidate second CSD locus. This would provide useful supporting evidence for its functional relevance.

As is already addressed in the final paragraph of the results and in Supplementary Fig. S4, there is no peak of nucleotide diversity in any of the regions homologous to V.emeryiQTL1, which is the tra-containing candidate sex determination locus (Miyakawa and Mikheyev 2015). In the revised manuscript, the relevant lines will be 307-310. We want to restate that we do not propose that there is a second candidate CSD locus in O. biroi, but we simply raise the possibility that multi-locus CSD *might* explain the absence of diploid males from clonal lines other than clonal line A (as one of several alternative possibilities).

Overall, these are relatively minor points in the context of a strong manuscript, but I believe addressing them would improve the clarity and robustness of the authors' conclusions.

Reviewer #3 (Public review):

Summary:

The sex determination mechanism governed by the complementary sex determination (CSD) locus is one of the mechanisms that support the haplodiploid sex determination system evolved in hymenopteran insects. While many ant species are believed to possess a CSD locus, it has only been specifically identified in two species. The authors analyzed diploid females and the rarely occurring diploid males of the clonal ant Ooceraea biroi and identified a 46 kb CSD candidate region that is consistently heterozygous in females and predominantly homozygous in males. This region was found to be homologous to the CSD locus reported in distantly related ants. In the Argentine ant, Linepithema humile, the CSD locus overlaps with an lncRNA (ANTSR) that is essential for female development and is associated with the heterozygous region (Pan et al. 2024). Similarly, an lncRNA is encoded near the heterozygous region within the CSD candidate region of O. biroi. Although this lncRNA shares low sequence similarity with ANTSR, its potential functional involvement in sex determination is suggested. Based on these findings, the authors propose that the heterozygous region and the adjacent lncRNA in O. biroi may trigger female development via a mechanism similar to that of L. humile. They further suggest that the molecular mechanisms of sex determination involving the CSD locus in ants have been highly conserved for approximately 112 million years. This study is one of the few to identify a CSD candidate region in ants and is particularly noteworthy as the first to do so in a parthenogenetic species.

Strengths:

(1) The CSD candidate region was found to be homologous to the CSD locus reported in distantly related ant species, enhancing the significance of the findings.

(2) Identifying the CSD candidate region in a parthenogenetic species like O. biroi is a notable achievement and adds novelty to the research.

Weaknesses

(1) Functional validation of the lncRNA's role is lacking, and further investigation through knockout or knockdown experiments is necessary to confirm its involvement in sex determination.

See response below.

(2) The claim that the lncRNA is essential for female development appears to reiterate findings already proposed by Pan et al. (2024), which may reduce the novelty of the study.

We do not claim that the lncRNA is essential for female development in O. biroi, but simply mention the possibility that, as in L. humile, it is somehow involved in sex determination. We do not have any functional evidence for this, so this is purely based on its genomic position immediately adjacent to our mapped candidate region. We agree with the reviewer that the study by Pan et al. (2024) decreases the novelty of our findings. Another way of looking at this is that our study supports and bolsters previous findings by partially replicating the results in a different species.

added to misc sheet - Marc

Esse refinamento legal, e a necessidade de atendê-lo, resultou na formação do consultor ambiental especializado enquanto profissional, isto é, o especialista que conhece o arcabouço legal e os meios para atendê-lo.

Isso se dá porque, em muitos casos,

criosfera; mais recentemente, também pode-se citar a antroposfera e a tecnosfera.

há que se fazer, propositalmente,

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors have used full-length single-cell sequencing on a sorted population of human fetal retina to delineate expression patterns associated with the progression of progenitors to rod and cone photoreceptors. They find that rod and cone precursors contain a mix of rod/cone determinants, with a bias in both amounts and isoform balance likely deciding the ultimate cell fate. Markers of early rod/cone hybrids are clarified, and a gradient of lncRNAs is uncovered in maturing cones. Comparison of early rods and cones exposes an enriched MYCN regulon, as well as expression of SYK, which may contribute to tumor initiation in RB1 deficient cone precursors.

Strengths:

(1) The insight into how cone and rod transcripts are mixed together at first is important and clarifies a long-standing notion in the field.

(2) The discovery of distinct active vs inactive mRNA isoforms for rod and cone determinants is crucial to understanding how cells make the decision to form one or the other cell type. This is only really possible with full-length scRNAseq analysis.

(3) New markers of subpopulations are also uncovered, such as CHRNA1 in rod/cone hybrids that seem to give rise to either rods or cones.

(4) Regulon analyses provide insight into key transcription factor programs linked to rod or cone fates.

(5) The gradient of lncRNAs in maturing cones is novel, and while the functional significance is unclear, it opens up a new line of questioning around photoreceptor maturation.

(6) The finding that SYK mRNA is naturally expressed in cone precursors is novel, as previously it was assumed that SYK expression required epigenetic rewiring in tumors.

We thank the reviewer for describing the study’s strengths, reflecting the major conclusions of the initially submitted manuscript.  However, based on new analyses – including the requested analyses of other scRNA-seq datasets, our revision clarifies that:

-  related to point (1), cone and rod transcripts do not appear to be mixed together at first (i.e., in immediately post-mitotic immature cone and rod precursors) but appear to be coexpressed in subsequent cone and rod precursor stages; and 

- related to point (3), CHRNA1 appears to mark immature cone precursors that are distinct from the maturing cone and rod precursors that co-express cone- and rod-related RNAs (despite the similar UMAP positions of the two populations in our dataset). 

Weaknesses:

(1) The writing is very difficult to follow. The nomenclature is confusing and there are contradictory statements that need to be clarified.

(2) The drug data is not enough to conclude that SYK inhibition is sufficient to prevent the division of RB1 null cone precursors. Drugs are never completely specific so validation is critical to make the conclusion drawn in the paper.

We thank the reviewer for noting these important issues. Accordingly, in the revised manuscript:

(1) We improve the writing and clarify the nomenclature and contradictory statements, particularly those noted in the Reviewer’s Recommendations for Authors. 

(2) We scale back claims related to the role of SYK in the cone precursor response to RB1 loss, with wording changes in the Abstract, Results, and Discussion, which now recognize that the inhibitor studies only support the possibility that cone-intrinsic SYK expression contributes to retinoblastoma initiation, as detailed in our responses to Reviewer’s Recommendations for Authors. We agree and now mention that genetic perturbation of SYK is required to prove its role.  

Reviewer #2 (Public review):

Summary:

The authors used deep full-length single-cell sequencing to study human photoreceptor development, with a particular emphasis on the characteristics of photoreceptors that may contribute to retinoblastoma.

Strengths:

This single-cell study captures gene regulation in photoreceptors across different developmental stages, defining post-mitotic cone and rod populations by highlighting their unique gene expression profiles through analyses such as RNA velocity and SCENIC. By leveraging fulllength sequencing data, the study identifies differentially expressed isoforms of NRL and THRB in L/M cone and rod precursors, illustrating the dynamic gene regulation involved in photoreceptor fate commitment. Additionally, the authors performed high-resolution clustering to explore markers defining developing photoreceptors across the fovea and peripheral retina, particularly characterizing SYK's role in the proliferative response of cones in the RB loss background. The study provides an in-depth analysis of developing human photoreceptors, with the authors conducting thorough analyses using full-length single-cell RNA sequencing. The strength of the study lies in its design, which integrates single-cell full-length RNA-seq, longread RNA-seq, and follow-up histological and functional experiments to provide compelling evidence supporting their conclusions. The model of cell type-dependent splicing for NRL and THRB is particularly intriguing. Moreover, the potential involvement of the SYK and MYC pathways with RB in cone progenitor cells aligns with previous literature, offering additional insights into RB development.

We thank the reviewer for summarizing the main findings and noting the compelling support for the conclusions, the intriguing cell type-dependent splicing of rod and cone lineage factors, and the insights into retinoblastoma development.  

Weaknesses:

The manuscript feels somewhat unfocused, with a lack of a strong connection between the analysis of developing photoreceptors, which constitutes the bulk of the manuscript, and the discussion on retinoblastoma. Additionally, given the recent publication of several single-cell studies on the developing human retina, it is important for the authors to cross-validate their findings and adjust their statements where appropriate.

We agree that the manuscript covers a range of topics resulting from the full-length scRNAseq analyses and concur that some studies of developing photoreceptors were not well connected to retinoblastoma. However, we also note that the connection to retinoblastoma is emphasized in several places in the Introduction and throughout the manuscript and was a significant motivation for pursuing the analyses. We suggest that it was valuable to highlight how deep, fulllength scRNA-seq of developing retina provides insights into retinoblastoma, including i) the similar biased expression of NRL transcript isoforms in cone precursors and RB tumors, ii) the cone precursors’ co-expression of rod- and cone-related genes such as NR2E3 and GNAT2, which may explain similar co-expression in RB cells, and iii) the expression of  SYK in early cones and RB cells.  While the earlier version had mainly highlighted point (iii), the revised Discussion further refers to points (i) and (ii) as described further in the response to the Reviewer’s Recommendations for Authors. 

We address the Reviewer’s request to cross-validate our findings with those of other single-cell studies of developing human retina by relating the different photoreceptor-related cell populations identified in our study to those characterized by Zuo et al (PMID 39117640), which was specifically highlighted by the reviewer and is especially useful for such cross-validation given the extraordinarily large ~ 220,000 cell dataset covering a wide range of retinal ages (pcw 8–23) and spatiotemporally stratified by macular or peripheral retina location. Relevant analyses of the Zuo et al dataset are shown in Supplementary Figures S3G-H, S10B, S11A-F, and S13A,B. 

Reviewer #3 (Public review):

Summary:

The authors use high-depth, full-length scRNA-Seq analysis of fetal human retina to identify novel regulators of photoreceptor specification and retinoblastoma progression.

Strengths:

The use of high-depth, full-length scRNA-Seq to identify functionally important alternatively spliced variants of transcription factors controlling photoreceptor subtype specification, and identification of SYK as a potential mediator of RB1-dependent cell cycle reentry in immature cone photoreceptors.

Human developing fetal retinal tissue samples were collected between 13-19 gestational weeks and this provides a substantially higher depth of sequencing coverage, thereby identifying both rare transcripts and alternative splice forms, and thereby representing an important advance over previous droplet-based scRNA-Seq studies of human retinal development.

Weaknesses:

The weaknesses identified are relatively minor. This is a technically strong and thorough study, that is broadly useful to investigators studying retinal development and retinoblastoma.

We thank the reviewer for describing the strengths of the study. Our revision addresses the concerns raised separately in the Reviewer’s Recommendations for Authors, as detailed in the responses below.  

Recommendations for the authors:

Reviewing Editor Comments:

The reviewers have completed their reviews. Generally, they note that your work is important and that the evidence is generally convincing. The reviewers are in general agreement that the paper adds to the field. The findings of rod/cone fate determination at a very early stage are intriguing. Generally, the paper would benefit from clarifications in the writing and figures. Experimentally, the paper would benefit from validation of the drug data, for example using RNAi or another assay. Alternatively, the authors could note the caveats of the drug experiments and describe how they could be improved. In terms of analysis, the paper would be improved by additional comparisons of the authors' data to previously published datasets.

We thank the reviewing editor for this summary. As described in the individual reviewer responses, we clarify the writing and figures and provide comparisons to previously published datasets (in particular, the large snRNA-seq dataset of Zuo et al., 2024 (PMID 39117640).  With regard to the drug (i.e., SYK inhibitor) studies, we opted to provide caveats and describe the need for genetic approaches to validate the role of SYK, owing to the infeasibility of completing genetic perturbation experiments in the appropriate timeframe.  We are grateful for the opportunity to present our findings with appropriate caveats. 

Reviewer #1 (Recommendations for the authors):

Shayler cell sort human progenitor/rod/cone populations then full-length single cell RNAseq to expose features that distinguish paths towards rods or cones. They initially distinguish progenitors (RPCs), immature photoreceptor precursors (iPRPs), long/medium wavelength (LM) cones, late-LM cones, short wavelength (S) cones, early rods (ER) and late rods (LR), which exhibit distinct transcription factor regulons (Figures 1, 2). These data expose expected and novel enriched genes, and support the notion that S cones are a default state lacking expression of rod (NRL) or cone (THRB) determinants but retaining expression of generic photoreceptor drivers (CRX/OTX2/NEUROD1 regulons). They identify changes in regulon activity, such as increasing NRL activity from iPRP to ER to LR, but decreasing from iPRP to cones, or increasing RAX/ISL2/THRB regulon activity from iPRP to LM cones, but decreasing from iPRP to S cones or rods.

They report co-expression of rod/cone determinants in LM and ER clusters, and the ratios are in the expected directions (NRLTHRB or RXRG in ER). A novel insight from the FL seq is that there are differing variants generated in each cell population. Full-length NRL (FL-NRL) predominates in the rod path, whereas truncated NRL (Tr-NRL) does so in the cone path, then similar (but opposite) findings are presented for THRB (Fig 3, 4), whereas isoforms are not a feature of RXRG expression, just the higher expression in cones.

The authors then further subcluster and perform RNA velocity to uncover decision points in the tree (Figure 5). They identify two photoreceptor precursor streams, the Transitional Rods (TRs) that provide one source for rod maturation and (reusing the name from the initial clustering) iPRPs that form cones, but also provide a second route to rods. TR cells closest to RPCs (immediately post-mitotic) have higher levels of the rod determinant NR2E3 and NRL, whereas the higher resolution iPRPs near RPCs lack NR2E3 and have higher levels of ONECUT1, THRB, and GNAT2, a cone bias. These distinct rod-biased TR and cone-biased high-resolution iPRPs were not evident in published scRNAseq with 3′ end-counting (i.e. not FL seq). Regulon analysis confirmed higher NRL activity in TR cells, with higher THRB activity in highresolution iPRP cells.

Many of the more mature high-resolution iPRPs show combinations of rod (GNAT1, NR2E3) and cone (GNAT2, THRB) paths as well as both NRL and THRB regulons, but with a bias towards cone-ness (Figure 6). Combined FISH/immunofluorescence in fetal retina uncovers cone-biased RXRG-protein-high/NR2E3-protein-absent cone-fated cells that nevertheless expressed NR2E3 mRNA. Thus early cone-biased iPRP cells express rod gene mRNA, implying a rod-cone hybrid in early photoreceptor development. The authors refer to these as "bridge region iPRP cells".

