Replace with: This normally occurs in a context where the action is “out of the ordinary”, and usually occurred when either a deserter fled and assumed a new life (as reported in figure c), or when they had subsequently been caught, tried and even executed as shown in figure d. More detailed reporting also often occurred when there were other notable vents related to desertion (such as theft, arson or violence).

Fig. c Example of a more detailed description of a case of desertion, 1 May 1899, De locomotief: Samarangsch handels- en advertentie-blad

Fig, d Newspaper description of the execution of a deserter, 21 December 1897, Soerabaijasch handelsblad

Replace with: This is highlighted in the above article (figure a), which describes how Chinese “agents” actively recruited deserters of the Dutch East Indies armed forces, and would assist them in desertion by offering large sums of money, clothes, and transportation to join the Chinese army.

Fig. a Article "Japan op Java" 16th February 1899, De Morgenpost

Replace with: The vast majority of articles list “rechtszaken” (see figure b) and detail the numbers of individuals who have deserted, usually with their name, age, occupation, and the punishment afforded to them.

Fig. b Example of 'Law suits' section of a newspaper, 17 March 1897, Vlaardingsche courant

Replace with: 'Flight as Fight': Exploring Historic Cases of Desertion from a Collective Labour Action Perspective

Replace with: The above figure details the spread of annotated articles over time. Of the 387 events tagged, we see that there are a few hits between 1810 and 1830, sporadically changing over time, with two peaks in desertion events between 1830 and 1840.

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

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Reply to the reviewers

Manuscript number: RC-2024-02491

Corresponding author(s): Gilbert, Vassart

1. General Statements [optional]

We thank referees 1 and 2 for their in-depth analysis of our manuscript. They see interest in our study, with questions to be answered. Referee 3 is essentially negative, considering that there is nothing new ("novel finding is missing"). We respectfully disagree with him/her, comforted by the opinion of referee 2 that "the authors developed a protocol to reproducibly generate fetal-like spheroids from adult tissue is an important advancement in the field and ... the manuscript should attract a significant amount of attention in the intestinal field" and we provide evidence in our answers that he/she did not read the manuscript with the same attention as referees 1 and 2 (see in particular answer to his/her question 5).

Here is a summary of the main reason why we consider that our study represents valuable new information in the field of intestinal regeneration.

It is based on the serendipitous observation that dissociation of adult intestinal tissue by collagenase generates stably replatable spheroids upon culture in matrigel. Surprisingly and contrary to canonical EDTA-generated intestinal organoids and fetal spheroids, these spheroids are not traced in Rosa26Tomato mice harboring a VilCre transgene, despite expressing robustly endogenous Villin. Our interpretation is that adult intestinal spheroids originate from a cell lineage, distinct from the main developmental intestinal lineage, in which the VilCre transgene is unexpectedly not expressed, probaly due to the absence of cis regulatory sequences required for expression in this lineage.

Adult spheroid transcriptome shares a gene signature with the YAP/TAZ signature commonly expressed in models of intestinal regeneration. This led us to look for VilCre negative crypts in the regenerating intestine of Lgr5/DTR mice in which Lgr5-positive stem cells have been ablated by diphtheria toxin. Numerous VilCre negative clones were observed, identifying a novel lineage of stem cells implicated in intestinal regeneration.

FACS purification and scRNAseq analysis of the rare VilCre negative cells present at homeostasis identified a population of cells with characteristics of quiescent stem cells.

In sum, we believe that our study demonstrates the existence of a hitherto undescribed stem cell lineage involved in intestinal regeneration. It points to the existence of a hierarchical model of intestinal regeneration in addition to the well-accepted plasticity model.

2. Description of the planned revisions

See section 3 below.

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

Here is a point-by-point reply to the queries of the three referees, with indication of the revisions introduced in the manuscript.

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

• *In this manuscript, Marefati et al report an Lgr5-independent lineage in the regenerating intestine using in vitro organoids and in vivo injury-coupled lineage tracing model. In organoids, collagenase/dispase dissociated resulted in "immortal spheroids" that maintain a cystic and undifferentiated phenotype in the absence of standard growth factors (Rspondin/Noggin/EGF). Bulk RNAseq of spheroids demonstrates downregulation of classical CBC signatures and upregulation of fetal spheroid, mesenchymal, inflammation and regenerative signatures. In mice, Villin-Cre lineage tracing revealed some Villin- negative progenies that lack reporter tracing throughout crypt-villus ribbons after injury.

*The authors proposed that there is Lgr5-independent population support the regenerative response upon CBC depletion. A major caveat of this study is the identification of this population is based on absence of VilCre expression. *

We respectfully disagree. It is precisely this characteristic that makes the interest of our study. Whereas mosaicism of transgene expression is widespread and usually of little significance, our study shows that the rare VilCre-negative cells in the intestinal epithelium are not randomly showing this phenotype: they give specifically birth to what we call adult spheroids and regenerating crypts, which cannot be due to chance. The absence of VilCre expression allows tracing these cells from the zygote stage of the various VilCre/Ros26 reporter mice. We have modified our text to emphasize this point.

*It is surprising that there is no characterisation of Lgr5 expression throughout the manuscript whilst claiming of a Lgr5- independent lineage. *

We understand the perplexity of the referee not to see direct Lgr5 expression data in our manuscript, given our title. However, our point is that it is the cells at the origin of adult spheroids and the regenerating crypts we have identified that are Lgr5-negative, not the spheroids or the regenerated crypts themselves. Those are downstream offspring that may, and indeed have, gained some Lgr5 expression (e.g. figure 3F). We believe that our data showing that VilCre-negative spheroids are not traced in Lgr5-CreERT2/Rosa reporter mice convincingly demonstrate absence of Lgr5 expression in the cells at the origin of adult spheroids (figure 4G). We think that this experiment is better evidence than attempts to show absence of two markers (Tom and Lgr5) in the rare "white" cells present in the epithelium. Regarding the Lgr5 status of cells at the origin of the regenerating "white" crypts that we have identified, the early appearance of these crypts following ablation of CBC (i.e. Lgr5+ve) cells is a strong argument that they originate from Lgr5-negative cells. Regarding the scRNAseq experiment, Lgr5 transcripts are notoriously low and difficult to measure reliably in CBCs (Haber et al 2017). However, blowing up the pertinent regions of the merged UMAP allows showing some Lgr5 transcripts in clusters 5,6 and none in cluster 1 of figure 8GH. Given the very low level of detection, we had chosen not to include these data in the manuscript, but we hope they may help answer the point of the referee (see portion of UMAP below, with Olfm4 as a control, together with the corresponding violin plot). Several markers that gave significant signals in the CBC cluster (Smoc2, Axin2, Slc12a2) were virtually undetectable in the Olfm4-low /Tom-negative cluster of our scRNAseq data (figure 8I) supporting our conclusion.

Although the research question is potentially interesting, the concept of epithelial reprogramming upon injury is well documented in the field. The data generated in this manuscript also seem to be preliminary and lack of detailed characterisation. Below are specific comments.

We do not question the existence of epithelial reprogramming upon injury. We believe our data show, in addition to this well demonstrated phenomenon, the existence of rare cells traced by absence of VilCre expression that are at the origin of a developmental cell lineage distinct from Lgr5+ stem cells and also implicated in regeneration.

• Expression of Lgr5 should be properly characterised throughout the manuscript in both organoid models and injury-induced regeneration in vivo.
• *

See above for a detailed answer to this point.

• An important question is the origin of these "Lgr5-independent" adult spheroids. They look and appear like fetal organoids, which could be induced by injury (e.g. upon collagenase/dispase dissociation). Have the authors tried to culture fetal spheroids in BCM over extensive period of time? Do they behave the same? This would be a great way to directly compare the collagenase/dispase-derived organoids with fetal origin. * *Fetal spheroids require ENR for survival and die in BCM. We have chosen to illustrate this point in Fig2A by showing that, contrary to adult spheroid, they die even when only Rspondin is missing.

• Fig 1C, Why is the replating spheroid culture time different between mesenchymal cells and conditioned medium? We took the earliest time showing convincingly the return to the organoid phenotype. This timing difference does not modify the conclusion that EDTA organoids becoming spheroid-like when exposed to factors originating from mesenchymal cells revert to the organoid phenotype when returned to ENR medium without mesenchymal influence.

• *It is unclear how the bulk RNA-seq data in Fig. 3 were compared. How long were the adult organoids and spheroids cultured for (how many passages)? Were they culture in the same condition of were they in ENR vs BCM? * Both EDTA organoids and spheroids displaying a stable phenotype were used in this experiment. Organoids were collected at passage 4, day 5; spheroids were collected at passage passage 9 day 3.

As stated in the legend to the figure: "...to allow pertinent comparison spheroids and organoids were cultured in the same ENR-containing medium...".

These are important information to consider when interpreting the results. For instance, are Ptgs1 & Ptgs2 expression in adult spheroids the same in ENR vs BCM? Are the gene signatures (regenerative, fetal and YAP) changed in adult spheroids culturing in ENR vs BCM?

We did compare bulk RNAseq of EDTA organoids to ENR-cultured spheroids, short term (passage 6, day 6) BCM-cultured spheroids and long term BCM-cultured (passage 26, day 6) spheroids. To avoid overloading the manuscript these data were not shown in the original manuscript. In summary the BCM-cultured spheroids display a similar phenotype as those cultured in ENR, but with further de-differentiation. See in revision plan folder the results for PTGS, some differentiation markers and fetal regenerative markers including YAP induced genes.

We have included a brief description of these data in the new version of the manuscript and added an additional supplementary file (Suppl table 2) presenting the whole data set.

• It is stated: "In agreement with their aptitude to grow indefinitely, adult spheroids express a set of upregulated genes overlapping significantly with an "adult tissue stem cell module" [159/721 genes; q value 2.11 e-94) (Fig.S2F)].". What is the definition of "indefinitely"? Are they referring to the Fig 1B where spheroid were passaged to P10? The authors should avoid the term "indefinitely" but use a more specific time scale, e.g. passages, months etc.

We agree that the term indefinitely should be avoided, as it is vague. We have introduced the maximum number of passages during which we have maintained the stable spheroid phenotype (26 passages). Also worth noting, the spheroids could be frozen and cultured repeatedly over many months.

SuppFig 3D: Row Z-Score is missing the "e" in Score.

Corrected

• Fig 4E: Figure legend says QNRQ instead of CNRQ. Corrected

• Fig 4G: The brightfield image of adult spheroids 5 days after 3x TAM injections doesn't look like a spheroid. It seems to be differentiating. True, the choice was not the best as the spheroids started to darken. When further replated, however, the offspring of these spheroids showing a clear phenotype remain negative 30 days after tamoxifen administration as shown on the figure. We are sorry, but for reasons explained in section 4 below, we cannot redo the experiment to get a better picture.

• Fig 4: Most mouse model data are missing the number of mice & their respective age used for organoid isolation. We have introduced these data in the legend.

• *Fig 4A-D, H-G: How was fluorescent signal of organoids quantified? *

The settings of fluo imaging or time of LacZ staining were the same for organoids and spheroid pictures. This has been added to the material and methods of the figure and an example is shown below for Rosa26Tomato.

*How many images? * 2 per animal per condition.

*Were there equal numbers of organoids? *

No, see number of total elements counted added to the figure

This all needs to be included in methods/figure legends.

We have introduced additional pertinent information in the material and methods section.

• Figure 4B-D, G-H: Which culturing conditions were used for adult spheroids? Original method or sandwich method? These data were obtained with the original protocol

• Fig 6D-E: Please add the timepoint after DT administration these samples are from. It is not listed in text or figure legend. These samples were those obtained from mice sacrificed at the end of the 5 day period as indicated in panel A. This has been emphasized in the legend of the figure.

• SuppFig 6D: again timepoint is missing. In this experiment all samples were untreated as indicated. This has been emphasized in the legend of the figure.

• SuppFig 6: How were the crypts of these mice (DT WT & DT HE) isolated? Was this via EDTA? This was RNA extracted from total uncultured EDTA-released material (crypts). This has been emphasized in the legend of the figure.

Also, what is the timepoint for isolation for these samples? Even if untreated, the timepoint adds context to the data. Please add more context to describing these different experiments, either in the figure legends or methods section.

All these experiments were from 2 month old animals. We have indicated this in the legend of the figure.

• SuppFig 6E: The quality of the heatmap resolution is too poor to read gene names. We have improved the resolution of the figure and hope the name of the genes are readable now.

• 5-7, are the regenerating crypt-villus units fully differentiated or are they maintained in the developmental state? Immunostaining of markers for stem cells (Lgr5), differentiated lineages (Alpi, Muc2, Lyz, ChgA etc.) and fetal state (Sca1, Trop2 etc) should be analysed in those "white" unrecombined crypt-villus units. The differentiation phenotype is shown by the clear presence of morphologically-identified Paneth and Goblet cells. We agree that specific immunostainings could have been performed to further explore this point. Regarding the fetal state, Clu expression was shown during the regeneration period (see figure 7D,E).

Unfortunately, for reasons explained in section 4 below, we are not in a position to perform these additional experiments.

• The following text needs clarification: "The kinetics of appearance of newly formed un-recombined ("white") crypts was studied after a single pulse of DT (Fig.7A). This demonstrated an increase at 48 hours, with further increase at day 10 and stable maintenance at day 30. The presence of newly formed white crypts one month after toxin administration indicates that the VilCre-negative lineage is developmentally stable and does not turn on the transgene during differentiation of the various epithelial lineages occurring after regeneration (Fig.7B).

*Comment: The "newly formed" is an overstatement, the data doesn't conclude that those are "new" crypts. *

Except if we do not understand the point, we think we can write that a fraction of "white" crypts must be "newly formed", since they are in excess of those present in untreated animals at the same time point.

*The end of the sentence states that these "white" crypts form developmentally stable lineages, thus these white crypts at day 30 could originate from the initial injury. *

As stated above, we consider that crypts found in excess of those present in untreated animals result from the initial injury.

*There was no characterisation of the various epitheial lineages. Are they fully differentiated? *

See above the point related to Paneth cells and Goblet cells.

Is Lgr5 expressed one month after toxin administration? Can the VilCre neg lineage give rise to CBCs?

We have tried hard to show presence or absence of Lgr5 in white crypts at the various times following DT administration. We tried double RFP / Lgr5-RNA scope labeling and double GFP/RFP immunolabeling. Unfortunately, we could not get these methods to produce convincing specific labeling of CBCs in homeostatic crypts, which explains why we could not reach a conclusion regarding the white crypts.

However, there is an indirect indication that "chronic" white crypts (i.e. those caused by DTR expression in CBC, plus those observed 30 days after DT administration) do not express Lgr5. Indeed, acute regeneration indicated by Clu expression at day 5 in Fig.7C is lower in white crypts than in red ones strongly suggesting that white crypts preexisting DT administration (the "chronic ones) do not express Lgr5DTR.

The relationship between white crypt generation and appearance of Clu-positive revival cells (Ayyaz et al., 2019) was then explored. In agreement with others and similar to what happens in the irradiation model, (Ayyaz et al., 2019; Yuan et al., 2023) Clu-positive cells were rare in crypts of untreated mice and their number transiently increased forty-eight hours after a single pulse of DT, and more so after three pulses of DT (Fig.7C,D).

Comment: Comparing 1 pulse at day 2 vs 3 pulses at day 5 makes the data hard to interpret. How is the Clu ISH level for 1 pulse at day 5? Are they equivalent?

After a single pulse of of DT, Clu is only transiently increased. As shown by Ayyaz et al it is back to the starting point at day 5 (supplementary figure 4 of Ayyaz et al).

Clu-positive cells were less frequently observed in white crypts (see "Total" versus "White" in Fig.7C). This fits with the hypothesis that Clu expression marks acutely regenerating crypts and that a proportion of the white crypts are chronically regenerating due to DTR expression in CBCs."

*Comment: I believe the authors suggested that the discrepancy of less Clu expression in white crypts is due to the ectopic expression of DTR in CBCs causing low grade injury without DT administration. This means that some white crypts could have been formed before the administration of DT, and thus are on a different regenerative timeline compared to the white crypts formed from DT administration. *

Yes, this is our interpretation. We have clarified it in the text.

Is there any proof of the chronic regeneration? Immunostaining of chronic regenerative markers such as Sca1, Anxa1 or Yap1 nuclear localization would support the claim. It'd be important to show only the white crypts, but not the RFP+ ones, show regenerative markers.

We think that the steady state higher number of white crypts in untreated Lgr5-DTR animals, compared to wild type siblings indicates chronical low-grade regeneration, which is supported by the RNAseq data (Suppl fig6). It must be noted, however, that this phenotype is mild compared to the well described fetal-like regeneration phenotype described in most injury models. Since these white crypts were made at undetermined earlier stages, the great majority of them are not expected to show markers of acute regeneration like Clu, Sca1....

