Nova redação do artigo 8º da Lei nº 7.853, de 24 de Outubro de 1989.

Deze stappen zijn uitgewerkt in HR Post-Keskin r.o. 9.2.)

Denk hierbij ook aan veiligheid v/d getuige

Here David is pointing out the hypocrisy of The Declaration of Independents. I find it very necessary that he points out that they don't know their own language. They don't mean ALL men they mean all WHITE men in The Declaration of Independence. They should have just said that because they have done it before.

David claims that African Americans have contributed to America than whites have. Which in his reasoning seems to be fair and understandable. Slave had a lot of blood sweat and tears which is just what America was built on. They were the reasons the slave owners made a profit and did most of the work.

Since the people of Boston aren't listening or doing anything why not to his appeal to the source? Heaven. Since he is a christian it would only seem like heaven would be the one listening over anyone else.

David Walker compared Israelites and the Helots to African Americans being slaves and I think the matter of time in between these events affects this the most. Slavery wasn't that long ago even now and at the time he was writing the appeal, it was still happening. He knows there should have been some kind of growth since there was so much time in between events. They should have learned to treat everyone equal. Especially if they are christian and treating others like this is just hypocrisy.

**Sólo tengo buenos comentarios de esta primer etapa de tu proyecto. **

En esta etapa mi evaluación es 100.

Veo muy bien logrado el proyecto. En esta estapa mi evaluación en redondo 100.

Un error que se peude arreglar facil. Lo dejamos pendiente par la siguiente etapa. La plicacion es sumanete interesante. Mi evaluacion es 98.

Рыбаки занимали видное место в городской среде (буквально). Достаточно взглянуть на живопись и гравюры. Их место в городской экономике требует дальнейшего исследования.

Рыболовство играло важную роль сразу после основания Петербурга, так как было сложно снабжать едой многочисленных строителей

eLife Assessment

This study provides a valuable new resource to investigate the molecular basis of the particular features characterizing the pipefish embryo. The authors found both unique and shared gene expression patterns in pipefish organs compared with other teleost fishes. The solid data collected in this unconventional model organism will give new insights into understanding the extraordinary adaptations of the Syngnathidae family and will be of interest in the domain of evolution of fish development.

Reviewer #1 (Public review):

Syngnathid fishes (seahorses, pipefishes, and seadragons) present very particular and elaborated features among teleosts and a major challenge is to understand the cellular and molecular mechanisms that permitted such innovations and adaptations. The study provides a valuable new resource to investigate the morphogenetic basis of four main traits characterizing syngnathids, including the elongated snout, toothlessness, dermal armor and male pregnancy. More particularly, the authors have focused on a late stage of pipefish organogenesis to perform single-cell RNA-sequencing (scRNA-seq) completed by in situ hybridization analyses to identify molecular pathways implicated in the formation of the different specific traits.<br /> The first set of data explores the scRNA-seq atlas composed of 35,785 cells from two samples of gulf pipefish embryos that authors have been able to classify into major cell types characterizing vertebrate organogenesis, including epithelial, connective, neural and muscle progenitors. To affirm identities and discover potential properties of clusters, authors primarily use KEGG analysis that reveals enriched genetic pathways in each cell types. After revisions, the authors have provided extended supplementary files to well interpret the dataset and some statements have been clarified. I thank the authors for the revisions/completions of ISH results compared to initial submission.

To conclude, the scRNA-seq dataset in this unconventional model organism will be useful for the community and will provide clues for future research to understand the extraordinary evolution of the Syngnathidae family.

Reviewer #2 (Public review):

Summary:

The authors present the first single-cell atlas for syngathid fishes, providing a resource for future evolution & development studies in this group.

Strengths:

The concept here is simple and I find the manuscript to be well written. I like the in situ hybridization of marker genes >> this is really nice. I also appreciate the gene co-expression analysis to identify modules of expression. There are no explicit hypotheses tested in the manuscript, but the discovery of these cell types should have value in this organism and in the determination of morphological novelties in seahorses and their relatives.

Weaknesses:

I think there are a few computational analyses that might improve the generality of the results.

(1) The cell types: The authors use marker gene analysis and KEGG pathways to identify cell types. I'd suggest a tool like SAMap (https://elifesciences.org/articles/66747) which compares single cell data sets from distinct organisms to identify 'homologous' cell types -- I imagine the zebrafish developmental atlases could serve as a reasonable comparative reference.

(2) Trajectory analyses: Authors suggest that their analyses might identify progenitor cell states and perhaps related differentiated states. They might explore cytoTRACE and/or pseudotime-based trajectory analyses to more fully delineate these ideas.

(3) Cell-cell communication: I think it's very difficult to identify 'tooth primordium' cell types, because cell types won't be defined by organ in this way. for instance dental glia will cluster with other glia, dental mesenchyme will likely cluster with other mesenchymal cell types. so the histology and ISH in most convincing in this regard. having said this, given the known signaling interactions in the developing tooth (and in development generally) the authors might explore cell-cell communication analysis (e.g., CellChat) to identify cell types that may be interacting.

Comments on revisions:

I feel essentially the same about this manuscript. it's a useful resource for future experimental forays into this unique system. The team made improvements to deal with comments from other reviewers related to quality of confirmatory in situ hybridization. This is good.

Regarding their response that one can't use CellChat if you're not working in mice or human, this is inaccurate. the assumption one makes is that ligand-receptor pairs and signaling pathways have conserved functions across animals (vertebrates). It's the same assumption the authors make when using the KEGG pathway to score enrichment of pathways in clusters. CellChat used in fishes in Johnson et al 2023 Nature Communications | ( 2023) 14:4891.

Reviewer #3 (Public review):

Summary:

This study established a single-cell RNA sequencing atlas of pipefish embryos. The results obtained identified unique gene expression patterns for pipefish-specific characteristics, such as fgf22 in the tip of the palatoquadrate and Meckel's cartilage, broadly informing the genetic mechanisms underlying morphological novelty in teleost fishes. The data obtained are unique and novel, potentially important in understanding fish diversity. Thus, I would enthusiastically support this manuscript if the authors improve it to generate stronger and more convincing conclusions than the current forms.

Weakness:

Regarding the expression of sfrp1a and bmp4 dorsal to the elongating ethmoid plate and surrounding the ceratohyal: Are their expression patterns spatially extended or broader compared to the pipefish ancestor? Is there a much closer species available to compare gene expression patterns with pipefish? Did the authors consider using other species closely related to pipefish for ISH? Sfrp1a and bmp4 may be expressed in the same regions of much more closely related species without face elongation. I understand that embryos of such species are not always accessible, but it is also hard to argue responsible genes for a specific phenotype by only comparing gene expression patterns between distantly related species (e.g., pipefish vs. zebrafish). Due to the same reason, I would not directly compare/argue gene expression patterns between pipefish and mice, although I should admit that mice gene expression patterns are sometimes helpful to make a hypothesis of fish evolution. Alternatively, can the authors conduct ISH in other species of pipefish? If the expression patterns of sfrp1a and bmp4 are common among fishes with face elongation, the conclusion would become more solid. If these embryos are not available, is it possible to reduce the amount of Wnt and BMP signal using Crispr/Cas, MO, or chemical inhibitor? I do think that there are several ways to test the Wnt and/or BMP hypothesis in face elongation.

Author response:

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

Public Reviews: 

Reviewer #1 (Public Review): 

Syngnathid fishes (seahorses, pipefishes, and seadragons) present very particular and elaborated features among teleosts and a major challenge is to understand the cellular and molecular mechanisms that permitted such innovations and adaptations. The study provides a valuable new resource to investigate the morphogenetic basis of four main traits characterizing syngnathids, including the elongated snout, toothlessness, dermal armor, and male pregnancy. More particularly, the authors have focused on a late stage of pipefish organogenesis to perform single-cell RNA-sequencing (scRNA-seq) completed by in situ hybridization analyses to identify molecular pathways implicated in the formation of the different specific traits. 

The first set of data explores the scRNA-seq atlas composed of 35,785 cells from two samples of gulf pipefish embryos that authors have been able to classify into major cell types characterizing vertebrate organogenesis, including epithelial, connective, neural, and muscle progenitors. To affirm identities and discover potential properties of clusters, authors primarily use KEGG analysis that reveals enriched genetic pathways in each cell types. While the analysis is informative and could be useful for the community, some interpretations appear superficial and data must be completed to confirm identities and properties. Notably, supplementary information should be provided to show quality control data corresponding to the final cell atlas including the UMAP showing the sample source of the cells, violin plots of gene count, UMI count, and mitochondrial fraction for the overall

dataset and by cluster, and expression profiles on UMAP of selected markers characterizing cluster identities. 

We thank the reviewer for these suggestions, and have added several figures and supplemental files in response. We added a supplemental UMAP showing the sample that each cell originated (S1). We also added supplemental violin plots for each sample showing the gene count, unique molecular identifier (UMI) count, mitochondrial fraction, and the doublet scores (S2). We added feature plots of zebrafish marker genes for these major cell types and marker genes identified from our dataset to the supplement (S3:S57). We also provided two supplemental files with marker genes. These changes should clarify the work that went into labeling the clusters. Although some of the cluster labels are general, we decided it would be unwise to label clusters with speculated specific annotations. We only gave specific annotations to clusters with concrete markers and/or in situ hybridization (ISH) results that cemented an annotation.  As shown in the new supplemental figures and files, certain clusters had clear, specific markers while others did not. Therefore, we used caution when we annotated clusters without distinct markers. 

The second set of data aims to correlate the scRNA-seq analysis with in situ hybridizations (ISH) in two different pipefish (gulf and bay) species to identify and characterize markers spatially, and validate cell types and signaling pathways active in them. While the approach is rational, the authors must complete the data and optimize labeling protocols to support their statements. One major concern is the quality of ISH stainings and images; embryos show a high degree of pigmentation that could hide part of the expression profile, and only subparts and hardly detectable tissues/stainings are presented. The authors should provide clear and good-quality images of ISH labeling on whole-mount specimens, highlighting the magnification regions and all other organs/structures (positive controls) expressing the marker of interest along the axis. Moreover, ISH probes have been designed and produced on gulf pipefish genome and cDNA respectively, while ISH labeling has been performed indifferently on bay or gulf pipefish embryos and larvae. The authors should specify stages and species on figure panels and should ensure sequence alignment of the probe-targeted sequences in the two species to validate ISH stainings in the bay pipefish. Moreover, spatiotemporal gene expression being a very dynamic process during embryogenesis, interpretations based on undefined embryonic and larval stages of pipefish development and compared to 3dpf zebrafish are insufficient to hypothesize on developmental specificities of pipefish features, such as on the absence of tooth primordia that could represent a very discrete and transient cell population. The ISH analyses would require a clean and precise spatiotemporal expression comparison of markers at the level of the entire pipefish and zebrafish specimens at well-defined stages, otherwise, the arguments proposed on teleost innovations and adaptations turn out to be very speculative. 

We are appreciative of the reviewer’s feedback. We primarily used the in situ hybridization (ISH) data as supplementary to the scRNAseq library and we are aware that further evidence is necessary to identify origins of syngnathid’s evolutionary novelties. Our goal was to provide clues for the developmental genetic basis of syngnathid derived features.  We hope that our study will inspire future investigations and are excited for the prospect that future research could include this reviewer’s ideas. 

All of the developmental stages and species information for the embryos used were in the figure captions as well as in supplemental file 6. Because we primarily used wild caught embryos, we did not have specific ages of most embryos. Syngnathid species are challenging to culture in the laboratory, and extracting embryos requires euthanizing the father which makes it difficult to obtain enough embryos for ISH. In addition, embryos do not survive long when removed from the brood pouch prematurely. We supplemented our ISH with bay pipefish caught off the Oregon coast because these fish have large broods. Wild caught pregnant male bay pipefish were immediately euthanized, and their broods were fixed. Because we did not have their age, we classified them based on developmental markers such as presence of somites and the extent of craniofacial elongation. Although these classification methods are not ideal, they are consistent with the syngnathid literature (Sommer et al. 2012). Since the embryos used for the ISH were primarily wild caught, we had a few different developmental stages represented in our ISH data. For our tooth primordia search, we used embryos from the same brood (therefore, same stage) for these experiments.