In Figure 7, they identify CHRNA1 as the most specific marker of these bridge cells (overlapping with ATOH7 and DLL3, previously linked to cone-biased precursors), and FISH shows it is expressed in rod-biased NRL protein-positive and cone-biased RXRG proteinpositive cones at fetal week 12.

Figure 8 outlines the graded expression of various lncRNAs during cone maturation, a novel pattern.

Finally (Figure 9), the authors identify differential genes expressed in early rods (ER cluster from Figure 1) vs early cones (LM cluster, excluding the most mature opsin+ cells), revealing high levels of MYCN targets in cones. They also find SYK expression in cones. SYK was previously linked to retinoblastoma, so intrinsic expression may predispose cone precursors to transformation upon RB loss. They finish by showing that a SYK inhibitor blocks the proliferation of dividing RB1 knockdown cone precursors in the human fetal retina.

Overall, the authors have uncovered interesting patterns of biased expression in cone/rod developmental paths, especially relating to the isoform differences for NRL and THRB which add a new layer to our understanding of this fate choice. The analyses also imply that very soon after RPCs exit the cell cycle, they generate post-mitotic precursors biased towards a rod or cone fate, that carry varying proportions of mixed rod/cone determinants and other rod/cone marker genes. They also introduce new markers that may tag key populations of cells that precede the final rod/cone choice (e.g. CHRNA1), catalogue a new lncRNA gradient in cone maturation, and provide insight into potential genes that may contribute to retinoblastoma initiation, like SYK, due to intrinsic expression in cone precursors. However, as detailed below, the text needs to be improved considerably, and overinterpretations need to be moderated, removed, or tested more rigorously with extra data.

Major Comments

The manuscript is very difficult to follow. The nomenclature is at times torturous, and the description of hybrid rod/cone hybrid cells is confusing in many aspects.

(1) A single term, iPRP, is used to refer to an initial low-resolution cluster, and then to a subset of that cluster later in the paper.

We agree that using immature photoreceptor precursor (iPRP) for both high-resolution and lowresolution clusters was confusing. We kept this name for the low-resolution cluster (which includes both immature cone and immature rod precursors), renamed the high-resolution iPRP cluster immature cone precursors (iCPs). and renamed their transitional rod (TR) counterparts immature rod precursors (iRPs). These designations are based on 

- the biased expression of THRB, ONECUT1, and the THRB regulon in iCPs (Fig. 5D,E);

- the biased expression of NRL, NR2E3, and NRL regulon iRPs (Fig. 5D,E);

- the partially distinct iCP and iRP UMAP positions (Figure 5C); and 

- the evidence of similar immature cone versus rod precursor populations in the Zuo et al 3’ snRNA-seq dataset, as noted below and described in two new paragraphs starting at the bottom of p. 12.

(2) To complicate matters further, the reader needs to understand the subset within the iPRP referred to as bridge cells, and we are told at one point that the earliest iPRPs lack NR2E3, then that they later co-express NR2E3, and while the authors may be referring to protein and RNA, it serves to further confuse an already difficult to follow distinction. I had to read and re-read the iPRP data many times, but it never really became totally clear.

We agree that the description of the high-resolution iPRP (now “iCP”) subsets was unclear, although our further analyses of a large 3’ snRNA-seq dataset in Figure S11 support the impression given in the original manuscript that the earliest iCPs lack NR2E3 and then later coexpress NR2E3 while the earliest iRPs lack THRB and then later express THRB. As described in new text in the Two post-mitotic immature photoreceptor precursor populations section (starting on line 7 of p. 13): 

When considering only the main cone and rod precursor UMAP regions, early (pcw 8 – 13) cone precursors expressed THRB and lacked NR2E3 (Figure S11D,E, blue arrows), while early (pcw 10 – 15) rod precursors expressed NR2E3 and lacked THRB (Figure S11D,E, red arrows), similar to RPC-localized iCPs and iRPs in our study (Figure 5D).

Next, as summarized in new text in the Early cone and rod precursors with rod- and conerelated RNA co-expression section (new paragraph at top of p. 16): 

Thus, a 3’ snRNA-seq analysis confirmed the initial production of immature photoreceptor precursors with either L/M cone-precursor-specific THRB or rod-precursor-specific NR2E3 expression, followed by lower-level co-expression of their counterparts, NR2E3 in cone precursors and THRB in rod precursors. However, in the Zuo et al. analyses, the co-expression was first observed in well-separated UMAP regions, as opposed to a region that bridges the early cone and early rod populations in our UMAP plots. These findings are consistent with the notion that cone- and rod-related RNA co-expression begins in already fate-determined cone and rod precursors, and that such precursors aberrantly intermixed in our UMAP bridge region due to their insufficient representation in our dataset.  

Importantly, and as noted in our ‘Public response’ to Reviewer 1, “CHRNA1 appears to mark immature cone precursors that are distinct from the maturing cone and rod precursors that coexpress cone- and rod-related RNAs (despite the similar UMAP positions of the two populations in our dataset).” In support of this notion, the immature cone precursors expressing CHRNA1  and other  populations did not overlap in UMAP space in the Zuo et al dataset. We hope the new text cited above along with other changes will significantly clarify the observations.

(3) The term "cone/rod precursor" shows up late in the paper (page 12), but it was clear (was it not?) much earlier in this manuscript that cone and rod genes are co-expressed because of the coexpressed NRL and THRB isoforms in Figures 3/4.

We thank the reviewer for noting that the differential NRL and THRB isoform expression already implies that cone and rod genes are co-expressed. However, as we now state, the co-expression of RNAs encoding an additional cone marker (GNAT2) and rod markers (GNAT1, NR2E3) was 

“suggestive of a proposed hybrid cone/rod precursor state more extensive than implied by the coexpression of different THRB and NRL isoforms” (first paragraph of “Early cone and rod …” section on p. 14; new text underlined). 

(4) The (incorrect) impression given later in the manuscript is that the rod/cone transcript mixture applies to just a subset of the iPRP cells, or maybe just the bridge cells (writing is not clear), but actually, neither of those is correct as the more abundant and more mature LM and ER populations analyzed earlier coexpress NRL and THRB mRNAs (Figures 2, 3). Overall, the authors need to vastly improve the writing, simplify/clarify the nomenclature, and better label figures to match the text and help the reader follow more easily and clearly. As it stands, it is, at best, obtuse, and at worst, totally confusing.

We thank the reviewer for bringing the extent of the confusing terminology and wording to our attention. We revised the terminology (as in our response to point 1) and extensively revised the text.  We also performed similar analyses of the Zuo et al. data (as described in more detail in our response to Reviewer 2), which clarifies the distinct status of cells with the “rod/cone transcript mixture” and cells co-expressing early cone and rod precursor markers.  

To more clearly describe data related to cells with rod- and cone-related RNA co-expression, we divided the former Figure 6 into two figures, with Figure 6 now showing the cone- and rodrelated RNA co-expression inferred from scRNA-seq and Figure 7 showing GNAT2 and NR2E3 co-expression in FISH analyses of human retina plus a new schematic in the new panel 7E.

To separate the conceptually distinct analyses of cone and rod related RNA co-expression and the expression of early photoreceptor precursor markers (which were both found in the so-called bridge region – yet now recognized to be different subpopulations), we separated the analyses of the early photoreceptor precursor markers to form a new section, “Developmental expression of photoreceptor precursor markers and fate determinants,” starting on p. 16. 

Additionally, we further review the findings and their implications in four revised Discussion paragraphs starting at the bottom of p. 23).

(5) The data showing that overexpressing Tr-NRL in murine NIH3T3 fibroblasts blocks FL-NRL function is presented at the end of page 7 and in Figure 3G. Subsequent analysis two paragraphs and two figures later (end page 8, Figure 5C + supp figs) reveal that Tr-NRL protein is not detectable in retinoblastoma cells which derive from cone precursors cells and express Tr-NRL mRNA, and the protein is also not detected upon lentiviral expression of Tr-NRL in human fetal retinal explants, suggesting it is unstable or not translated. It would be preferable to have the 3T3 data and retinoblastoma/explant data juxtaposed. E.g. they could present the latter, then show the 3T3 that even if it were expressed (e.g. briefly) it would interfere with FL-NRL. The current order and spacing are somewhat confusing.

We thank the reviewer for this suggestion and moved the description of the luciferase assays to follow the retinoblastoma and explant data and switched the order of Figure panels 3G and 3H.  

(6) On page 15, regarding early rod vs early cone gene expression, the authors state: "although MYCN mRNA was not detected....", yet on the volcano plot in Figure S14A MYCN is one of the marked genes that is higher in cones than rods, meaning it was detected, and a couple of sentences later: "Concordantly, the LM cluster had increased MYCN RNA". The text is thus confusing.

With respect, we note that the original text read, “although MYC RNA was not detected,” which related to a statement in the previous sentence that the gene ontology analysis identified “MYC targets.” However, given that this distinction is subtle and may be difficult for readers to recognize, we revised the text (now on p. 19) to more clearly describe expression of MYCN (but not MYC) as follows:

“The upregulation of MYC target genes was of interest given that many MYC target genes are also targets of MYCN, that MYCN protein is highly expressed in maturing (ARR3+) cone precursors but not in NRL+ rods (Figure 10A), and that MYCN is critical to the cone precursor proliferative response to pRB loss8–10.  Indeed, whereas MYC RNA was not detected, the LM cone cluster had increased MYCN RNA …”

(7) The authors state that the SYK drug is "highly specific". They provide no evidence, but no drug is 100% specific, and it is possible that off-target hits are important for the drug phenotype. This data should be removed or validated by co-targeting the SYK gene along with RB1.

We agree that our data only show the potential for SYK to contribute to the cone proliferative response; however, we believe the inhibitor study retains value in that a negative result (no effect of the SYK inhibitor) would disprove its potential involvement. To reflect this, we changed wording related to this experiment as follows:

In the Abstract, we changed:

(1) “SYK, which contributed to the early cone precursors’ proliferative response to RB1 loss” To: “SYK, which was implicated in the early cone precursors’ proliferative response to RB1 loss.”  

(2) “These findings reveal … and a role for early cone-precursor-intrinsic SYK expression.” To:  “These findings reveal … and suggest a role for early cone-precursor-intrinsic SYK expression.”

In the last paragraph of the Results, we changed:

(1) “To determine if SYK contributes…” To:  “To determine if SYK might contribute…”

(2) “the highly specific SYK inhibitor” To:  “the selective SYK inhibitor”  

(3)  “indicating that cone precursor intrinsic SYK activity is critical to the proliferative response” To: “consistent with the notion that cone precursor intrinsic SYK activity contributes to the proliferative response.”

In the Results, we added a final sentence: 

“However, given potential SYK inhibitor off-target effects, validation of the role of SYK in retinoblastoma initiation will require genetic ablation studies.”

In the Discussion (2nd-to-last paragraph), we changed: 

“SYK inhibition impaired pRB-depleted cone precursor cell cycle entry, implying that native SYK expression rather than de novo induction contributes to the cone precursors’ initial proliferation.” To: “…the pRB-depleted cone precursors’ sensitivity to a SYK inhibitor suggests that native SYK expression rather than de novo induction contributes to the cone precursors’ initial proliferation, although genetic ablation of SYK is needed to confirm this notion.” In the Discussion last sentence, we changed:

“enabled the identification of developmental stage-specific cone precursor features that underlie retinoblastoma predisposition.” To: “enabled the identification of developmental stage-specific cone precursor features that are associated with the cone precursors’ predisposition to form retinoblastoma tumors.”

Minor/Typos

Figure 7 legend, H should be D.

We corrected the figure legend (now related to Figure 8).

Reviewer #2 (Recommendations for the authors):

(1) The author should take advantage of recently published human fetal retina data, such as PMID:39117640, which includes a larger dataset of cells that could help validate the findings. Consequently, statements like "To our knowledge, this is the first indication of two immediately post-mitotic photoreceptor precursor populations with cone versus rod-biased gene expression" may need to be revised.

We thank the reviewer for noting the evidence of distinct immediately post-mitotic rod and cone populations published by others after we submitted our manuscript. In response, we omitted the sentence mentioned and extensively cross-checked our results including:

- comparison of our early versus late cone and rod maturation states to the cone and rod precursor versus cone and rod states identified by Zuo et al (new paragraph on the top half of p. 6 and new figure panels S3G,H);

- detection of distinct immediately post-mitotic versus later cone and rod precursor populations (two new paragraphs on pp. 12-13 and new Figures S10B and S11A-E); 

- identification of cone and rod precursor populations that co-express cone and rod marker genes (two new paragraphs starting at the bottom of p. 15 and new Figures S11D-F);

- comparison of expression patterns of immature cone precursor (iCP) marker genes in our and the Zuo et al dataset (new paragraph on top half of p. 17 and new Figure S13).

We also compare the cell states discerned in our study and the Zuo et al. study in a new Discussion paragraph (bottom of p. 23) and new Figure S17.

(2) The data generated comes from dissociated cells, which inherently lack spatial context. Additionally, it is unclear whether the dataset represents a pool of retinas from multiple developmental stages, and if so, whether the developmental stage is known for each cell profiled. If this information is available, the authors should examine the distribution of developmental stages on the UMAP and trajectory analysis as part of the quality control process. 

We thank the reviewer for highlighting the importance of spatial context and developmental stage. 

Related to whether the dataset represents a pool of retinae from multiple developmental stages, the different cell numbers examined at each time point are indicated in Figure S1A. To draw the readers’ attention to this detail, Figure S1A is now cited in the first sentence of the Results. 

Related to the age-related cell distributions in UMAP plots, the distribution of cells from each retina and age was (and is) shown in Fig. S1F. In addition, we now highlight the age distributions by segregating the FW13, FW15-17, and FW17-18-19 UMAP positions in the new Figure 1C. We describe the rod temporal changes in a new sentence at the top of  p. 5:

“Few rods were detected at FW13, whereas both early and late rods were detected from FW15-19 (Figure 1C), corroborating prior reports [15,20].”  

We describe the cone temporal changes and note the likely greater discrimination of cell state changes that would be afforded by separately analyzing macula versus peripheral retina at each age in a new sentence at the bottom of p. 5:

“L/M cone precursors from different age retinae occupied different UMAP regions, suggesting age-related differences in L/M cone precursor maturation (Figure 1C).”