Fig 7D-E: What are the timepoints of harvest for HE-WT-HE 1 pulse DT mice and HE- HE-HE PBS injected mice?

We have added this information in the figure.

• *Fig 8-9: Regarding the CBC-like Olfm4 low population, what is the status of Lgr5? This should be shown in the figure since the argument is that this is an Lgr5-independent lineage. * See response to the second point.

And what about the regenerative, Yap, mesenchymal and inflammatory signatures? Are they enriched in the white crypts similar to the in vitro spheroids?

In a portion of white crypts, those we believe are newly formed after CBC ablation (see above), there is a transient increase in Clu, which may be considered a marker of Yap activation. In the CBC-like Olfm4 low cells, as seen by scRNAseq, there is nothing like an actively regenerating phenotype. This is expected, since these cells are coming from homeostatic untreated VilCre/Rosa26Tom animals and are supposed to be quiescent "awaiting to be activated".

Reviewer #1 (Significance (Required)):

Strengths: The study employed a range of in vitro and in vivo models to test the hypothesis.

• *

*Limitations: Unfortunately, the models chosen did not provide sufficient evidence to draw the conclusions. Injury induced reprogramming, both in vivo and in vitro, has been well documented in the field. The new message here is to show that such reprogrammed state is continuous rather than transient; instead of regenerating Lgr5+ stem cells, it can continue to differentiate to all cell lineages in Lgr5-independent manner.

*

We respectfully disagree with this analysis of our results. What we show is not "that such reprogrammed state is continuous rather than transient; instead of regenerating Lgr5+ stem cells, it can continue to differentiate to all cell lineages in Lgr5-independent manner", but that a quiescent stem cell line, not previously identified, is activated to regenerate a portion of crypts following CBC ablation. These cells are not reprogrammed, they correspond to a developmental lineage waiting to be activated and keep their VilCre-negative state at least of 30 days. We believe that their "by default tracing" (VilCre negative from the zygote stage) is as strong an evidence for the existence of such a lineage as positive lineage tracing would be. The increase in crypts originating from this lineage after CBC ablation indicates that it is implicated in regeneration. We do not question the well-demonstrated plasticity-associated reprogramming taking place during regeneration; we simply suggest that this would coexist with the involvement of the quiescent VilCre-negative lineage we have identified.

*However, through the manuscript, there was no immunostaining of Lgr5 and other differentiation markers. The conclusion is an overstatement without solid proof. * We have provided the best answer we could to this point in our answer to the second question of the referee hereabove.

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

In this manuscript, the Marefati et al. developed a novel approach to generate spheroids from adult intestinal epithelium using a collagenase/dispase based protocol. Adult spheroids were found to be distinct from classic budding-type organoids normally generated from EDTA based release of the crypt epithelium. Transcriptional profiling indicated that adult spheroids were undifferentiated and similar to regenerating crypts or fetal spheroids. To identify the cell of origin that generates adult spheroids, the authors labelled epithelial cells with VilCreERT-LSL-Tom, VilCre-LSL-GFP and Lgr5CreERT- LSLTom mice. From these experiments the authors conclude that that spheroids are only generated from Vil-Cre negative and Lgr5 negative cells. Next the authors deleted the anti- apoptotic gene Mcl1 using Vil-CreERT mice. This led to a strong apoptotic response throughout the crypt epithelium and tissues processed from knockout mice readily generated spheroids, and in vivo, replenishment of the gut epithelium was mediated by unrecombined cells. In a second model, CBCs were ablated using Lgr5DTR mice and VilCre negative cells were found again to contribute to regeneration of the crypt epithelium. Finally based on the absence of Vil-Cre reporter activity, the authors were able to sort out and perform scRNAseq to profile VilCre negative cells. These cells were found to be quiescent, express the stem cell marker Olfm4 and were also abundant in ribosomal gene expression.

• *

The fact that the authors developed a protocol to reproducibly generate fetal-like spheroids from adult tissue is an important advancement in the field. Previous reports have shown that treatment with various small molecule inhibitors can revert budding organoids into a spheroid morphology, but this manuscript demonstrates that spheroids can also be generated from otherwise untreated cells. This new methodology will provide new tools to dissect the molecular determinants of fetal/regenerative cells in the gut. Based on this, the manuscript should attract a significant amount of attention in the intestinal field.

• *

As pointed out by the authors themselves the study has important limitations that diminish enthusiasm. The primary issue relates to the inability of the team to identify markers of VilCre neg cells other than the fact that these cells are Olfm4+ and quiescent. Nonetheless, for the reasons stated above the manuscript should reach the target audience within the research community, if the authors can address the specific points below related to issues with methodology as well as defining more precisely the characteristics and growth requirements of adult spheroid cultures.

Thank you for this positive analysis of our study.

Major comments

The main conclusion of the study is that Vil-Cre neg cells are rare quiescent Olfm4+ crypt cells. If this is the case, then standard EDTA treatment should release these cells as well. Consequently, spheroids should also emerge from isolated crypts grown in the absence of ENR. If this is not the case how do the authors explain this?

We have tried hard to generate spheroids by culturing EDTA organoids in medium lacking ENR and by treating EDTA organoids with collagenase/dispase, without success. Therefore, we are left with the conclusion that spheroid-generating cells must be more tightly attached to the matrix than those released by EDTA, and that it is their release from this attachment by collagenase that triggers a regeneration-like phenotype. This hypothesis is supported by several models of regeneration in other tissues as indicated in our references (Gilbert et al., 2010; Machado et al., 2021; Montarras et al., 2005).

From the text the authors appear to suggest that growth of adult spheroids is dependent initially on "material" released by collagenase/dispase treatment. An obvious candidate would be mesenchymal cells, which are known to secrete factors such as Wnts and PGE2 that drive spheroid morphology. To test this, the authors should treat spheroid cultures with Porcupine and/or PGE2 inhibitors.

We followed similar reasoning, considering that spheroids express strongly Ptgs1 ,2 (Figure 3A). We thought their phenotype might be maintained by autocrine prostaglandin action. We tested aspirin, a Ptgs inhibitor, which was without effect on the spheroid phenotype. Besides, we explored a wide variety of conditions to test whether they would affect the spheroid phenotype [Aspirin-see above, cAMP agonists/antagonists, YapTaz inhibitors (verteporfin and CA3), valproic acid, Notch inhibitors (DAPT, DBZ, LY511455), all-trans retinoic acid, NFkB inhibitors (TCPA, BMS), TGFbeta inhibitor (SB431542)]. As these results were negative, we did not include them in the manuscript.

• If these inhibitors block growth then this would suggest that either stromal cells or autocrine signalling involving these pathways is important. Overall, more in-depth analysis of the growth requirements of adult spheroids is required.*

Figure 1d indicates that adult spheroids can be propagated for at least 10 passages. The abstract mentions they are "immortal". The text itself does not address this issue. More precise information as to how long spheroids can be propagated is required. If these cultures can be propagated for 10 passages or more it becomes important to determine what nutrients/mitogens in the basal media are driving growth? Alternatively, what is the evidence that spheroid cultures are completely devoid of mesenchymal cells. The text only mentions that "Upon replating, these spheroids could be stably cultured free of mesenchymal cells (Fig.1B)". No validation is shown to support this.

We agree that "immortal" is not a good way to characterize our spheroids, as also pointed out by referee nr 1. We have changed that in the text, indicating the maximal number of replating we tested was 26 and replacing immortal by stably replatable. Of note, the spheroids could frozen/thawed and recultured many times.

Related to the question whether mesenchymal cells could still contaminate the spheroid cultures, we can provide the following answers:

• No fibroblasts could be seen in replated cultures and multiple spheroids could be repeatedly propagated from a single starting spheroid.
• The bulk RNAseq experiment comparing organoids to ENR or BCM cultured spheroids show, despite expression of several mesenchymal markers (see matrisome in Fig3), absence of significant expression of Pdgfra (see in revision plan folder for CP20Millions results from the raw data of new suppl table 2, with Clu, Tacstd2 and Alpi shown as controls).
• Regarding the nutrients/mitogens in the medium driving spheroid growth, we did not explore the point further than showing that they grow in basal medium (i.e. advanced DMEM), given that the presence of Matrigel makes it difficult to pinpoint what is really needed. In Figure 2, the authors describe the growth requirements for adult spheroids and indicate that spheroids grown in ENR or EN became dark and shrink. The representative images showing this are clear, but this analysis should be quantified.

Added to the manuscript.

In SF3, the gene expression profile of organoids from the sandwich method only partially overlaps with that of organoids from the old protocol. What are the gene expression differences between the 2 culture systems? Secondly, the sandwich method appears to sustain growth of Tom+ spheroids based on RNAseq and the IF images. This suggest that Vil-Cre negative cells are not necessarily the only source of adult spheroids and thus this experiment seems to indicate that any cell may be converted to grow as a spheroid under the right conditions. These points should be addressed.

Looking back to our data in order to answer the point raised by the referee, we realized that we had inadvertently-compared organoids to ENR-cultured spheroids generated by the first protocol to BCM-cultured spheroids generated by the sandwich method. We have corrected this error in a new version of suppl fig3. This shows increased correspondence between genes up- or downregulated in the spheroids obtained in the two protocols (from 49/48% to 57/57% (Venn diagram on the new figure). We agree that, even after this correction, the spheroids obtained with the two protocols present sizeable differences in their transcriptome. However, considering the very different way these spheroids were obtained and cultured initially, we do not believe this to be unexpected. The important point in our opinion is that the core of the up- and down-regulated genes typical of the de-differentiation phenotype of adult spheroids is very similar, as shown in the heatmap (which was made with the correct samples!). Also, a key observation is that that both kind of spheroids survive and can be replated in basal medium. As already stated, this characteristic is only seen rare cases [spheroids obtained from rare FACS-purified cells (Smith et al 2018) or helminth-infected intestinal tissue (Nusse et al.2018)]. Together with the observation that the majority of them is not traced by VilCre constitutes what we consider the halmark of the spheroids described in our study. As shown in figure 4E (old protocol) and Suppl Fig.3 (sandwich protocol) both red and white spheroids were extremely low in VilCre expression. As stated in the text, the fact that some spheroids are nevertheless red is most probably related to the extreme sensitivity of the Rosa26Tom marker to recombination (Liu et al., 2013), but this does not mean that there are two phenotypically different kind of spheroids. It means that the arbitrary threshold of Rosa26Tom recombination introduces an artificial subdivision of spheroids with no phenotypical significance.

Regarding the point made by the referee that "that any cell may be converted to grow as a spheroid under the right conditions", we agree and have shown with others that organoids acquire indeed a spheroid phenotype when cultured for instance in fibroblasts-conditioned medium (see suppl fig1B and (Lahar et al., 2011; Roulis et al., 2020) quoted in the manuscript). However, these spheroids cannot be propagated in basal medium, and revert to an organoid phenotype when put back in ENR (Suppl fig1B).

*In Figure 4, the authors conclude that spheroids do not originate from Lgr5 cell derived clones even after 30days post Tam induction. Does this suggest that in vivo and under homeostatic conditions VilCre neg cells are derived from a distinct stem cell pool or are themselves a quiescent stem cell. Given the rarity of VilCre neg cells, the latter seems unlikely.

*

Despite their rarity, we believe VilCre-negative cells observed under homeostatic conditions are themselves quiescent stem cells. Actually, if they were derived from a larger stem cell pool, this pool should also be VilCre-negative. And we do not see such larger number of VilCre-neg cells under homeostatic conditions.

The problem with the original assertion is that Lgr5-CreERT mice are mosaic and therefore not all Lgr5+ cells are labelled in this model. "White" spheroids may thus derive from cells that in turn derive from these unlabelled Lgr5 cells.

We had considered the possibility that mosaicism [very low for VilCre (Madison et al., 2002); in the 40-50% range for Lgr5CreERT2 (Barker & Clevers. Curr Protoc Stem Cell Biol. 2010 Chapter 5)] could explain our data. We think, however that we can exclude this possibility on the basis that spheroids do not conform to the expected ratio of unrecombined cells, given the observed level of mosaicism. Indeed, for VilCre, a few percent, at most, of unrecombined cells in the epithelium translates into almost 100% unrecombined spheroids. For Lgr5CreERT2 mice, the mosaicism level is in the range of 40%, which is what we observe for EDTA organoids (Figure 4G), while spheroids were in their vast majority unrecombined.

We have included a discussion about the possible role of mosaicism in the new version.

ATACseq experiments were briefly mentioned in the manuscript but unfortunately little information was extracted from this experiment. What does this experiment reveal about the chromatin landscape of adult spheroids relative to normal organoids?

We only performed this experiment to search for an explanation to the paradoxical absence of expression of the VilCre transgene in spheroids, despite robust expression of endogenous villin (Suppl Fig.4). We chose to show the chromatin landscape of a gene equally expressed in both organoids and spheroids (Krt19), a gene specifically expressed in spheroids (Tacstd2) and the endogenous Villin gene also expressed in both. We believe that the observation of a clear difference in pattern of the chromatin accessibility around the endogenous villin gene in organoids and spheroids provides an explanation to the observed results. The cis regulatory sequences needed for expression of the endogenous villin gene seem to be different in organoids and spheroids, which may explain why the regulatory sequences present in the transgene (only 12.4kb) might not allow expression of the transgene in spheroids. We have added a sentence in the manuscript clarifying this point. Missing is obviously the chromatin landscape around the VilCre transgene, but this is beyond reach in such kind of experiments.

Reviewer #2 (Significance (Required)):

The fact that the authors developed a protocol to reproducibly generate fetal-like spheroids from adult tissue is an important advancement in the field. Previous reports have shown that treatment with various small molecule inhibitors can revert budding organoids into a spheroid morphology, but this manuscript demonstrates that spheroids can also be generated from otherwise untreated cells. This new methodology will provide new tools to dissect the molecular determinants of fetal/regenerative cells in the gut. Based on this, the manuscript should attract a significant amount of attention in the intestinal field.

Reviewer #3 (Evidence, reproducibility and clarity (Required)): CR-2024-02491

An Lgr5-independent developmental lineage is involved in mouse intestinal regeneration

Marefati et al.

Homeostatic maintenance of the intestinal epithelium has long been thought to rely upon Wnt signaling responsive Lgr5-expressing stem cells that reside at the crypt base.

However, myriad reported mechanisms or populations have been reported to underlie epithelial regeneration after injury. Many groups have reported that reacquisition of a fetal- link intestinal phenotype is an import part of the regenerative response, however the originating cell type has not been definitively identified. Herein, the authors demonstrate that cells from adult homeostatic intestine can generate immortal spheroids that resemble fetal spheroids and are derived independent of Lgr5+ intestinal stem cells (ISCs). The authors then draw the conclusion that this indicates that a hierarchical stem cell model applies to regeneration of the intestinal epithelium, in addition to the plasticity model.

• *

Comments:

1. Please indicate what species is used for studies in Fig 1.

All experiments were performed in Mus musculus.

Please clarify if Figure 2 studies utilize Matrigel or not.

Yes

RNA-seq analyses of adult intestinal generated spheroids lack the granularity of single cell analyses and thus it is unclear if this is a homogeneous population or if the population has diversity across it (i.e., enteroids/organoids have a high level of diversity). Many of the conclusions from the RNA-seq study are broad and generalized-for example Fig 3F indicates that markers of the +4 ISC populations (Bmi1, tert, lrig1, hopx) were all expressed similarly in adult spheroids as compared to adult organoids. However, while this may be true in the bulk-RNA-seq analyses, clearly scRNA-seq would provide a better foundation to make this statement, as enteroids/organoids are comprised of heterogeneous subpopulations. . .and it might indicate that these +4 markers have only very low expression in the spheroids. Based upon these concerns, misconclusions are likely to be drawn.

We agree and it would be certainly worthwhile to perform scRNAseq of adult spheroid populations. This would certainly be worth doing in future studies to explore the possible heterogeneity of adult spheroids. We nevertheless believe that our scRNAseq performed on homeostatic intestinal tissue from VilCre/Rosa26Tom mice identify Olfm4-low VilCre-neg cells that are likely at the origin of adult spheroids and display a quite homogenous phenotype.

*The language around Figure 4 results is confusing. Please define "white" and "red". It might be simpler to designate recombined versus not recombined lineage.

*

We have clarified this in the figure.

The hypothesis that collagenase/dispase solution acts as a proxy for injury is not demonstrated and backed by data. Thus, it is difficult to make the conclusion that this approach could represent a "stable avatar" of intestinal regenerating cells. It is clear that subpopulations of crypt-based cells generate spheroids in culture without collagenase/dispase (see the cited reference Smith et al, 2018).