We understand the concern for the degree of pigmentation in the samples. We completed numerous bleach trials before embarking on the in situ hybridization experiments. After completing a bleach trial with a probe created from the gene tnmd for ISH_,_ we noticed that the bleached embryos were missing expression domains found in the unbleached embryos. We were, therefore, concerned that using bleached embryos for our experiments would result incorrect conclusions about the expression domains of these genes. We sparingly used bleaching at older stages, hatched larvae, where it was fundamentally necessary to see staining. As stated above, the primary goal of this manuscript was to generate and annotate the first scRNA-seq atlas in a syngnathid, and the ISHs were utilized to support inferred cluster annotations only through a positive identification of marker gene expression in expected tissues/cells. Therefore, the obscuring of gene expression by pigmentation would have resulted in the absence of evidence for a possible cluster annotation, not an incorrect annotation.

For the ease of viewing the ISHs, we improved annotations and clarity. We increased the brightness and contrast of images. In the original submission, we had to lower the image resolution to make the submission file smaller. We hope that these improvements plus the true image quality improves clarity of ISH results. We also included alignments in our supplementary files of bay pipefish sequences to the Gulf pipefish probes to showcase the high degree of sequence similarity. 

Sommer, S., Whittington, C. M., & Wilson, A. B. (2012). Standardised classification of pre-release development in male-brooding pipefish, seahorses, and seadragons (Family Syngnathidae). BMC Developmental Biology, 12, 12–15. 

To conclude, whereas the scRNA-seq dataset in this unconventional model organism will be useful for the community, the spatiotemporal and comparative expression analyses have to be thoroughly pushed forward to support the claims. Addressing these points is absolutely necessary to validate the data and to give new insights to understand the extraordinary evolution of the Syngnathidae family. 

We really appreciate the reviewer’s enthusiasm for syngnathid research, and hope that the additional files and explanation of the supporting role of the ISHs have adequately addressed their concerns. We share the reviewer’s enthusiasm and are excited for future work that can extend this study. 

Reviewer #2 (Public Review):

Summary: 

The authors present the first single-cell atlas for syngnathid fishes, providing a resource for future evolution & development studies in this group. 

Strengths: 

The concept here is simple and I find the manuscript to be well written. I like the in situ hybridization of marker genes - this is really nice. I also appreciate the gene co-expression analysis to identify modules of expression. There are no explicit hypotheses tested in the manuscript, but the discovery of these cell types should have value in this organism and in the determination of morphological novelties in seahorses and their relatives.  

We are grateful for this reviewer’s appreciation of the huge amount of work that went into this study, and we agree that the in situ hybridizations (ISHs) support the scRNAseq study as we intended. We appreciate that the reviewer thinks that this work will add value to the syngnathid field.

Weaknesses: 

I think there are a few computational analyses that might improve the generality of the results. 

(1) The cell types: The authors use marker gene analysis and KEGG pathways to identify cell types. I'd suggest a tool like SAMap (https://elifesciences.org/articles/66747) which compares single-cell data sets from distinct organisms to identify 'homologous' cell types - I imagine the zebrafish developmental atlases could serve as a reasonable comparative reference. 

We appreciate the reviewer’s request, and in fact we would have loved to integrate our dataset with zebrafish. However, syngnathid’s unique craniofacial development makes it challenging to determine the appropriate stage for comparison. While 3 days post fertilization (dpf) zebrafish data were appropriate for comparisons of certain cell types (e.g. epidermal cells), it would have been problematic for other cell types (e.g. osteoblasts) that are not easily detectable until older zebrafish stages. Therefore, determining equivalent stages between these species is difficult and contains potential for error. Future research should focus on trying to better match stages across syngnathids and zebrafish (and other fish species such as stickleback). Studies of this nature promise to uncover the role of heterochrony in the evo-devo of syngnathid’s unique snouts.

(2) Trajectory analyses: The authors suggest that their analyses might identify progenitor cell states and perhaps related differentiated states. They might explore cytoTRACE and/or pseudotime-based trajectory analyses to more fully delineate these ideas.

We thank the reviewer for this suggestion! We added a trajectory analysis using cytoTRACE to the manuscript. It complemented our KEGG analysis well (L172-175; S73) and has improved the manuscript.

(3) Cell-cell communication: I think it's very difficult to identify 'tooth primordium' cell types, because cell types won't be defined by an organ in this way. For instance, dental glia will cluster with other glia, and dental mesenchyme will likely cluster with other mesenchymal cell types. So the histology and ISH is most convincing in this regard. Having said this, given the known signaling interactions in the developing tooth (and in development generally) the authors might explore cell-cell communication analysis (e.g., CellChat) to identify cell types that may be interacting. 

We agree! It would have been a wonderful addition to the paper to include a cell-cell communication analysis. One limitation of CellChat is that it only includes mouse and human orthologs. Given concerns of reviewer #3 for mouse-syngnathid comparisons, we decided to not pursue CellChat for this study. We are looking forward to future cell communication resources that include teleost fishes.

Reviewer #3 (Public Review): 

Summary: 

This study established a single-cell RNA sequencing atlas of pipefish embryos. The results obtained identified unique gene expression patterns for pipefish-specific characteristics, such as fgf22 in the tip of the palatoquadrate and Meckel's cartilage, broadly informing the genetic mechanisms underlying morphological novelty in teleost fishes. The data obtained are unique and novel, potentially important in understanding fish diversity. Thus, I would enthusiastically support this manuscript if the authors improve it to generate stronger and more convincing conclusions than the current forms. 

Thank you, we appreciate the reviewer’s enthusiasm!

Weaknesses: 

Regarding the expression of sfrp1a and bmp4 dorsal to the elongating ethmoid plate and surrounding the ceratohyal: are their expression patterns spatially extended or broader compared to the pipefish ancestor? Is there a much closer species available to compare gene expression patterns with pipefish? Did the authors consider using other species closely related to pipefish for ISH? Sfrp1a and bmp4 may be expressed in the same regions of much more closely related species without face elongation. I understand that embryos of such species are not always accessible, but it is also hard to argue responsible genes for a specific phenotype by only comparing gene expression patterns between distantly related species (e.g., pipefish vs. zebrafish). Due to the same reason, I would not directly compare/argue gene expression patterns between pipefish and mice, although I should admit that mice gene expression patterns are sometimes helpful to make a hypothesis of fish evolution. Alternatively, can the authors conduct ISH in other species of pipefish? If the expression patterns of sfrp1a and bmp4 are common among fishes with face elongation, the conclusion would become more solid. If these embryos are not available, is it possible to reduce the amount of Wnt and BMP signal using Crispr/Cas, MO, or chemical inhibitor? I do think that there are several ways to test the Wnt and/or BMP hypothesis in face elongation. 

We appreciate the reviewer’s suggestion, and their recognition for challenges within this system. In response to this comment, we completed further in situ hybridization experiments in threespine stickleback, a short snouted fish that is much more closely related to syngnathids than is zebrafish, to make comparisons with pipefish craniofacial expression patterns (S76-S79). We added ISH data for the signaling genes (fgf22, bmp4, and sfrp1a) as well as prdm16. Through adding this additional ISH results, we speculated that craniofacial expression of bmp4, sfrp1a, and prdm16 is conserved across species. However, compared to the specific ceratohyal/ethmoid staining seen in pipefish, stickleback had broad staining throughout the jaws and gills. These data suggest that pipefish have co-opted existing developmental gene networks in the development of their derived snouts. We added this interpretation to the results and discussion of the manuscript (L244-L248; L262-277; L444-470).

Recommendations for the authors:  

Reviewing Editor (Recommendations for the Authors): 

We hope that the eLife assessment, as well as the revisions specified here, prove helpful to you for further revisions of your manuscript. 

Revisions considered essential: 

(1) Marker genes and single-cell dataset analyses. While these analyses have been performed to a good standard in broad terms, there is a majority view here that cell type annotations and trajectory analyses can be improved. In particular, there is question about the choice of marker genes for the current annotation. For one it can depend on the use of single marker genes (see tnnti1 example for clusters 17 and 31). Here, we recommend incorporating results from SAMap and trajectory analysis (e.g., cytoTRACE or standard pseudotime).

Because of the reviewer comments, we became aware that we insufficiently communicated how cell clusters were annotated. We did mention in the manuscript that we did not use single marker genes to annotate clusters, but instead we used multiple marker genes for each cluster for the annotation process. We used both marker genes derived from our dataset and marker genes identified from zebrafish resources for cluster annotation. We chose single marker genes for each cluster for visualization purposes and for in situ hybridizations. However, it is clear from the reviewers’ comments that we needed to make more clear how the annotations were performed. To make this effort more clear in our revision, we included two new supplementary files – one with Seurat derived marker genes and one with marker genes derived from our DotPlot method. We also included extensive supplementary figures highlighting different markers. Using Daniocell, we identified 6 zebrafish markers per major cell type and showed their expression patterns in our atlas with FeaturePlots. We also included feature plots of the top 6 marker genes for each cluster. We hope that the addition of these 40+ plots (S3:S57) to the supplement fully addresses these concerns. 

We appreciated the suggestion of cytotrace from reviewer #2! We ran cytotrace on three major cell lineages (neural, muscle, and connective; S73) which complemented our KEGG analysis in suggesting an undifferentiated fate for clusters 8, 10, and 16. We chose to not run SAMap because it is a scRNA-seq library integration tool. Although we compared our lectin epidermal findings to 3 dpf zebrafish scRNA-seq data, we did not integrate the datasets out of concern that we could draw erroneous conclusions for other cell types.  Future work that explores this technical challenge may uncover the role of heterochrony in syngnathid craniofacial development. We detail these changes more fully in our responses to reviewers.

(2) The claims regarding evolutionary novelty and/or the genes involved are considered speculative. In part, this comes from relying too heavily on comparisons against zebrafish, as opposed to more closely related species. For example, the discussion regarding C-type lectin expression in the epidermis and KEGG enrichment (lines 358 - 364) seems confusing. Another good example here is the discussion on sfrp1a (lines 258 - 261). Here, the text seems to suggest craniofacial sfrp1a expression (or specifically ethmoid expression?) is connected to the development of the elongated snout in pipefish. However, craniofacial expression of sfrp1a is also reported in the arctic charr, which the authors grouped into fishes with derived craniofacial structures. Separately, sfrp2 expression was also reported in stickleback fish, for example. Do these different discussions truly support the notion that sfrp1a expression is all that unique in pipefish, rather than that pipefish and zebrafish are only distantly related and that sfrp1a was a marker gene first, and co-opted gene second? The authors should respond to the comments in the public review related to this aspect, and include more informative comparison and discussion. 

A much more nuanced discussion with appropriate comparisons and caveats would be strongly recommended here.  

We appreciate this insight and used it as a motivator to complete and add select comparative ISH data to this manuscript. We added in situ hybridization experiments from stickleback fish for craniofacial development genes (sfrp_1a, prdm16, bmp4_, and fgf22; S76-S79).  After adding stickleback ISH to the manuscript, we were able to make comparisons between pipefish and stickleback patterns and draw more informed conclusions (L244-L248; L262-277; L444-470). We added additional nuance to the discussion of the head, tooth (L485-489), and male pregnancy (L358-L391) sections to address concerns of study limitations. We describe in more detail these additional data in response to reviewers.

(3) In situ hybridization results: as already included above, there is generally weak labeling of species, developmental stages, and other markings that can provide context. The collective feeling here is that as it is currently presented, the ISH results do not go too far beyond simply illustrative purposes. To take these results further, more detailed comparison may be needed. At a minimum, far better labeling can help avoid making the wrong impression. 

Based on the reviewers’ comments, we made changes to improve ISH clarity and add select comparative ISH findings. ISH was used to further interpretation of the scRNAseq atlas. All the developmental stages and species information for the embryos used were in the figure captions as well as in supplemental file 4. Since we primarily used wild caught embryos, we did not have specific ages of most embryos. The technical challenges of acquiring and staging Syngnathus embryos are detailed above. Because we did not have their age, we classified them based on developmental markers (such as presence of somites and the extent of craniofacial elongation). Although these classification methods are not ideal, they are consistent with the syngnathid literature (Sommer et al. 2012).  

We followed reviewer #1’s recommendations by adding an annotated graphic of a pipefish head, aligning bay and Gulf pipefish sequences for the probe regions, expanding out our supplemental figures for ISH into a figure for each probe, and improving labeling. These changes improved the description of the ISH experiments and have increased the quality of the manuscript.