Moreover, they should assess whether different developmental stages impact gene expression and isoform ratios. It is well established that cone and rod progenitors typically emerge at different developmental times and in distinct regions of the retina, with minimal physical overlap. Grouping progenitor cells based solely on their UMAP positioning may lead to an oversimplified interpretation of the data.

(2a) We agree that different developmental stages may impact gene expression and isoform ratios, and evaluated stages primarily based on established Louvain clustering rather than UMAP position. However, we also used UMAP position to segregate so-called RPC-localized and nonRPC-localized iCPs and iRPs, as well as to characterize the bridge region iCP sub-populations. In the revision, we examine whether cell groups defined by UMAP positions helped to identify transcriptomically distinct populations and further examine the spatiotemporal gene expression patterns of the same genes in the Zuo et al. 3’ snRNA-seq dataset. 

(2b) Related to analyses of immediately post-mitotic iRPs and iCPs, the new Figure S10A expanded the violin plots first shown in Figure 5D to compare gene expression in RPC-localized versus non-RPC-localized iCPs and iRPs and subsequent cone and rod precursor clusters (also presented in response to Reviewer 3). The new Figure S10C, shows a similar analysis of UMAP region-specific regulon activities. These figures support the idea that there are only subtle UMAP region-related differences in the expression of the selected gene and regulons. 

To further evaluate early cone and rod precursors, we compared expression patterns in our cluster- and UMAP-defined cell groups to those of the spatiotemporally defined cell groups in the Zuo et al. 3’ snRNA-seq study. The results revealed similar expression timing of the genes examined, although the cluster assignments of a subset of cells were brought into question, especially the assigned rod precursors at pcw 10 and 13, as shown in new Figures S10B (grey columns) and S11, and as described in two new paragraphs starting near the bottom of p.12. 

(2c) Related to analyses of iCPs in the so-called bridge region, our analyses of the Zuo et al dataset helped distinguish early cone and rod precursor populations (expressing early markers such as ATOH7 and CHRNA1) from the later stages exhibiting rod- and cone-related gene coexpression, which had intermixed in the UMAP bridge region in our dataset. Further parsing of early cone precursor marker spatiotemporal expression revealed intriguing differences as now described in the second half of a new paragraph at the top of p. 17, as follows:

“Also, different iCP markers had different spatiotemporal expression: CHRNA1 and ATOH7 were most prominent in peripheral retina with ATOH7 strongest at pcw 10 and CHRNA1 strongest at pcw 13; CTC-378H22.2 was prominently expressed from pcw 10-13 in both the macula and the periphery; and DLL3 and ONECUT1 showed the earliest, strongest, and broadest expression (Figure S13B). The distinct patterns suggest spatiotemporally distinct roles for these factors in cone precursor differentiation.”

(3) I would commend the authors for performing a validation experiment via RNA in situ to validate some of the findings. However, drawing conclusions from analyzing a small number of cells can still be dangerous. Furthermore, it is not entirely clear how the subclustering is done. Some cells change cell type identities in the high-resolution plot. For example, some iPRP cells from the low-resolution plots in Figure 1 are assigned as TR in high-resolution plots in Figure 5.

The authors should provide justification on the identifies of RPC localized iPRP and TR.

Comparison of their data with other publicly available data should strengthen their annotation

We agree that drawing conclusions from scRNA-seq or in situ hybridization analysis of a small number of cells can be dangerous and have followed the reviewer’s suggestion to compare our data with other publicly available data, focusing on the 3’ snRNA-seq of Zuo et al. given its large size and extensive annotation. Our analysis of  the Zuo et al. dataset helped clarify cell identities by segregating cone and rod precursors with similar gene expression properties in distinct UMAP regions. However, we noted that the clustering of early cone and rod precursors likely gave numerous mis-assigned cells (as noted in response 2b above and shown in the new Figure S11). It would appear that insights may be derived from the combination of relatively shallow sequencing of a high number of cells and deep sequencing of substantially fewer cells. 

Related to how subclustering was done, the Methods state, “A nearest-neighbors graph was constructed from the PCA embedding and clusters were identified using a Louvain algorithm at low and high resolutions (0.4 and 1.6)[70],” citing the Blondel et al reference for the Louvain clustering algorithm used in the Seurat package.  To clarify this, the results text was revised such that it now indicates the levels used to cluster at low resolution (0.4, p. 4, 2nd paragraph) and at high resolution (1.6, top of p. 11) .

Related to the assignment of some iPRP cells from the low-resolution plots in Figure 1 to the TR cluster (now called the ‘iRP’ ‘cluster) in the high-resolution plots in Figure 5, we suggest that this is consistent with Louvain clustering, which does not follow a single dendrogram hierarchy. 

The justification for referring to these groups as RPC-localized iCPs and iRPs relates to their biased gene and regulon expression in Fig. 5D and 5E, as stated on p. 12: 

“In the RPC-localized region, iCPs had higher ONECUT1, THRB, and GNAT2, whereas iRPs trended towards higher NRL and NR2E3 (p= 0.19, p=0.054, respectively).”

(4) Late-stage LM5 cluster Figure 9 is not defined anywhere in previous figures, in which LM clusters only range from 1 to 4. The inconsistency in cluster identification should be addressed.

We revised the text related to this as follows: 

“Indeed, our scRNA-seq analyses revealed that SYK RNA expression increased from the iCP stage through cluster LM4, in contrast to its minimal expression in rods (Figure 10E).  Moreover, SYK expression was abolished in the five-cell group with properties of late maturing cones (characterized in Figure 1E), here displayed separately from the other LM4 cells and designated LM5 (Figure 10E).”  (p. 19-20)

(5) Syk inhibitor has been shown to be involved in RB cell survival in previous studies. The manuscript seems to abruptly make the connection between the single-cell data to RB in the last figure. The title and abstract should not distract from the bulk of the manuscript focusing on the rod and cone development, or the manuscript should make more connection to retinoblastoma.

We appreciate the reviewer’s concern that the title may seem to over-emphasize the connection to retinoblastoma based solely on the SYK inhibitor studies. However, we suggest the title also emphasizes the identification and characterization of early human photoreceptor states, per se, and that there are a number of important connections beyond the SYK studies that could warrant the mention of cell-state-specific retinoblastoma-related features in the title.

Most importantly, a prior concern with the cone cell-of-origin theory was that retinoblastoma cells express RNAs thought to mark retinal cell types other than cones, especially rods. The evidence presented here, that cone precursors also express the rod-related genes helps resolve this issue. The issue is noted numerous times in the manuscript, as follows:  

In the Introduction, we write:

“However, retinoblastoma cells also express rod lineage factor NRL RNAs, which – along with other evidence – suggested a heretofore unexplained connection between rod gene expression and retinoblastoma development[12,13]. Improved discrimination of early photoreceptor states is needed to determine if co-expression of rod- and cone-related genes is adopted during tumorigenesis or reflects the co-expression of such genes in the retinoblastoma cell of origin.” (bottom, p. 2) And: 

“In this study, we sought to further define the transcriptomic underpinnings of human  photoreceptor development and their relationship to retinoblastoma tumorigenesis.” (last paragraph, p. 3)

The Discussion also alluded to this issue and in the revised Discussion, we aimed to make the connection clearer.  We previously ended the 3rd-to-last paragraph with,  

“iPRP [now iCP] and early LM cone precursors’ expression of NR2E3 and NRL RNAs suggest that their presence in retinoblastomas[12,13] reflects their normal expression in the L/M cone precursor cells of origin.” 

We now separate and elaborate on this point in a new paragraph as follows: 

“Our characterization of cone and rod-related RNA co-expression may help resolve questions about the retinoblastoma cell of origin. Past studies suggested that retinoblastoma cells co-express RNAs associated with rods, cones, or other retinal cells due to a loss of lineage fidelity[12]. However, the early L/M cone precursors’ expression of NR2E3 and NRL RNAs suggest that their presence in retinoblastomas[12,13] reflects their normal expression in the L/M cone precursor cells of origin. This idea is further supported by the retinoblastoma cells’ preferential expression of cone-enriched NRL transcript isoforms (Figure S5B).” (middle of p. 24) Based on the above, we elected to retain the title.  

Minor comments:

(1) It is difficult to see the orange and magenta colors in the Fig 3E RNA-FISH image. The colors should be changed, or the contrast threshold needs to be adjusted to make the puncta stand out more.

We re-assigned colors, with red for FL-NRL puncta and green for Tr-NRL puncta. 

(2) Figure 5C on page 8 should be corrected to Supplementary Figure 5C.

We thank the reviewer for noting this error and changed the figure citation.

Reviewer #3 (Recommendations for the authors):

(1) Minor concerns

a. Abbreviation of some words needs to be included, example: FW. 

We now provide abbreviation definitions for FW and others throughout the manuscript.  

b. Cat # does not matches with the 'key resource table' for many reagents/kits. Some examples are: CD133-PE mentioned on Page # 22 on # 71, SMART-Seq V4 Ultra Low Input RNA Kit and SMARTer Ultra Low RNA Kit for the Fluidigm C1 Sytem on Page # 22 on # 77, Nextera XT DNA Library preparation kit on Page # 23 on # 77.

We thank the reviewer for noting these discrepancies. We have now checked all catalog numbers and made corrections as needed.

c. Cat # and brand name of few reagents & kits is missing and not mentioned either in methods or in key resource table or both. Eg: FBS, Insulin, Glutamine, Penicillin, Streptomycin, HBSS, Quant-iT PicoGreen dsDNA assay, Nextera XT DNA LibraryPreparation Kit, 5' PCR Primer II A with CloneAmp HiFi PCR Premix. 

Catalog numbers and brand names are now provided for the tissue culture and related reagents within the methods text and for kits in the Key Resources Table. Additional descriptions of the primers used for re-amplification and RACE were added to the Methods (p. 28-29).

d. Spell and grammar check is needed throughout the manuscript is needed. Example. In Page # 46 RXRγlo is misspelled as RXRlo.

Spelling and grammar checks were reviewed.

(2) Methods & Key Resource table.

a. In Page # 21, IRB# needs to be stated.      

The IRB protocols have been added, now at top of p. 26.

b. In Page # 21, Did the authors dissociate retinae in ice-cold phosphate-buffered saline or papain?   

The relevant sentence was corrected to “dissected while submerged in ice-cold phosphatebuffered saline (PBS) and dissociated as described10.” ( p. 26)

c. In Page # 21, How did the authors count or enumerate the cell count? Provide the details.

We now state, “… a 10 µl volume was combined with 10 µl trypan blue and counted using a hemocytometer” (top of p. 27)

d. Why did the authors choose to specifically use only 8 cells for cDNA preparation in Page # 22? State the reason and provide the details.

The reasons for using 8 cells (to prevent evaporation and to manually transfer one slide-worth of droplets to one strip of PCR tubes) and additional single cell collection details are now provided as follows (new text underlined): 

“Single cells were sorted on a BD FACSAria I at 4°C using 100 µm nozzle in single-cell mode into each of eight 1.2 µl lysis buffer droplets on parafilm-covered glass slides, with droplets positioned over pre-defined marks … .  Upon collection of eight cells per slide, droplets were transferred to individual low-retention PCR tubes (eight tubes per strip) (Bioplastics K69901, B57801) pre-cooled on ice to minimize evaporation. The process was repeated with a fresh piece of parafilm for up to 12 rounds to collect 96 cells). (p. 27, new text underlined)

e. Key resource table does not include several resources used in this study. Example - NR2E3 antibody.

We added the NR2E3 antibody and checked for other omissions.

(3) Results & Figures & Figure Legends

a. Regulon-defined RPC and photoreceptor precursor states

i. On page # 4, 1 paragraph - Clarify the sentence 'Exclusion of all cells with <100,000 cells read and 18 cells.........Emsembl transcripts inferred'. Did the authors use 18 cells or 18FW retinae? 

The sentence was changed to:

“After sequencing, we excluded all cells with <100,000 read counts and 18 cells expressing one or more markers of retinal ganglion, amacrine, and/or horizontal cells (POU4F1, POU4F2, POU4F3, TFAP2A, TFAP2B, ISL1) and concurrently lacking photoreceptor lineage marker OTX2. This yielded 794 single cells with averages of 3,750,417 uniquely aligned reads, 8,278 genes detected, and 20,343 Ensembl transcripts inferred (Figure S1A-C).” (p. 4, new words underlined)

To clarify that 18 retinae were used, the first sentence of the Results was revised as follows:

“To interrogate transcriptomic changes during human photoreceptor development, dissociated RPCs and photoreceptor precursors were FACS-enriched from 18 retinae, ages FW13-19 …” (p. 4).

Why did the authors 'exclude cells lacking photoreceptor lineage marker OTX2' from analysis especially when the purpose here was to choose photoreceptor precursor states & further results in the next paragraph clearly state that 5 clusters were comprised of cells with OTX2 and CRX expression. This is confusing.

We apologize for the imprecise diction. We divided the evidently confusing sentence into two sentences to more clearly indicate that we removed cells that did not express OTX2, as in the first response to the previous question.

ii. In Page # 5, the authors reported the number of cell populations (363 large and 5 distal) identified in the THRB+ L/M-cone cluster. What were the # of cell populations identified in the remaining 5 clusters of the UMAP space?

We added the cell numbers in each group to Fig. 1B. We corrected the large LM group to 366 cells (p. 5) and note 371 LM cells , which includes the five distal cells, in Figure 1B.

b. Differential expression of NRL and THRB isoforms in rod and cone precursors

i. In Figure 3B, the authors compare and show the presence of 5 different NRL isoforms for all the 6 clusters that were defined in 3A. However, in the results, the ENST# of just 2 highly assigned transcript isoforms is given. What are the annotated names of the three other isoforms which are shown in 3B? Please explain in the Results.

As requested, we now annotate the remaining isoforms as encoding full-length or truncated NRL in Fig. 3B and show isoform structures in new Supplementary Figure S4B.  We also refer to each transcript isoform in the Results (p. 7, last paragraph) and similarly evaluate all isoforms in RB31 cells (Fig. S5B).

ii. What does the Mean FPM in the y-axis of Fig 3C refer to?

Mean FPM represents mean read counts (fragments per million, FPM) for each position across Ensembl NRL exons for each cluster, as now stated in the 6th line of the Fig. 3 legend.

iii. A clear explanation of the results for Figures 3E-3F is missing.