* *Smith et al demonstrate clearly the possibility to obtain spheroids with properties probably similar to ours from EDTA derived intestinal crypt cells. However they need to prepurify them by FACS. Besides, Nusse et al describe spheroids similar to ours after infection of the intestine by helminths (Nusse et al. 2018). In our case, and for most labs preparing enteroids with the EDTA protocol, the result is close to 100% organoids. Even if we treat EDTA organoids with collagenase, we do not obtain spheroids. This brought us to the conclusion that spheroid-generating cells must be more tightly attached to the matrix than CBCs and that it is their release from the matrix that activates the spheroid regeneration-like phenotype. This hypothesis is supported by several models of regeneration in other tissues as indicated in our references (Gilbert et al., 2010; Machado et al., 2021; Montarras et al., 2005)

A study based on the absence of recombination in a VilCre lineage tracing scenario is not well-established to be strong experimental approach, as there are many reasons why recombination may not cells may not be lineage marked. In order to use this system as the authors intend, they first need to demonstrate that villin is not expressed in the discrete cell population that they are targeting. For the presented observational studies, this would be difficult to do. While they do demonstrate differences in chromatin accessibility between cells from organoids versus spheroids (fig s4), some of these differences could merely be due to the bulk analytical nature of the study and the lack of comparing stem cell populations from spheroids to stem cell populations from organoids-since the spheroids are likely homogenous versus the organoids that only have a small fraction of stem cells-and thus represent a mix of stem cell and differentiated cell populations. The authors do not demonstrate that villin protein expression varies in these cells.

If it were found that villin is not expressed in their "novel" population, then one would expect that the downstream use of villin-based recombination would demonstrate the same recombination potential (i.e., Mcl1 would not be recombined). Both recombination studies in Fig 6 are difficult to interpret, and thus it is not clear if these studies support the stated conclusions. Quantification of number of crypts that are negative should be reported as a percentage of recombined crypts.

We are sorry but there seems to be a complete misunderstanding of our data regarding the point raised by the referee. The important point of our initial observation is that despite robust expression of villin in spheroids, the VilCre transgene is not expressed (see figure 4E). This in our opinion makes absence of VilCre expression (or of Rosa marker recombination) a trustful marker of a new developmental lineage. All the data in figure 4 constitute an answer.

*The reasoning about heterogeneity of cell type in organoids versus probable homogeneity of spheroids is well taken. However, as the endogenous villin gene is expressed in all cells of both organoids and spheroids, it is highly significant that only spheroids do not express the transgene. *

We performed the ATACseq experiment to search for an explanation to the paradoxical absence of expression of the VilCre transgene in spheroids, despite robust expression of endogenous villin (Suppl Fig.4). We chose to show the chromatin landscape of a gene equally expressed in both organoids and spheroids (Krt19), a gene specifically expressed in spheroids (Tacstd2) and the endogenous Villin gene also expressed in both. We believe that the observation of a clear difference in pattern of the chromatin accessibility around the endogenous villin gene in organoids and spheroids provides an explanation to the observed results. The cis regulatory sequences needed for expression of the endogenous villin gene seem to be different in organoids and spheroids, which may explain why the regulatory sequences present in the transgene (only 12.4kb) might not allow expression of the transgene in spheroids. We have added a sentence in the manuscript clarifying this point. Missing is obviously the chromatin landscape around the VilCre transgene, but this is beyond reach in such kind of experiments.

*Figure 8 indicates that the cell population identified by scRNA-seq may be quiescent. Companion IF or IHC should be conducted to confirm this finding, as well as other conclusions from the informatics conducted.

*

We agree that additional experiments could be performed to support this point. We are unfortunately not in a position to perform these experiments (see section 4 below).

Clearly the data is intriguing, however, the conclusion is strong and is an over interpretation of the presented data. There are a number of validation or extension data that would enhance the overall interpretation of the study: 1. validation of scRNA-seq or bulk RNA-seq concepts by protein staining of intestinal tissues in the damage model will serve as a secondary observation. 2. identification of the ISC that they are defining is critical and important. There is already the notion that this cell type exists and it has been shown with various different markers. 3. expand the analyses of the fetal-like expression profiling to injured intestines to demonstrate that the lineage negative cells indeed express fetal-like proteins. 4. expand the discussion of the Clu+ cell type. Is this cell the previously described revival cell? If so, how does this body of work provide unique aspects to the field?

We agree that all these suggested experiments could be performed and would be of interest. However, we consider that they would not modify the main message of our study and would only constitute an expansion of the present work. As already stated, we are not in the position to perform them (see section 4).

*There is some level of conflicting data, with the stem population being proliferative in culture stimulated by the stromal cells, but quiescent in vivo and also based upon scRNA- seq data in Fig 9.

*

We do not see any conflict in our observation regarding this point. The observation that cells that are quiescent in vivo become proliferative when subjected to culture (with or without addition of stromal cells) is routinely made in a multitude of cell culture systems. In particular, it has been shown that intestinal tissue dissociation activates the Yap/Taz pathway, resulting in proliferation (Yu et al. Hippo Pathway Regulation of Gastrointestinal Tissues. Annual Review of Physiology, 2015 Volume 77, 201-227).

Many of the findings have been previously reported: Population that grows as spheroids (Figure 2), Population that is Wnt independent (Figure 2), Lgr5 independent regenerative growth of the intestine (figure 3F, Figure 4), Clu+ ISCs drive regeneration (Figure 7).

Whereas these individual findings have indeed been reported, it was in a different context. We strongly disagree with the underlying suggestion that our study would not bring new information. We have identified here a developmental lineage involved in intestinal regeneration that has not been described up to now.

Minor comments:

1. • The statement that spheroids must originate from collagenase/dispase digested material might be an overstatement. As spheroids generation from EDTA treated intestines have been previously reported (Smith et al, 2018). * See answer to point 4 above. *Overall while the study includes an extensive amount of work and different approaches, a foundationally supported novel finding is missing. Many of the statements have already been demonstrated by others in the fields. In addition, one of the most intriguing aspects of the study is that the stromal population impacts this stem cell population, however, interactions and factors stimulating the crosstalk are not addressed.

*

Reviewer #3 (Significance (Required)):

Overal while the study includes an extensive amount of work and different approaches, a foundationally supported novel finding is missing. Many of the statements have already been demonstrated by others in the fields. In addition, one of the most intriguing aspects of the study is that the stromal population impacts this stem cell population, however, interactions and factors stimulating the crosstalk are not addressed.

We can only disagree.

4. Description of analyses that authors prefer not to carry out

• *

We have answered most questions raised by the referees by explaining our view, by clarifying individual points and, in several cases, by providing additional information that was not included in the original manuscript.

In a limited number of cases when additional experiments were suggested, we were unfortunately obliged to write that we are not in a position to perform them. This is because my lab is closing after more than fifty years of uninterrupted activity. There will unfortunately be nobody to perform additional experiments.

Nevertheless, as written by referees 1 and 2, we believe that the revised manuscript, as it stands, contains data that will be of interest to the people in the field and may be the bases for future developments. We hope editors will find interest in publishing it.

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Referee #3

Evidence, reproducibility and clarity

RC-2024-02491

An Lgr5-independent developmental lineage is involved in mouse intestinal regeneration Marefati et al.

Homeostatic maintenance of the intestinal epithelium has long been thought to rely upon Wnt signaling responsive Lgr5-expressing stem cells that reside at the crypt base. However, myriad reported mechanisms or populations have been reported to underlie epithelial regeneration after injury. Many groups have reported that reacquisition of a fetal-link intestinal phenotype is an import part of the regenerative response, however the originating cell type has not been definitively identified. Herein, the authors demonstrate that cells from adult homeostatic intestine can generate immortal spheroids that resemble fetal spheroids and are derived independent of Lgr5+ intestinal stem cells (ISCs). The authors then draw the conclusion that this indicates that a hierarchical stem cell model applies to regeneration of the intestinal epithelium, in addition to the plasticity model.

Comments:

1. Please indicate what species is used for studies in Fig 1.
2. Please clarify if Figure 2 studies utilize Matrigel or not.
3. RNA-seq analyses of adult intestinal generated spheroids lack the granularity of single cell analyses and thus it is unclear if this is a homogeneous population or if the population has diversity across it (i.e., enteroids/organoids have a high level of diversity). Many of the conclusions from the RNA-seq study are broad and generalized-for example Fig 3F indicates that markers of the +4 ISC populations (Bmi1, tert, lrig1, hopx) were all expressed similarly in adult spheroids as compared to adult organoids. However, while this may be true in the bulk-RNA-seq analyses, clearly scRNA-seq would provide a better foundation to make this statement, as enteroids/organoids are comprised of heterogeneous subpopulations. . .and it might indicate that these +4 markers have only very low expression in the spheroids. Based upon these concerns, misconclusions are likely to be drawn.
4. The language around Figure 4 results is confusing. Please define "white" and "red". It might be simpler to designate recombined versus not recombined lineage.
5. The hypothesis that collagenase/dispase solution acts as a proxy for injury is not demonstrated and backed by data. Thus, it is difficult to make the conclusion that this approach could represent a "stable avatar" of intestinal regenerating cells. It is clear that subpopulations of crypt-based cells generate spheroids in culture without collagenase/dispase (see the cited reference Smith et al, 2018).
6. A study based on the absence of recombination in a VilCre lineage tracing scenario is not well-established to be strong experimental approach, as there are many reasons why recombination may not cells may not be lineage marked. In order to use this system as the authors intend, they first need to demonstrate that villin is not expressed in the discrete cell population that they are targeting. For the presented observational studies, this would be difficult to do. While they do demonstrate differences in chromatin accessibility between cells from organoids versus spheroids (fig s4), some of these differences could merely be due to the bulk analytical nature of the study and the lack of comparing stem cell populations from spheroids to stem cell populations from organoids-since the spheroids are likely homogenous versus the organoids that only have a small fraction of stem cells-and thus represent a mix of stem cell and differentiated cell populations. The authors do not demonstrate that villin protein expression varies in these cells. If it were found that villin is not expressed in their "novel" population, then one would expect that the downstream use of villin-based recombination would demonstrate the same recombination potential (i.e., Mcl1 would not be recombined). Both recombination studies in Fig 6 are difficult to interpret, and thus it is not clear if these studies support the stated conclusions. Quantification of number of crypts that are negative should be reported as a percentage of recombined crypts.
7. Figure 8 indicates that the cell population identified by scRNA-seq may be quiescent. Companion IF or IHC should be conducted to confirm this finding, as well as other conclusions from the informatics conducted.
8. Clearly the data is intriguing, however, the conclusion is strong and is an over interpretation of the presented data. There are a number of validation or extension data that would enhance the overall interpretation of the study:
   • a. validation of scRNA-seq or bulk RNA-seq concepts by protein staining of intestinal tissues in the damage model will serve as a secondary observation.
   • b. identification of the ISC that they are defining is critical and important. There is already the notion that this cell type exists and it has been shown with various different markers.
   • c. expand the analyses of the fetal-like expression profiling to injured intestines to demonstrate that the lineage negative cells indeed express fetal-like proteins.
   • d. expand the discussion of the Clu+ cell type. Is this cell the previously described revival cell? If so, how does this body of work provide unique aspects to the field?
9. There is some level of conflicting data, with the stem population being proliferative in culture stimulated by the stromal cells, but quiescent in vivo and also based upon scRNA-seq data in Fig 9.
10. Many of the findings have been previously reported: Population that grows as spheroids (Figure 2), Population that is Wnt independent (Figure 2), Lgr5 independent regenerative growth of the intestine (figure 3F, Figure 4), Clu+ ISCs drive regeneration (Figure 7).

Minor comments:

1. The statement that spheroids must originate from collagenase/dispase digested material might be an overstatement. As spheroids generation from EDTA treated intestines have been previously reported (Smith et al, 2018).

Overall while the study includes an extensive amount of work and different approaches, a foundationally supported novel finding is missing. Many of the statements have already been demonstrated by others in the fields. In addition, one of the most intriguing aspects of the study is that the stromal population impacts this stem cell population, however, interactions and factors stimulating the crosstalk are not addressed.

Significance

Overall while the study includes an extensive amount of work and different approaches, a foundationally supported novel finding is missing. Many of the statements have already been demonstrated by others in the fields. In addition, one of the most intriguing aspects of the study is that the stromal population impacts this stem cell population, however, interactions and factors stimulating the crosstalk are not addressed.

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Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

In this manuscript, the Marefati et al. developed a novel approach to generate spheroids from adult intestinal epithelium using a collagenase/dispase based protocol. Adult spheroids were found to be distinct from classic budding-type organoids normally generated from EDTA based release of the crypt epithelium. Transcriptional profiling indicated that adult spheroids were undifferentiated and similar to regenerating crypts or fetal spheroids. To identify the cell of origin that generates adult spheroids, the authors labelled epithelial cells with VilCreERT-LSL-Tom, VilCre-LSL-GFP and Lgr5CreERT-LSLTom mice. From these experiments the authors conclude that that spheroids are only generated from Vil-Cre negative and Lgr5 negative cells. Next the authors deleted the anti-apoptotic gene Mcl1 using Vil-CreERT mice. This led to a strong apoptotic response throughout the crypt epithelium and tissues processed from knockout mice readily generated spheroids, and in vivo, replenishment of the gut epithelium was mediated by unrecombined cells. In a second model, CBCs were ablated using Lgr5DTR mice and VilCre negative cells were found again to contribute to regeneration of the crypt epithelium. Finally based on the absence of Vil-Cre reporter activity, the authors were able to sort out and perform scRNAseq to profile VilCre negative cells. These cells were found to be quiescent, express the stem cell marker Olfm4 and were also abundant in ribosomal gene expression.

The fact that the authors developed a protocol to reproducibly generate fetal-like spheroids from adult tissue is an important advancement in the field. Previous reports have shown that treatment with various small molecule inhibitors can revert budding organoids into a spheroid morphology, but this manuscript demonstrates that spheroids can also be generated from otherwise untreated cells. This new methodology will provide new tools to dissect the molecular determinants of fetal/regenerative cells in the gut. Based on this, the manuscript should attract a significant amount of attention in the intestinal field.

As pointed out by the authors themselves the study has important limitations that diminish enthusiasm. The primary issue relates to the inability of the team to identify markers of VilCre neg cells other than the fact that these cells are Olfm4+ and quiescent. Nonetheless, for the reasons stated above the manuscript should reach the target audience within the research community, if the authors can address the specific points below related to issues with methodology as well as defining more precisely the characteristics and growth requirements of adult spheroid cultures.

Major comments

The main conclusion of the study is that Vil-Cre neg cells are rare quiescent Olfm4+ crypt cells. If this is the case, then standard EDTA treatment should release these cells as well. Consequently, spheroids should also emerge from isolated crypts grown in the absence of ENR. If this is not the case how do the authors explain this?

From the text the authors appear to suggest that growth of adult spheroids is dependent initially on "material" released by collagenase/dispase treatment. An obvious candidate would be mesenchymal cells, which are known to secrete factors such as Wnts and PGE2 that drive spheroid morphology. To test this, the authors should treat spheroid cultures with Porcupine and/or PGE2 inhibitors. If these inhibitors block growth then this would suggest that either stromal cells or autocrine signalling involving these pathways is important. Overall, more in-depth analysis of the growth requirements of adult spheroids is required.

Figure 1d indicates that adult spheroids can be propagated for at least 10 passages. The abstract mentions they are "immortal". The text itself does not address this issue. More precise information as to how long spheroids can be propagated is required. If these cultures can be propagated for 10 passages or more it becomes important to determine what nutrients/mitogens in the basal media are driving growth? Alternatively, what is the evidence that spheroid cultures are completely devoid of mesenchymal cells. The text only mentions that "Upon replating, these spheroids could be stably cultured free of mesenchymal cells (Fig.1B)". No validation is shown to support this.

In Figure 2, the authors describe the growth requirements for adult spheroids and indicate that spheroids grown in ENR or EN became dark and shrink. The representative images showing this are clear, but this analysis should be quantified.

In SF3, the gene expression profile of organoids from the sandwich method only partially overlaps with that of organoids from the old protocol. What are the gene expression differences between the 2 culture systems? Secondly, the sandwich method appears to sustain growth of Tom+ spheroids based on RNAseq and the IF images. This suggest that Vil-Cre negative cells are not necessarily the only source of adult spheroids and thus this experiment seems to indicate that any cell may be converted to grow as a spheroid under the right conditions. These points should be addressed.