We would have loved to complete detailed comparative studies as suggested, but doing such a complete analysis was not feasible for this study. Therefore, we completed an additional focused analysis. We followed reviewer #3’s idea and added ISHs from threespine stickleback, a short snouted fish, for 4 genes (sfrp1a, prdm16, fgf22, and bmp4). While more extensive ISHs tracking all marker genes through a variety of developmental stages in pipefish and stickleback would have provided crucial insights, we feel that it is beyond the scope of this study and would require a significant amount of additional work. We, thus, primarily interpreted the ISH results as illustrative data points in our discussion. As we state in the response to reviewer 1, the generation and annotation of the first scRNA-seq atlas in a syngnathid is the primary goal of this manuscript.  The ISHs were utilized primarily to support inferred cluster annotations if a positive identification of marker gene expression in expected tissues/cells occurred. 

Reviewer #1 (Recommendations For The Authors): 

While the scRNA-seq dataset offers a valuable resource for evo-devo analyses in fish and the hypotheses are of interest, critical aspects should be strengthened to support the claims of the study. 

Concerning the scRNA-seq dataset, the major points to be addressed are listed below: 

- Supplementary file 3 reports the single markers used to validate cluster annotations. To confirm cluster identities, more markers specific to each cluster should be highlighted and presented on the UMAP. 

We recognize the reviewer’s concern and had in reality used numerous markers to annotate the clusters. Based upon the reviewer’s comment we decided to make this clear by creating feature plots for every cluster with the top 6 marker genes. These plots showcase gene specificity in UMAP space. We also added feature plots for zebrafish marker genes for key cell types. Through these changes and the addition of 54 supplementary figures (S3:S57), we hope that it is clear that numerous markers validated cluster identity.

For example, as clusters 17 and 37 share the same tnnti1 marker, which other markers permit to differentiate their respective identity. 

This is a fair point. Cluster 17 and 37 both are marked by a tnni1 ortholog.

Different paralogous co-orthologs mark each cluster (cluster 17: LOC125989146; cluster 37: LOC125970863). In our revision to the above comment, additional (6) markers per cluster were highlighted which should remedy this concern. 

- L146: the low number of identified cartilaginous cells (only 2% of total connective tissue cells) appears aberrant compared to bone cell number, while Figure 1 presents a welldeveloped cartilaginous skeleton with poor or no signs of ossification. Please discuss this point. 

We also found this to be interesting and added a brief discussion on this subject to the results section (L147-L149). Single cell dissociations can have variable success for certain cell types. It is possible that the cartilaginous cells were more difficult to dissociate than the osteoblast cells.

- L162: pax3a/b are not specific to muscle progenitors as the genes are also expressed in the neural tube and neural crest derivatives during organogenesis. Please confirm cluster 10 identity.  

Thank you for the reminder, we added numerous feature plots that explored zebrafish (from Daniocell) and pipefish markers (identified in our dataset). Examining zebrafish satellite muscle markers (myog, pabpc4, and jam2a) shows a strong correspondence with cluster #10.

- L198: please specify in the text the pigment cell cluster number. 

We completed this change.

- L199: it is not clear why considering module 38 correlated to cluster 20 while modules 2/24 appear more correlated according to the p-value color code. 

We thank the reviewer for pointing this confusing element out! Although the t-statistic value for module 38 (3.75) is lower than the t-statistics for modules 2 and 24 (5.6 and 5.2, respectively), we chose to highlight module 38 for its ‘connectivity dependence’ score. In our connectivity test, we examined whether removing cells from a specific cell cluster reduced the connectivity of a gene network. We found that removing cluster 20 led to a decrease in module 38’s connectivity (-.13, p=0) while it led to an increase in modules 2 and 24’s connectivity (.145, p=1; .145, p=9.14; our original supplemental files 9-10). Therefore, the connectivity analysis showed that module 38’s structure was more dependent on cluster 20 than in comparison with modules 2 and 24. Although you highlighted an interesting quandary, we decided that this is tangential to the paper and did not add this discussion to the manuscript. 

- Please describe in the text Figure 4A. 

Completed, we thank the reviewer for catching this! 

Concerning embryo stainings, the major points to be addressed are listed below: 

- Figure 1: please enhance the light/contrast of figures to highlight or show the absence of alcian/alizarin staining. Mineralized structures are hardly detectable in the head and slight differences can be seen between the two samples. The developmental stage should be added. Please homogenize the scale bar format (remove the unit on panels E and, G as the information is already in the text legend). It would be useful to illustrate the data with a schematic view of the structures presented in panels B, and E, and please annotate structures in the other panels.  

We thank the reviewer for these suggestions to improve our figure. We increased the brightness and contrast for all our images. We also added an illustration of the head with labels of elements. As discussed, we used wild caught pregnant males and, therefore, do not know the exact age of the specimens. However, we described the developmental stage based on morphological observations. Slight differences in morphology between samples is expected. We and others have noticed that

developmental rate varies, even within the same brood pouch, for syngnathid embryos. We observed several mineralization zones including in the embryos including the upper and lower jaws, the mes(ethmoid), and the pectoral fin. We recognize the cartilage staining is more apparent than the bone staining, though increasing image brightness and contrast did improve the visibility of the mineralization front.

- All ISH stainings and images presented in Figures 4-6/ Figures S2-3 should be revised according to comments provided in the public review. 

We thank the reviewer for providing thorough comments, we provided an in-depth response to the public review. We made several improvements to the manuscript to address their concerns. 

- Figure 4: Figure 4B should be described before 4C in the text or inverse panels / L222 the Meckel's cartilage is not shown on Figure 4C. The schematic views in H should be annotated and the color code described / the ISH data must be completed to correlate spatially clusters to head structures. 

We thank the reviewer for pointing this out, we fixed the issues with this figure and added annotations to the head schematics.

- Figure 5: typo on panels 'alician' = alcian. 

We completed this change. 

- Figures S2-3: data must be better presented, polished / typo in captions 'relavant'= relevant. 

Thank you for this critique, we created new supplementary figures to enhance interpretation of the data (S59-S71). In these new figures, we included a feature plot for each gene and respective ISHs.

- Figure S3: soat2 = no evidence of muscle marker neither by ISH presented nor in the literature. 

We realized this staining was not clear with the previous S2/S3 figures. Our new changes in these supplementary figures based on the reviewer’s ideas made these ISH results clearer. We observed soat2 staining in the sternohyoideus muscle (panel B in S71).

Other points: 

- The cartilage/bone developmental state (Alcian/alizarin staining) and/or ISH for classical markers of muscle development (such as pax3/myf5) could be used to clarify the This could permit the completion of a comparative analysis between the two species and the interpretation of novel and adaptative characters.  

We appreciate this idea! We thought deeply about a well characterized comparative analysis between pipefish and zebrafish for this study. We discussed our concerns in our public response to reviewer 2. We found that it was challenging to stage match all cell types, and were concerned that we could make erroneous conclusions. For example, our pipefish samples were still inside the male brood pouch and possessed yolk sacs. However, we found osteoblast cells in our scRNAseq atlas, and in alizarin staining. Although zebrafish literature notes that the first zebrafish bone appears at 3 dpf (Kimmel et al. 1995), osteoblasts were not recognized until 5 dpf in two scRNAseq datasets (Fabian et al. 2022; Lange et al. 2023). A 5dpf zebrafish is considered larval and has begun hunting. Therefore, we chose to not integrate our data out of concern that osteoblast development may occur at different timelines between the fishes. 

Fabian, P., Tseng, K.-C., Thiruppathy, M., Arata, C., Chen, H.-J., Smeeton, J., Nelson, N., & Crump, J. G. (2022). Lifelong single-cell profiling of cranial neural crest diversification in zebrafish. Nature Communications 2022 13:1, 13(1), 1–13. 

Lange, M., Granados, A., VijayKumar, S., Bragantini, J., Ancheta, S., Santhosh, S., Borja, M., Kobayashi, H., McGeever, E., Solak, A. C., Yang, B., Zhao, X., Liu, Y., Detweiler, A. M., Paul,

S., Mekonen, H., Lao, T., Banks, R., Kim, Y.-J., … Royer, L. A. (2023). Zebrahub – Multimodal Zebrafish Developmental Atlas Reveals the State-Transition Dynamics of Late-Vertebrate Pluripotent Axial Progenitors. BioRxiv, 2023.03.06.531398. 

Kimmel, C., Ballard, S., Kimmel, S., Ullmann, B., Schilling, T. (1995). Stages of Embryonic Development of the Zebrafish. Developmental Dynamics 203:253:-310.

'in situs' in the text should be replaced by 'in situ experiments'.  

We made this change (L395, L663, L666, L762).

- Lines 562-565: information on samples should be added at the start of the result section to better apprehend the following scRNA-seq data.

We thank the reviewer for pointing out this issue. Although we had a few sentences on the samples in the first paragraph of the result section, we understand that it was missing some critical pieces of information. Therefore, we added these additional details to the beginning of the results section (L126-L132). 

- Lines 629-665: PCR with primers designed on gulf pipefish genome could be performed in parallel on bay and gulf cDNA libraries, and amplification products could be sequenced to analyze alignment and validate the use of gulf pipefish ISH probes in bay pipefish embryos. Probe production could also be performed using gulf primers on bay pipefish cDNA pools. 

After the submission of this manuscript, a bay pipefish genome was prepared by our laboratory. We used this genome to align our probes, these alignments demonstrate strong sequence conservation between the species. We included these alignments in our supplemental files.

- L663: the bleaching step must be optimized on pipefish embryos. 

We understand this concern and had completed several bleach optimization experiments prior to publication. Although we found that bleaching improved visibility of staining, we noticed with the probe tnmd that bleached embryos did not have complete staining of tendons and ligaments. The unbleached embryos had more extensive staining than the bleached embryos. We were concerned that bleaching would lead to failures to detect expression domains (false negatives) important for our analysis. Therefore, we did not use bleaching with our in situs experiments (except with hatched fish with a high degree of pigmentation). 

- Indicate the number of specimens analyzed for each labeling condition.  

We thank the reviewer for noticing this issue. We added this information to the methods (L766-767).

- Describe the fixation and pre-treatment methods previous to ISH and skeleton stainings

We thank the reviewer for pointing out this issue, we added these descriptions (L765-766; L772-774). 

Reviewer #3 (Recommendations For The Authors): 

(1) If sfrp1a expression is observed also in other fish species with derived craniofacial structures, it's important to discuss this more in the Discussion. This could be a common mechanism to modify craniofacial structures, although functional tests are ultimately required (but not in this paper, for sure). Can lines 421-428 involve the statement "a prolonged period of chondrocyte differentiation" underlies craniofacial diversity?

This is a great idea, and we added a sentence that captures this ethos (L451-452).

(2) Lines 334-346 need to be rephrased. It's hard to understand which genes are expressed or not in pipefish and zebrafish. Did "23 endocytosis genes" show significant enrichment in zebrafish epidermis, or are they expressed in zebrafish epidermis? 

We thank the reviewer for this comment, we re-phrased this section for clarity (L365-368).

(3) Figure 4 is missing the "D" panel and two "E" panels. 

We thank the reviewer for noticing this, we fixed this figure.

(4) Line 302: "whole-mount" or "whole mount"

We thank the reviewer for the catch!

important

An example of colonialism in tech is mining for materials like cobalt, used in electronics. Workers in poor countries face unsafe conditions and low pay, while big companies in rich nations make most of the profits.

I like how sweet this line is. It's almost like a lesson to little kids to always be nice and gentle to people.

In socialism, the government owns businesses, decides what to make, and sets prices. It uses profits to support public services or give money to people, like Alaska’s oil payouts. Public schools and sewer systems show how this works well for shared resources. Many countries use a mix of socialism and capitalism.

De l'Éducation des Parents au Soutien à la Parentalité: Tensions et Controverses

I. Introduction: Interactions Enfant-Parent-École et la Question Parentale

**Interactions Enfant-Parent-École : Un Système d'Attentes Réciproques (5:00): **

La relation entre parents et école est marquée par des attentes mutuelles, notamment en ce qui concerne la réussite scolaire. Cette interaction est fortement influencée par des sujets partagés comme la réussite scolaire, le comportement et le bien-être des enfants.