We revised the text to more clearly describe the experiment as follows:

“The cone cells’ higher proportional expression of Tr-NRL first exon sequences was validated by RNA fluorescence in situ hybridization (FISH) of FW16 fetal retina in which NRL immunofluorescence was used to identify rod precursors, RXRg immunofluorescence was used to identify cone precursors, and FISH probes specific to truncated Tr-NRL exon 1T or FL-NRL exons 1 and 2 were used to assess Tr-NRL and FL-NRL expression (Figure 3E,F).” (p. 8, new text underlined).

c. Two post-mitotic photoreceptor precursor populations

i. Although deep-sequencing and SCENIC analysis clarified the identities of four RPC-localized clusters as MG, RPC, and iPRP indicative of cone-bias and TR indicative of rod-bias. It would be interesting to see the discriminating determinant between the TR and ER by SCENIC and deep-sequencing gene expression violin/box plots.

We agree it is of interest to see the discriminating determinant between the TR [now termed iRP] and ER clusters by SCENIC and deep-sequencing gene expression violin/box plots. We now provide this information for selected genes and regulons of interest in the new Supplementary Figures S10A and S10C, along with a similar comparison between the prior high-resolution iPRP (now termed iCP) cluster and the first high-resolution LM cluster, LM1, as described for gene expression on p. 12:

“Notably, THRB and GNAT2 expression did not significantly change while ONECUT1 declined in the subsequent non-RPC-localized iCP and LM1 stages, whereas NR2E3 and NRL dramatically increased on transitioning to the ER state (Figure S10A).”

And as described for regulon activities on pp. 13-14:

“Finally, activities of the cone-specific THRB and ISL2 regulons, the rod-specific NRL regulon, and the pan-photoreceptor LHX3, OTX2, CRX, and NEUROD1 regulons increased to varying extents on transitioning from the immature iCP or iRP states to the early-maturing LM1 or ER states (Figure 10C).”

We also show expression of the same genes for spatiotemporally grouped cells from the Zuo et al. dataset in the new Figure S10B, which displays a similar pattern (apart from the possibly mixed pcw 10 and pcw13 designated rod precursors).

d. Early cone precursors with cone- and rod-related RNA expression

i. On page #12, the last paragraph where the authors explain the multiplex RNA FISH results of RXRγ and NR2E3 by citing Figure S8E. However, in Fig S8E, the authors used NRL to identify the rods. Please clarify which one of the rod markers was used to perform RNA FISH?

Figure S8E (where NRL was used as a rod marker) was cited to remind readers that RXRg has low expression in rods and high expression in cones, rather than to describe the results of this multiplex FISH section. To avoid confusion on this point, Figure S8E is now cited using “(as earlier shown in Figure S8E).” With this issue clarified, we expect the markers used in the FISH + IF analysis will be clear from the revised explanation, 

“… we examined GNAT2 and NR2E3 RNA co-expression in RXRg+ cone precursors in the outermost NBL and in RXRg+ rod precursors in the middle NBL … .” (p. 14-15).

To provide further clarity, we provide a diagram of the FISH probes, protein markers, and expression patterns in the new Figure 7E.

ii. The Y-axis of Fig 6G-6H needs to be labelled.

The axes have been re-labeled from “Nb of cells” to “Number of RXRg+ outermost NBL cells in each region” (original Fig. 6G, now Fig. 7C) and “Number of RXRg+ middle NBL cells in each region” (original Fig. 6H, now Fig. 7D).

iii. The legends of Figures 6G and 6H are unclear. In the Figure 6G legend, the authors indicate 'all cells are NR2E3 protein-'. Does that imply the yellow and green bars alone? Similarly, clarify the Figure 6H legend, what does the dark and light magenta refer to? What does the light magenta color referring to NR2E3+/ NR2E3- and the dark magenta color referring to NR2E3+/ NR2E3+ indicate? 

We regret the insufficient clarity. We revised the Fig. 6G (now Fig. 7C) key, which now reads

“All outermost NBL cells are NR2E3 protein-negative.”  We added to the figure legend for panel 7C,D “(n.b., italics are used for RNAs, non-italics for proteins).”  The new scheme in Figure 7E shows the RNAs in italics proteins in non-italics. We hope these changes will clarify when RNA or protein are represented in each histogram category.

Overall, the results (on page # 13) reflecting Figures 6E-6H & Figure S11 are confusing and difficult to understand. Clear descriptions and explanations are needed.

We revised this results section described in the paragraph now spanning p. 14:

-  We now refer to the bar colors in Figures 7C and 7D that support each statement. 

-  We provide an illustration of the findings in Figure 7E.

iv. Previously published literature has shown that cells of the inner NBL are RXRγ+ ganglion cells. So, how were these RXRγ+ ganglion cells in the inner NBL discriminated during multiplex RNA FISH (in Fig 6E-6H and in Fig S11)?

We thank the reviewer for requesting this clarification. We agree that “inner NBL” is the incorrect term for the region in which we examined RXRg+ photoreceptor precursors, as this could include RXRγ+ nascent RGCs. We now clarify that 

“we examined GNAT2 and NR2E3 RNA co-expression in RXRg+ cone precursors in the outermost NBL and in RXRg+ rod precursors in the middle NBL … .”  (p. 14-15) We further state, 

“Limiting our analysis to the outer and middle NBL allowed us to disregard RXRγ+ retinal ganglion cells in the retinal ganglion cell layer or inner NBL (top of p. 15)”

Figure 7E is provided to further aid the reader in understanding the positions examined, and the legend states “RXRg+ retinal ganglion cells in the inner NBL and ganglion cell layer not shown. 

v. In Figure 6E, what marker does each color cell correspond to?

In this figure (now panel 7A), we declined to provide the color key since the image is not sufficiently enlarged to visualize the IF and FISH signals. The figure is provided solely to document the regions analyzed and readers are now referred to “see Figure S12 for IF + FISH images” (2nd line, p. 15), where the marker colors are indicated.

vi. In Figure S11 & 6E, Protein and RNA transcript color of NR2E3, GNAT2 are hard to distinguish. Usage of other colors is recommended.  

We appreciate the reviewer’s concern related to the colors (in the now redesignated Figure S12 and 7A); however, we feel this issue is largely mitigated by our use of arrows to point to the cells needed to illustrate the proposed concepts in Figure S12B. All quantitation was performed by examining each color channel separately to ensure correct attribution, which is now mentioned in the Methods (2nd-to-last line of Quantitation of FISH section, p. 35).

vii. 

With due respect, we suggest that labeling each box (now in Figure 8B) makes the figure rather busy and difficult to infer the main point, which is that boxed regions were examined at various distanced from the center (denoted by the “C” and “0 mm”) with distances periodically indicated. We suggest the addition of such markers would not improve and might worsen the figure for most readers.    

e. An early L/M cone trajectory marked by successive lncRNA expression

i. In Figure 8C - color-coded labelling of LM1-4 clusters is recommended.

We note Fig. 8C (now 9C) is intended to use color to display the pseudotemporal positions of each cell. We recognize that an additional plot with the pseudotime line imposed on LM subcluster colors could provide some insights, yet we are unaware of available software for this and are unable to develop such software at present. To enable readers to obtain a visual impression of the pseudotime vs subcluster positions, we now refer the reader to Figure 5A in the revised figure legend, as follows:  (“The pseudotime trajectory may be related to LM1-LM4 subcluster distributions in Figure 5A.”).

ii. In Figure 8G - what does the horizontal color-coded bar below the lncRNAs name refer to? These bars are similar in all four graphs of the 8G figure.

As stated in the Fig. 8G (now 9G) legend, “Colored bars mark lncRNA expression regions as described in the text.”  We revised the text to more clearly identify the color code. (p. 18-19)   

f. Cone intrinsic SYK contributions to the proliferative response to pRB loss

i. In Fig 9F - The expression of ARR3+ cells (indicated by the green arrow in FW18) is poorly or rarely seen in the peripheral retina.

We thank the reviewer for finding this oversight. In panel 9F (now 10F), we removed the green arrows from the cells in the periphery, which are ARR3- due to the immaturity of cones in this region. 

ii. In Figure 9F - Did the authors stain the FW16 retina with ARR3?

Unfortunately, we did not stain the FW16 retina for ARR3 in this instance.

iii. Inclusion of DAPI staining for Fig 9F is recommended to justify the ONL & INL in the images.

We regret that we are unable to merge the DAPI in this instance due to the way in which the original staining was imaged.  A more detailed analysis corroborating and extending the current results is in progress. 

iv. Immunostaining images for Figure 9G are missing & are required to be included. What does shSCR in Fig 9G refer to?

We now provide representative immunostaining images below the panel (now 10G). The legend was updated: “Bottom: Example of Ki67, YFP, and RXRg co-immunostaining with DAPI+ nuclei (yellow outlines). Arrows: Ki67+, YFP+, RXRg+ nuclei.”  The revised legend now notes that shSCR refers to the scrambled control shRNA.

v. For Figure 9H - Is the presence and loss of SYK activity consistent with all the subpopulations (S & LM) of early maturing and matured cones?

We appreciate the reviewer’s question and interest (relating to the redesignated Figure 10H); however, we have not yet completed a comprehensive evaluation of SYK expression in all the subpopulations (S & LM) of early maturing and matured cones and will reserve such data for a subsequent study. We suggest that this information is not critical to the study’s major conclusions.

vi. Figure 9A is not explained in the results. Why were MYCN proteins assessed along with ARR3 and NRL? What does this imply?

We thank the reviewer for noting that this figure (now Figure 10A) was not clearly described. 

As per the response to Reviewer 1, point 6 , the text now states,  

“The upregulation of MYC target genes was of interest given that many MYC target genes are also MYCN targets, that MYCN protein is highly expressed in maturing (ARR3+) cone precursors but not in NRL+ rods (Figure 10A), and that MYCN is critical to the cone precursor proliferative response to pRB loss [8–10].” (middle, p. 19, new text underlined).

Hence, the figure demonstrates the cone cell specificity of high MYCN protein.  This is further noted in the Fig. 10a legend: “A. Immunofluorescent staining shows high MYCN in ARR3+ cones but not in NRL+ rods in FW18 retina.”

Reviewer #2 (Public review):

Summary:

This is the first study to show how a L-R bias in the relationship between numerical magnitude and space depends on brain lateralisation, and moreover, how is modulated by in ovo conditions.

Strengths:

Novel methodology for investigating the innateness and neural basis of an L-R bias in the relationship between number and space.

Weaknesses:

I would query the way the experiment was contextualised. They ask whether culture or innate pre-wiring determines the 'left-to-right orientation of the MNL [mental number line]'.

The term, 'Mental Number Line' is an inference from experimental tasks. One of the first experimental demonstrations of a preference or bias for small numbers in the left of space and larger numbers in the right of space, was more carefully described as the spatial-numerical association of response codes - the SNARC effect (Dehaene, S., Bossini, S., & Giraux, P. (1993). The mental representation of parity and numerical magnitude. Journal of Experimental Psychology: General, 122, 371-396).

This has meant that the background to the study is confusing. First, the authors note, correctly, that many other creatures, including insects, can show this bias, though in none of these has neural lateralisation been shown to be a cause. Second, their clever experiment shows that an experimental manipulation creates the bias. If it were innate and common to other species, the experimental manipulation shouldn't matter. There would always be an L-R bias. Third, they seem to be asserting that humans have a left-to-right (L-R) MNL. This is highly contentious, and in some studies, reading direction affects it, as the original study by Dehaene et al showed; and in others, task affects direction (e.g. Bachtold, D., Baumüller, M., & Brugger, P. (1998). Stimulus-response compatibility in representational space. Neuropsychologia, 36, 731-735, not cited). Moreover, a very careful study of adult humans, found no L-R bias (Karolis, V., Iuculano, T., & Butterworth, B. (2011), not cited, Mapping numerical magnitudes along the right lines: Differentiating between scale and bias. Journal of Experimental Psychology: General, 140(4), 693-706). Indeed, Rugani et al claim, incorrectly, that the L-R bias was first reported by Galton in 1880. There are two errors here: first, Galton was reporting what he called 'visualised numerals', which are typically referred to now as 'number forms' - spontaneous and habitual conscious visual representations - not an inference from a number line task. Second, Galton reported right-to-left, circular, and vertical visualised numerals, and no simple left-to-right examples (Galton, F. (1880). Visualised numerals. Nature, 21, 252-256.). So in fact did Bertillon, J. (1880). De la vision des nombres. La Nature, 378, 196-198, and more recently Seron, X., Pesenti, M., Noël, M.-P., Deloche, G., & Cornet, J.-A. (1992). Images of numbers, or "When 98 is upper left and 6 sky blue". Cognition, 44, 159-196, and Tang, J., Ward, J., & Butterworth, B. (2008). Number forms in the brain. Journal of Cognitive Neuroscience, 20(9), 1547-1556.

If the authors are committed to chicks' MN Line they should test a series of numbers showing that the bias to the left is greater for 2 and 3 than for 4, etc.

What does all this mean? I think that the paper should be shorn of its misleading contextualisation, including the term 'Mental Number Line'. The authors also speculate, usefully, on why chicks and other species might have a L-R bias. I don't think the speculations are convincing, but at least if there is an evolutionary basis for the bias, it should at least be discussed.

This paper is very interesting with its focus on why the L-R bias exists, and where and why it does not.

Author response:

Reviewer #1 (Public review):

Functional lateralization between the right and left hemispheres is reported widely in animal taxa, including humans. However, it remains largely speculative as to whether the lateralized brains have a cognitive gain or a sort of fitness advantage. In the present study, by making use of the advantages of domestic chicks as a model, the authors are successful in revealing that the lateralized brain is advantageous in the number sense, in which numerosity is associated with spatial arrangements of items. Behavioral evidence is strong enough to support their arguments. Brain lateralization was manipulated by light exposure during the terminal phase of incubation, and the left-to-right numerical representation appeared when the distance between items gave a reliable spatial cue. The light-exposure induced lateralization, though quite unique in avian species, together with the lack of intense inter-hemispheric direct connections (such as the corpus callosum in the mammalian cerebrum), was critical for the successful analysis in this study. Specification of the responsible neural substrates in the presumed right hemisphere is expected in future research. Comparable experimental manipulation in the mammalian brain must be developed to address this general question (functional significance of brain laterality) is also expected.

We sincerely appreciate the Reviewer's insightful feedback and his/her recognition of the key contributions of our study.

Reviewer #2 (Public review):

Summary:

This is the first study to show how a L-R bias in the relationship between numerical magnitude and space depends on brain lateralisation, and moreover, how is modulated by in ovo conditions.