In Figure 4, the authors conclude that spheroids do not originate from Lgr5 cell derived clones even after 30days post Tam induction. Does this suggest that in vivo and under homeostatic conditions VilCre neg cells are derived from a distinct stem cell pool or are themselves a quiescent stem cell. Given the rarity of VilCre neg cells, the latter seems unlikely. The problem with the original assertion is that Lgr5-CreERT mice are mosaic and therefore not all Lgr5+ cells are labelled in this model. "White" spheroids may thus derive from cells that in turn derive from these unlabelled Lgr5 cells.

ATACseq experiments were briefly mentioned in the manuscript but unfortunately little information was extracted from this experiment. What does this experiment reveal about the chromatin landscape of adult spheroids relative to normal organoids?

Significance

The fact that the authors developed a protocol to reproducibly generate fetal-like spheroids from adult tissue is an important advancement in the field. Previous reports have shown that treatment with various small molecule inhibitors can revert budding organoids into a spheroid morphology, but this manuscript demonstrates that spheroids can also be generated from otherwise untreated cells. This new methodology will provide new tools to dissect the molecular determinants of fetal/regenerative cells in the gut. Based on this, the manuscript should attract a significant amount of attention in the intestinal field.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

In this manuscript, Marefati et al report an Lgr5-independent lineage in the regenerating intestine using in vitro organoids and in vivo injury-coupled lineage tracing model. In organoids, collagenase/dispase dissociated resulted in "immortal spheroids" that maintain a cystic and undifferentiated phenotype in the absence of standard growth factors (Rspondin/Noggin/EGF). Bulk RNAseq of spheroids demonstrates downregulation of classical CBC signatures and upregulation of fetal spheroid, mesenchymal, inflammation and regenerative signatures. In mice, Villin-Cre lineage tracing revealed some Villin-negative progenies that lack reporter tracing throughout crypt-villus ribbons after injury. The authors proposed that there is Lgr5-independent population support the regenerative response upon CBC depletion. A major caveat of this study is the identification of this population is based on absence of VilCre expression. It is surprising that there is no characterisation of Lgr5 expression throughout the manuscript whilst claiming of a Lgr5-independent lineage. Although the research question is potentially interesting, the concept of epithelial reprogramming upon injury is well documented in the field. The data generated in this manuscript also seem to be preliminary and lack of detailed characterisation. Below are specific comments.

• Expression of Lgr5 should be properly characterised throughout the manuscript in both organoid models and injury-induced regeneration in vivo.
• An important question is the origin of these "Lgr5-independent" adult spheroids. They look and appear like fetal organoids, which could be induced by injury (e.g. upon collagenase/dispase dissociation). Have the authors tried to culture fetal spheroids in BCM over extensive period of time? Do they behave the same? This would be a great way to directly compare the collagenase/dispase-derived organoids with fetal origin.
• Fig 1C, Why is the replating spheroid culture time different between mesenchymal cells and conditioned medium?
• It is unclear how the bulk RNA-seq data in Fig. 3 were compared. How long were the adult organoids and spheroids cultured for (how many passages)? Were they culture in the same condition of were they in ENR vs BCM? These are important information to consider when interpreting the results. For instance, are Ptgs1 & Ptgs2 expression in adult spheroids the same in ENR vs BCM? Are the gene signatures (regenerative, fetal and YAP) changed in adult spheroids culturing in ENR vs BCM?
• It is stated: "In agreement with their aptitude to grow indefinitely, adult spheroids express a set of upregulated genes overlapping significantly with an "adult tissue stem cell module" [159/721 genes; q value 2.11 e-94) (Fig.S2F)].". What is the definition of "indefinitely"? Are they referring to the Fig 1B where spheroid were passaged to P10? The authors should avoid the term "indefinitely" but use a more specific time scale, e.g. passages, months etc.
• SuppFig 3D: Row Z-Score is missing the "e" in Score.
• Fig 4E: Figure legend says QNRQ instead of CNRQ.
• Fig 4G: The brightfield image of adult spheroids 5 days after 3x TAM injections doesn't look like a spheroid. It seems to be differentiating.
• Fig 4: Most mouse model data are missing the number of mice & their respective age used for organoid isolation.
• Fig 4A-D, H-G: How was fluorescent signal of organoids quantified? How many images? Were there equal numbers of organoids? This all needs to be included in methods/figure legends.
• Figure 4B-D, G-H: Which culturing conditions were used for adult spheroids? Original method or sandwich method?
• Fig 6D-E: Please add the timepoint after DT administration these samples are from. It is not listed in text or figure legend.
• SuppFig 6D: again timepoint is missing.
• SuppFig 6: How were the crypts of these mice (DT WT & DT HE) isolated? Was this via EDTA? Also, what is the timepoint for isolation for these samples? Even if untreated, the timepoint adds context to the data. Please add more context to describing these different experiments, either in the figure legends or methods section.
• SuppFig 6E: The quality of the heatmap resolution is too poor to read gene names.
• Fig.5-7, are the regenerating crypt-villus units fully differentiated or are they maintained in the developmental state? Immunostaining of markers for stem cells (Lgr5), differentiated lineages (Alpi, Muc2, Lyz, ChgA etc.) and fetal state (Sca1, Trop2 etc) should be analysed in those "white" unrecombined crypt-villus units.
• The following text needs clarification:

"The kinetics of appearance of newly formed un-recombined ("white") crypts was studied after a single pulse of DT (Fig.7A). This demonstrated an increase at 48 hours, with further increase at day 10 and stable maintenance at day 30. The presence of newly formed white crypts one month after toxin administration indicates that the VilCre-negative lineage is developmentally stable and does not turn on the transgene during differentiation of the various epithelial lineages occurring after regeneration (Fig.7B). Comment: The "newly formed" is an overstatement, the data doesn't conclude that those are "new" crypts. The end of the sentence states that these "white" crypts form developmentally stable lineages, thus these white crypts at day 30 could originate from the initial injury. There was no characterisation of the various epitheial lineages. Are they fully differentiated? Is Lgr5 expressed one month after toxin administration? Can the VilCre neg lineage give rise to CBCs?

The relationship between white crypt generation and appearance of Clu-positive revival cells (Ayyaz et al., 2019) was then explored. In agreement with others and similar to what happens in the irradiation model, (Ayyaz et al., 2019; Yuan et al., 2023) Clu-positive cells were rare in crypts of untreated mice and their number transiently increased forty-eight hours after a single pulse of DT, and more so after three pulses of DT (Fig.7C,D). Comment: Comparing 1 pulse at day 2 vs 3 pulses at day 5 makes the data hard to interpret. How is the Clu ISH level for 1 pulse at day 5? Are they equivalent?

Clu-positive cells were less frequently observed in white crypts (see "Total" versus "White" in Fig.7C). This fits with the hypothesis that Clu expression marks acutely regenerating crypts and that a proportion of the white crypts are chronically regenerating due to DTR expression in CBCs." Comment: I believe the authors suggested that the discrepancy of less Clu expression in white crypts is due to the ectopic expression of DTR in CBCs causing low grade injury without DT administration. This means that some white crypts could have been formed before the administration of DT, and thus are on a different regenerative timeline compared to the white crypts formed from DT administration. Is there any proof of the chronic regeneration? Immunostaining of chronic regenerative markers such as Sca1, Anxa1 or Yap1 nuclear localization would support the claim. It'd be important to show only the white crypts, but not the RFP+ ones, show regenerative markers. - Fig 7D-E: What are the timepoints of harvest for HE-WT-HE 1 pulse DT mice and HE-HE-HE PBS injected mice? - Fig 8-9: Regarding the CBC-like Olfm4 low population, what is the status of Lgr5? This should be shown in the figure since the argument is that this is an Lgr5-independent lineage. And what about the regenerative, Yap, mesenchymal and inflammatory signatures? Are they enriched in the white crypts similar to the in vitro spheroids?

Significance

Strengths: The study employed a range of in vitro and in vivo models to test the hypothesis.

Limitations: Unfortunately, the models chosen did not provide sufficient evidence to draw the conclusions. Injury induced reprogramming, both in vivo and in vitro, has been well documented in the field. The new message here is to show that such reprogrammed state is continuous rather than transient; instead of regenerating Lgr5+ stem cells, it can continue to differentiate to all cell lineages in Lgr5-independent manner. However, through the manuscript, there was no immunostaining of Lgr5 and other differentiation markers. The conclusion is an overstatement without solid proof.

Yet https://docs.chrultrabook.com/docs/reverting/

Version 2 of this preprint has been peer-reviewed and recommended by Peer Community in Archaeology.<br /> See the peer reviews and the recommendation.

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Tbl. 2 / Table 2

A second question arises from the development we see in Fig. 8, with significantly more articles on amok being published in the first half of the 1860s and in the late 1870s and 1880s.

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eLife assessment

This valuable paper presents findings showing that different brain regions were best described by a distinct accumulation model, which all differed from the model that best described the rat's choices. These findings are solid because the authors present a very strong methodological approach. This work will be of interest to a wide neuroscientific audience.

Reviewer #1 (Public Review):

The authors use neural recordings from three different brain areas to assess whether the type of evidence accumulation dynamics in those regions are (1) similar to one another, and (2) similar to best-fitting evidence accumulation dynamics to behavioral choice alone. This is an important theoretical question because it relates to the 'linking hypothesis' that relates neurophysiological data to psychological phenomena. Although the standard evidence accumulation dynamic in describing choice has been the gradual accumulation of evidence, the authors find that those dynamics are not represented equally in all brain regions. Such results suggest that more nuanced computational models are needed to explain how brain areas interact to produce decisions, and the focus of theoretical development should shift away from explaining behavioral patterns alone and more toward explaining both brain and behavioral interactions. Given that the authors simply test the assumption that the same dynamics that best explain behavior should also explain neural data, they accomplish their objective using a sophisticated methodology and find evidence *against* this assumption: they find that each region was best described by a distinct accumulation model, which all differed from the model that best described the rat's choices.

I thought this was an excellent paper with a clear scientific objective, direct analysis to achieve that objective, and a very strong methodological approach to leave little doubt that the conclusions they drew from their analyses were as reasonable and accurate as possible.

Reviewer #2 (Public Review):

The neural dynamics underlying decision-making have long been studied across different species (e.g., primates and rodents) and brain areas (e.g., parietal cortex, frontal eye fields, striatum). The key question is to what extent neural firing rates covary with evidence accumulation processes as proposed by evidence accumulation models. It is often assumed that the evidence-accumulation process at the neural level should mirror the evidence-accumulation process at the behavioral level. The current paper shows that the neural dynamics of three rat brain regions (the FOF, ADS, and PCC) all show signatures of evidence accumulation, but in distinct ways. Especially the role of the FOF appears to be distinct, due to its dependence on early evidence compared to the other regions. This sheds new light and a new interpretation of the role of the FOF in decision-making - previously, it has been described as a region encoding the choice that is currently being committed to; this new analysis suggests it is instead strongly influenced by early evidence.

A major strength of the paper is that the results are achieved through joint modelling of the behavioral and neural data, combined with information on the physical stimulus at hand. Joint models were shown to provide more information on the underlying processes compared to behavioral or neural models alone. Especially the inclusion of the neural data seemed to have greatly improved the quality of inferences. This is a key contribution that illustrates that the sophisticated modelling of multiple sources of data at the same time, pays off in terms of the quality of inferences. Yet, it should be added here, that due to the nature of the task, the behavioral data contained only choices, and not response times, which tend to contain more information regarding the evidence accumulation process than choice alone. It would be interesting to additionally discuss how choice decision times can be modeled with the proposed modelling framework.

A main limitation of the paper is that it does not appear to address a seemingly logical follow-up question: If these three brain regions individually accumulate evidence in distinct manners, how do these multiple brain regions then each contribute to a final choice? The joint models fit each region's data separately, so how well does each region individually 'explain' or 'predict' behavior, and how does the combined neural activity of the regions lead to manifest behavior? I would be very interested in the authors' perspectives on these questions.

There are some remaining questions regarding the specific models used, that I was hoping the authors could clarify. Specifically, in equations 10-11, I was wondering to what extent there might be a collinearity issue. Equation 10 proposes that the firing rates of neurons can vary across time due to two mechanisms: (1) The dependence of the firing rate on the accumulated evidence, and (2) a time-varying trial average (as detailed in Equation 11). If firing rates of the neuron indeed covary with the accumulated evidence and therefore increase across time, how can the effects of mechanisms 1 and 2 be disentangled? Relatedly, the independent noise models model each neuron separately and thereby include many more parameters, each informed by less data. Is it possible that the relatively poor cross-validation of the independent noise model may be a consequence of the overfitting of the independent noise model?<br /> Another related question is how robust the parameter recovery properties of these models are under a wider range of data-generating parameter settings. I greatly appreciate the inclusion of a parameter recovery study (Figure S1C) using a single synthetic dataset, but it could be made even stronger by simulating multiple datasets with a wider range of parameter settings. Such a simulation study would help understand how robust and reliable the estimated parameters of all models are. Similarly, it would be helpful if also the \theta_{y} parameters are shown, which aren't shown in Figure S1C.

An aspect of the paper that initially raised confusion with me is that the models fit on the choice data and stimulus information alone, make different predictions for the evidence accumulation dynamics in different regions (e.g., Figure 5A, 6A) and also led to different best-fitting parameters in different regions (Figure S9A). It took me a while to realize that this is due to the data being pooled across different rats and sessions - as such, the behavioral choice data are not the same across regions, and neither is the resulting fit models. This could easily be clarified by adding a few notes in the captions of the relevant figures.

Combined, this manuscript represents an interesting and welcome contribution to an ongoing debate on the neural dynamics of decision-making across different brain regions. It also introduced new joint modelling techniques that can be used in the field and raised new questions on how the concurrent activity of neurons across different brain regions combined leads to behavior.

Author response

Reviewer #1 (Public Review):

The authors use neural recordings from three different brain areas to assess whether the type of evidence accumulation dynamics in those regions are (1) similar to one another, and (2) similar to best-fitting evidence accumulation dynamics to behavioral choice alone. This is an important theoretical question because it relates to the 'linking hypothesis' that relates neurophysiological data to psychological phenomena. Although the standard evidence accumulation dynamic in describing choice has been the gradual accumulation of evidence, the authors find that those dynamics are not represented equally in all brain regions. Such results suggest that more nuanced computational models are needed to explain how brain areas interact to produce decisions, and the focus of theoretical development should shift away from explaining behavioral patterns alone and more toward explaining both brain and behavioral interactions. Given that the authors simply test the assumption that the same dynamics that best explain behavior should also explain neural data, they accomplish their objective using a sophisticated methodology and find evidence *against* this assumption: they find that each region was best described by a distinct accumulation model, which all differed from the model that best described the rat's choices.

I thought this was an excellent paper with a clear scientific objective, direct analysis to achieve that objective, and a very strong methodological approach to leave little doubt that the conclusions they drew from their analyses were as reasonable and accurate as possible.

We thank the reviewer for their time and appreciate their generous comments.

Reviewer #2 (Public Review):

The neural dynamics underlying decision-making have long been studied across different species (e.g., primates and rodents) and brain areas (e.g., parietal cortex, frontal eye fields, striatum). The key question is to what extent neural firing rates covary with evidence accumulation processes as proposed by evidence accumulation models. It is often assumed that the evidence-accumulation process at the neural level should mirror the evidence-accumulation process at the behavioral level. The current paper shows that the neural dynamics of three rat brain regions (the FOF, ADS, and PCC) all show signatures of evidence accumulation, but in distinct ways. Especially the role of the FOF appears to be distinct, due to its dependence on early evidence compared to the other regions. This sheds new light and a new interpretation of the role of the FOF in decision-making - previously, it has been described as a region encoding the choice that is currently being committed to; this new analysis suggests it is instead strongly influenced by early evidence.

A major strength of the paper is that the results are achieved through joint modelling of the behavioral and neural data, combined with information on the physical stimulus at hand. Joint models were shown to provide more information on the underlying processes compared to behavioral or neural models alone. Especially the inclusion of the neural data seemed to have greatly improved the quality of inferences. This is a key contribution that illustrates that the sophisticated modelling of multiple sources of data at the same time, pays off in terms of the quality of inferences. Yet, it should be added here, that due to the nature of the task, the behavioral data contained only choices, and not response times, which tend to contain more information regarding the evidence accumulation process than choice alone. It would be interesting to additionally discuss how choice decision times can be modeled with the proposed modelling framework.

We thank the reviewer for their generous views on our work. We agree that adding decision times, which could readily be added to our framework, will likely further constrain the inference of the latent model. We are currently pursuing such topics using this framework and appropriate data. We have altered a passage in our Discussion, where we note the various extensions of our model one could pursue, to include response time within the set of behavioral measurements one might include.