**L'Emprise Scolaire et la Transformation des Parents en Coachs (6:15): **

La massification de l'accès à l'école a engendré une "emprise scolaire", où la question de l'école domine les interactions parents-enfants. Les parents se transforment en "coachs scolaires", centrés sur la performance de leurs enfants, ce qui peut avoir un impact négatif sur la relation parent-enfant.

**Symptômes Émergents et Mal-être des Enfants (10:00): **

L'augmentation des troubles psychiques chez les enfants et adolescents, manifestée par des symptômes comme le retrait scolaire (hikikomori) et la surconsommation de psychotropes, met en lumière les difficultés croissantes rencontrées par les jeunes et interroge le rôle des parents et des institutions dans leur bien-être.

II. Histoire de la Relation entre Pouvoirs Publics et Parents

L'Émergence de l'Éducation des Parents (15:00):

Dès le 19ème siècle, l'idée d'éduquer les parents à leur rôle, notamment en matière de maternage, prend forme pour lutter contre la mortalité infantile et garantir le bien-être des enfants.

Cette préoccupation s'intensifie au 20ème siècle, avec la création d'institutions dédiées à l'éducation des parents.

L'École des Parents et la Défense du Rôle Parental (19:00):

Créée dans un contexte de crise idéologique dans les années 30, l'École des Parents vise à soutenir les parents face à l'intrusion perçue de l'État dans l'éducation des enfants.

Elle est initialement portée par une élite catholique et conservatrice, défendant une vision traditionnelle de la famille.

L'Après-Guerre et le Marché du Conseil aux Parents (24:00):

Après la Seconde Guerre mondiale, un véritable marché du conseil aux parents se développe, avec des figures comme Benjamin Spock, Françoise Dolto et Laurence Pernoud, qui publient des ouvrages et donnent des conseils aux parents.

L'accent est mis sur la valorisation des connaissances des mères et l'importance de l'écoute et de la compréhension de l'enfant.

III. Le Tournant de la Parentalité et l'Émergence d'une Politique Publique

L'Apparition du Concept de "Parentalité" (27:50):

Dans les années 90, le concept de "parentalité" émerge, influencé par la Convention Internationale des Droits de l'Enfant et le rôle croissant des institutions internationales dans la promotion du bien-être des enfants.

Le Soutien à la Parentalité : Définition et Objectifs (31:20):

Le soutien à la parentalité est défini comme un ensemble de mesures visant à informer, soutenir, conseiller et former les parents dans leur rôle. Il se distingue des politiques de l'enfance en ciblant les parents plutôt que les enfants.

Diversité des Mesures et Tensions Idéologiques (34:00):

Le soutien à la parentalité se traduit par une variété de mesures, allant de l'information générale au conseil individuel en passant par des programmes de formation.

Cependant, des tensions idéologiques émergent entre des approches universalistes et des initiatives ciblant les parents en difficulté.

IV. Controverses et Débats Autour de la Parentalité

Le Déterminisme Parental et la Responsabilisation des Parents (46:00):

Une vision déterministe de la parentalité tend à attribuer la responsabilité des problèmes rencontrés par les enfants aux déficits parentaux. Cette approche risque d'individualiser et de psychologiser les difficultés sociales, en négligeant les contextes socio-économiques dans lesquels les familles évoluent.

Débats Autour des Neurosciences et de la Psychologie Positive (48:00):

L'influence croissante des neurosciences et de la psychologie positive dans le domaine de la parentalité suscite des débats. La focalisation sur les trois premières années de l'enfant et l'insistance sur l'importance des interactions précoces peuvent occulter les influences sociales et culturelles qui façonnent la parentalité.

Parentalité Positive vs Autorité Parentale (50:00):

La promotion de la parentalité positive, prônant la bienveillance et l'écoute, est parfois confrontée à des discours valorisant l'autorité et la discipline. La question de la limite et de la punition dans l'éducation des enfants divise les experts et les parents.

V. Conclusion : Penser les Cultures de la Parentalité et les Inégalités

Le Double Bind de la Parentalité (58:00):

Les parents sont confrontés à un "double bind" : ils sont encouragés à s'investir intensément dans l'éducation de leurs enfants, mais risquent d'être critiqués s'ils en font "trop" ou "pas assez". Il est essentiel de reconnaître la diversité des cultures de la parentalité et de ne pas imposer un modèle unique.

L'Importance des Contextes Socio-économiques (59:00):

Les conditions de vie des familles, leurs ressources économiques, leurs conditions de travail et de logement, influencent profondément la manière dont les parents exercent leur rôle. Il est crucial de tenir compte de ces inégalités et de ne pas responsabiliser les parents sans prendre en considération les contextes dans lesquels ils évoluent.

Acetylocholina, streszczenie

for - democracy - erosion from two forces - dark money and - dark tech - Otto Scharmer

for - 2024 US election results - many Americans voted for down-ballot candidates but not national candidates - suggested no options available to voters - Otto Scharmer

for - Indyweb dev - Presencing Institute - U-lab - natural application - weaving together these subnets with mindplexes via open source SRG complexity mapping tools in the Indyweb

for - to - a proposal for a love-based intervention for addressing the emergence of fascism, populism, polarization and the climate crisis - Roger Hallam

to - a proposal for a love-based intervention for addressing the emergence of fascism, populism, polarization and the climate crisis - Roger Hallam - https://hyp.is/wUDpaKsAEe-DM9fteMUtzw/www.youtube.com/watch?v=AiKWCHAcS7E

for - stats - most people in America agree on 85 - 90% of issues - unpack why and how the 10 - 15% is made so divisive

for - voting - false dichotomy - no real choice - need for a third otion

voting - false dichotomy - no real choice - need for a third option - We are not going back can imply one of two possibilities: - we are staying still (status quo) - we are moving forward in a new direction (emergence) - We are going to Make America Great Again (going back) - We can't go back because we have degraded our world beyond that possibility - We can't stay still because it is untenable - We can only move forward - But that option was not given - This goes to the heart of Yanis Varoufakis, Michel Bauwen and many other's claim that there is only one party in control of both the Democrats and the Republicans, the elites

to - Youtube - interview - 2008 was the West's 1991 moment - Yanis Varoufakis - https://hyp.is/BZ88pKj5Ee-k86snmHsbnQ/www.youtube.com/watch?v=8nTBWf4JgYQ

for - to - examples of Neo feudalism - Techno Feudalism - What killed Capitalism - Yanis Varoufakis - to - example of Neo feudalism - Youtube - interview - 2008 was the West's 1991 moment - Yanis Varoufakis

to - examples of Neo feudalism - Techno Feudalism: What killed capitalism - Yanis Varoufakis - https://hyp.is/9S3SGKj4Ee-btAdw5i_vLg/www.youtube.com/watch?v=Fhgm5b8BR0k - Youtube - interview - 2008 was the West's 1991 moment - Yanis Varoufakis - https://hyp.is/BZ88pKj5Ee-k86snmHsbnQ/www.youtube.com/watch?v=8nTBWf4JgYQ

for - adjacency - "dismantled from within" - polarization - consequences of - shortermism - executive orders - destruction every time other party wins election - melancholia

adjacency - between - "dismantled from within" - polarization - executive order - consequences of - shortermism - destruction every time other party takes over - melancholia - adjacency relationship - When I read the words "dismantle from within", I made an association with how destructive policies are when polarization means leads to an inability to find a middle ground - New executive orders are issued to undo the executive orders of the previous term - This shortermism of every election cycle when no compromise can be found is collectively melancholy

for - to - Substack article - A global history of societal regulation - metacrisis, polycrisis - role of the commons and cosmolocal coordination - Michel Bauwens

to Substack article - A global history of societal regulation - metacrisis, polycrisis - role of the commons and cosmolocal coordination - Michel Bauwens - https://hyp.is/wlywbqkTEe-ROXfhSmA3bA/4thgenerationcivilization.substack.com/p/a-global-history-of-societal-regulation

for - to - example of Status Quoism - Why Harris lost to Trump - from Youtube - So Trump won? What's next? - Roger Hallam

to - Why Harris lost to Trump - from Youtube - So Trump won? What's next? - Roger Hallam - https://hyp.is/aRrY5KsEEe-yezeexHETqg/www.youtube.com/watch?v=AiKWCHAcS7E

for - Deep Humanity - Common Human Denominators (CHD)

Amen. This is the focus of Me2We2All- amplifying aliveness through more skillful connected learning and adaptation.

Bingo a

"Linking islands of coherence into connected competence"

Opportunity for action/impact based on connecting and enactivating our own, feasible islands of coherence.

"Connecting local coherence into competence greater than"

Active inference is natural (and mathematical) way to describe and model the predicted landscape and adapt to the emergent surprise

Amen.

Love this! Too often the hunt for consensus gets in the ways of the other form of legitimacy - competence.

Great critique. Enactivating change management through "corrective standards and regulation" distorts surprising moments from opportunities for distributed learnign into a compliance checklist for heirarchy

Temps Forts de la Vidéo

La transcription de la vidéo "Envisager la coéducation comme relation de reconnaissance mutuelle - Chloé Riban" permet d'identifier plusieurs temps forts, marqués par des interventions et des réflexions clés :

1. Introduction et Contexte (0:00 - 3:17):

• Chloé Riban se présente et situe son travail de recherche, basé sur sa thèse et ses travaux actuels.
• Elle met en avant l'importance du terrain ethnographique pour comprendre les relations école-famille.
• Elle décrit le contexte socio-économique du quartier où elle a mené son enquête, marqué par la précarité.
• Elle évoque ses terrains de recherche actuels, notamment un café des parents et une université populaire de parents.

2. Repères Historiques et Théoriques (3:17 - 10:27):

• Rappel historique de l'évolution des relations école-parents, de l'absence de communication initiale à l'émergence de la coéducation.
• Présentation des influences théoriques de ses recherches, incluant le concept de "différent" de Pierre Perrier et les approches systémiques et intersectionnelles.
• Discussion sur la définition de la coéducation et ses différentes interprétations.

3. La Demande de Coéducation et ses Destinataires (10:27 - 17:05):

• Questionnement sur l'universalité de la demande de coéducation et mise en évidence de son ciblage sur certains types de parents, notamment ceux perçus comme "éloignés" de l'école.
• Analyse des attentes normées des professionnels en matière de coéducation, basées sur une figure de parent idéal.
• Mise en lumière des représentations et des perceptions souvent négatives des professionnels sur les pratiques familiales des élèves en difficulté.

4. Regards Croisés sur l'Investissement Parental (17:05 - 28:35):

• Débat animé entre les participants sur la question de l'éloignement et du désinvestissement des parents en REP, avec des témoignages divergents et nuancés.
• Chloé Riban souligne la pertinence des observations des participants et annonce aborder la question de l'investissement parental du point de vue des parents.

5. Comprendre l'Investissement Parental en Contexte (28:35 - 36:39):

• Description de la confiance et des attentes élevées des parents, notamment des mères de famille populaires, envers l'institution scolaire.
• Énumération des multiples formes d'investissement parental dans la scolarité, souvent invisibles aux yeux des professionnels.

6. Obstacles et Difficultés des Parents Face à l'École (36:39 - 41:46):

• Identification des obstacles à la confiance initiale, tels que la méconnaissance du système scolaire, la peur du jugement, les difficultés de communication et les contraintes temporelles.
• Mise en évidence du rapport dissymétrique entre les mères et l'école, marqué par le doute, le sentiment d'être mises en cause et la crainte de ne pas correspondre à l'image de la "bonne mère".

7. Le Quotidien des Mères de Famille Populaires (41:46 - 46:42):

• Analyse du quotidien des mères, ancré dans les tâches de "care", les solidarités familiales et une charge mentale importante.
• Évocation des vulnérabilités cumulées, des parcours de vie heurtés et des stigmates sociaux subis.

8. Stratégies Parentales: Oscillations entre Proximité et Distance (46:42 - 49:09):

• Description des tactiques développées par les mères pour faire face à l'école, oscillant entre la participation et le retrait, la recherche de valorisation et la protection contre le jugement.

9. Enjeux de Reconnaissance et Recherche de Parité (49:09 - 50:37):

• Mise en lumière des enjeux de reconnaissance sous-jacents à la participation scolaire et à l'investissement parental.
• Soulignement de l'importance pour les mères de se sentir sur un pied d'égalité avec les enseignants et de voir leurs paroles et leurs regards reconnus.