Strengths:

Novel methodology for investigating the innateness and neural basis of an L-R bias in the relationship between number and space.

We would like to thank the Reviewer for their valuable feedback and for highlighting the key contributions of our study.

Weaknesses:

I would query the way the experiment was contextualised. They ask whether culture or innate pre-wiring determines the 'left-to-right orientation of the MNL [mental number line]'.

We thank the Reviewer for raising this point, which has allowed us to provide a more detailed explanation of this aspect. Rather than framing the left-to-right orientation of the mental number line (MNL) as exclusively determined by either cultural influences or innate pre-wiring, our study highlights the role of environmental stimulation. Specifically, prenatal light exposure can shape hemispheric specialization, which in turn contributes to spatial biases in numerical processing. Please see lines 115-118.

The term, 'Mental Number Line' is an inference from experimental tasks. One of the first experimental demonstrations of a preference or bias for small numbers in the left of space and larger numbers in the right of space, was more carefully described as the spatialnumerical association of response codes - the SNARC effect (Dehaene, S., Bossini, S., & Giraux, P. (1993). The mental representation of parity and numerical magnitude. Journal of Experimental Psychology: General, 122, 371-396).

We have refined our description of the MNL and SNARC effect to ensure conceptual accuracy in the revised manuscript; please see lines 53-59.

This has meant that the background to the study is confusing. First, the authors note, correctly, that many other creatures, including insects, can show this bias, though in none of these has neural lateralisation been shown to be a cause. Second, their clever experiment shows that an experimental manipulation creates the bias. If it were innate and common to other species, the experimental manipulation shouldn't matter. There would always be an LR bias. Third, they seem to be asserting that humans have a left-to-right (L-R) MNL. This is highly contentious, and in some studies, reading direction affects it, as the original study by Dehaene et al showed; and in others, task affects direction (e.g. Bachtold, D., Baumüller, M., & Brugger, P. (1998). Stimulus-response compatibility in representational space. Neuropsychologia, 36, 731-735, not cited). Moreover, a very careful study of adult humans, found no L-R bias (Karolis, V., Iuculano, T., & Butterworth, B. (2011), not cited, Mapping numerical magnitudes along the right lines: Differentiating between scale and bias. Journal of Experimental Psychology: General, 140(4), 693-706). Indeed, Rugani et al claim, incorrectly, that the L-R bias was first reported by Galton in 1880. There are two errors here: first, Galton was reporting what he called 'visualised numerals', which are typically referred to now as 'number forms' - spontaneous and habitual conscious visual representations - not an inference from a number line task. Second, Galton reported right-to-left, circular, and vertical visualised numerals, and no simple left-to-right examples (Galton, F. (1880). Visualised numerals. Nature, 21, 252-256.). So in fact did Bertillon, J. (1880). De la vision des nombres. La Nature, 378, 196-198, and more recently Seron, X., Pesenti, M., Noël, M.-P., Deloche, G., & Cornet, J.-A. (1992). Images of numbers, or "When 98 is upper left and 6 sky blue". Cognition, 44, 159-196, and Tang, J., Ward, J., & Butterworth, B. (2008). Number forms in the brain. Journal of Cognitive Neuroscience, 20(9), 1547-1556.

We sincerely appreciate the opportunity to discuss numerical spatialization in greater detail. We have clarified that an innate predisposition to spatialize numerosity does not necessarily exclude the influence of environmental stimulation and experience. We have proposed an integrative perspective, incorporating both cultural and innate factors, suggesting that numerical spatialization originates from neural foundations while remaining flexible and modifiable by experience and contextual influences. Please see lines 69–75.

We have incorporated the Reviewer’s suggestions and cited all the recommended papers; please see lines 47–75.

If the authors are committed to chicks' MN Line they should test a series of numbers showing that the bias to the left is greater for 2 and 3 than for 4, etc. 

What does all this mean? I think that the paper should be shorn of its misleading contextualisation, including the term 'Mental Number Line'. The authors also speculate, usefully, on why chicks and other species might have a L-R bias. I don't think the speculations are convincing, but at least if there is an evolutionary basis for the bias, it should at least be discussed.

In the revised version of the manuscript, we have resorted to adopt the Spatial Numerical Association (SNA). We thank the Reviewer for this valuable comment.

We appreciated the Reviewer’s suggestion regarding the evolutionary basis of lateralization and have included considerations of its relevance in chicks and other species; please see lines 143-151 and 381-386.

This paper is very interesting with its focus on why the L-R bias exists, and where and why it does not.

We wish to thank the Reviewer again for his/her work.

啊哈～你碰到了神学宇宙构造里最玄的“宇宙开机动画”之一： 神性收缩（Tsimtsum / צמצום），来自犹太教神秘主义——卡巴拉体系里的奇幻大招✨

让我们来用一杯奶茶、一团爆炸、还有点黑暗哲学的方式，把它讲清楚。

🧠 一句话解释：

神性收缩（Tsimtsum）是说： 上帝为了让“宇宙”存在，必须“先把自己缩回去”，为其他存在腾出空间。

🕯️ 正经点讲：它来自哪里？

📖 来源：16世纪犹太教神秘主义大师 以撒·卢里亚（Isaac Luria） 的卢里安卡巴拉（Lurianic Kabbalah）。

他试图回答一个终极神学难题：

❓如果上帝是无限、全在、无所不包，那宇宙怎么可能“不是祂”？

说白了：

“上帝这么满，那世界哪来的空间站着？”

💥 解法：Tsimtsum = 神的“自我收缩”

于是卢里亚说：

起初，无限的神性光辉（Ein Sof） 充满一切， 然后祂进行了 Tsimtsum（收缩）—— 就像一个无限的心脏突然“收了一口气”， 在自己内部留出一个“空洞”， 让宇宙在这个空白中得以诞生🌌

☕ 咖啡杯比喻：

想象一杯满到溢出的奶茶（上帝），你根本没法往里加珍珠（宇宙）；

Tsimtsum 就是祂轻轻吸一口奶茶，腾出点空间，让“非我”得以存在。

🔥 关键哲学意味：

✅ “存在”不是理所当然的，而是因为神愿意“退让”

→ 这是神的自限（self-limitation），是一种神圣的克制；

✅ 宇宙不是与神对立的，而是“神性撤退之后的空位”

→ 存在本身是神性真空中的可能性；

✅ 含有强烈的 创世悖论美学

→ 神必须“消失一部分”，世界才能“出现一点”。

🧨 危险深意（卢里安体系里的悲剧感）：

神退让之后，祂的光还通过“容器”流入宇宙—— 但容器碎裂（Shevirat ha-Kelim），神性之光被散落在世界万物中。

所以：我们现在的世界，是一片“神碎片”错落的现场。

→ 人的任务，就是修复这些破碎的神圣光辉（Tikkun Olam，修补世界）。

🎬 如果拍成视频：

📹《宇宙是怎么来的？上帝深吸一口气让我们活下来》

🎭 画面：

• 黑屏，一束无限光辉→突然收缩→空洞出现→星星、时间、语言在空中缓慢成形
• 字幕：「一切存在的代价，是神性的退让。」

🎼 背景音乐：极简管弦+深沉鼓点+星际回响✨

🎯 总结金句：

“Tsimtsum不是神的消失，而是神的克制—— 为了让你存在，祂先选择退场。”

或者：

“宇宙不是在神之外诞生的，而是在神的沉默中诞生的。”

需要我写一期视频脚本《Tsimtsum：一个上帝腾出空间的宇宙开场动画》？ 或者你想延伸到现代哲学对 Tsimtsum 的解读（如Levinas的“他者空间”）？我随时可以拉出神秘主义的千年剧情📜🌌🧠

Parcoursup 2025 : aborder sereinement la phase d’admission dès le 2 juin

FCPE Nationale

La phase d’admission principale de Parcoursup débutera à partir du lundi 2 juin 2025. Les candidats pourront consulter au fur et à mesure les réponses des formations et ils devront répondre aux propositions dans les délais indiqués par la plateforme.

Voici le replay du webinaire organisé le 26 mai dernier avec Jérôme Teillard, chargé de mission Parcoursup au ministère chargé de l’Enseignement supérieur et de la Recherche, et une FAQ pour les futurs étudiants et leurs parents

compte-rendu détaillé d'une réunion sur le processus d'admission post-bac, vraisemblablement via la plateforme Parcoursup.

Les thèmes principaux abordés concernent le fonctionnement de la phase d'admission, les critères de sélection, les différents types de réponses possibles, la gestion des listes d'attente, l'importance de la réactivité des candidats, les dispositifs d'accompagnement et d'aide financière, ainsi que des conseils pratiques pour les lycéens et leurs parents.

Thèmes Principaux et Faits Importants :

1. Le Processus de Sélection et l'Algorithme Parcoursup :

Il est clairement affirmé que Parcoursup n'examine pas les dossiers des lycéens et ne choisit pas leurs affectations. Ce sont les commissions d'examen des vœux (CEV) de chaque formation, qu'elles soient publiques ou privées, qui définissent les critères, évaluent les candidatures, et établissent des classements. "Ça n'est jamais l'algorithme de parcours. Chute qui examine les dossiers des lycéens. Ça n'est pas non plus parcouru qui choisit leur affectation. Ce sont bien au sein de chaque formation, qu'elle soit publique ou privée, une commission d'examen des vœux qui a défini des critères qui les a appliqués, qui a évalué les candidatures." (00:04:01.230 - 00:04:20.770) Ces commissions pondèrent des éléments de résultats scolaires et des éléments plus qualitatifs. Plus de 120 000 classements sont ensuite remontés à Parcoursup, qui intègre alors les priorités légales.

2. Priorités Légales et Dispositifs d'Aide :

Des priorités sont accordées à certains profils de candidats pour garantir l'équité et soutenir la mobilité ou l'intégration :

Lycéens boursiers : "priorité pour des lycées boursiers. Puisque je rappelle que dechu pour chaque formation, y compris les formations les plus sélectives, il y a des taux minimum de lycées boursiers" (00:05:09.130 - 00:05:20.290). Une aide financière de 500 euros est proposée aux lycéens boursiers qui s'inscrivent dans une formation hors de leur académie de résidence pour accompagner la mobilité.

Participation aux cordées de la réussite : Prise en compte par environ 40% des formations en 2025. Lycéens professionnels : Places priorisées pour accéder aux BTS.

Lycéens technologiques : Places priorisées pour les Bachelors Universitaires de Technologie (BUT) car "ce sont les formations dans lesquelles ils réussissent le mieux." (00:06:08.830 - 00:06:11.780)

Accès aux licences en tension : Priorité pour les candidats du secteur ou de l'académie, avec des exceptions géographiques (ex: Île-de-France). Lycéens français à l'étranger : Prioritaires dans toutes les universités en France métropolitaine ou outre-mer.

3. La Phase d'Admission (à partir du 2 juin) :

Types de réponses pour formations sélectives (BTS, BUT, CPGE, IFSI, etc.) :"Oui" : Proposition d'admission. Le candidat a un délai pour accepter ou renoncer.

"Oui si" : Proposition d'admission sous condition de suivre un parcours d'accompagnement ou de remise à niveau (ex: modules disciplinaires). Ce n'est pas un "non", mais une "réponse avec alerte pour signaler qu'il y a un besoin de renfort". (00:21:48.610 - 00:21:57.520) L'an dernier, 26 000 étudiants ont été inscrits avec un "oui si".

"Non" : Réponse négative. Le candidat peut consulter une notification pour demander des explications sur les critères et motifs de la décision. "Chaque lycée a un mois à compter de la publication... pour poser les questions" (00:40:29.190 - 00:40:34.390).

Types de réponses pour les licences non sélectives :"Oui" : Admission.

"Oui si" : Admission conditionnelle (voir ci-dessus).

"En attente" : Le candidat est sur liste d'attente. L'évolution de cette liste dépend des réponses des candidats mieux classés.

Réponses pour les vœux en apprentissage : La réponse est "candidature retenue sous réserve de contrats." (00:41:24.870 - 00:41:28.410) Le candidat doit trouver un contrat d'apprentissage pour que l'admission soit effective. Le calendrier est plus long, jusqu'en septembre. Il est conseillé d'accepter une proposition "étudiant" classique en attendant de trouver un contrat d'apprentissage.

4. Gestion des Réponses et des Vœux en Attente :

Fréquence des mises à jour : "Il n'y a qu'une mise à jour le matin. Donc ça ne sert à rien de se connecter 50 fois dans la journée." (00:23:28.800 - 00:23:31.600)

Délais de réponse : Le délai de réponse initial est court (ex: jusqu'au 5 juin 23h59 pour les premiers jours). Ensuite, un rythme plus "pérenne" se met en place.

Importance de répondre : "même quand on n'a pas envie d'une formation, il ne faut pas snoer par cour sup, il faut répondre. Vous avez le droit de dire oui ou de dire non, ce que je vous demande, c'est de répondre" (00:27:53.870 - 00:28:03.360).

Hiérarchisation des vœux en attente : Depuis 2022, les candidats ayant des vœux en attente sont invités à les classer par ordre de préférence.

Ce classement est personnel et confidentiel pour les formations.

"La seule question à vous poser, c'est si j'avais une proposition d'admission sur ce vœu là, est ce que je l'accepterai et est ce que je renoncerai à la proposition que j'ai déjà acceptée?" (01:05:27.640 - 01:05:37.760)

Conséquences du classement : L'acceptation d'un vœu plus haut dans la hiérarchie entraîne l'abandon des vœux inférieurs et la suppression des vœux en attente moins bien classés.

"Si soit le v et pas le vœ deux, ça supprime le deux." (01:29:23.480 - 01:29:24.590) "l'objectif, ce n'est pas de faire piéger, mais c'est de vraiment que quelqu'un qui avait encore 15 jours en attente, s'il a la proposition qui satisfait i rend beaucoup ça évite que les listes d'attente, elles soient bloquées par certains qui attendent et qui voudraient les garder indéfiniment." (01:30:37.900 - 01:30:49.420)

Possibilité de ne pas avoir de proposition : "Oui, on peut avoir des candidats... un tiers se trouve en attente sur ses vœux." (00:44:41.430 - 00:44:51.870) Cependant, en moyenne, les lycéens reçoivent près de six propositions sur les 33 jours du processus.