A main limitation of the paper is that it does not appear to address a seemingly logical follow-up question: If these three brain regions individually accumulate evidence in distinct manners, how do these multiple brain regions then each contribute to a final choice? The joint models fit each region's data separately, so how well does each region individually 'explain' or 'predict' behavior, and how does the combined neural activity of the regions lead to manifest behavior? I would be very interested in the authors' perspectives on these questions.

We could not share the reviewers view and interest in this question with any more excitement than we already do! Unfortunately, the experiments necessary for answering this question in a satisfying way have not yet been performed (e.g. simultaneous multi-region population recordings). Additionally, our analysis approach, as presented currently, would require some technical alterations to deal with data at that scale. Both efforts are underway, but we feel as though the current manuscript describes the basic modeling framework one would need to use to address these questions if/when such data exists. We have added some text to the Discussion to highlight these exciting future directions:

“An exciting future application of our modeling framework is to model multiple, independent accumulators in several brain regions which collectively give rise to the animal’s behavior. Such a model would provide incredible insight into how the brain collectively gives rise to behavioral choices.”

There are some remaining questions regarding the specific models used, that I was hoping the authors could clarify. Specifically, in equations 10-11, I was wondering to what extent there might be a collinearity issue. Equation 10 proposes that the firing rates of neurons can vary across time due to two mechanisms: (1) The dependence of the firing rate on the accumulated evidence, and (2) a time-varying trial average (as detailed in Equation 11). If firing rates of the neuron indeed covary with the accumulated evidence and therefore increase across time, how can the effects of mechanisms 1 and 2 be disentangled? Relatedly, the independent noise models model each neuron separately and thereby include many more parameters, each informed by less data. Is it possible that the relatively poor cross-validation of the independent noise model may be a consequence of the overfitting of the independent noise model?

Thank you for this important observation. Please see our response to the essential revisions above which addresses this issue. In short, although it is true that firing rates increase with time (with accumulating evidence) they do so in a way that depends on the stimulus, and so just as often as they increase with time, they decrease.

Regarding the poor cross-validation of the independent noise model, we apologize for confusion here — both the shared and independent noise model have exactly the same number of parameters. They only differ in that the latent process for a trial contains unique noise instantiation per trial for the independent noise model and the same instantiating for the shared model. The number of parameters is the same. See above for our response to this issue, and how the manuscript was modified in light of this confusion.

Another related question is how robust the parameter recovery properties of these models are under a wider range of data-generating parameter settings. I greatly appreciate the inclusion of a parameter recovery study (Figure S1C) using a single synthetic dataset, but it could be made even stronger by simulating multiple datasets with a wider range of parameter settings. Such a simulation study would help understand how robust and reliable the estimated parameters of all models are. Similarly, it would be helpful if also the \theta_{y} parameters are shown, which aren't shown in Figure S1C.

We agree that understanding the model fitting behavior under a wider set of parameter settings is valuable. We fit our model to additional sets of parameter settings and included an additional supplemental figure (Figure 1 — figure supplement 2) to illustrate these results. In short, we found that parameter recovery was robust across the different parameter settings we tested. We also updated Figure S1C with the neural parameters. We included the following in the Results to note that parameter recovery was robust:

“We verified that our method was able to recover the parameters that generated synthetic physiologically-relevant spiking and choices data (Figure 1 — figure supplement 1), and that parameter recovery was robust across a range of parameter values (Figure 1 — figure supplement 2)).” 

An aspect of the paper that initially raised confusion with me is that the models fit on the choice data and stimulus information alone, make different predictions for the evidence accumulation dynamics in different regions (e.g., Figure 5A, 6A) and also led to different best-fitting parameters in different regions (Figure S9A). It took me a while to realize that this is due to the data being pooled across different rats and sessions - as such, the behavioral choice data are not the same across regions, and neither is the resulting fit models. This could easily be clarified by adding a few notes in the captions of the relevant figures.

Thanks for pointing this out. We agree that this tends to be a point of confusion, and we have added clarification prior to Fig 3, where the choice model is first introduced:

“We stress that because of this, each fitted choice model uses different behavioral choice data, and thus the fitted parameters vary from fitted model to fitted model.”

Combined, this manuscript represents an interesting and welcome contribution to an ongoing debate on the neural dynamics of decision-making across different brain regions. It also introduced new joint modelling techniques that can be used in the field and raised new questions on how the concurrent activity of neurons across different brain regions combined leads to behavior.

We appreciate the very generous views on our work!

metaphor the narrator compares the old man¨s eye to that of the vulture, using this metaphor to emphasize his revulsion towards it.

Repetition the narrator repeats key phrases like the eye and rid myself to reinforce his obsession.

hyperbole - this exaggerates the narrator΅s heightened senses and paranoia

The repetition of the sound creates a rhythmic, unsettling quality

Personification - the narrator personifies his desire to rid myself of the eye as if it is a separate entity

Author response:

eLife assessment

This useful study reports on the discovery of an antimicrobial agent that kills Neisseria gonorrhoeae. Sensitivity is attributed to a combination of DedA assisted uptake of oxydifficidin into the cytoplasm and the presence of a oxydifficidin-sensitive RplL ribosomal protein. Due to the narrow scope, the broader antibacterial spectrum remains unclear and therefore the evidence supporting the conclusions is incomplete with key methods and data lacking. This work will be of interest to microbiologists and synthetic biologists.

General comment about narrow scope: The broader antibacterial spectrum of oxydifficidin has been reported previously (S B Zimmerman et al., 1987). The main focus of this study is on its previously unreported potent anti-gonococcal activity and mode of action. While it is true that broad-spectrum antibiotics have historically played a role in effectively controlling a wide range of infections, we and others believe that narrow-spectrum antibiotics have an overlooked importance in addressing bacterial infections. Their advantage lies in their ability to target specific pathogens without markedly disrupting the human microbiota.

We are troubled by the statement that our paper is narrow in scope and that evidence supporting our conclusions is incomplete. We do not feel the reviews as presented substantiate drawing this conclusion about our work.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Kan et al. report the serendipitous discovery of a Bacillus amyloliquefaciens strain that kills N. gonorrhoeae. They use TnSeq to identify that the anti-gonococcal agent is oxydifficidin and show that it acts at the ribosome and that one of the dedA gene products in N. gonorrhoeae MS11 is important for moving the oxydifficidin across the membrane.

Strengths:

This is an impressive amount of work, moving from a serendipitous observation through TnSeq to characterize the mechanism by which Oxydifficidin works.

Weaknesses:

(1) There are important gaps in the manuscript's methods.

The requested additions to the method describing bacterial sequencing and anti-gonococcal activity screening will be made. However, we do not think the absence of these generic methods reduces the significance of our findings.

(2) The work should evaluate antibiotics relevant to N. gonorrhoeae.

(1) It is not clear to us why reevaluating the activity of well characterized antibiotics against known gonorrhoeae clinical strains would add value to this manuscript. The activity of clinically relevant antibiotics against antibiotic-resistant N. gonorrhoeae clinical isolates is well described in the literature. Our use of antibiotics in this study was intended to aid in the identification of oxydifficidin’s mode of action. This is true for both Tables 1 and 2.

(2) If the reviewer insists, we would be happy to include MIC data for the following clinically relevant antibiotics: ceftriaxone (cephalosporin/beta-lactam), gentamicin (aminoglycoside), azithromycin (macrolide), and ciprofloxacin (fluoroquinolone).

(3) The genetic diversity of dedA and rplL in N. gonorrhoeae is not clear, neither is it clear whether oxydifficidin is active against more relevant strains and species than tested so far.

(1) We thank the reviewer for this suggestion. We aligned the DedA sequence from strain MS11 with DedA proteins from 220 N. gonorrhoeae strains that have high-quality assemblies in NCBI. The result showed that there are no amino acid changes in this protein. Using the same method, we observed several single amino acid changes in RplL. This included changes at A64, G25 and S82 in 4 strains with one change per strain. These sites differ from R76 and K84, where we identified changes that provide resistance to oxydifficidin. Notably, in a similar search of representative Escherichia, Chlamydia, Vibrio, and Pseudomonas NCBI deposited genomes, we did not identify changes in RplL at position R76 or K84.

(2) While the usefulness of screening more clinically relevant antibiotics against clinical isolates as suggested in comment 2 was not clear to us, we agree that screening these strains for oxydifficidin activity would be beneficial. We have ordered Neisseria gonorrhoeae strain AR1280, AR1281 (CDC), and Neisseria meningitidis ATCC 13090. They will be tested when they arrive.

Reviewer #2 (Public Review):

Summary:

Kan et al. present the discovery of oxydifficidin as a potential antimicrobial against N. gonorrhoeae, including multi-drug resistant strains. The authors show the role of DedA flippase-assisted uptake and the specificity of RplL in the mechanism of action for oxydifficidin. This novel mode of action could potentially offer a new therapeutic avenue, providing a critical addition to the limited arsenal of antibiotics effective against gonorrhea.

Strengths:

This study underscores the potential of revisiting natural products for antibiotic discovery of modern-day-concerning pathogens and highlights a new target mechanism that could inform future drug development. Indeed there is a recent growing body of research utilizing AI and predictive computational informatics to revisit potential antimicrobial agents and metabolites from cultured bacterial species. The discovery of oxydifficidin interaction with RplL and its DedA-assisted uptake mechanism opens new research directions in understanding and combating antibiotic-resistant N. gonorrhoeae. Methodologically, the study is rigorous employing various experimental techniques such as genome sequencing, bioassay-guided fractionation, LCMS, NMR, and Tn-mutagenesis.

Weaknesses:

The scope is somewhat narrow, focusing primarily on N. gonorrhoeae. This limits the generalizability of the findings and leaves questions about its broader antibacterial spectrum. Moreover, while the study demonstrates the in vitro effectiveness of oxydifficidin, there is a lack of in vivo validation (i.e., animal models) for assessing pre-clinical potential of oxydifficidin. Potential SNPs within dedA or RplL raise concerns about how quickly resistance could emerge in clinical settings.

(1) Spectrum/narrow scope: The broader antibacterial spectrum of oxydifficidin has been reported previously (S B Zimmerman et al., 1987). The focus of this study is on its previously unreported potent anti-gonococcal activity and its mode of action. While it is true that broad-spectrum antibiotics have historically played a role in effectively controlling a wide range of infections, we and others believe that narrow-spectrum antibiotics have an overlooked importance in addressing bacterial infections. Their advantage lies in their ability to target specific pathogens without markedly disrupting the human microbiota.

(2) Animal models: We acknowledge the reviewer’s insight regarding the importance of in vivo validation to enhance oxydifficidin’s pre-clinical potential. However, due to the labor-intensive process needed to isolate oxydifficidin, obtaining a sufficient quantity for animal studies is beyond the scope of this study. Our future work will focus on optimizing the yield of oxydifficidin and developing a topical mouse model for subsequent investigations.

(3) Potential SNPs: Please see our response to Reviewer #1’s comment 3. We acknowledge that potential SNPs within dedA and rplL raise concerns regarding clinical resistance, which is a common issue for protein-targeting antibiotics. Yet, as pointed out in the manuscript, obtaining mutants in the lab was a very low yield endeavor.

Reviewer #3 (Public Review):

Summary:

The authors have shown that oxydifficidin is a potent inhibitor of Neisseria gonorrhoeae. They were able to identify the target of action to rplL and showed that resistance could occur via mutation in the DedA flippase and RplL.

Strengths:

This was a very thorough and clearly argued set of experiments that supported their conclusions.

Weaknesses:

There was no obvious weakness in the experimental design. Although it is promising that the DedA mutations resulted in attenuation of fitness, it remains an open question whether secondary rounds of mutation could overcome this selective disadvantage which was untried in this study.

We thank the reviewer for the positive comment. We agree that investigating factors that could compensate for the fitness attenuation caused by DedA mutation would enhance our understanding of the role of DedA.

eLife assessment

This study provides valuable new insights into the trade-offs associated with the evolution of drug resistance in the yeast S. cerevisiae, based on a solid approach to evolving and phenotyping hundreds of independent strains. The authors identify distinct phenotypic clusters, defined by their growth across defined conditions, which suggest that tradeoffs are diverse but at the same time could be limited to a few classes according to the underlying resistance mechanisms. The methodologies used align with the current state-of-the-art, and the data and analysis are solid as they broadly support the claims, with only a few minor weaknesses remaining after revision. This work will interest molecular biologists working on the evolution of new phenotypes and microbiologists studying multi-drug therapy.

Reviewer #1 (Public Review):

Summary:

In their manuscript, Schmidlin, Apodaca et al try to answer fundamental questions about the evolution of new phenotypes and the trade-offs associated with this process. As a model, they use yeast resistance to two drugs, fluconazole and radicicol. They use barcoded libraries of isogenic yeasts to evolve thousands of strains in 12 different environments. They then measure the fitness of evolved strains in all environments and use these measurements to enumerate patterns in fitness trade-offs. They identify only six major clusters corresponding to different trade-off profiles, suggesting the vast genotypic landscape of evolved mutants translates to a highly constrained phenotypic space. They sequence over a hundred evolved strains and find that mutations in the same gene can result in different phenotypic profiles.

Overall, the authors deploy innovative methods to scale up experimental evolution experiments, and in many aspects of their approach tried to minimize experimental variation.

Weaknesses:

(1) The main objective of the authors is to characterize the extent of phenotypic diversity in terms of resistance trade-offs between fluconazole and radicicol. To minimize noise in the measurement of relative fitness, the authors only included strains with at least 500 barcode counts across all time points in all 12 experimental conditions, resulting in a set of 774 lineages passing this threshold. As the authors remark, this will bias their datasets for lineages with high fitness in all 12 environments, as all these strains must be fit enough to maintain a high abundance. One of the main observations of the authors is phenotypic space is constrained to a few clusters of roughly similar relative fitness patterns, giving hope that such clusters could be enumerated and considered to design antimicrobial treatment strategies. However, by excluding all lineages that fit in only one or a few environments, they conceal much of the diversity that might exist in terms of trade-offs and set up an inclusion threshold that might present only a small fraction of phenotypic space with characteristics consistent with generalist resistance mechanisms or broadly increased fitness. The general conclusions of the authors regarding the evolution of trade-offs might thus be more focused on multi-drug resistant phenotypes.

(2) Most large-scale pooled competition assays using barcodes are usually stopped after ~25 to avoid noise due to the emergence of secondary mutations. The authors measure fitness across ~40 generations, which is almost the same number of generations as in the evolution experiment. This raises the possibility of secondary mutations biasing abundance values, which would not have been detected by the whole genome sequencing as it was performed before the competition assay. Previous studies approximated the fraction of lineages that could be overtaken by secondary mutations (Venkataram and Dunn et al 2016). In their calculations, Venkataram and Dunn et al defined adaptive mutations in their data as having a selection coefficient of 5% and highly adaptive mutations at around 10%. From this and an estimation of the mutation rate, they estimate that the fraction of lineages overtaken by adaptive mutations is negligible (10^4) after 32 generations. However, the effects on fitness observed by the authors here tend to be much stronger than 5-10%, with relative fitness advantages above 1 and often reaching 2. This could result in a much higher chance of lineages being overtaken at 40 generations.

(3) The approach used by the authors to identify and visualize clusters of phenotypes among lineages does not seem to consider the uncertainty in the measurement of their relative fitness. As can be seen from Figure S4, the inter-replicate difference in measured fitness can often be quite large. From these graphs, it is also possible to see that some of the fitness measurements do not correlate linearly (ex.: Med Flu, Hi Rad Low Flu), meaning that taking the average of both replicates might not be the best approach. Because the clustering approach used does not seem to take this variability into account, it becomes difficult to evaluate the strength of the clustering, especially because the UMAP projection does not include any representation of uncertainty around the position of lineages.

(4) The authors make the decision to use UMAP and a Gaussian mixed model as well as validation data to identify unique clusters, which is one of their main objectives. The choice of 7 clusters as the cutoff for the multiple Gaussian model is not well explained. Based on Figure S6A, BIC starts leveling off at 6 clusters, not 7, and going to 8 clusters would provide the same reduction as going from 6 to 7. This choice also appears arbitrary in Figure S6B, where BIC levels off at 9 clusters when only highly abundant lineages are considered. All of the data presented in the validations is presented to fit within the 6 clusters structure but does not include evidence against alternative scenarios for additional relevant clusters as might be suggested by Figure S6.

(5) Large-scale barcode sequencing assays can often be noisy and are generally validated using growth curves or competition assays. Reconstructing some of the specific mutants they identified to validate their phenotypes would also have been a good addition. If the phenotypic clusters identified cannot be reproduced outside of the sequencing assay, then their relevance are they as a model for multi-drug resistance scenarios might be reduced.