10. Vers une Coéducation Basée sur la Reconnaissance Mutuelle (50:37 - 58:44):

• Présentation des pistes concrètes pour une coéducation plus inclusive et respectueuse des parents, inspirées des travaux de Catherine Urtique de Lâtre et de Pierre Perrier.
• Introduction du concept de "palabre" d'Isabelle Stengers comme modèle alternatif au débat, favorisant la reconnaissance mutuelle et la construction collective du sens.

11. Échanges et Questions (58:44 - 1:15:53):

• Session de questions-réponses et d'échanges riches entre Chloé Riban et les participants, abordant des points clés comme l'accueil des parents, la communication, la transition entre cycles, la place des parents dans les décisions scolaires et les ressources disponibles.

Ces temps forts, articulés autour de l'analyse des relations école-famille et de la coéducation, offrent un aperçu des enjeux complexes et des pistes de réflexion pour une collaboration plus inclusive et respectueuse de la diversité des parents et de leurs réalités.

Recognition can change the world. Signals need to be valid and trustworthy, but we're so close to making a huge difference in the world through recognition of things that are already there, just hidden in plain sight.

This comparison reveals the trade-off in predation risk and habitat selection, with shoreline habitats offering forage benefits but higher risks for prime moose.

This explains that spatial predation patterns are tied to specific habitat characteristics, connecting environmental features with predator-prey dynamics.

This finding highlights a major ecological difference in predation risk based on habitat preference and life stage, emphasizing how geography impacts predator-prey interactions.

Ripley’s K analysis is used here to quantify clustering of carcass locations. The clustering results support the hypothesis that senescent and prime moose experience predation differently.

Pattern analysis and kernel density estimates are crucial for understanding spatial predation patterns. The distinction between senescent and prime moose sets up the analysis of how prey life stages impact predation zones.

This is very surprising. You would think that wolves would target the moose of senescence more often.

Habitat decisions can affect accessibility of moose to wolves and their ability to evade predation.

eLife Assessment

This important study investigates how working memory load influences the Stroop effect from a temporal dynamics perspective. Solid evidence is provided that the working memory load influences the Stroop effect in the late-stage stimulus-response mapping instead of the early sensory stage. This study will be of interest to both neuroscientists and psychologists who work on cognitive control.

Reviewer #1 (Public review):

Summary:

This study investigates an intriguing question in cognitive control from a temporal dynamics perspective: why does concurrent verbal working memory load eliminate the color-word Stroop effect? Through a series of thorough data analyses, the authors propose that verbal working memory load occupies the stimulus-response mapping resources represented by theta-band activity, thereby disrupting the mapping process for task-irrelevant distractors. This reduces the response tendency to the distractors, ultimately leading to the elimination of the Stroop effect.

Strengths:

The behavioral and neural evidence presented in the manuscript is solid, and the findings have valuable theoretical implications for research on Stroop conflict processing.

Weaknesses:

There are several areas where the manuscript could be improved.

Major Comments:

(1) In the Results section, the rationale behind selecting the beta band for the central (C3, CP3, Cz, CP4, C4) regions and the theta band for the fronto-central (Fz, FCz, Cz) regions is not clearly explained in the main text. This information is only mentioned in the figure captions. Additionally, why was the beta band chosen for the S-ROI fronto-central region and the theta band for the S-ROI central region? Was this choice influenced by the MVPA results?

(2) In the Data Analysis section, line 424 states: "Only trials that were correct in both the memory task and the Stroop task were included in all subsequent analyses. In addition, trials in which response times (RTs) deviated by more than three standard deviations from the condition mean were excluded from behavioral analyses." The percentage of excluded trials should be reported. Also, for the EEG-related analyses, were the same trials excluded, or were different criteria applied?

(3) In the Methods section, line 493 mentions: "A 400-200 ms pre-stimulus time window was selected as the baseline time window." What is the justification in the literature for choosing the 400-200 ms pre-stimulus window as the baseline? Why was the 200-0 ms pre-stimulus period not considered?

(4) Is the primary innovation of this study limited to the methodology, such as employing MVPA and RSA to establish the relationship between late theta activity and behavior?

(5) On page 14, lines 280-287, the authors discuss a specific pattern observed in the alpha band. However, the manuscript does not provide the corresponding results to substantiate this discussion. It is recommended to include these results as supplementary material.

(6) On page 16, lines 323-328, the authors provide a generalized explanation of the findings. According to load theory, stimuli compete for resources only when represented in the same form. Since the pre-memorized Chinese characters are represented semantically in working memory, this explanation lacks a critical premise: that semantic-response mapping is also represented semantically during processing.

(7) The classic Stroop task includes both a manual and a vocal version. Since stimulus-response mapping in the vocal version is more automatic than in the manual version, it is unclear whether the findings of this study would generalize to the impact of working memory load on the Stroop effect in the vocal version.

(8) While the discussion section provides a comprehensive analysis of the study's results, the authors could further elaborate on the theoretical and practical contributions of this work.

Reviewer #2 (Public review):

Summary:

Li et al. explored which stage of Stroop conflict processing was influenced by working memory loads. Participants completed a single task (Stroop task) and a dual task (the Sternberg working memory task combined with the Stroop task) while their EEG data was recorded. They adopted the event-related potential (ERP), and multivariate pattern analyses (MVPA) to investigate the interaction effect of task (single/dual) and congruency (congruent/incongruent). The results showed that the interaction effect was significant on the sustained potential (SP; 650-950 ms), the late theta (740-820 ms), and beta (920-1040 ms) power but not significant on the early P1 potential (110-150 ms). They used the representational similarity analyses (RSA) method to explore the correlation between behavioral and neural data, and the results revealed a significant contribution of late theta activity.

Strengths:

(1) The experiment is well-designed.

(2) The data were analyzed in depth from both time and frequency domain perspectives by combining several methods.

Weaknesses:

(1) As the researchers mentioned, a previous study reported a diminished Stroop effect with concurrent working memory tasks to memorize meaningless visual shapes rather than memorize Chinese characters as in the study. My main concern is that lower-level graphic processing when memorizing visual shapes also influences the Stroop effect. The stage of Stroop conflict processing affected by the working memory load may depend on the specific content of the concurrent working memory task. If that's the case, I sense that the generalization of this finding may be limited.

(2) The P1 and N450 components are sensitive to congruency in previous studies as mentioned by the researchers, but the results in the present study did not replicate them. This raised concerns about data quality and needs to be explained.

Author response:

Reviewer #1 (Public review):

Comment 1: In the Results section, the rationale behind selecting the beta band for the central (C3, CP3, Cz, CP4, C4) regions and the theta band for the fronto-central (Fz, FCz, Cz) regions is not clearly explained in the main text. This information is only mentioned in the figure captions. Additionally, why was the beta band chosen for the S-ROI central region and the theta band for the S-ROI fronto-central region? Was this choice influenced by the MVPA results?

We thank the reviewer for the question regarding the rationale for the S-ROI selection in our study. The beta band was chosen for the central region due to its established relevance in motor control (Engel & Fries, 2010), movement planning (Little et al., 2019) and motor inhibition (Duque et al., 2017). The fronto-central theta band (or frontal midline theta) was a widely recognized indicator in cognitive control research (Cavanagh & Frank, 2014), associated with conflict detection and resolution processes. Moreover, recent empirical evidence suggested that the fronto-central theta reflected the coordination and integration between stimuli and responses (Senoussi et al., 2022). Although we have described the cognitive processes linked to these different frequencies in the introduction and discussion sections, along with the potential patterns of results observed in Stroop-related studies, we did not specify the involved cortical areas. Therefore, we have specified these areas in the introduction to enhance the clarity of the revised version (in the fourth paragraph of the Introduction section).

Regarding whether the selection of S-ROIs was influenced by the MVPA results, we would like to clarify here that we selected the S-ROIs based on prior research and then conducted the decoding analysis. Specifically, we first extracted the data representing different frequency indicators (three F-ROIs and three S-ROIs) as features, followed by decoding to obtain the MVPA results. Subsequently, the time-frequency analysis, combined with the specific time windows during which each frequency was decoded, provided detailed interaction patterns among the variables for each indicator. The specifics of feature selection are described in the revised version (in the first paragraph of the Multivariate Pattern Analysis section).

Comment 2: In the Data Analysis section, line 424 states: “Only trials that were correct in both the memory task and the Stroop task were included in all subsequent analyses. In addition, trials in which response times (RTs) deviated by more than three standard deviations from the condition mean were excluded from behavioral analyses.” The percentage of excluded trials should be reported. Also, for the EEG-related analyses, were the same trials excluded, or were different criteria applied?

We thank the reviewer for this suggestion. Beyond the behavioral exclusion criteria, trials with EEG artifacts were also excluded from the data for the EEG-related analyses. We have now reported the percentage of excluded trials for both behavioral and EEG data analyses in the revised version (in the second paragraph of the EEG Recording and Preprocessing section and the first paragraph of the Behavioral Analysis section).

Comment 3: In the Methods section, line 493 mentions: “A 400-200 ms pre-stimulus time window was selected as the baseline time window.” What is the justification in the literature for choosing the 400-200 ms pre-stimulus window as the baseline? Why was the 200-0 ms pre-stimulus period not considered?

We thank the reviewer for this question and would like to provide the following justification. First, although a baseline ending at 0 ms is common in ERP analyses, it may not be suitable for time-frequency analysis. Due to the inherent temporal smoothing characteristic of wavelet convolution in time-frequency decomposition, task-related early activities can leak into the pre-stimulus period (before 0 ms) (Cohen, 2014). This means that extending the baseline to 0 ms will include some post-stimulus activity in the baseline window, thereby increasing baseline power and compromising the accuracy of the results. Second, an ideal baseline duration is recommended to be around 10-20% of the entire trial of interest (Morales & Bowers, 2022). In our study, the epoch duration was 2000 ms, making 200-400 ms an appropriate baseline length. Third, given that the minimum duration of the fixation point before the stimulus in our experiment was 400 ms, we chose the 400 ms before the stimulus as the baseline point to ensure its purity. In summary, considering edge effects, duration requirements, and the need to exclude other influences, we selected a baseline correction window of -400 to -200 ms. To enhance the clarity of the revised version, we have provided the rationale for the selected time windows along with relevant references (in the first paragraph of the Time-frequency analysis section).

Comment 4: Is the primary innovation of this study limited to the methodology, such as employing MVPA and RSA to establish the relationship between late theta activity and behavior?

We thank the reviewer for this insightful question and would like to clarify that our research extends beyond mere methodological innovation; rather, it utilized new methods to explore novel theoretical perspectives. Specifically, our research presents three levels of innovation: methodological, empirical, and theoretical. First, methodologically, MVPA overcame the drawbacks of traditional EEG analyses based on specific averaged voltage intensities, providing new perspectives on how the brain dynamically encoded particular neural representations over time. Furthermore, RSA aimed to identify which indicators among the decoded were directly related to behavioral representation patterns. Second, in terms of empirical results, using these two methods, we have identified for the first time three EEG markers that modulate the Stroop effect under verbal working memory load: SP, late theta, and beta, with late theta being directly linked to the elimination of the behavioral Stroop effect. Lastly, from a theoretical perspective, we proposed the novel idea that working memory played a crucial role in the late stages of conflict processing, specifically in the stimulus-response mapping stage (the specific theoretical contributions are detailed in the second-to-last paragraph of the Discussion section).

Comment 5: On page 14, lines 280-287, the authors discuss a specific pattern observed in the alpha band. However, the manuscript does not provide the corresponding results to substantiate this discussion. It is recommended to include these results as supplementary material.

We thank the reviewer for this suggestion. We added a new figure along with the corresponding statistical results that displayed the specific result patterns for the alpha band (Supplementary Figure 1).

Comment 6: On page 16, lines 323-328, the authors provide a generalized explanation of the findings. According to load theory, stimuli compete for resources only when represented in the same form. Since the pre-memorized Chinese characters are represented semantically in working memory, this explanation lacks a critical premise: that semantic-response mapping is also represented semantically during processing.

We thank the reviewer for this insightful suggestion. We fully agree with the reviewer’s perspective. As stated in our revised version, load theory suggests that cognitive resources are limited and dependent on a specific type (in the second paragraph of the Discussion section). The previously memorized Chinese characters are stored in working memory in the form of semantic representations; meanwhile the stimulus-response mapping should also be represented semantically, leading to resource occupancy. We have included this logical premise in the revised version (in the third-to-last paragraph of the Discussion section).