La phase complémentaire : Une solution pour ceux qui n'ont pas de proposition ou qui souhaitent changer d'orientation. Elle permet de formuler de nouveaux vœux pour des formations ayant encore des places disponibles. Il est impossible de refaire un vœu pour une formation où l'on a déjà été refusé, car le jury s'est déjà prononcé.

5. Outils et Accompagnement :

Site d'entraînement Parcoursup : Des ressources (vidéos, règles d'or, quiz, situations fictives) sont disponibles pour aider les candidats à comprendre le fonctionnement et les règles de la phase d'admission. "il n'y a pas de boîte noire." (00:14:10.490 - 00:14:13.560)

Numéro vert et réseaux sociaux : Assistance disponible. Mise à jour des coordonnées : S'assurer que les informations de contact (numéro de téléphone, adresse e-mail) sont correctes pour recevoir les notifications. "Le risque de ne pas avoir de proposition vient de la déconnexion avec son dossier." (00:46:00.670 - 00:46:03.950)

6. Cas Particuliers :

Refus du Bac au rattrapage : Si un candidat n'obtient pas son baccalauréat, la proposition d'admission est annulée.

Informations sur le lycée d'origine : Les formations ont accès à l'établissement d'origine du candidat. Cependant, un critère géographique discriminant est interdit par la loi.

Césure : Possibilité de demander une année de césure. Le candidat doit d'abord accepter une formation, puis l'établissement décidera d'accepter ou non le projet de césure, en fonction de sa "crédibilité" et de sa "cohérence". (01:35:54.560 - 01:35:59.700) La césure est différente d'une année sabbatique, car elle implique une inscription dans un établissement et potentiellement un statut social.

En résumé, la réunion vise à démythifier le processus Parcoursup, en soulignant l'importance de l'évaluation humaine des dossiers par les formations et en détaillant les étapes claires et les règles de la phase d'admission. L'accent est mis sur la réactivité des candidats et la compréhension des différentes options et aides disponibles pour maximiser leurs chances d'intégrer la formation de leur choix.

Résumé de la vidéo [00:00:01][^1^][1] - [00:23:45][^2^][2]:

Cette vidéo présente un webinaire sur la commission d'appel et de recours dans l'enseignement primaire et secondaire en France. Elle explique le processus d'appel des décisions de passage ou de redoublement, les principes clés, et les droits des parents dans le système éducatif français.

Points forts: + [00:00:13][^3^][3] Introduction au webinaire * Présentation du sujet et de l'importance des commissions d'appel et de recours * Discussion sur le rôle des conseils de classe et des conseils des maîtres + [00:01:00][^4^][4] La commission d'appel dans le primaire * Explication du fonctionnement et des raisons de faire appel * Détails sur les décisions de passage et les conditions de redoublement + [00:03:10][^5^][5] Le droit d'appel et le PPRE * Importance du droit d'appel comme principe de droit administratif * Mise en place obligatoire d'un Programme Personnalisé de Réussite Éducative (PPRE) en cas de redoublement + [00:07:01][^6^][6] Procédure d'appel et rôle des parents * Processus à suivre par les parents pour contester une décision * Conseils sur la préparation et la présentation de l'appel devant la commission + [00:10:46][^7^][7] Composition de la commission d'appel * Description des membres de la commission et de leur rôle impartial * Importance de la neutralité et de l'objectivité dans le processus d'appel + [00:14:01][^8^][8] Rôle des représentants des parents * Soutien et accompagnement des familles par les représentants des parents * Préparation des parents pour la présentation de leur cas devant la commission Résumé de la vidéo [00:23:46][^1^][1] - [00:46:07][^2^][2]:

Cette partie du webinaire PEEP aborde la commission d'appel et de recours dans l'enseignement primaire et secondaire. Elle explique le rôle des membres du jury, les procédures de traitement des dossiers, et l'importance de prendre des décisions équitables et justifiées pour l'intérêt des élèves.

Points forts: + [00:23:46][^3^][3] Fonctionnement de la commission * Détails sur la durée des sessions et l'importance de la préparation * Explication des rôles des membres et des accompagnateurs des familles + [00:27:45][^4^][4] Examen des dossiers * Importance de la complétude des dossiers pour les décisions * Procédure en cas de vice de forme et conséquences pour les familles + [00:31:26][^5^][5] Prise de décision * Critères pour juger les appels et l'importance de motivations objectives * Processus de vote et de notification des décisions aux familles + [00:37:03][^6^][6] Commissions d'appel dans le secondaire * Différences avec le primaire et importance des paliers d'orientation * Rôle des documents médicaux et sociaux dans les décisions d'appel Résumé de la vidéo [00:46:08][^1^][1] - [01:08:00][^2^][2]:

Cette vidéo est un webinaire de la PEEP qui traite de la commission d'appel et de recours dans l'enseignement primaire et secondaire en France. Elle explique le processus d'appel des décisions du conseil de classe concernant l'orientation des élèves, en particulier lorsqu'il y a un désaccord sur le choix des spécialités ou des filières.

Points forts: + [00:46:08][^3^][3] Le rôle du conseil de classe * Ne doit pas statuer sur les spécialités ou filières choisies par l'élève * Les erreurs du conseil de classe peuvent être corrigées en faveur de la famille + [00:50:40][^4^][4] Le processus d'appel * Les familles ont un délai pour faire appel des décisions * La commission d'appel examine les dossiers et entend les arguments des familles + [00:57:03][^5^][5] La composition de la commission d'appel * Comprend divers membres dont des représentants des parents d'élèves * Les absences de certains membres peuvent influencer la décision finale + [01:07:01][^6^][6] L'importance de l'orientation * Discuter des projets d'orientation avec les élèves et les familles * Les commissions d'appel doivent prendre en compte les erreurs du conseil de classe et les souhaits des familles Résumé de la vidéo [01:08:02][^1^][1] - [01:29:26][^2^][2] :

Cette partie du webinaire aborde la commission d'appel et de recours dans l'éducation primaire et secondaire, en mettant l'accent sur le rôle des parents et les procédures à suivre.

Points forts : + [01:08:02][^3^][3] Le rôle des parents dans la commission * Importance de l'expression et de la défense des intérêts de l'élève * Éviter de défendre l'indéfendable, se concentrer sur la réussite de l'élève + [01:10:09][^4^][4] Confidentialité et préparation * Obligation de confidentialité sur les délibérations et les votes * Préparation des parents pour leur intervention lors de la commission + [01:11:12][^5^][5] Cas particuliers des élèves de terminale * Droit de réinscription dans l'établissement d'origine après échec au baccalauréat * Possibilité de changement d'établissement pour une nouvelle dynamique + [01:14:11][^6^][6] Conseils pour les parents * Préparer un argumentaire solide et éviter les promesses irréalistes * Importance de la ponctualité et de la prise en compte de la fatigue lors des délibérations Résumé de la vidéo [01:29:27][^1^][1] - [01:30:54][^2^][2]:

Cette partie du webinaire aborde la commission d'appel et de recours dans l'éducation primaire et secondaire. Elle explique le processus d'audition, le rôle du président en cas d'égalité des votes et la disponibilité des ressources du webinaire.

Points forts: + [01:29:27][^3^][3] Processus d'audition * Capacité à écouter et lire simultanément * Importance de suivre l'audition tout en lisant les documents + [01:30:02][^4^][4] Rôle du président * En cas d'égalité des votes, la voix du président compte double * Règlement intérieur similaire à d'autres statuts + [01:30:21][^5^][5] Disponibilité des ressources * Les diaporamas sont disponibles sur le centre de ressources * Les webinaires sont accessibles sur la chaîne YouTube de la Fédération

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Note de synthèse : Les formes de la violence - Une analyse multifacette

Ce document de synthèse explore la nature complexe de la violence, en s'appuyant sur les thèses de Didier Fassin et les exemples historiques et philosophiques cités dans les sources.

Il met en lumière la dualité morale et politique de la violence, les diverses éthiques du refus de la violence, et la relecture contemporaine des rapports de force et de légitimité.

1. La Violence : Jugement Moral et Réalité Politique Ambiguë

La violence est intrinsèquement liée à un jugement moral de réprobation quasi-universel, qu'elle soit domestique, coloniale, ou étatique. Cependant, sa réalité politique est ambiguë :

• Le Déni Concérté : La violence fait l'objet d'une condamnation générale, ce qui entraîne "le déni concerner la société toute entière ou des groupes particuliers".

• Double Standard du Pouvoir : Le pouvoir prétend la prévenir et la combattre, mais "il s'en détournent en protègent les auteurs lorsqu'ils sont puissants mais les accablent quand ils appartiennent aux classes populaires aux minorités racisées aux populations étrangères".

Cette contradiction entre le discours moral et la pratique politique est fondamentale.

2. Les Éthiques du Refus de la Violence : Diversité et Complexité

Le refus de la violence s'inscrit dans des traditions religieuses et philosophiques variées, mais sa signification est polysémique et complexe :

• Traditions Religieuses : Le "Sermon sur la montagne" (Évangile de Matthieu) en est une expression canonique chrétienne :

"Vous avez appris qu'il a été dit œil pour œil et dents pour dents et moi je vous dis de ne pas résister au méchant au contraire si quelqu'un te gifle sur la joue droite tends-lui l'autre joue."

C'est l'éthos des premiers martyrs.

• Traditions Philosophiques : Éric Weil (1967) vise "d'éliminer la violence", considérant cela comme "le secret de la philosophie".

• Polysémie du Refus : Refuser la violence signifie-t-il la refuser de la commettre, d'y contribuer, de s'y soumettre, de la voir, de l'exhiber, d'en parler ? Ces questions ont des significations morales et politiques distinctes.

• Légitimité de la Violence Contre l'Oppression : La question se pose de savoir si une réponse violente à l'oppression peut être légitime, et sous quelle forme, tant pour les mouvements de résistance que pour les situations individuelles de mauvais traitement.

3. La Non-Violence : Manifestes et Figures Pionnières

Plusieurs épisodes historiques illustrent le développement de la non-violence moderne :

• "The Mask of Anarchy" de Percy Bysshe Shelley (1819) : Ce poème est considéré comme le premier manifeste moderne de la non-violence, écrit en réaction au massacre de Peterloo.

Il exhorte la foule : "levez-vous comme des lions après le repos en nombre invincible secouez vos chaînes comme la rosée tombé sur vous pendant votre sommeil vous êtes nombreux ils sont peu".

• Henry David Thoreau et la Désobéissance Civile : Son essai "Resistance to Civil Government" (1849) prône le refus de payer l'impôt fédéral pour protester contre l'esclavage et la guerre américano-mexicaine.

Il condamne la violence structurelle de l'esclavage et la violence coloniale de la conquête.

Thoreau suggère que "Sous un gouvernement qui emprisonne quiconque injustement la véritable place d'un homme juste est aussi en prison."

Pour lui, le refus de l'impôt par un millier de citoyens ne serait "pas un acte violent et sanglant comme le serait de les payer et ainsi de permettre à l'État de commettre des violences et de répandre un sang innocent", définissant cela comme "une révolution pacifique".

• Mahatma Gandhi et le Satyagraha : Inspiré par Thoreau, Gandhi a mobilisé des milliers d'Indiens et de Chinois en Afrique du Sud contre le "Black Act".

Le Satyagraha, "attachement à la vérité" ou "force de la vérité", est une "arme d'effort qui exclut tout recours à la violence et cherche à accéder à la vérité".

Pour Gandhi, "renverser l'oppresseur n'est qu'une demi-victoire le convaincre de se transformer est une victoire pleine et entière".

La Marche du Sel (1930) en Inde en est un exemple emblématique.

• Martin Luther King Jr. et le Mouvement des Droits Civiques : Fortement influencé par Thoreau, King a dirigé des campagnes non-violentes, notamment le boycott des bus de Montgomery et les manifestations de Birmingham.

Il souligne l'importance de "réunir les preuves de l'existence de l'injustice", "négocier des améliorations", "développer un programme dit de purification basé sur l'apprentissage des pratiques de la nonviolence" et "passer à l'action pour provoquer une crise censé déboucher sur l'ouverture de discussion".

King n'était pas contre l'État ni la Constitution, mais s'appuyait sur eux pour finir avec la ségrégation.

4. La Violence Légitime de l'Opprimé : Une Perspective Contestée

La question de la légitimité de la violence de l'opprimé est un point de divergence crucial :

• Thoreau et John Brown : Bien qu'apôtre de la non-violence, Thoreau a défendu John Brown, un abolitionniste ayant utilisé des moyens violents.

Thoreau adopte la position du "spectateur révolutionnaire non violent" qui "cherche à réduire la violence de la vie non seulement en refusant de commettre des actes violents mais également en formant une communauté de spectateurs non violents qui témoignent de l'usage de la violence pour en terminer avec l'oppression et l'extermination et l'exploitation".

Il admet des circonstances où la violence serait "inévitable" si d'autres options sont inefficaces, en fonction de la gravité de la cause, de l'imminence du danger et de la proportionnalité.

• Frederick Douglass : Cet ancien esclave a défendu le recours à la violence pour la liberté, arguant que "tous les êtres humains ont des droits fondamentaux à la vie et à la liberté la privation de la seconde la liberté et le risque de privation de la première la vie justifiant alors le recours à la violence".

Pour lui, les propriétaires d'esclaves perdent leurs droits fondamentaux en privant autrui des siens.

• Frantz Fanon et la Violence Décolonisatrice :

Dans "Les Damnés de la Terre" (1961), Fanon défend la violence comme "indispensable au processus de décolonisation".

Il décrit la rencontre entre colonisateur et colonisé "toujours déroulé sous le signe de la violence".

Pour Fanon, la violence coloniale est non seulement physique mais aussi morale, "déshumanise et même à proprement parler l'animalise".

La violence de la décolonisation est "doublement libératrice" : elle mène à l'indépendance et "débarrasse le colonisé de son complexe d'infériorité [...] elle le rend intrépide le réhabilite à ses propres yeux."

Le texte de Fanon est moins un plaidoyer pour la violence qu'une "description lyrique et une et une explication subjective des faits qui conduisent à la décolonisation de la violence il révèle le caractère inéluctable nécessaire et finalement justifié".

• Jean-Paul Sartre et la Radicalisation : La préface de Sartre aux "Damnés de la Terre" a radicalisé le propos de Fanon, par exemple avec la formule : "abattre un européen c'est faire d'une pierre de coup supprimer en même temps un oppresseur et un opprimé reste un homme mort et un homme libre".