Reviewer #1 (Public Review):

Summary:

In their manuscript, Schmidlin, Apodaca, et al try to answer fundamental questions about the evolution of new phenotypes and the trade-offs associated with this process. As a model, they use yeast resistance to two drugs, fluconazole and radicicol. They use barcoded libraries of isogenic yeasts to evolve thousands of strains in 12 different environments. They then measure the fitness of evolved strains in all environments and use these measurements to examine patterns in fitness trade-offs. They identify only six major clusters corresponding to different trade-off profiles, suggesting the vast genotypic landscape of evolved mutants translates to a highly constrained phenotypic space. They sequence over a hundred evolved strains and find that mutations in the same gene can result in different phenotypic profiles.

Overall, the authors deploy innovative methods to scale up experimental evolution experiments, and in many aspects of their approach tried to minimize experimental variation.

Weaknesses:

(1) One of the objectives of the authors is to characterize the extent of phenotypic diversity in terms of resistance trade-offs between fluconazole and radicicol. To minimize noise in the measurement of relative fitness, the authors only included strains with at least 500 barcode counts across all time points in all 12 experimental conditions, resulting in a set of 774 lineages passing this threshold. This corresponds to a very small fraction of the starting set of ~21 000 lineages that were combined after experimental evolution for fitness measurements. As the authors briefly remark, this will bias their datasets for lineages with high fitness in all 12 environments, as all these strains must be fit enough to maintain a high abundance. One of the main observations of the authors is phenotypic space is constrained to a few clusters of roughly similar relative fitness patterns, giving hope that such clusters could be enumerated and considered to design antimicrobial treatment strategies. However, by excluding all lineages that fit in only one or a few environments, they conceal much of the diversity that might exist in terms of trade-offs and set up an inclusion threshold that might present only a small fraction of phenotypic space with characteristics consistent with generalist resistance mechanisms or broadly increased fitness. This has important implications regarding the general conclusions of the authors regarding the evolution of trade-offs.

(2) Most large-scale pooled competition assays using barcodes are usually stopped after ~25 to avoid noise due to the emergence of secondary mutations. The authors measure fitness across ~40 generations, which is almost the same number of generations as in the evolution experiment. This raises the possibility of secondary mutations biasing abundance values, which would not have been detected by the whole genome sequencing as it was performed before the competition assay.

(3) The approach used by the authors to identify and visualize clusters of phenotypes among lineages does not seem to consider the uncertainty in the measurement of their relative fitness. As can be seen from Figure S4, the inter-replicate difference in measured fitness can often be quite large. From these graphs, it is also possible to see that some of the fitness measurements do not correlate linearly (ex.: Med Flu, Hi Rad Low Flu), meaning that taking the average of both replicates might not be the best approach. Because the clustering approach used does not seem to take this variability into account, it becomes difficult to evaluate the strength of the clustering, especially because the UMAP projection does not include any representation of uncertainty around the position of lineages. This might paint a misleading picture where clusters appear well separate and well defined but are in fact much fuzzier, which would impact the conclusion that the phenotypic space is constricted.

(4) The authors make the decision to use UMAP and a gaussian mixed model to cluster and represent the different fitness landscapes of their lineages of interest. Their approach has many caveats. First, compared to PCA, the axis does not provide any information about the actual dissimilarities between clusters. Using PCA would have allowed a better understanding of the amount of variance explained by components that separate clusters, as well as more interpretable components. Second, the advantages of dimensional reduction are not clear. In the competition experiment, 11/12 conditions (all but the no drug, no DMSO conditions) can be mapped to only three dimensions: concentration of fluconazole, concentration of radicicol, and relative fitness. Each lineage would have its own fitness landscape as defined by the plane formed by relative fitness values in this space, which can then be examined and compared between lineages. Third, the choice of 7 clusters as the cutoff for the multiple Gaussian model is not well explained. Based on Figure S6A, BIC starts leveling off at 6 clusters, not 7, and going to 8 clusters would provide the same reduction as going from 6 to 7. This choice also appears arbitrary in Figure S6B, where BIC levels off at 9 clusters when only highly abundant lineages are considered. This directly contradicts the statement in the main text that clusters are robust to noise, as more a stringent inclusion threshold appears to increase and not decrease the optimal number of clusters. Additional criteria to BIC could have been used to help choose the optimal number of clusters or even if mixed Gaussian modeling is appropriate for this dataset.

(5) Large-scale barcode sequencing assays can often be noisy and are generally validated using growth curves or competition assays. Having these types of results would help support the accuracy of the main assay in the manuscript and thus better support the claims of the authors.

Reviewer #2 (Public Review):

Summary:

Schmidlin & Apodaca et al. aim to distinguish mutants that resist drugs via different mechanisms by examining fitness tradeoffs across hundreds of fluconazole-resistant yeast strains. They barcoded a collection of fluconazole-resistant isolates and evolved them in different environments with a view to having relevance for evolutionary theory, medicine, and genotype-phenotype mapping.

Strengths:

There are multiple strengths to this paper, the first of which is pointing out how much work has gone into it; the quality of the experiments (the thought process, the data, the figures) is excellent. Here, the authors seek to induce mutations in multiple environments, which is a really large-scale task. I particularly like the attention paid to isolates with are resistant to low concentrations of FLU. So often these are overlooked in favour of those conferring MIC values >64/128 etc. What was seen is different genotype and fitness profiles. I think there's a wealth of information here that will actually be of interest to more than just the fields mentioned (evolutionary medicine/theory).

Weaknesses:

Not picking up low fitness lineages - which the authors discuss and provide a rationale as to why. I can completely see how this has occurred during this research, and whilst it is a shame I do not think this takes away from the findings of this paper. Maybe in the next one!

In the abstract the authors focus on 'tradeoffs' yet in the discussion they say the purpose of the study is to see how many different mechanisms of FLU resistance may exist (lines 679-680), followed up by "We distinguish mutants that likely act via different mechanisms by identifying those with different fitness tradeoffs across 12 environments". Whilst I do see their point, and this is entirely feasible, I would like a bit more explanation around this (perhaps in the intro) to help lay-readers make this jump. The remainder of my comments on 'weaknesses' are relatively fixable, I think:

In the introduction I struggle to see how this body of research fits in with the current literature, as the literature cited is a hodge-podge of bacterial and fungal evolution studies, which are very different! So example, the authors state "previous work suggests that mutants with different fitness tradeoffs may affect fitness through different molecular mechanisms" (lines 129-131) and then cite three papers, only one of which is a fungal research output. However, the next sentence focuses solely on literature from fungal research. Citing bacterial work as a foundation is fine, but as you're using yeast for this I think tailoring the introduction more to what is and isn't known in fungi would be more appropriate. It would also be great to then circle back around and mention monotherapy vs combination drug therapy for fungal infections as a rationale for this study. The study seems to be focused on FLU-resistant mutants, which is the first-line drug of choice, but many (yeast) infections have acquired resistance to this and combination therapy is the norm.

Methods: Line 769 - which yeast? I haven't even seen mention of which species is being used in this study; different yeast employ different mechanisms of adaptation for resistance, so could greatly impact the results seen. This could help with some background context if the species is mentioned (although I assume S. cerevisiae). In which case, should aneuploidy be considered as a mechanism? This is mentioned briefly on line 556, but with all the sequencing data acquired this could be checked quickly?

I think the authors could be bolder and try and link this to other (pathogenic) yeasts. What are the implications of this work on say, Candida infections?

Author response:

We thank you for the opportunity to provide a concise response. The criticisms are accurately summarized in the eLife assessment:

the study fails to engage prior literature that has extensively examined the impact of variance in offspring number, implying that some of the paradoxes presented might be resolved within existing frameworks.

The essence of our study is to propose the adoption of the Haldane model of genetic drift, based on the branching process, in lieu of the Wright-Fisher (WF) model, based on sampling, usually binomial.  In addition to some extensions of the Haldane model, we present 4 paradoxes that cannot be resolved by the WF model. The reviews suggest that some of the paradoxes could be resolved by the WF model, if we engage prior literature sufficiently.

We certainly could not review all the literature on genetic drift as there must be thousands of them. Nevertheless, the literature we do not cover is based on the WF model, which has the general properties that all modifications of the WF model share.  (We should note that all such modifications share the sampling aspect of the WF model. To model such sampling, N is imposed from outside of the model, rather than self-generating within the model.  Most important, these modifications are mathematically valid but biologically untenable, as will be elaborated below. Thus, in concept, the WF and Haldane models are fundamentally different.)

In short, our proposal is general with the key point that the WF model cannot resolve these (and many other) paradoxes.  The reviewers disagree (apparently only partially) and we shall be specific in our response below.

We shall first present the 4th paradox, which is about multi-copy gene systems (such as rRNA genes and viruses, see the companion paper). Viruses evolve both within and between hosts. In both stages, there are severe bottlenecks.  How does one address the genetic drift in viral evolution? How can we model the effective population sizes both within- and between- hosts?  The inability of the WF model in dealing with such multi-copy gene systems may explain the difficulties in accounting for the SARS-CoV-2 evolution. Given the small number of virions transmitted between hosts, drift is strong which we have shown by using the Haldane model (Ruan, Luo, et al. 2021; Ruan, Wen, et al. 2021; Hou, et al. 2023). 

As the reviewers suggest, it is possible to modify the WF model to account for some of these paradoxes. However, the modifications are often mathematically convenient but biologically dubious. Much of the debate is about the progeny number, K.  (We shall use haploid model for this purpose but diploidy does not pose a problem as stated in the main text.) The modifications relax the constraint of V(k) = E(k) inherent in the WF sampling.  One would then ask how V(k) can be different from E(k) in the WF sampling even though it is mathematically feasible (but biologically dubious)?  Kimura and Crow (1963) may be the first to offer a biological explanation.  If one reads it carefully, Kimura's modification is to make the WF model like the Haldane model. Then, why don't we use the Haldane model in the first place by having two parameters, E(k) and V(k), instead of the one-parameter WF model?

The Haldane model is conceptually simpler. It allows the variation in population size, N, to be generated from within the model, rather than artificially imposed from outside of the model.  This brings us to the first paradox, the density-dependent Haldane model. When N is increasing exponentially as in bacterial or yeast cultures, there is almost no drift when N is very low and drift becomes intense as N grows to near the carrying capacity.  We do not see how the WF model can resolve this paradox, which can otherwise be resolved by the Haldane model.

The second and third paradoxes are about how much mathematical models of population genetic can be detached from biological mechanisms. The second paradox about sex chromosomes is rooted in the realization of V(k) ≠ E(k).  Since E(k) is the same between sexes but V(k) is different, how does the WF sampling give rise to V(k) ≠ E(k)? We are asking a biological question that troubled Kimura and Crow (1963) alluded above. The third paradox is acknowledged by two reviewers. Genetic drift manifested in the fixation probability of an advantageous mutation is 2s/V(k).  It is thus strange that the fundamental parameter of drift in the WF model, N (or Ne), is missing.  In the Haldane model, drift is determined by V(k) with N being a scaling factor; hence 2s/V(k) makes perfect biological sense,

We now answer the obvious question: If the model is fundamentally about the Haldane model, why do we call it the WF-Haldane model? The reason is that most results obtained by the WF model are pretty good approximations and the branching process may not need to constantly re-derive the results.  At least, one can use the WF results to see how well they fit into the Haldane model. In our earlier study (Chen, et al. (2017); Fig. 3), we show that the approximations can be very good in many (or most) settings.

We would like to use the modern analogy of gas-engine cars vs. electric-motor ones. The Haldane model and the WF model are as fundamentally different concepts as the driving mechanisms of gas-powered vs electric cars.  The old model is now facing many problems and the fixes are often not possible.  Some fixes are so complicated that one starts thinking about simpler solutions. The reservations are that we have invested so much in the old models which might be wasted by the switch. However, we are suggesting the integration of the WF and Haldane models. In this sense, the WF model has had many contributions which the new model gratefully inherits. This is true with the legacy of gas-engine cars inherited by EVs.

The editors also issue the instruction: while the modified model yields intriguing theoretical predictions, the simulations and empirical analyses are incomplete to support the authors' claims. 

We are thankful to the editors and reviewers for the thoughtful comments and constructive criticisms. We also appreciate the publishing philosophy of eLife that allows exchanges, debates and improvements, which are the true spirits of science publishing.

References for the provisional author responses

Chen Y, Tong D, Wu CI. 2017. A New Formulation of Random Genetic Drift and Its Application to the Evolution of Cell Populations. Mol. Biol. Evol. 34:2057-2064.

Hou M, Shi J, Gong Z, Wen H, Lan Y, Deng X, Fan Q, Li J, Jiang M, Tang X, et al. 2023. Intra- vs. Interhost Evolution of SARS-CoV-2 Driven by Uncorrelated Selection-The Evolution Thwarted. Mol. Biol. Evol. 40.

Kimura M, Crow JF. 1963. The measurement of effective population number. Evolution:279-288.

Ruan Y, Luo Z, Tang X, Li G, Wen H, He X, Lu X, Lu J, Wu CI. 2021. On the founder effect in COVID-19 outbreaks: how many infected travelers may have started them all? Natl. Sci. Rev. 8:nwaa246.

Ruan Y, Wen H, He X, Wu CI. 2021. A theoretical exploration of the origin and early evolution of a pandemic. Sci Bull (Beijing) 66:1022-1029.

Review comments

eLife assessment 

This study presents a useful modification of a standard model of genetic drift by incorporating variance in offspring numbers, claiming to address several paradoxes in molecular evolution.

It is unfortunate that the study fails to engage prior literature that has extensively examined the impact of variance in offspring number, implying that some of the paradoxes presented might be resolved within existing frameworks.

We do not believe that the paradoxes can be resolved.

In addition, while the modified model yields intriguing theoretical predictions, the simulations and empirical analyses are incomplete to support the authors' claims. 

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

The authors present a theoretical treatment of what they term the "Wright-Fisher-Haldane" model, a claimed modification of the standard model of genetic drift that accounts for variability in offspring number, and argue that it resolves a number of paradoxes in molecular evolution. Ultimately, I found this manuscript quite strange.

The notion of effective population size as inversely related to the variance in offspring number is well known in the literature, and not exclusive to Haldane's branching process treatment. However, I found the authors' point about variance in offspring changing over the course of, e.g. exponential growth fairly interesting, and I'm not sure I'd seen that pointed out before.

Nonetheless, I don't think the authors' modeling, simulations, or empirical data analysis are sufficient to justify their claims. 

Weaknesses: 

I have several outstanding issues. First of all, the authors really do not engage with the literature regarding different notions of an effective population. Most strikingly, the authors don't talk about Cannings models at all, which are a broad class of models with non-Poisson offspring distributions that nonetheless converge to the standard Wright-Fisher diffusion under many circumstances, and to "jumpy" diffusions/coalescents otherwise (see e.g. Mohle 1998, Sagitov (2003), Der et al (2011), etc.). Moreover, there is extensive literature on effective population sizes in populations whose sizes vary with time, such as Sano et al (2004) and Sjodin et al (2005).

Of course in many cases here the discussion is under neutrality, but it seems like the authors really need to engage with this literature more. 

The most interesting part of the manuscript, I think, is the discussion of the Density Dependent Haldane model (DDH). However, I feel like I did not fully understand some of the derivation presented in this section, which might be my own fault. For instance, I can't tell if Equation 5 is a result or an assumption - when I attempted a naive derivation of Equation 5, I obtained E(K_t) = 1 + r/c*(c-n)*dt. It's unclear where the parameter z comes from, for example. Similarly, is equation 6 a derivation or an assumption? Finally, I'm not 100% sure how to interpret equation 7. I that a variance effective size at time t? Is it possible to obtain something like a coalescent Ne or an expected number of segregating sites or something from this? 

Similarly, I don't understand their simulations. I expected that the authors would do individual-based simulations under a stochastic model of logistic growth, and show that you naturally get variance in offspring number that changes over time. But it seems that they simply used their equations 5 and 6 to fix those values. Moreover, I don't understand how they enforce population regulation in their simulations---is N_t random and determined by the (independent) draws from K_t for each individual? In that case, there's no "interaction" between individuals (except abstractly, since logistic growth arises from a model that assumes interactions between individuals). This seems problematic for their model, which is essentially motivated by the fact that early during logistic growth, there are basically no interactions, and later there are, which increases variance in reproduction. But their simulations assume no interactions throughout! 