Comment 7: The classic Stroop task includes both a manual and a vocal version. Since stimulus-response mapping in the vocal version is more automatic than in the manual version, it is unclear whether the findings of this study would generalize to the impact of working memory load on the Stroop effect in the vocal version.

We fully agree with the reviewer’s point that the verbal version of the Stroop task differs from the manual version in terms of the degree of automation in the stimulus-response mapping. Specifically, the verbal version relies on mappings that are established through daily language use, while the manual version involves arbitrary mappings created in the laboratory. Therefore, the stimulus-response mapping in the verbal response version is more automated and less likely to be suppressed. However, our previous research indicated that the degree of automation in the stimulus-response mapping was influenced by practice (Chen et al., 2013). After approximately 128 practice trials, semantic conflict almost disappears, suggesting that the level of automation in stimulus-response mapping for the verbal Stroop task is comparable to that of the manual version (Chen et al., 2010). Given that participants in our study completed 144 practice trials (in the Procedure section), we believe these findings can be generalized to the verbal version.

Comment 8: While the discussion section provides a comprehensive analysis of the study’s results, the authors could further elaborate on the theoretical and practical contributions of this work.

We thank the reviewer for the constructive suggestions. We recognize that the theoretical and practical contributions of the study were not thoroughly elaborated in the original manuscript. Therefore, we have now provided a more detailed discussion. Specifically, the theoretical contributions focus on advancing load theory and highlighting the critical role of working memory in conflict processing. The practical contributions emphasize the application of load theory and the development of intervention strategies for enhancing inhibitory control. A more detailed discussion can be found in the revised version (in the second-to-last paragraph of the Discussion section).

Reviewer #2 (Public review):

Comment 1: As the researchers mentioned, a previous study reported a diminished Stroop effect with concurrent working memory tasks to memorize meaningless visual shapes rather than memorize Chinese characters as in the study. My main concern is that lower-level graphic processing when memorizing visual shapes also influences the Stroop effect. The stage of Stroop conflict processing affected by the working memory load may depend on the specific content of the concurrent working memory task. If that’s the case, I sense that the generalization of this finding may be limited.

We thank the reviewer for this insightful concern. As mentioned in the manuscript, this may be attributed to the inherent characteristics of Chinese characters. In contrast to English words, the processing of Chinese characters relies more on graphemic encoding and memory (Chen, 1993). Therefore, the processing of line patterns essentially occupies some of the resources needed for character processing, which aligns with our study’s hypothesis based on dimensional overlap. Additionally, regarding the results, even though the previous study presents lower-level line patterns, the results still showed that the working memory load modulated the later theta band. We hypothesize that, regardless of the specific content of the pre-presented working memory load, once the stimulus disappears from view, these loads are maintained as representations in the working memory platform. Therefore, they do not influence early perceptual processing, and resource competition only occurs once the distractors reach the working memory platform. Lastly, previous study has shown that spatial loads, which do not overlap with either the target or distractor dimensions, do not influence conflict effect (Zhao et al., 2010). Taken together, we believe that regardless of the specific content of the concurrent working memory tasks, as long as they occupy resources related to irrelevant stimulus dimensions, they can influence the late-stage processing of conflict effect. Perhaps our original manuscript did not convey this clearly, so we have rephrased it in a more straightforward manner (in the second paragraph of the Discussion section).

Comment 2: The P1 and N450 components are sensitive to congruency in previous studies as mentioned by the researchers, but the results in the present study did not replicate them. This raised concerns about data quality and needs to be explained.

We thank the reviewer for this insightful concern. For P1, we aimed to convey that the early perceptual processing represented by P1 is part of the conflict processing process. Therefore, we included it in our analysis. Additionally, as mentioned in the discussion, most studies find P1 to be insensitive to congruency. However, we inappropriately cited a study in the introduction that suggested P1 shows differences in congruency, which is among the few studies that hold this perspective. To prevent confusion for readers, we have removed this citation from the introduction.

As for N450, most studies have indeed found it to be influenced by congruency. In our manuscript, we did not observe a congruency effect at our chosen electrodes and time window. However, significant congruency effects were detected at other central-parietal electrodes (CP3, CP4, P5, P6) during the 350-500 ms interval. The interaction between task type and consistency remained non-significant, consistent with previous results. Furthermore, with respect to the location of the electrodes chosen, existing studies on N450 vary widely, including central-parietal electrodes and frontal-central electrodes (for a review, see Heidlmayr et al., 2020). We speculate that this phenomenon may be related to the extent of practice. With fewer total trials, the task may involve more stimulus conflicts, engaging more frontal brain areas. On the other hand, with more total trials, the task may involve more response conflicts, engaging more central-parietal brain areas (Chen et al., 2013; van Veen & Carter, 2005). Due to the extensive practice required in our study, we identified a congruency N450 effect in the central-parietal region. We apologize for not thoroughly exploring other potential electrodes in the previous manuscript, and we have revised the results and interpretations regarding N450 accordingly in the revised version (in the N450 section of the ERP results and the third paragraph of the Discussion section).

Reference

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Chen, M. J. (1993). A Comparison of Chinese and English Language Processing. In Advances in Psychology (Vol. 103, pp. 97–117). North-Holland. https://doi.org/10.1016/S0166-4115(08)61659-3

Chen, X. F., Jiang, J., Zhao, X., & Chen, A. (2010). Effects of practice on semantic conflict and response conflict in the Stroop task. Psychol. Sci., 33, 869–871.

Chen, Z., Lei, X., Ding, C., Li, H., & Chen, A. (2013). The neural mechanisms of semantic and response conflicts: An fMRI study of practice-related effects in the Stroop task. NeuroImage, 66, 577–584. https://doi.org/10.1016/j.neuroimage.2012.10.028

Cohen, M. X. (2014). Analyzing Neural Time Series Data: Theory and Practice. The MIT Press. https://doi.org/10.7551/mitpress/9609.001.0001

Duprez, J., Gulbinaite, R., & Cohen, M. X. (2020). Midfrontal theta phase coordinates behaviorally relevant brain computations during cognitive control. NeuroImage, 207, 116340. https://doi.org/10.1016/j.neuroimage.2019.116340

Duque, J., Greenhouse, I., Labruna, L., & Ivry, R. B. (2017). Physiological Markers of Motor Inhibition during Human Behavior. Trends in Neurosciences, 40(4), 219–236. https://doi.org/10.1016/j.tins.2017.02.006

Engel, A. K., & Fries, P. (2010). Beta-band oscillations—Signalling the status quo? Current Opinion in Neurobiology, 20(2), 156–165. https://doi.org/10.1016/j.conb.2010.02.015

Heidlmayr, K., Kihlstedt, M., & Isel, F. (2020). A review on the electroencephalography markers of Stroop executive control processes. Brain and Cognition, 146, 105637. https://doi.org/10.1016/j.bandc.2020.105637

Little, S., Bonaiuto, J., Barnes, G., & Bestmann, S. (2019). Human motor cortical beta bursts relate to movement planning and response errors. PLOS Biology, 17(10), e3000479. https://doi.org/10.1371/journal.pbio.3000479

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van Veen, V., & Carter, C. S. (2005). Separating semantic conflict and response conflict in the Stroop task: A functional MRI study. NeuroImage, 27(3), 497–504. https://doi.org/10.1016/j.neuroimage.2005.04.042

Zhao, X., Chen, A., & West, R. (2010). The influence of working memory load on the Simon effect. Psychonomic Bulletin & Review, 17(5), 687–692. https://doi.org/10.3758/PBR.17.5.687

This was in the context of controlling donetsk in minsk

OMG. He said that Ukraine is an ARTIFICIAL creation by lenin due to the era of cultural openness that lenin allowed

Putin ssaid this in reference to a return to communism by leftist parties. Putin backs the ultra nationalist parties meaning something close to the Tsardom. So no you just misattributed a quote to mislead readers.

Yes they do. You hve never read a book about Russian national identity in your life

Putins tucker carlson interview, Russian ideologues and philosphers, Russia department of defense, Russian media, Russian generals, Russia's actions, the promotion of the common origins theory, Russias behavior in minsk, Russisa highlighting the 2008 Russian world project a goal to revassalize neighbors, y ea no evidence. No you just refuse to discuss the evidence

that little brain of yours. Chechnya Georgia Moldova and Syria were first

Yea thats how you stop an empie from starting world war three. You de fang it

WHat a genius so we just let Russia take Ukraine. How did that go in Syria, Chechnya, Georgia, Moldova, etc

This was after MInsk Failed, Russia realized there was no way to vassalize Ukraine wihtout a war Ukraine refused to give the Donbas controlled. by russia veto power over their politics

This is false USA renegged on Nato Membership and gaveRussia assurances in Minsk

THe United state blocked ukraine from joining nato under OBama Trump and Biden to appease your dady putin, and in minsk russia didn't even care a bout that their priority was to re vassalize Ukraine

Then why didnt they invade poland or threaten finalnd with nukes

THis is false especially after 2008 ther was an American European divide that occured, and under Trump

LOL you said Russia wouldnt invade and that Syrian intervention wouldn't have happened. You were wrong, and you continue to be wrong

Connecting idea to promote a narratve seems more like propaganda. Iraq and Vietnam were American invasions Ukraine is a Russia invasion and the social determinents were fundementally different... How is this an American miscalculation. The united State didn't invade..

meant to show the importance of the issue by getting people's attention

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Reply to the reviewers

The authors do not wish to provide a response at this time.

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Referee #4

Evidence, reproducibility and clarity

Summary

This study analyses the shugoshin gene (SGO1) of the single-celled basidiomycete Cryptococcus neoformans. The function of Sgo proteins has been studied in various organisms, including yeasts (budding and fission yeast, C. albicans), flies, frog eggs, mammals, and plants. In general, Sgo proteins function as adapter proteins that recruit activities, such as the PP2A phosphatase, the chromosomal passenger complex (CPC), kinesins, or condensin to the pericentromeric region and kinetochores. This is required for proper biorientation of sister kinetochores at metaphase, correction of erroneous microtubule-kinetochore attachments, signaling by the spindle-assembly checkpoint (SAC), and protection of centromeric cohesin from removal by separase at meiosis and a non-proteolytic pathway in mammalian mitosis. These processes are interconnected, which has made distinguishing different functions of Sgo proteins a challenging and ongoing task.

The authors use plate assay to show that proliferation of the sgo1 mutant is sensitive to microtubule-depolymerizing drugs. Double mutants with a deletion of the SAC component MAD2 or a non-essential kinetochore subunit are even more sensitive. The sgo1 mutant fails to halt cell division, re-budding, and DNA replication in the presence of a MT drug, suggesting that it is defective in inducing or maintaining SAC activity.

Live-cell imaging is used to analyze the kinetochores recruitment of several proteins involved in error correction and/or SAC activity in the presence of a MT drug. They conclude that sgo1 mutants fail to maintain the SAC kinase Bub1 at kinetochores. Furthermore, sgo1 mutants fail to maintain at kinetochores the Aurora B kinase, which is required for error correction and SAC activity. Conversely, sgo1 mutants recruit higher levels of the PP1 phosphatase, which is known to oppose Aurora B in several processes. The authors suggest that the sgo1 mutant is defective in SAC function because it shifts the balance between Aurora B and PP1 towards the latter.

While the kinase activity of Bub1 promotes the SAC, it is not essential. However, kinase activity is required for recruitment of Sgo1 to centromeres/kinetochores. Bub1 phosphorylates histone H2A to which Sgo1 is thought to bind via its conserved SGO domain. The authors analyzed a BUB1 kinase-dead mutant and find it to be defective in SAC activity, while a mutation in Sgo1's SGO domain is SAC proficient. The authors conclude that Sgo1 recruitment in Cryptococcus differs from the conventional, Bub1-dependent mechanism.

Finally, the author present experiments suggesting that Sgo1 localizes to spindle pole bodies (SPBs) and the spindle, whereas it accumulates at the pericentromere in other organisms.

Major comments

This work might be seen against the background of a large body of work on Sgo proteins in various organisms, including detailed and mechanistic studies in yeast and animals. The author should better explain what we might learn from a less-commonly studied microorganism in which detailed mechanistic studies are much harder. They briefly mention that Cryptococcus has an unconventional kinetochore but do not elaborate. Studying Sgo in such a context is certainly interesting and would give this work a unique angle.