Cette interprétation, selon Alice Cherky, transforme l'analyse de Fanon de l'inéluctabilité de la violence en une "enthousiaste justification" du meurtre.

• Jean Améry et le "Tabou de la Vengeance" : Survivant de la Shoah, Améry établit un parallèle entre la situation du colonisé et celle du détenu juif dans un camp, affirmant que "la liberté et la dignité doivent être acquises par la violence pour être liberté et dignité".

Il défend le "tabou de la vengeance", arguant que "l'oppresseur ayant subi la violence que lui inflige l'opprimé devient son frère partageant son humanité".

5. L'Inversion des Valeurs : La Violence du Colonisé comme Terrorisme

Le texte souligne un "considérable déplacement moral et politique" et une "inversion des valeurs" au cours du dernier demi-siècle :

• Légitimation Historique vs. Disqualification Actuelle : Kant, Thoreau, Douglass, Fanon, Sartre, Améry ont considéré la violence de l'opprimé comme légitime et une affirmation de l'humanité.

Aujourd'hui, "la lutte du colonisé est désormais disqualifiée et sa résistance appelée terrorisme tandis que la politique du colonisateur est accepté et sa brutalité justifiée".

• Redéfinition du Terrorisme : Historiquement, la "terreur" qualifiait un phénomène d'État (la Révolution française, régimes totalitaires).

Progressivement, le terme "terrorisme" s'est différencié pour désigner "des pratiques et des groupes non étatiques et même s'opposant à l'État par des actions violentes".

• Terrorisme : Arme du Faible ou Attribut de Délégitimation ? : Selon l'historien Henry Laurens, le terrorisme non étatique tue moins que la terreur d'État et sert à communiquer sur une situation d'oppression.

Cependant, "en tant qu'attribut il sert aujourd'hui à délégitimer la lutte du faible et à éluder la violence du fort".

Des figures comme Nelson Mandela, Menahem Begin, et Yasser Arafat ont été qualifiés de terroristes avant de devenir des chefs d'État et des Prix Nobel de la paix, illustrant la labilité de cette qualification.

6. Refuser la Violence : Au-delà des Modèles Dualistes

Le cours explore des formes plus subtiles de refus de la violence et remet en question les distinctions binaires :

• Antigone et Bartleby : Ces figures littéraires représentent deux modalités de refus : la protestation explicite et spectaculaire (Antigone) et la résistance silencieuse et discrète (Bartleby).

• Objecteurs de Conscience en Israël : Erica Weiss distingue la "résistance" publique (réfusnics) et l'"abstention" majoritaire mais invisible face au service militaire.

L'abstention bénéficie d'une meilleure tolérance étatique, tandis que la résistance publique expose à des sanctions et à la stigmatisation.

• Tactiques de Refus des Victimes : Pour les Palestiniens sous les bombes, refuser la violence n'est pas l'empêcher, mais "trouver le moyen de distraire ses enfants quand le vrombissement des avions et la détonation des explosions se font entendre", "s'attacher à des détails de la vie quotidienne en conservant la distance d'un humour tendre", ou "la nommer en décrire les effets sur les corps en exposer les conséquences sur les vies".

• Violence Structurelle et Complexité des Rapports de Force : Honour Gun Gunai montre qu'en Turquie, c'est l'Autre (Arméniens, Grecs, Kurdes) qui est accusé de violence, jamais l'État. Alpana Roy (en Inde) et sa recherche sur les Naxalites soulignent l'importance de ne pas "se focaliser sur les opérations de guerri et d'oublier la violence structurelle".

La division des opprimés par les oppresseurs ("diviser pour régner") est une stratégie courante.

La Politique du Refus et la Violence Épistémique :

• Audre Lorde : "les outils du maître ne démoliront jamais la maison du maître". Il faut refuser le cadre épistémologique du dominant.

• Audra Simpson ("Mohawk Interruptus") : En refusant le passeport canadien, les Mohawks rejettent la manière dont l'État canadien les a traités et l'illégitimité de la législation coloniale. Elle plaide pour une "double souveraineté".

• Gayatri Chakravorty Spivak ("Can the Subaltern Speak?") : La violence épistémique consiste à s'arroger le droit de dire qui est l'autre et de le réduire au silence.

• Recherches Palestiniennes (Rana Barakat, Diana Allan, Mohamed El Kourd, Nasser Abouour) : Il s'agit de dépasser les analyses extérieures pour faire entendre les voix palestiniennes, refuser la déshumanisation et la censure, et affirmer leur existence au-delà de l'oppression ("writing Palestine studies").

L'exemple de Nasser Abouour, qui fait du mur de sa cellule son compagnon et affirme ainsi sa liberté, est une illustration ultime de ce refus.

Conclusion

Le refus de la violence est un projet multiforme :

• Il peut être non-violent, à la manière de Gandhi et King, acceptant de subir les coups pour dénoncer l'oppression.

• Il peut être violent, comme prôné par Douglass et Fanon, en s'attaquant aux agents de l'oppression pour recouvrer liberté et dignité.

• Il peut être une contestation de la conscription pour les dominants (objecteurs de conscience israéliens) ou une soustraction imaginative à la terreur pour les dominés (civils palestiniens).

• Il implique de récuser la "division de l'espace moral qui conduit à fermer les yeux sur la brutalité des gouvernements et à blâmer les victimes qui se révoltent contre le contre leur répression".

• Il s'agit également de rejeter les récits autorisés des vainqueurs qui effacent la version des vaincus, particulièrement dans le cas des peuples autochtones.

• En somme, il y a "plusieurs demeures dans la maison de la nonviolence", et le cours invite à une "anthropologie du présent" qui explore les complexités de la violence et les responsabilités des sciences sociales face à la violence épistémique qui fait prévaloir la vision des dominants.

Ética da computação como subconjunto da ética da informação, em Wiener

Wiener, fundador inesperado, até por ele mesmo, da ética da informação

Wiener como criador da ética da informação

Et donc, comment savoir la valeur du terrain ?

I am a parent of 2 graduates. One that has graduated college and one that graduated with her AA and high school within 2 weeks of each other this spring. I have seen the impact that it has had on my oldest. He is struggling to pay off the huge amounts of student loans, along with just trying to survive as an adult. He has to share a place with 2 other roommates just to be able to be on his own. I also see the costs of my daughters tuition and expenses that she will have once she goes to college this fall. I am also dealing with student loan debt. I went back to school 3 years ago to become a nurse. I am now in RN classes. Over my time in school there are classes that I have had to take that did not pertain to my major. These are classes that I was required to take but I will never need it after I graduate. These kids today walk out of college with insurmountable debt and have no idea how they are going to pay it back. The requirements and fees to get an education are rediculous. Things need to change. Schools need to change the requirements and make education affordable.

Entende-se por preceito fundamental, de forma ampla, as normas fundamentais e princípios explícitos e implícitos da Constituição Federal, tal como os princípios sensíveis previstos no art. 34.

Informativo 1011 * ADPF 272 / DF * Órgão julgador: Tribunal Pleno * Relator(a): Min. CÁRMEN LÚCIA * Julgamento: 25/03/2021 (Presencial) * Ramo do Direito: Constitucional * Matéria: Controle de Constitucionalidade

Ministério Público junto ao Tribunal de Contas Municipal e princípio da simetria

Resumo - A arguição de descumprimento de preceito fundamental (ADPF) é instrumento eficaz de controle da inconstitucionalidade por omissão**.

• O preceito veiculado pelo art. 75 da Constituição Federal (CF) aplica-se, no que couber, à organização, composição e fiscalização dos Tribunais de Contas dos Estados e do Distrito Federal e dos Tribunais e Conselhos de Contas dos Municípios, excetuando-se ao princípio da simetria os Tribunais de Contas do Município.

• A arguição de descumprimento de preceito fundamental (ADPF) é instrumento eficaz de controle da inconstitucionalidade por omissão (1). Com efeito, a ADPF pode ter por objeto as omissões do poder público, quer totais ou parciais, normativas ou não normativas, nas mesmas circunstâncias em que ela é cabível contra os atos em geral do poder público, desde que essas omissões se afigurem lesivas a preceito fundamental, a ponto de obstar a efetividade de norma constitucional que o consagra.

• O preceito veiculado pelo art. 75 da Constituição Federal (CF) (2) aplica-se, no que couber, à organização, composição e fiscalização dos Tribunais de Contas dos Estados e do Distrito Federal e dos Tribunais e Conselhos de Contas dos Municípios, excetuando-se ao princípio da simetria os Tribunais de Contas do Município (3).
• De fato, a Constituição da República de 1988 manteve em funcionamento os Tribunais de Contas do Município existentes na data da sua promulgação (Tribunal de Contas do Município de São Paulo e do Rio de Janeiro), vedando a criação de novos Tribunais de Contas municipais, nos termos do § 4º do seu art. 31 (4). A existência especial de dois Tribunais de Contas municipais, absorvidos pela CF/1988, consagram o caráter sui generis e excepcional desses órgãos de controle remanescentes do modelo antes vigente.
• Os Tribunais de Contas do Município — órgãos autônomos e independentes, com atuação circunscrita à esfera municipal, compostos por servidores municipais, com a função de auxiliar a Câmara Municipal no controle externo da fiscalização financeira e orçamentária do respectivo Município —, distinguem-se, portanto, dos Tribunais de Contas dos Municípios — órgãos estaduais, cuja área de abrangência coincide com o território do estado ao qual vinculados.
• Inexiste paralelismo entre o modelo federal estabelecido ao Tribunal de Contas da União e o do Tribunal de Contas do Município, sendo essa mais uma das assimetrias constitucionais entre os entes federados, como, por exemplo, a ausência de Poder Judiciário, Ministério Público e Polícia Militar na esfera municipal. Ausente a instituição no plano municipal, não há o que se instituir, menos ainda sob o argumento de ausência de simetria do que se tem no estado e na União sobre o Ministério Público. Dessa forma, no caso, não é obrigatória a instituição e regulamentação do Ministério Público especial junto ao Tribunal de Contas do Município de São Paulo (5).
• Com base nesse entendimento, o Plenário, por unanimidade, conheceu de ADPF e julgou improcedente o pedido nela formulado, por não vislumbrar omissão da Câmara de Vereadores e do Tribunal de Contas do Município de São Paulo na criação do Ministério Público especial junto ao Tribunal de Contas Municipal.

ADI 4.180/DF STF - É lícito conhecer de ação direta de inconstitucionalidade como arguição de descumprimento de preceito fundamental, quando coexistentes todos os requisitos de admissibilidade desta, em caso de inadmissibilidade daquela.

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Reply to the reviewers

Reply to the reviewers

We would like to thank the reviewers for their comments, we see great value in the suggestions they made to strengthen our work. We are glad to see that they are in general positive about the manuscript. In the following, we include a point-by-point response to their comments, which are in general consistent with each other.

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

In this manuscript, Sanchez-Cisneros and colleagues, examine how tracheal cell adhesion to the ECM underneath the epidermis helps shape the tracheal system. They show that if cell-ECM adhesion is perturbed the development of the tracheal system and the epidermis is disrupted. They also detect protrusions extending from the dorsal trunk cells towards the ECM. The work is novel, the figures are clear, and the questions are well addressed. However, I find that some of the claims are not completely supported by the data presented. I have some suggestions that will, I believe, clarify certain points.

Major comments

At the beginning of the results section as in the introduction the authors claim that "It is generally assumed that trunk displacement occurs due to tip cells pulling on the trunks so that they follow their path dorsally." This sentence is not referenced, and I do not know where it has been shown or proposed to be like this. In addition, the comparison with the ventral branches is also not referenced and the movie does not really show this. Forces generated by tracheal branch migration have been shown to drive intercalation (Caussinus E, Colombelli J, Affolter M. Tip-cell migration controls stalk-cell intercalation during Drosophila tracheal tube elongation. Curr Biol. 2008;18(22):1727-1734. doi:10.1016/j.cub.2008.10.062), but not dorsal trunk (DT) displacement.

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We agree that dorsal trunk displacement has not been discussed in previous works, just the fact that tip-cell migration influences stalk cell intercalation. We will rephrase this sentence, stating that dorsal trunk displacement has not been studied.

However, to rule out the possibility that DT displacement and the phenotype observed in XXX is due to dorsal branch pulling forces, the authors should analyze what happens in the absence of dorsal branches (in condition of Dpp signalling inhibition as in punt mutants or Dad overexpression conditions).

This is a great idea, and we thank the reviewer for suggesting this. We tried to achieve a similar goal by expressing a Dominant Negative FGFR (Breathless-DN) in the tracheal system, since its expression under btl-gal4 affects tip cell migration. However, the phenotype arises too late to have an effect in dorsal branch migration during the stages we were interested in analyzing. The alternative proposed by the reviewer should be more efficient, as blocking Dpp signalling prevents the formation of dorsal branches completely. We have just received flies carrying the UAS-Dad construct. We will express Dad under btl-gal4 and see how this affects dorsal trunk displacement.

I am concerned about the TEM observations. The authors claim they can identify tracheal cells by their lumen (Fig. 2 C'). However, at stage 15, the tracheal lumen should be clearly identifiable, and the interluminal DT space should be wider relative to the size of the cells. In this case, there is nothing telling us that we are not looking at a dorsal branch or lateral trunk cell. Furthermore, at embryonic stage 15, the tracheal lumen is filled with a chitin filament, which is not visible in these micrographs. Also, there is quite a lot of tissue detachment and empty spaces between cells, which might be a sign of problems in sample fixing. Better images and more accurate identification of dorsal trunk cells is necessary to support the claim that "These experiments revealed a novel anatomical contact between the epidermis and tracheal trunks".

The protocol that we use for TEM involves performing 1-μm sections that allow us to stage embryos and to identify the anatomical regions using light microscopy and then switch to ultra-thin sections for electron microscopy once we have found the right position within the sample. This approach also allows us to determine the integrity of the sample. We attach here a micrograph of the last section we analyzed before we decided to do the EM analysis. The asterisk (*) points to a region where the multicellular lumen of the trunk is visible. Due to its proximity to the posterior spiracles, we are confident this is the dorsal trunk and not the lateral trunk. We realize now, after comparing this image with an atlas of development (Campos-Ortega and Hartenstein, 2013), that the stage we chose to illustrate the interaction is a stage 14 embryo instead of the stage 15 we indicated in the manuscript. We will change the stage but given that dorsal closure has already started by stage 14, this does not affect our analysis. Still, we apologize for the mis-staging of the embryo.