The authors also attempt to show that changing variance in reproductive success occurs naturally during exponential growth using a yeast experiment. However, the authors are not counting the offspring of individual yeast during growth (which I'm sure is quite hard). Instead, they use an equation that estimates the variance in offspring number based on the observed population size, as shown in the section "Estimation of V(K) and E(K) in yeast cells". This is fairly clever, however, I am not sure it is right, because the authors neglect covariance in offspring between individuals. My attempt at this derivation assumes that I_t | I_{t-1} = \sum_{I=1}^{I_{t-1}} K_{i,t-1} where K_{i,t-1} is the number of offspring of individual i at time t-1. Then, for example, E(V(I_t | I_{t-1})) = E(V(\sum_{i=1}^{I_{t-1}} K_{i,t-1})) = E(I_{t-1})V(K_{t-1}) + E(I_{k-1}(I_{k-1}-1))*Cov(K_{i,t-1},K_{j,t-1}). The authors have the first term, but not the second, and I'm not sure the second can be neglected (in fact, I believe it's the second term that's actually important, as early on during growth there is very little covariance because resources aren't constrained, but at carrying capacity, an individual having offspring means that another individuals has to have fewer offspring - this is the whole notion of exchangeability, also neglected in this manuscript). As such, I don't believe that their analysis of the empirical data supports their claim. 

Thus, while I think there are some interesting ideas in this manuscript, I believe it has some fundamental issues:

first, it fails to engage thoroughly with the literature on a very important topic that has been studied extensively. Second, I do not believe their simulations are appropriate to show what they want to show. And finally, I don't think their empirical analysis shows what they want to show. 

References: 

Möhle M. Robustness results for the coalescent. Journal of Applied Probability. 1998;35(2):438-447. doi:10.1239/jap/1032192859 

Sagitov S. Convergence to the coalescent with simultaneous multiple mergers. Journal of Applied Probability. 2003;40(4):839-854. doi:10.1239/jap/1067436085 

Der, Ricky, Charles L. Epstein, and Joshua B. Plotkin. "Generalized population models and the nature of genetic drift." Theoretical population biology 80.2 (2011): 80-99 

Sano, Akinori, Akinobu Shimizu, and Masaru Iizuka. "Coalescent process with fluctuating population size and its effective size." Theoretical population biology 65.1 (2004): 39-48 

Sjodin, P., et al. "On the meaning and existence of an effective population size." Genetics 169.2 (2005): 1061-1070 

Reviewer #2 (Public Review): 

Summary: 

This theoretical paper examines genetic drift in scenarios deviating from the standard Wright-Fisher model. The authors discuss Haldane's branching process model, highlighting that the variance in reproductive success equates to genetic drift. By integrating the Wright-Fisher model with the Haldane model, the authors derive theoretical results that resolve paradoxes related to effective population size. 

Strengths: 

The most significant and compelling result from this paper is perhaps that the probability of fixing a new beneficial mutation is 2s/V(K). This is an intriguing and potentially generalizable discovery that could be applied to many different study systems. 

The authors also made a lot of effort to connect theory with various real-world examples, such as genetic diversity in sex chromosomes and reproductive variance across different species. 

Weaknesses: 

One way to define effective population size is by the inverse of the coalescent rate. This is where the geometric mean of Ne comes from. If Ne is defined this way, many of the paradoxes mentioned seem to resolve naturally. If we take this approach, one could easily show that a large N population can still have a low coalescent rate depending on the reproduction model. However, the authors did not discuss Ne in light of the coalescent theory. This is surprising given that Eldon and Wakeley's 2006 paper is cited in the introduction, and the multiple mergers coalescent was introduced to explain the discrepancy between census size and effective population size, superspreaders, and reproduction variance - that said, there is no explicit discussion or introduction of the multiple mergers coalescent. 

The Wright-Fisher model is often treated as a special case of the Cannings 1974 model, which incorporates the variance in reproductive success. This model should be discussed. It is unclear to me whether the results here have to be explained by the newly introduced WFH model, or could have been explained by the existing Cannings model. 

The abstract makes it difficult to discern the main focus of the paper. It spends most of the space introducing "paradoxes". 

The standard Wright-Fisher model makes several assumptions, including hermaphroditism, non-overlapping generations, random mating, and no selection. It will be more helpful to clarify which assumptions are being violated in each tested scenario, as V(K) is often not the only assumption being violated. For example, the logistic growth model assumes no cell death at the exponential growth phase, so it also violates the assumption about non-overlapping generations. 

The theory and data regarding sex chromosomes do not align. The fact that \hat{alpha'} can be negative does not make sense. The authors claim that a negative \hat{alpha'} is equivalent to infinity, but why is that? It is also unclear how theta is defined. It seems to me that one should take the first principle approach e.g., define theta as pairwise genetic diversity, and start with deriving the expected pair-wise coalescence time under the MMC model, rather than starting with assuming theta = 4Neu. Overall, the theory in this section is not well supported by the data, and the explanation is insufficient. 

{Alpha and alpha' can both be negative.  X^2 = 0.47 would yield x = -0.7}

Reviewer #3 (Public Review): 

Summary: 

Ruan and colleagues consider a branching process model (in their terminology the "Haldane model") and the most basic Wright-Fisher model. They convincingly show that offspring distributions are usually non-Poissonian (as opposed to what's assumed in the Wright-Fisher model), and can depend on short-term ecological dynamics (e.g., variance in offspring number may be smaller during exponential growth). The authors discuss branching processes and the Wright-Fisher model in the context of 3 "paradoxes": (1) how Ne depends on N might depend on population dynamics; (2) how Ne is different on the X chromosome, the Y chromosome, and the autosomes, and these differences do match the expectations base on simple counts of the number of chromosomes in the populations; (3) how genetic drift interacts with selection. The authors provide some theoretical explanations for the role of variance in the offspring distribution in each of these three paradoxes. They also perform some experiments to directly measure the variance in offspring number, as well as perform some analyses of published data. 

Strengths: 

(1) The theoretical results are well-described and easy to follow. 

(2) The analyses of different variances in offspring number (both experimentally and analyzing public data) are convincing that non-Poissonian offspring distributions are the norm. 

(3) The point that this variance can change as the population size (or population dynamics) change is also very interesting and important to keep in mind. 

(4) I enjoyed the Density-Dependent Haldane model. It was a nice example of the decoupling of census size and effective size. 

Weaknesses: 

(1) I am not convinced that these types of effects cannot just be absorbed into some time-varying Ne and still be well-modeled by the Wright-Fisher process. 

(2) Along these lines, there is well-established literature showing that a broad class of processes (a large subset of Cannings' Exchangeable Models) converge to the Wright-Fisher diffusion, even those with non-Poissonian offspring distributions (e.g., Mohle and Sagitov 2001). E.g., equation (4) in Mohle and Sagitov 2001 shows that in such cases the "coalescent Ne" should be (N-1) / Var(K), essentially matching equation (3) in the present paper. 

(3) Beyond this, I would imagine that branching processes with heavy-tailed offspring distributions could result in deviations that are not well captured by the authors' WFH model. In this case, the processes are known to converge (backward-in-time) to Lambda or Xi coalescents (e.g., Eldon and Wakely 2006 or again in Mohle and Sagitov 2001 and subsequent papers), which have well-defined forward-in-time processes. 

(4) These results that Ne in the Wright-Fisher process might not be related to N in any straightforward (or even one-to-one) way are well-known (e.g., Neher and Hallatschek 2012; Spence, Kamm, and Song 2016; Matuszewski, Hildebrandt, Achaz, and Jensen 2018; Rice, Novembre, and Desai 2018; the work of Lounès Chikhi on how Ne can be affected by population structure; etc...) 

(5) I was also missing some discussion of the relationship between the branching process and the Wright-Fisher model (or more generally Cannings' Exchangeable Models) when conditioning on the total population size. In particular, if the offspring distribution is Poisson, then conditioned on the total population size, the branching process is identical to the Wright-Fisher model. 

(6) In the discussion, it is claimed that the last glacial maximum could have caused the bottleneck observed in human populations currently residing outside of Africa. Compelling evidence has been amassed that this bottleneck is due to serial founder events associated with the out-of-Africa migration (see e.g., Henn, Cavalli-Sforza, and Feldman 2012 for an older review - subsequent work has only strengthened this view). For me, a more compelling example of changes in carrying capacity would be the advent of agriculture ~11kya and other more recent technological advances. 

Recommendations for the authors: 

Reviewing Editor Comments: 

The reviewers recognize the value of this model and some of the findings, particularly results from the density-dependent Haldane model. However, they expressed considerable concerns with the model and overall framing of this manuscript.

First, all reviewers pointed out that the manuscript does not sufficiently engage with the extensive literature on various models of effective population size and genetic drift, notably lacking discussion on Cannings models and related works.

Second, there is a disproportionate discussion on the paradoxes, yet some of the paradoxes might already be resolved within current theoretical frameworks. All three reviewers found the modeling and simulation of the yeast growth experiment hard to follow or lacking justification for certain choices. The analysis approach of sex chromosomes is also questioned. 

The reviewers recommend a more thorough review of relevant prior literature to better contextualize their findings. The authors need to clarify and/or modify their derivations and simulations of the yeast growth experiment to address the identified caveats and ensure robustness. Additionally, the empirical analysis of the sex chromosome should be revisited, considering alternative scenarios rather than relying solely on the MSE, which only provides a superficial solution. Furthermore, the manuscript's overall framing should be adjusted to emphasize the conclusions drawn from the WFH model, rather than focusing on the "unresolved paradoxes", as some of these may be more readily explained by existing frameworks. Please see the reviewers' overall assessment and specific comments. 

Reviewer #2 (Recommendations For The Authors): 

In the introduction -- "Genetic drift is simply V(K)" -- this is a very strong statement. You can say it is inversely proportional to V(K), but drift is often defined based on changes in allele frequency. 

Page 3 line 86. "sexes is a sufficient explanation."--> "sex could be a sufficient explanation" 

The strongest line of new results is about 2s/V(K). Perhaps, the paper could put more emphasis on this part and demonstrate the generality of this result with a different example. 

The math notations in the supplement are not intuitive. e.g., using i_k and j_k as probabilities. I also recommend using E[X] and V[X]for expectation and variance rather than \italic{E(X)} to improve the readability of many equations. 

Eq A6, A7, While I manage to follow, P_{10}(t) and P_{10} are not defined anywhere in the text. 

Supplement page 7, the term "probability of fixation" is confusing in a branching model. 

E.q. A 28. It is unclear eq. A.1 could be used here directly. Some justification would be nice. 

Supplement page 17. "the biological meaning of negative..". There is no clear justification for this claim. As a reader, I don't have any intuition as to why that is the case.

hi

eLife assessment

This paper describes an important advance in an in vitro neural culture system to generate mature, functional, diverse, and geometrically consistent cultures, in a 384-well format with defined dimensions and the absence of the necrotic core, which persists for up to 300 days. The well-based format and conserved geometry make it a promising tool for arrayed screening studies. Some of the evidence is incomplete and could benefit from a more direct head-to-head comparison with more standard culture methods and standardization of cell seeding density as well as further data on reproducibility in each well and for each cell line.

Author response:

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

eLife assessment:

Franke et al. explore and characterize the color response properties in the mouse primary visual cortex, revealing specific color opponent encoding strategies across the visual field. The data is solid; however, the evidence supporting some conclusions is incomplete. In its current form, the paper makes a useful contribution to how color is coded in mouse V1. Significance would be enhanced with some additional analyses and a clearer discussion of the limitations of the data presented.

We thank the reviewers for appreciating our manuscript. We have rewritten the conclusions of the paper to be more conservative and now more explicitly focus on color processing in mouse V1, rather than comparing V1 to the retina. Additionally, we discuss the limitations of our approach in detail in the Discussion section. Finally, we have addressed all comments from the reviewers below.

Referee 1 (Remarks to the Author):

In this study, Franke et al. explore and characterize color response properties across primary visual cortex, revealing specific color opponent encoding strategies across the visual field. The authors use awake 2P imaging to define the spectral response properties of visual interneurons in layer 2/3. They find that opponent responses are more pronounced at photopic light levels, and that diversity in color opponent responses exists across the visual field, with green ON/ UV OFF responses more strongly represented in the upper visual field. This is argued to be relevant for the detection of certain features that are more salient when using chromatic space, possibly due to noise reduction. In the revised version, Franke et al. have addressed the potential pitfalls in the discussion, which is an important point for the non-expert reader. Thus, this study provides a solid characterization of the color properties of V1 and is a valuable addition to visual neuroscience research.

My remaining concerns are based more on the interpretation. I’m still not convinced by the statement "This type of color-opponency in the receptive field center of V1 neurons was not present in the receptive field center of retinal ganglion cells and, therefore, is likely computed by integrating center and surround information downstream of the retina." and I would suggest rewording it in the abstract.

As discussed previously and now nicely added to the discussion, it is difficult to make a direct comparison given the different stimulus types used to characterize the retina and V1 recordings and the different levels of adaptation in both tissues. I will leave this point to the discussion, which allows for a more nuanced description of the phenomenon. Why do I think this is important? In the introduction, the authors argue that "the discrepancy [of previous studies] may be due to differences in stimulus design or light levels." However, while different light levels can be tested in V1, this cannot be done properly in the retina with 2P experiments. To address this, one would have to examine color-opponency in RGC terminals in vivo, which is beyond the scope of this study. Addressing these latter points directly in the discussion would, in my opinion, only strengthen the study.

We thank the reviewer for the feedback. We removed the sentence mentioned by the reviewer from the abstract, as well as from the summary of our results in the Introduction. Additionally, we now phrase the interpretation of the retinal results more conservatively and specifically highlight in the Discussion that comparing ex-vivo retinal to in-vivo cortical data is challenging. With these changes, we believe that the focus of the paper is explicitly defined to be on the neuronal representation of color in mouse visual cortex, rather than on the comparison of retinal and cortical color processing.

Minor:

In the abstract, the second sentence says that we already know the mechanisms in primates.

Unfortunately, I do not think this is true. First, primates refers to an order with several species, which might have adaptations to their color-processing. Second, I’m aware of several characterizations in "primates" that have led to convincing models (as referenced), but in my opinion, this is far from a true understanding the mechanisms, especially since very little is known about foveal color processing due to the difficulties of these experiments. Similarly in the introduction. "Primates" is indirectly defined as a species. Perhaps some rewording is needed here as well, since we know how different cone distributions can be in rodents (see Peichl’s work).

Thanks. We have reworded the Abstract and Introduction towards indicating that many studies have been performed in primate species, without suggesting that the mechanisms are described.

The legend in Fig. 2 has a "Fig. ???"

Fixed.

Referee 2 (Remarks to the Author):

Franke et al. characterize the representation of color in the primary visual cortex of mice, highlighting how this changes across the visual field. Using calcium imaging in awake, head-fixed mice, they characterize the properties of V1 neurons (layer 2/3) using a large center-surround stimulation where green and ultra-violet colors were presented in random combinations. Clustering of responses revealed a set of functional cell-types based on their preference to different combinations of green and UV in their center and surround. These functional types were demonstrated to have different spatial distributions across V1, including one neuronal type (Green-ON/UV-OFF) that was much more prominent in the posterior V1 (i.e. upper visual field). Modelling work suggests that these neurons likely support the detection of predator-like objects in the sky.

Strengths: The large-scale single-cell resolution imaging used in this work allows the authors to map the responses of individual neurons across large regions of the visual cortex. Combining this large dataset with clustering analysis enabled the authors to group V1 neurons into distinct functional cell types and demonstrate their relative distribution in the upper and lower visual fields. Modelling work demonstrated the different capacity of each functional type to detect objects in the sky, providing insight into the ethological relevance of color opponent neurons in V1.

We thank the reviewer for appreciating our study.

Weaknesses: While the study presents convincing evidence about the asymmetric distribution of color-opponent neurons in V1, the paper would greatly benefit from a more in-depth discussion of the caveats related to the conclusions drawn about their origin. This is particularly relevant regarding the conclusion drawn about the contribution of color opponent neurons in the retina. The mismatch between retinal color opponency and V1 color opponency could imply that this feature is not solely inherited from the retina, however, there are other plausible explanations that are not discussed here. Direct evidence for this statement remains weak.

Thanks for this comment. We removed the retinal findings from the abstract, as well as from the summary of our results in the Introduction. In addition, we now phrase the interpretation of the retinal results more conservatively and specifically highlight in the Discussion that comparing ex-vivo retinal to in-vivo cortical data is challenging. With these changes, we believe that the focus of the paper is explicitly defined to be on the neuronal representation of color in mouse visual cortex, rather than on the comparison of retinal and cortical color processing.

In addition, the paper would benefit from adding explicit neuron counts or percentages to the quadrants of each of the density plots in Figures 2-5. The variance explained by the principal components does not capture the percentage of color opponent cells. Additionally, there appear to be some remaining errors in the figure legend and labels that have not been addressed (e.g. ’??’ in Fig 2 legend).