One of the challenges of working on Sgo function is to distinguish its functions in the interconnected processes of biorientation, error correction, and SAC activity. For instance, the authors show that sgo1 mutants fail to arrest at metaphase in response to microtubule depolymerization. Does this failure lead to the loss of Bub1/Aurora from kinetochores or is this loss the reason for the inability to arrest? Furthermore, error correction leads to SAC activation, which in turn leads to accumulation of proteins relevant to error correction. The authors should discuss these issues.

The authors might want to refrain from making detailed claims about the mechanism of Sgo recruitment to subcellular structures, while the experiments presented are not detailed enough to reject more conventional models. The authors detect Sgo on SPBs and the spindle, which does not mean that it is absent from kinetochores or the pericentromere. I note that vertebrate Sgo1 has originally been identified as a microtubule-binding protein.

Recruitment of Sgo to the pericentromere is more complex than implied by the authors. Bub1's kinase activity is important but not essential for SAC function. It is required for Sgo recruitment even in cells containing a phosphomimic version of histone H2A, and Sgo proteins have additional binding partners at the pericentromere, including cohesin and HP1.

Minor comments

The time course experiments are difficult to interpret. It would be preferably to show separate curves for large-budded cells and e.g. Bub1 or Aurora B at kinetochores. It is difficult to see what fraction of cells arrested at metaphase and what fraction recruited Bub1/Aurora B.

I cannot judge the live imaging experiments. Materials and Methods mentions the removal of outliers and the bridging of gaps in the imaging but lacks information on how these procedures might affect the data presented. Furthermore, the graphs showing statistics are unconventional. They present the means of three experiments (open circles) and all the individual data points (colored circles), while the (very small) error bars refer to the means (that is what I assume). It would be preferable to separately show the individual data points and their respective means and then use an ANOVA to compare them.

The live -cell imaging experiments could be presented as montages (or, indeed, movies) to capture changes over time. Also, the quantification might be presented as percentages or intensities over time - not just a single timepoint. After all, the authors claim that the relevant proteins are first recruited to but then lost from kinetochores in the sgo1 mutant.

Referee Cross-Commenting

I agree with the comments made by the other reviewers. In particular, all reviewers indicate that claims about a new mechanism (Bub1-independent role of Sgo1) should be toned down or backed-up by new experiments. Same for the relevance of the localization of Sgo1 to spindles and SPBs. In agreement with reviewer #2, I would strongly recommend describing and discussing the use of C. neoformans. I still feel that presentation, analysis, and statistics of the live-imaging experiments should be improved.

Significance

This study is interesting to researchers working on mechanisms of chromosome segregation in microorganisms and the functions of Sgo proteins. As stated above, the author could make their study more appealing if they explained the unique features of Cryptococcus with regards to chromosome segregation. This reviewer works on mechanisms controlling chromosome segregation including Sgo proteins but is not familiar with the Cryptococcus system.

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Referee #3

Evidence, reproducibility and clarity

This study investigates the spindle assembly checkpoint (SAC) in a budding yeast Cryptococcus neoformans. The authors propose that Sgo1, the shugoshin homolog in the above species, maintains the SAC activity independently of the Bub1 kinase activity, which is usually critical for the full SAC activity in other species.

Major comments:

The main conclusion of the manuscript may not appear convincing because of the following reasons (1) The spotting assay in Fig. 5B can be interpreted as Sgo1 depends on the Bub1 kinase activity as in other species, but Sgo1 also has additional roles in chromosome segregation, which leads to the stronger sensitivity to TBZ in sgo1∆ compared to bub1-kd. (2) Both bub1-kd and sgo1∆ show defects in maintaining SAC arrest (Fig. 2D). If Sgo1 does not depend on the Bub1 kinase activity to maintain SAC arrest, Bub1 is maintaining the arrest through other pathways. Therefore, if the authors repeated this experiment with sgo1∆ bub1-kd double mutants, there would be an additive effect and the cells would arrest less efficient compared to single mutants. On the other hand, if Sgo1 depends on Bub1, the double mutant will behave similarly to single mutants. I strongly recommend the authors to perform this experiment. (3) Along the same line; if it is not through Sgo1, how does the Bub1 kinase activity support the SAC arrest? (4) The authors conclude that the SPB localization is critical for Sgo1 functions. However, there are some dotty Sgo1 signals between the SPBs in metaphase (Fig. 6A) that could be centromeric localization. I would recommend the authors to repeat this experiment in bub1-kd mutant to see if they detect similar centromeric enrichment of Sgo1. If not, this indicate that there is a Sgo1 pool at the centromere that depends on Bub1, similar to other species. (5) The only convincing supporting evidence is the Sgo1-K382A mutant showing no TBZ sensitivity, but this data is not sufficient to draw the conclusion (and having this conclusion as their tile) because the SGO motif is not so conserved and this mutation may not be completely abolishing the interaction with H2ApT120.

Minor comments:

Line 180-181: Mad2 is the master regulator of SAC, and mad2 mutants cancel the SAC completely in other systems. However, it appears that sgo1∆mad2∆ has slightly "stronger" SAC compared to mad2∆. This is an interesting observation, and I recommend the authors to speculate why deleting sgo1 made the SAC stronger in mad2∆ cells. Also, this contradicts with the TBZ sensitivity that sgo1∆mad2∆ shows higher sensitivity compared to mad2∆. This would also be a nice discussion point.

Line 210: The evidence presented to make this point is flawed. Fig. 2D shows that the sgo1 mutant starts with a percent large-budded cells with signal below the other two (see 40 min). It would make sense then that the peak for sgo1∆ cells is lower at the inflection point the authors are referring to around 160min. A better argument is that bub1-kd and sgo1∆ act like each other, not that there is a particular difference. This again points out the possibility that Sgo1 and the Bub1 kinase activity is in the same pathway for the SAC signaling.

Line 330: Is the centromeric localization actually killed in Sgo1-K382A mutant? Tagging the mutant protein with GFP would be informative (also see comments above regarding Sgo1 localization experiment in bub1-kd background).

Line 385: The resolution used in this microscopy is not enough to tell whether or not Sgo1 localizes to centromeres in this species. Perhaps the kinetochore pool of Sgo1 is buried in the spindle signal the authors see. I understand the technical difficulty but an experimental system to completely dissociate kinetochores from SPB (like nda3-cs mutant in pombe) is required to make this conclusion.

Line 418: Again, this conclusion is supported by weak evidence. Perhaps Sgo1 localization is more diffuse than expected along the spindle and concentrated at SPBs, but it is premature to conclude that there is not a pool of Sgo1 that does not localize in the proximity of centromeres in this particular species.

• There are no error bars in the Figure 3F and Supp Figure 4B. Are these experiments repeated?
• The quantification of the intensity of the fluorescent signals is performed on the maximum intensity Z-projected images, and it is recommended to use sum-projected images for accurate measurement.

Significance

The authors investigate fundamental mechanisms that support faithful chromosome segregation, using a non-typical model organism. It is very important to study diverse model systems to comprehensively understand what the conserved mechanisms are in each lineage.

our expertise: mitosis and meiosis, mouse oocytes, spindle, chromosome segregation, centromere, confocal microscopy, genetics, cell biology

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Referee #2

Evidence, reproducibility and clarity

The authors characterized the mitotic function of Shugoshin SGO1 in a basidiomycete budding yeast Cryptococcus neoformans. Sgo1 is well conserved protein, which ensures the maintenance of spindle assembly checkpoint signals in response to unattached kinetochore. Based on their results the authors propose that Shugoshin monitors the kinetochore-MT attachments by maintaining higher Aurora B and lower PP1 levels at kinetochores. Shugoshin localizes to centromeres/kinetochores during mitosis in most species, however Shugoshin in C. neoformans specifically localizes to SPBs and along the mitotic spindle. Two of the distinctive findings are the unique localization of Sgo1 and the Bub1 kinase independent checkpoint maintenance function of Sgo1.

The experiments are performed thoroughly with appropriate controls. suitable statistical measures, and results are presented in a manner that is logical and easy to understand. Although results are interesting for the centromere and chromosome biology communities, mechanistic studies for localization of Sgo1 and how the unique Bub1 independent kinase function of Sgo1 mediates its function has not been adequately addressed. Also, I was not very clear of the unique advantage of pursuing studies with this yeast? How are these studies advancing our understanding of chromosome segregation in human health and disease? Are there other differences between findings from C. neoformans and budding/fission yeast wrt to chromosome segregation?

Major Comments:

1. Localization of Sgo1 to CEN or not? Authors report that Sgo1 is recruited to SPBs and along the mitotic spindle (lines 332-362). They have also observed Sgo1 signal at the periphery of the kinetochore. In other systems, Sgo1 has been associated with CEN and peri-CEN chromatin (Deng and Kuo 2018 G3 8:2901-2911; Garcia-Nieto et al. 2023 NSMB 30: 853-859). Cell biology is not most definitive to rule out kinetochore localization, ChIP experiments are most definitive can these be done?
2. Localization of Sgo1 to SPB. As highlighted in summary of the review, recruitment of Sgo1 to the SPBs is an interesting observation for C. neoformans, however, the molecular mechanisms involved in this novel function of Sgo1 remains unknown. Which factors mediate the localization of Sgo1 to the SPBs during the cell cycle? The recruitment of Sgo1 to SPBs could be influenced or mediated by the geometric changes in the mitotic spindle that occur during the mitosis. It is possible that orientation of mitotic spindle relative to the kinetochore during the mitotic cell cycle may dictate the recruitment of Sgo1 to the SPBs. There is precedent from S. cerevisiae where kinetochore protein Ndc10 was found to be associated with SPBs and along mitotic spindle (Bouck and Bloom, 2005 PNAS 102: 5408-5413). It is likely that similar mechanisms might be involved for Sgo1. One of the candidate genes mediating localization of Sgo1 to the SPBs could be the components of CPCs (Abad et al. 2022 JCB 221:e202108156). Authors should at least examine one of them that will help provide a mechanistic insight into the novel role of Sgo1 at the SPBs.
3. Bub1 independent function of Sgo1. It would be useful to provide the evolutionarily timescale for the gain or loss of Bub1-independent SAC function of Sgo1. An evolutionary comparison between basidiomycetes and ascomycetes would help readers understand the biological reasoning and significance of Bub1-independent SAC function of Sgo1.
4. Bub1 independent function of Sgo1. What is the localization of Sgo1 in bub1-kd strain?
5. Authors have established a relationship between Sgo1, Bub1 and Aurora B (lines 247-248). However, such observations could also be mediated by other molecular factors.. It will improve their manuscript and conclusions if they provide further evidence for in vivo interaction of Sgo1 with Bub1 or Aurora B in C. neoformans using Co-IP.

Minor Comments:

1. Figure 1D: The figure is very crowded and difficult to understand. Authors should use letters to show the statistical significance instead of drawing multiple lines.
2. Figures 1B and 5D: The sgo1null strain is not sensitive to 3 ug of thiabendazole in Figure 1B but it is in figure 5D. Please explain.
3. Figures 1 and 2: Authors have used 1 ug of nocodazole in figure 1 and it is 2.5 ug in figure 2. Please explain.
4. Figure 2C-2E: Is GFP-Bub1 signals in sgo1∆ similar to GFP-bub1-kd signals? To clarify the GFP-bub1-kd signals in Figure 2, the authors should show pictures of GFP-bub1-kd signals in Figure 2C, as well as GFP-bub1-kd intensity in Figure 2E. They need to explain how they score the diffused GFP-Bub1 signals in the whole cells of 280 min post-nocodazole treatment.
5. In line 253-256, the authors incubated the Aurora B-overexpressed cells (galactose medium) at permissive temperature, but Aurora B-depleted cells (glucose medium) at non-permissive temperature. What is the purpose for incubations at different temperatures? Do the Aurora B-overexpressed cells show a ts phenotype?
6. Reproducibility of localization pattern of GFP-Sgo1 in Figure 6. Can the signals be quantified?
7. Figure 5: Since Sgo1 in C. neoformans has unique localization pattern, it is possible that the target of phosphorylation by Bub1 is different, rather than binding to phosphorylated H2A. The authors should at least discuss the possibility.
8. Authors have used a linker protein Bgi1 as a control (lines 148-150). However, there are other controls, which are more suitable than Bgi1. Perhaps authors should use a protein that interacts with microtubules. One of the candidates in this experiment would be evolutionarily conserved Ndc80.
9. Bub1 independent function of Sgo1. It is unclear which factors are involved in Bub1-independent function of Sgo1.. It is possible that histone H3 or its variant CENP-A might have some role in Sgo1 recruitment as has been shown for S. cerevisiae and other systems (Luo et al. 2016 Genetics 204:1029-1043; Wu et al. 2023 JMCB, mjad061; Mishra et al. 2018 Cell Cycle 17:11-23). Authors can try to deplete CENP-A and examine the localization of Sgo1 at the kinetochore and at the SPBs in Bub1 and its mutant strains.