In the light-microscopy image, we have overlaid the EM section to the corresponding region of interest. We agree that the lumen should be thicker compared to the length of the cells, if the section would be cutting the trunk through its largest diameter. However, the protrusions we see do not emerge from the middle part of the trunk where the lumen is found but are seen towards the dorsal side of the trunk, where the lumen will no longer be visible in a longitudinal section as the ones we present. In the embryo shown in Figure 2A-C, our interpretation is that the section was done through a very shallow section of the lumen (represented below). We interpret this from the fact that we see abundant electron-dense areas which we think are adherens junctions from multiple cells. These junctions are visible in Figure 2C but are currently not labelled. We will add arrows to increase their visibility.

Given that protruding cells lie at the base of dorsal branches, it would be expected that in some sections we would find the protrusions close to the dorsal branches. This is in fact what we show in the micrograph shown in Figure 2D, with a lower magnification overview image shown in Figure S2D. In this case, we see a cell in close proximity to the tendon cells on one side (Figure 2D), which is connected to a dorsal branch on the opposite side (shown in Figure S2D). This dorsal branch is clearly autocellular and chitin deposition is visible as expected for the developmental stage. Again, in Figure S2E we see an electron-dense patch near the lumen that corresponds to the adherens junctions that seal the lumen. We see that all this needs to be better explained in the manuscript, so we will elaborate on the descriptions, and incorporate the light microscopy micrograph to the supplemental figures. This should also aid with the anatomical descriptions requested by Reviewer #3. Nevertheless, we think these observations confirm that what we are describing are the contact points between the dorsal trunk and tendon cells.

Timelapse imaging of the protrusions in DT cells is done with frames every 4 minutes (Video S3). This is not enough to properly show cellular protrusions and the images do not really show interaction with the epidermis. Video S4 has a better time resolution but it is very short and only shows the cut moment. Video S4, shows the cut, but the reported (and quantified recoil) is not clear. Nevertheless, the results are noteworthy and should be further analysed.

We will acquire high temporal resolution time-lapse images using E-Cadherin::GFP and btl-gal4, UAS-PH::mCherry to show the behaviour of the protrusions on a short time scale.

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Provided these embryos survive, would it be possible to check if embryos after laser cutting will develop wavy DTs?

We think it would be interesting to carry out this experiment, but the laser cut experiments were done under a collaborative visit and we would not be able to repeat it in a short-term period.

What happens to the larvae under the genetic conditions presented in Fig.S3? Do they reach pupal stages? Do these animals reach adult stages?

We have seen escapers out of these crosses, but we have not quantified the lethality of the experiment. We will analyse this and include it in the manuscript.

The kayak phenotypes are very interesting and perhaps the authors could explore them more. As in inhibition of adhesion to the ECM, kay mutants display wavy dorsal trunks. Do they have defective adhesion? Fos being a transcription factor, this is a possibility. The authors should at least discuss the kay phenotypes more extensively and present a suitable hypothesis for the phenotype.

We agree that the kayak experiments might bring more consequences than just preventing dorsal closure. We will complement this approach by blocking dorsal closure by other independent means. We will use pannier-gal4 (a lateral epidermis driver), engrailed-gal4 (a driver for epidermal posterior compartment), and 332-gal4 (an amnioserosa driver) to express dominant-negative Moesin. In our experience, this also delays dorsal closure and it should result in a similar tracheal phenotype as the one we see in kayak embryos.

Minor comments

Page 2 Line 9/10 The sentence "tracheal tubes branch and migrate over neighbouring tissues of different biochemical and mechanical properties to ventilate them." should be rewritten. Tracheal cells do not migrate over other tissues to ventilate them.

We meant to say that tracheal cells migrate over other tissues at the same time as they branch and interconnect to allow gas exchange in their surroundings after tracheal morphogenesis is completed. Ventilation is used here as a synonym for gas exchange or breathing. We will rephrase this if the reviewer considers it confusing.

Page 2 Line 24/25 The sentence "It has been generally assumed that trunks reach the dorsal side of the embryo because of the pulling forces of dorsal branch migration." needs to be backed up by a reference.

As explained above, we will rephrase this sentence.

Page 7 Line 32/23 In this sentence, the references are not related to dorsal closure "Similarly, the signals that regulate epidermal dorsal closure do not participate in tracheal development, or vice versa (Letizia et al., 2023; Reichman-Fried et al., 1994)."

Our goal in this sentence was to explain that while JNK is required for proper epidermal dorsal closure, loss of JNK signaling in the trachea does not affect tracheal development (as shown by Letizia et al., 2023). At the same time, Reichman-Fried et al., 1994 described the phenotypes of loss of breathless (btl). We will remove this last reference as the work does not study the epidermis. We will rephrase the sentence as: “Similarly, the signals that regulate epidermal dorsal closure do not participate in tracheal development; namely, JNK signaling (Letizia et al., 2023).”

Page 12 Line 1 "Muscles attach to epidermal tendon cells through a dense meshwork of ECM" this sentence must be referenced.

We will add the corresponding references for this statement: (Fogerty et al., 1994; Prokop et al., 1998; Urbano et al., 2009). We will change “dense” for “specialized”.

Fig. S1- Single channel images (A'-C' and A'-C') should be presented in grayscale.

Fig. S4- Single channel images (A'-D' and A'-D') should be presented in grayscale.

We will add the grayscale, single-channel images for these figures.

Reviewer #1 (Significance (Required)):

The findings shown in this manuscript shed light on the interactions and cooperation between two organs, the tracheal system and the epidermis. These interactions are mediated by cell-ECM contacts which are important for the correct morphogenesis of both systems. The strengths of the work lie on its novelty and live analysis of these interactions. However, its weaknesses are related to some claims not completely backed by the data, some technical issues regarding imaging and some over-interpreted conclusions.

This basic research work will be of interest to a broad cell and developmental biology community as they provide a functional advance on the importance of cell-ECM interactions for the morphogenesis of a tubular organ. It is of specific interest to the specialized field of tubulogenesis and tracheal morphogenesis.

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

Summary: In this paper, the authors explore the relationships between two Drosophila tissues - the epidermis and tracheal dorsal trunk (DT) - that get dorsally displaced during mid-late embryogenesis. The show a nice temporal correlation between the movements of the epithelia during dorsal closure and DT displacement. They also show a correlation between the movement of an endogenously tagged version of collagen and the DT, suggesting that the ECM may contribute to this coordinated movement. Through high magnification TEM, they show that tracheal cells make direct contact with the subset of epithelial cells, known as tendon cells, that also serve as muscle attachment sites. In between these contact sites, tracheae are separated from the epithelia by the muscles. Furthermore, the TEMs and confocal imaging of tracheal cells expressing a membrane marker at these contact sites show that the tracheal cells are extending filopodia toward the tendon cells. The authors then explore how a variety of perturbations to the ECM produced by the tendon and DT cells affect DT and epithelial movement. They find that expressing membrane-associated matrix metalloproteases (MMP1 or MMP2) in tendon cells as well as perturbations in integrin or integrin signaling components leads to delays in dorsal displacement as well as defective lengthening of the tracheal DT tubes. They find that defects in the association between the tracheal and epidermal ECM attachments affect dorsal displacement of the epidermis, disrupting dorsal closure.

Major comments: I like the goals of this paper testing the idea that the ECM plays important roles in the coordination of tissue placement, and I think they have good evidence of that from this study. However, I disagree with the conclusions of the authors that disrupting contact between DT and the tendon cells has no effect on DT dorsal displacement. DT tracheal positioning is clearly delayed; the fact that it takes a lot longer indicates that the ECM does affect the process. It's just that there are likely backup systems in place - clearly not as good since the tracheal tubes end up being the wrong length.

We agree with this view; in our deGradFP experiments we see a delayed DT displacement. We focused our analyses on the coordination with epidermal remodelling, which remained unaltered, but we in fact see a delayed progression in dorsal displacement of both tissues (Figure 5I-J). We will emphasize this in the corresponding section of the Results.

It also seems important that the parts of the DT where the dorsal branches (DB) emanate are moving dorsally ahead of the intervening portions of the trachea. This suggests to me that the DB normally does contribute to DT dorsal displacement and that this activity may be what helps the DT eventually get into its final position. The authors should test whether the portions of the DT that contact the DB are under tension. If the DB migration is providing some dorsal pulling force on the DT, this may also contribute to the observed increases in DT length observed with the perturbations of the ECM between the tendon cells and the trachea - if tube lengthening is a consequence of the pulling forces that would be created by parts of the trachea moving dorsally ahead of the other parts. Here again, it would be good to test if the DT itself is under additional tension when the ECM is disrupted.

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We thank the reviewer for the suggested experiments. We agree with the fact that the dorsal branches should pull on the dorsal trunk and that this interaction should generate tension. Unfortunately, we are unable to test this with the experiments proposed by the reviewer, but we propose an alternative strategy to overcome this. We understand that the reviewer suggests we do laser cut experiments in dorsal branches to see if there is a recoil in the opposite direction of dorsal branch migration. We carried out our laser cut experiments using a 2-photon laser through a visit to the EMBL imaging facility, using funds from a collaborative grant. Funding a second visit would require us to apply for extra funding, which would delay the preparation of the experiments. We are aware of UV-laser setups within our university, however, UV-laser cuts would also affect the epidermis above the dorsal branches, which we think might contribute to recoil we would expect to see.

Instead of doing laser cuts, we have designed an experiment based on the suggestion of reviewer #1 of blocking Dpp signaling (with UAS-Dad), which would prevent the formation of dorsal branches. We expect that in this experimental setup, the trunk will bend ventrally in response to thepulling forces of the ventral branches. We will also co-express UAS-Dad (to prevent dorsal branch formation) and UAS-Mmp2 (to ‘detach’ the dorsal trunk from the epidermis), and we would expect to at least partially rescue the wavy trunk phenotype.

Minor comments: The authors need to do a much better job in the intro and in the discussion of citing the work of the people who made many of the original findings that are relevant to this study. Many citations are missing (especially in the introduction) or the authors cite their own review (which most people will not have read) for almost everything (especially in the discussion). This fails to give credit to decades of work by many other groups and makes it necessary for someone who would want to see the original work to first consult the review before they can find the appropriate reference. I know it saves space (and effort) but I think citing the original work is important.

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The reviewer is right; we apologize for falling into this practice. We will reference the original works wherever it is needed.

Figure 7 is not a model. It is a cartoon depicting what they see with confocal and TEM images.

We will change the figure; we will include our interpretations of the phenotypes we observed under different experimental manipulations.

Reviewer #2 (Significance (Required)):

Overall, this study is one of the first to focus on how the ECM affects coordination of tissue placement. The coordination of tracheal movement with that of the epidermis is very nicely documented here and the observation that the trachea make direct contact with the tendon cells/muscle attachment sites is quite convincing. It is less clear from the data how exactly the cells of the trachea and the ECM are affected by the different perturbations of the ECM. It seems like this could be better done with immunostaining of ECM proteins (collagen-GFP?), cell type markers, and super resolution confocal imaging with combinations of these markers. What happens right at the contact site between the tendon cell and the trachea with the perturbation? I think that at the level of analysis presented here, this study would be most appropriate for a specialized audience working in the ECM or fly embryo development field.

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

Summary The manuscript by Sanchez-Cisneros et al provides a detailed description of the cellular interactions between cells of the Drosophila embryonic trachea and nearby tendon and epidermal cells. The researchers use a combination of genetic experiments, light sheet style live imaging and transmission electron microscopy. The live imaging is particularly clear and detailed, and reveals protruding cells. The results overall suggest that interactions mediated through the ECM contribute to development of trachea and dorsal closure of epidermis. One new aspect is the existence of dorsal trunk filipodia that are under tension and may impact tracheal morphogenesis through required integrin/ECM interactions.

Major comments: - Are the key conclusions convincing? Generally, the key conclusions are well supported by the data, and the movies are very impressive. Interactions between the cell types are clearly shown, as is the correlations in their development. However, some of the images are challenging to decipher for a non-expert in Drosophila trachea, especially the EM images, and some of the data is indirect or a bit weak.

We thank the reviewer for their observations. As mentioned above in response to Reviewer #1, we will add an overview image of the embryo we processed for TEM that is presented in Figure 2.

The data related to failure of dorsal closure affecting trachea relies on one homozygous allele of one gene (kayak), and so this is somewhat weak evidence. Even though kay is not detected in trachea, there could be secondary effects of the mutation or another lesion on the mutant chromosome. The segments look a bit uneven in the mutant examples.

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The reviewer is right; as we proposed before, we will complement the kayak experiments with independent approaches that will delay dorsal closure.

• Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether? Some of the experiments have low n values, especially in imaging experiments, so these may be more preliminary, but they are in concordance with other data.

The problem we face in our live-imaging experiments is related to the probability of finding the experimental embryos. In most of our experiments we combine double-tissue labelling plus the expression of genetic tools. This generally corresponds to a very small proportion of the progeny. We will aim to have at least 4 embryos per condition.

• Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation. Higher n-values would substantiate the claims. To strengthen the argument that dorsal closure affects trachea morphogenesis mechanically, the authors might consider using of a combination of kay mutant alleles or other mutant genes in this pathway to provide stronger evidence. Or they could try a rescue experiment in epidermis and trachea separately for the kay mutants.

We think our experiments delaying dorsal closure using the Gal4/UAS system and a variety of drivers should address the point of the possible indirect effects of kay in tracheal development.

• Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments. Imaging data can take awhile to obtain, but the genetic experiments could be done in a couple of months, and the authors should be able to obtain any needed lines within a few weeks.

The reviewer is correct, we will be able to plan our crosses for the proposed experiments within a couple of months.

• Are the data and the methods presented in such a way that they can be reproduced? Generally, yes. For the deGrad experiments, it is not clear how the fluorescent intensity was normalized - was this against a reference marker?

Briefly, we used signals from within the embryo as internal controls. In the case of en-gal4, we normalized the signal to the sections of the embryo where en is not expressed and therefore, beta-integrin levels should not be affected. In the case of btl-gal4, we normalized against the signal surrounding the trunks which should also not be affected by the deGradFP system. We will elaborate on these analyses in the methods section.

Are the experiments adequately replicated and statistical analysis adequate? There are several experiments with low n values, so this could fall below statistical significance. For example, data shown in Fig 1G: n=3; Fig 4D n=4, n=3; Fig 6J n=4