Thank you for this suggestion. We believe that adding the numbers or percentages to the figure panels would make them too crowded. Instead, we have now mentioned in the Results section and the legends that the percentages of variance explained by the color (off-diagonal) and luminance axis (diagonal) correlate with the number of neurons located in the color (top left and bottom right) and luminance contrast quadrants (top right and bottom left), respectively. Together with the number of neurons in each plot stated in the legends and the scale bar indicating the number of neurons per gray level, we hope this approach provides clarity for the reader to interpret the panels. Additionally, we have fixed the broken reference in the legend of Fig. 2.

Overall, this study will be a valuable resource for researchers studying color vision, cortical processing, and the processing of ethologically relevant information. It provides a useful basis for future work on the origin of color opponency in V1 and its ethological relevance.

General Suggestions:

-  Please add possible caveats of using ETA method to the discussion section. For example, it is unclear to what extent ON/OFF cells are being overlooked by using ETA method.

We now discuss the limitations of the ETA approach in the Discussion section.

- The caveats of using the percentage of variance explained in the retina as evidence against V1 solely inheriting color-opponency from retinal output neurons are not adequately addressed. For example, could the mismatch in explained variance of the color axis between V1 and RGCs be explained by a subset of non-color opponent RGCs projecting elsewhere (not dLGN-V1) or that color opponent cells project to a larger number of neurons in V1 than non-color opponent cells? We suggest adding a paragraph to the discussion to address this issue.

We have removed these conclusions from the paper, more carefully interpret the retinal results and mention that comparing ex-vivo retina data with in-vivo cortical data is challenging.

- Please clarify how the different response types shown in Figure 5e-f lead to differences in noise detection and thereby differences in predator discriminability. For example, why does Gon/UVoff not respond to the noise scene while Goff/UVoff does?

We added this to the Results section.

- Please clarify the relationship between ETA amplitude, neural response probability, and neural response amplitude. For example, do color-opponent cells have equal absolute neural response amplitudes to the different colors?

Thank you for bringing up this point. The ETA is obtained by summing the stimulus sequences that elicit an event (i.e., response), weighted by the amplitude of the response. Consequently, the absolute amplitude of the ETA correlates with the calcium amplitude. Importantly, the ETA amplitudes of different stimulus conditions are comparable because they were estimated on the same normalized calcium trace. Therefore, comparing the absolute amplitudes of ETAs of color-opponent neurons reveals the response magnitude of the cells to different colors. We have now included this information in the Results section.

Abstract: - "more than a third of neurons in mouse V1 are color-opponent in their receptive field center". It is unclear what data supports this statement. Can you please provide a statement in the manuscript that supports this directly using the number of neurons?

We added the following sentence to the Results section: Nevertheless, a substantial fraction of neurons (33.1%) preferred color-opponent stimuli and scattered along the off-diagonal in the upper left and lower right quadrants, especially for the RF center.

Figure 2: - There is a ?? in the figure legend. Which figure should this refer to? - please provide explicit neuron counts/percentages for each quadrant in b.

We fixed the figure reference. We believe that adding the numbers or percentages to the figure panels would make them too crowded. Instead, we have now mentioned in the Results section and the legends that the percentages of variance explained by the color (off-diagonal) and luminance axis (diagonal) correlate with the number of neurons located in the color (top left and bottom right) and luminance contrast quadrants (top right and bottom left), respectively. Together with the number of neurons in each plot stated in the legends and the scale bar indicating the number of neurons per gray level, we hope this approach provides clarity for the reader to interpret the panels.

Figure 3: - Fig 3: Color scheme makes it very difficult to differentiate the different conditions, especially when printed.

Thanks we changed the color scheme.

- Add explicit neuron counts/percentages for each quadrant in b.

See above.

Figure 4: - Add explicit neuron counts/percentages for each quadrant in b.

See above.

Figure 5: - Add explicit neuron counts/percentages for each quadrant in c.

See above.

Methods: - "we modeled each response type to have a square RF with 10 degrees visual angle in diameter". There appears to be a mismatch between this statement and Figure 5e where 18 degrees is reported.

Thanks we fixed that.

Referee 3 (Remarks to the Author):

This paper studies chromatic coding in mouse primary visual cortex. Calcium responses of a large collection of cells are measured in response to a simple spot stimulus. These responses are used to estimate chromatic tuning properties - specifically sensitivity to UV and green stimuli presented in a large central spot or a larger still surrounding region. Cells are divided based on their responses to these stimuli into luminance or chromatic sensitive groups. The results are interesting and many aspects of the experiments and conclusions are well done; several technical concerns, however, limit the support for several main conclusions,

Limitations of stimulus choice The paper relies on responses to a large (37.5 degree diameter) modulated spot and surround region. This spot is considerably larger than the receptive fields of both V1 cells and retinal ganglion cells (it is twice the area of the average V1 receptive field). As a result, the spot itself is very likely to strongly activate both center and surround mechanisms, and responses of cells are likely to depend on where the receptive fields are located within the spot

(and, e.g., how much of the true neural surround samples the center spot vs the surround region). Most importantly, the surrounds of most of the recorded cells will be strongly activated by the central spot. This brings into question statements in the paper about selective activation of center and surround (e.g. page 2, right column). This in turn raises questions about several subsequent analyses that rely on selective center and surround activation.

Thank you for this comment. A similar point was raised by a reviewer in the first round of revision. We agree with the reviewers that it is critical to discuss both the rationale behind our stimulus design and its limitations to facilitate better interpretation by the reader.

To be able to record from many V1 neurons simultaneously, we used a stimulus size of 37.5 degree visual angle in diameter, which is slightly larger than center RFs of single V1 neurons (between 20 - 30 degrees visual angle depending on the stimulus, see here). The disadvantage of this approach is that the stimulus is only roughly centered on the neurons’ center RFs. To reduce the impact of potential stimulus misalignment on our results, we used the following steps: { For each recording, we positioned the monitor such that the mean RF across all neurons lies within the center of the stimulus field of view.

We confirmed that this procedure results in good stimulus alignment for the large majority of recorded neurons within individual recording fields by using a sparse noise stimulus (Suppl. Fig. 1a-c). Specifically, we found that for 83% of tested neurons, more than two thirds of their center RF, determined by the sparse noise stimulus, overlapped with the center spot of the color noise stimulus.

For analysis, we excluded neurons without a significant center STA, which may be caused by misalignment of the stimulus.

Together, we believe these points strongly suggest that the center spot and the surround annulus of the noise stimulus predominantly drive center (i.e. classical RF) and surround (i.e. extraclassical RF), respectively, of the recorded V1 neurons. This is further supported by the fact that color response types identified using an automated clustering method were robust across mice (Suppl. Fig. 6c), indicating consistent stimulus centering.

Nevertheless, we cannot exclude the possibility that the stimulus was misaligned for a subset of the recorded neurons used in our analysis. We agree with the reviewer that such misalignment might have caused the center stimulus to partially activate the surround. To further address this issue beyond the controls we have already implemented, one could compare the results of our approach with an approach that centers the stimulus on individual neurons. However, we believe that performing these additional experiments is beyond the scope of the current study.

To acknowledge the experimental limitations of our study and the concerns brought up by the reviewer, we have added the steps we perform to reduce the effects of stimulus misalignment in the Results section and discuss the problem of stimulus alignment in the Discussion in a separate section. With this, we believe our manuscript explains both the rationale behind our stimulus design as well as important limitations of the approach.

Comparison with retina A key conclusion of the paper is that the chromatic tuning in V1 is not inherited from retinal ganglion cells. This conclusion comes from comparing chromatic tuning in a previously-collected data set from retina with the present results. But the retina recordings were made using a considerably smaller spot, and hence it is not clear that the comparison made in the paper is accurate. For example, the stimulus used for the V1 experiments almost certainly strongly stimulates both center and surround of retinal ganglion cells. The text focuses on color opponency in the receptive field centers of retinal ganglion cells, but center-surround opponency seems at least as relevant for such large spots. This issue needs to be described more clearly and earlier in the paper.

Thanks for this comment. We removed the retinal findings from the abstract, as well as from the summary of our results in the Introduction. In addition, we now phrase the interpretation of the retinal results more conservatively and specifically highlight in the Discussion that comparing ex-vivo retinal to in-vivo cortical data is challenging. With these changes, we believe that the focus of the paper is explicitly defined to be on the neuronal representation of color in mouse visual cortex, rather than on the comparison of retinal and cortical color processing.

Limitations associated with ETA analysis One of the reviewers in the previous round of reviews raised the concern that the ETA analysis may not accurately capture responses of cells with nonlinear receptive field properties such as On/Off cells. This possibility and whether it is a concern should be discussed.

Thanks for this comment. We now discuss the limitation of using an ETA analysis in the

Discussion section.

Discrimination performance poor Discriminability of color or luminance is used as a measure of population coding. The discrimination performance appears to be quite poor - with 500-1000 neurons needed to reliably distinguish light from dark or green from UV. Intuitively I would expect that a single cell would provide such discrimination. Is this intuition wrong? If not, how do we interpret the discrimination analyses?

Thank you for raising this point. The plots in Fig. 2c (and Figs. 3-5) show discriminability in bits, with the discrimination accuracy in % highlighted by the dotted horizontal lines. For 500 neurons, the discriminability is approx. 0.8 bits, corresponding to 95% accuracy. Even for 50 neurons, the accuracy is significantly above chance level. We now mention in the legends that the dotted lines indicate decoding accuracy in %.

eLife assessment

This study presents an important set of results illuminating how movement sequences are planned. Using several different behavioural manipulations and analysis methods, the authors present compelling evidence that multiple future movements are planned simultaneously with execution, and that these future movement plans influence each other. The work will be of great interest to those studying motor control.

Reviewer #1 (Public Review):

Mehrdad Kashefi et al. investigated the availability of planning future reaches while simultaneously controlling the execution of the current reach. Through a series of experiments employing a novel sequential arm reaching paradigm they developed, the authors made several findings: 1) participants demonstrate the capability to plan future reaches in advance, thereby accelerating the execution of the reaching sequence, 2) planning processes for future movements are not independent one another, however, it's not a single chunk neither, 3) Interaction among these planning processes optimizes the current movement for the movement that comes after for it.

The question of this paper is very interesting, and the conclusions of this paper are well supported by data.

Reviewer #2 (Public Review):

In this work, Kashefi et al. investigate the planning of sequential reaching movements and how the additional information about future reaches affects planning and execution. This study, carried out with human subjects, extends a body of research in sequential movements to ask important questions: How many future reaches can you plan in advance? And how do those future plans interact with each other?

The authors designed several experiments to address these questions, finding that information about future targets makes reaches more efficient in both timing and path curvature. Further, with some clever target jump manipulations, the authors show that plans for a distant future reach can influence plans for a near future reach, suggesting that the planning for multiple future reaches is not independent. Lastly, the authors show that information about future targets is acquired parafoveally--that is, subjects tend to fixate mainly on the target they are about to reach to, acquiring future target information by paying attention to targets outside the fixation point.

The study opens up exciting questions about how this kind of multi-target planning is implemented in the brain. As the authors note in the manuscript, previous work in monkeys showed that preparatory neural activity for a future reaching movement can occur simultaneously with a current reaching movement, but that study was limited to the monkey only knowing about two future targets. It would be quite interesting to see how neural activity partitions preparatory activity for a third future target, given that this study shows that the third target's planning may interact with the second target's planning.

[Editors' note: The authors fully addressed the reviewers' comments on the original manuscript.]

Version 5 of this preprint has been peer-reviewed and recommended by Peer Community in Zoology.<br /> See the peer reviews and the recommendation.

based on power relations

something non-fixed

I do not think it is just Oaxacans who think that mental health is for crazies. A lot of people in the hispanic culture also believe or do not believe that mental health is a thing.

Micro aspects of everyday life influenced by digital world.

It starts mattering when you think of taking a break from the Internet or even your smartphone.

Compared to the US, India has mere 31.7% smartphone users,

Daily routine of students and working people.

Prozac is still a very popular drug for depression. Not only is it the most prescribed, but you can catch a commercial every time on day time tv. It is one of the medicines that has the most propaganda.

The world could become more educated on trans people. I know there is a lot of information that would help people understand how to address them better as well as be more mindful

We live in a society where sex rules. A lot of things are sexualized, and women are over sexualized to the point where they are not seen as people but items.

I don't get the origin of item 4 of this list.

Coming from the Latin community - I can say that mental illnesses are not recognized. A lot of the time they are pinned to nervous attacks or anxiety and even still, anxiety is not a real thing.

I do believe that people usually feel distress in different types of ways - panic attacks, sadness, anger. Large events that can also contribute to internal dysfunctions can also be too much of something, like winning the lottery.

It is hard to be faced with suicide. It is personal when it involves someone close to you, but also when it is something you see on the daily basis. Working with the public as a doctor, nurse, or counselor, and dealing with people that have tried to commit suicide multiple times can take a toll on someone.

That is a lot of money for someone especially the top 1 percent especially when most of the population is considered in poverty.

Suicide has always existed - we have learned that it follows the same pattern. It does occur spontaneously and usually is cause to reoccurring events or developed over time. It is not just one thing that plays a part into the act but a multitude of things which can be very complicated.

It is interesting to see that 352% increase in usage of antidepressants. That's quite a large increase.

This makes a good point. There are a lot patients who are given prescriptions for depression that might not or could not be depressed. Which can lead to more issues or lead to depression.

34 children out of 340 that were left inside of psychiatric hospitals. Although 34 does not seem like a large number, it does pose the question of how many children are too many to be kept in a location where they do not belong?

Children are placed in medical facilities and left there for days even when it is not medically necessary. It is sad to know that the children are left in psychiatric hospitals without reason

My Ther bik

MyThe bik

Smith Corona Typewriter Repaired Flying Carriage ! Fixed Escapement Pawl by [[Phoenix Typewriter]]

Pawl was frozen on escapement. He also took out a piece of the rubber which he planned to replace with some surgical tubing to add a small layer of spacing.

This repair is similar to part of that at Smith Corona Typewriter Power Spacer Escapement Repair Adjustment No Silencer Model

Smith Corona Typewriter Power Spacer Escapement Repair Adjustment No Silencer Model by [[Phoenix Typewriter]]

Transparent Peer Review

---

Download the complete Review Process [PDF] including:

• reviews
• authors' reply
• editorial decisions

</br>

SCM typewriter escapement and margin lock/release by [[Nis Kildegaard]]

この表記が正しい?スペースありが正しい?

MyStのポスターはどれですか?あとMySTだと思います

あー、奥にあるのがMySTなのか、ぱっと見わかりにくいです。

今年も、だけど、はじめて?

BoF

私は聞き慣れない単語なので、簡単な説明がほしいです https://www.enago.jp/academy/conference-publications/

表記揺れ

NumPy 2.0 かなと。表記揺れあり

Python内に?

リンクが欲しい。これかな https://anywidget.dev/

少し前に似た文章があるので、どちらか1つにしてほしい

リンクがあってもよさそう https://jupyterbook.org/

書かれた、がどこにかかっているのかわかりにくいです。 トルでも意味は通じそう

Jupyter Notebookとスペース有りが正しいと思います

ちょっとキャプションのこれは意味が伝わらなかったです。

ユーザーというのは、NumPyを使用するユーザーという意味ですか?ちょっとわかりにくい

開発で?

正確には「事前にプロポーザルを提出し、その中で選ばれた」とかかなと

これもあまりキーノートの内容とは関係ない感じがする。なにをきっかけに再確認したのか?

この余談がキーノートに入っている理由がちょっとわからない

Considering that there is some form of aggression regarding critical race theory, it might be profitable from a publisher's viewpoint to not endorse a product that has gotten significant backlash. Or perhaps, the opposite could be true. I know that there is a second voice from the opposite side that is strongly for critical race theory. I think I would just consider that whichever decision I make will cause divisiveness when this topic is so controversial.

Seeing a report by the literal American Library Association's department would be a good indicator to a publisher if a work will be successful or not. This section states that books with LGBTQ or anti-racism themes were some of the top challenged books. If I were a publisher, I wouldn't want my investment to face any sort of challenge or opposition. I would want to maximize reward and minimize struggle.

Being a publisher, it could seem intimidating to endorse a novel if there was a greater chance that it would be shunned by the public. I can understand to some extent why white authors could be prioritized over those of color for fear of lack of business.

https://new.reddit.com/r/ObsidianMD/comments/1efd8he/are_there_any_working_academics_on_here/

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Resource: RRID:Addgene_12260

Curator: @olekpark

SciCrunch record: RRID:Addgene_12260

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What is this?