Referee Cross-Commenting

Summary: The data supporting an unusual role of Sgo1 as indicated in the title is not supported by the data. Two main conclusions of the paper are the unique localization of Sgo1 and the Bub1 kinase independent role of Sgo1. With respect to localization: Molecular evidence to rule out lack of Sgo1 at centromeric and pericentromeric regions is needed (ChIP, FRET) is needed. Same is true for SPB association is this affected in spb mutants?

Bub1 kinase independent role of Sgo1 needs further experimentation.

Discussion about the significance of studying C. neoformans needs to be included.

Significance

The experiments are performed thoroughly with appropriate controls. suitable statistical measures, and results are presented in a manner that is logical and easy to understand. Although results are interesting for the centromere and chromosome biology communities, mechanistic studies for localization of Sgo1 and how the unique Bub1 independent kinase function of Sgo1 mediates its function has not been adequately addressed. Also, I was not very clear of the unique advantage of pursuing studies with this yeast? How are these studies advancing our understanding of chromosome segregation in human health and disease? Are there other differences between findings from C. neoformans and budding/fission yeast wrt to chromosome segregation?

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Referee #1

Evidence, reproducibility and clarity

The authors present a thorough characterization of the mitotic protein Shugoshin (Sgo1) in the context of Cryptococcus neoformans, an interesting and medically important fungal species. Sgo1 function in chromosome segregation has been well-studied in other eukaryotic species: it recruits the error correction kinase Aurora B and Protein Phosphatase 2 to the centromere and promotes the loading of cohesion at the centromeric locus. The primary function of these proteins is to enable the bipolar attachment of sister chromatids to the spindle apparatus. In this manuscript, Polysetty et al mainly investigate the "secondary" function of Sgo1 in Spindle Assembly Checkpoint (SAC) signaling. They show that: (1) sgo1Δ cells fail to maintain a prolonged SAC response to spindle poisons, because they cannot achieve or maintain normal Bub1 recruitment at unattached kinetochores. (2) The lower Bub1 recruitment is mainly due to the loss of centromeric Aurora B recruitment and increased PP1 recruitment to the kinetochore. There are some interesting departures from the behavior Sgo1 known in other eukaryotes. (3) Sgo1 localizes to the spindle poles in C neoformans with smaller amounts dispersed along the spindle. (4) Furthermore, Sgo1 localization at the centromeres does not require Bub1 kinase activity. Based on these and other observations, the authors advance a model for how Sgo1 is recruited to the centromeres and how it promotes strong SAC response to spindle poisons.

Main comments:

1. In interpreting the effects of thiabendazole on colony growth, the authors should also consider the role of Sgo1 in promoting bipolar attachments by establishing centromeric cohesion and proper geometry (Indjeian and Murray Current Biology 2007, Verzijlbergen et al. eLife 2014). In the absence of Sgo1, cells are more likely to divide with wrongly attached chromosomes and become highly aneuploid, which may promote mortality. The relative importance of Sgo1 function in biorientation and SAC signaling will have to be established using known separation of function mutations in Sgo1.
2. Unless I am missing something, the phenotypes of Sgo1 related to the SAC are completely consistent with the established model of Aurora B and PP1 roles in regulating SAC signaling. Sgo1 promotes Aurora B activity at the kinetochore, and the increased Aurora B activity can promote SAC signaling by: (1) delaying SAC silencing by creating unattached kinetochores during error correction, (2) suppressing PP1 recruitment to the kinetochore, and (3) potentially phosphorylating MELT motif-proximal residues to promote Bub1 recruitment as has been observed in Drosophila (Audette et al MBoC 2021). Conversely, Sgo1 deletion will result in hyperstabilization of even wrong kinetochore-microtubule attachments, higher PP1 recruitment and, therefore, weaker SAC signaling. These points should be discussed when interpreting the results in this study.
3. Phenotypes related to the sgo1-K382A mutations are interesting, and they suggest that Sgo1 recruitment may be independent of Bub1 kinase activity. However, the data need to be strengthened to fully support this conclusion. First, the multiple sequence alignment for Sgo1 shows lysine residues at position 381 and 382 in Cryptococcus. This makes me wonder if the point mutation is sufficient to abolish the Sgo1-pH2A interaction. Second, the expression levels of the mutant should be compared with the wild type to confirm that the phenotype is not due to over-expression/stabilization of the mutant protein. I understand that the thiabendazole resistance of Bub1-kd is quite interesting, but Haspin kinases are also involved in loading Sgo1 at the centromere. The authors could investigate their role in Crypotcoccus to define the alternative mechanism of centromere specific Sgo1 loading.
4. In Figure 6, the authors find strong Sgo1 colocalization at the spindle poles and what they describe as "spindle-like location along the pole-to-pole axis" (lines 358-360). The authors should consider the possibility that this is the pool of centromere associated Sgo1 because it colocalizes reasonably well with CENPA. This interpretation obviates the necessity of the complex model in Figure 6E explaining Sgo1 loading at the centromeres. Given that there must be Sgo1 at the centromeres, the null hypothesis has to be that this Sgo1 is associated with centromeres rather than microtubules. The authors could use biochemical methods to test the hypothesis. Alternatively, BiFC ro FRET based assays may be useful if the authors want to persist with cell biology experiments.

Minor points:

1. The authors use gene repression and over-expression using the Gal1 promoter, but do not assay protein levels for the degree of over-expression. This is fine in many cases because the phenotypes are consistent with over-expression/repression. However, it needs to be confirmed when interpreting the effects of point mutations, e.g., Bub1-kd, Sgo1-K382, etc.
2. I found the model in 5A confusing because the arrows are used to indicate both protein recruitment and promotion/expression of downstream proteins/events. Adding to the confusion is the inverted sgo1 → Aur B direction. The authors should simplify this important panel.
3. The model in Figure 6E is missing some information: gradients in Sgo1 and Ipl1 pools are shown, but they don't line up with the spindle direction and are not otherwise indicated on the spindle. Importantly, this model is necessary only if the authors can conclusively show that the Sgo1 along the spindle axis is associated with microtubule and not the centromere.
4. The authors examine Sgo1's role in loading cohesin at the centromere in Figure S6. An interesting experiment would be to test the thiabendazole sensitivity of Scc1 over-expressing cells to understand whether it can suppress the effects of sgo1Δ.

Referee Cross-Commenting

I think Reviewer #2's comment above summarizes the all the comments effectively. I don't have anything else to add.

Significance

This is a well-constructed and well-executed study. However, the interpretation of many of the results and how strongly these results depart from what's already known in other systems is debatable. Almost all the results describing CnSgo1 involvement in SAC signaling reinforce the established model that Sgo1 recruits Aurora B, which suppresses the recruitment of PP1, the main antagonist for Bub1 recruitment. Thus, Δsgo1 cells are unable to mount a strong SAC response because increased PP1 recruitment antagonizes SAC signaling. In my view, the novel findings are the Bub1 kinase activity-independent loading of Sgo1 at the centromere and the surprising Sgo1 localization at the spindle poles. The manuscript will generate wider interest if the authors dissected the molecular basis of these unexpected observations rather than its established functions. Doing so will require a functional and molecular dissection of Sgo1.

eLife Assessment

This study presents a useful modification of a standard model of genetic drift by incorporating variance in reproductive success, claiming to address several paradoxes in molecular evolution. However, some of the claimed "paradoxes" seem to be overstatements, as previous literature has pointed out the limitations of the standard model and proposed more advanced models to address those limitations. While the modified model presented in this paper yields some intriguing theoretical predictions, the analysis and simulations presented are incomplete to support the authors' strong claims, and it is unclear how much the model helps explain empirical observations.

Reviewer #1 (Public review):

The revision by Ruan et al clarifies several aspects of the original manuscript that were difficult to understand, and I think it presents some useful and interesting ideas. I understand that the authors are distinguishing their model from the standard Wright-Fisher model in that the population size is not imposed externally, but is instead a consequence of the stochastic reproduction scheme. Here, the authors chose a branching process but in principle any Markov chain can probably be used. Within this framework, the authors are particularly interested in cases where the variance in reproductive success changes through time, as explored by the DDH model, for example. They argue with some experimental results that there is a reason to believe that the variance in reproductive success does change over time.

One of the key aspects of the original manuscript that I want to engage with is the DDH model. As the authors point out, their equations 5 and 6 are assumptions, and not derived from any principles. In essence, the authors are positing that that the variance in reproductive success, given by 6, changes as a function of the current population size. There is nothing "inherent" to a negative binomial branching mechanism that results in this: in fact, the the variance in offspring number could in principle be the same for all time. As relates to models that exist in the literature, I believe that this is the key difference: unlike Cannings models, the authors allow for a changing variance in reproduction through time.

This is, of course, an interesting thing to consider, and I think that the situation the authors point out, in which drift is lower at small population sizes and larger at large population sizes, is not appreciated in the literature. However, I am not so sure that there is anything that needs to be resolved in Paradox 1. A very strong prediction of that model is that Ne and N could be inversely related, as shown by the blue line in Fig 3b. This suggests that you could see something very strange if you, for example, infer a population size history using a Wright-Fisher framework, because you would infer a population *decline* when there is in fact a population *expansion*. However, as far as I know there are very few "surprising population declines" found in empirical data. An obvious case where we know there is very rapid population growth is human populations; I don't think I've ever seen an inference of recent human demographic history from genetic data that suggests anything other than a massive population expansion. While I appreciate the authors empirical data supporting their claim of Paradox 1 (more on the empirical data later), it's not clear to me that there's a "paradox" in the literature that needs explaining so much as this is a "words of caution about interpreting inferred effective population sizes". To be clear, I think those words of caution are important, and I had never considered that you might be so fundamentally misled as to infer decline when there is growth, but calling it a "paradox" seems to suggest that this is an outstanding problem in the literature, when in fact I think the authors are raising a *new* and important problem. Perhaps an interesting thing for the authors to do to raise the salience of this point would be to perform simulations under this model and then infer effective population sizes using e.g. dadi or psmc and show that you could identify a situation in which the true history is one of growth, but the best fit would be one of decline

The authors also highlight that their approach reflects a case where the population size is determined by the population dynamics themselves, as opposed to being imposed externally as is typical in Cannings models. I agree with the authors that this aspect of population regulation is understudied. Nonetheless, several manuscripts have dealt with the case of population genetic dynamics in populations of stochastically fluctuating size. For example, Kaj and Krone (2003) show that under pretty general conditions you get something very much like a standard coalescent; for example, combining their theorem 1 with their arguments on page 36 and 37, they find that exchangeable populations with stochastic population dynamics where the variance does not change with time still converge to exactly the coalescent you would expect from Cannings models. This is strongly suggestive that the authors key result isn't about stochastic population dynamics per se, but instead related to arguing that variance in reproductive success could change through time. In fact, I believe that the result of Kaj and Krone (2003) is substantially more general than the models considered in this manuscript. That being said, I believe that the authors of this manuscript do a much better job of making the implications for evolutionary processes clear than Kaj and Krone, which is important---it's very difficult to understand from Kaj and Krone the conditions under which effective population sizes will be substantially impacted by stochastic population dynamics.

I also find the authors exposition on Paradox 3 to be somewhat strange. First of all, I'm not sure there's a paradox there at all? The authors claim that the lack of dependence of the fixation probability on Ne is a paradox, but this is ultimately not surprising---fixation of a positively selected allele depends mostly on escaping the boundary layer, which doesn't really depend on the population size (see Gillespie's book "The Causes of Molecular Evolution" for great exposition on boundary layer effects). Moreover, the authors *use a Cannings-style argument* to get gain a good approximation of how the fixation probability changes when there is non-Poisson reproduction. So it's not clear that the WFH model is really doing a lot of work here. I suppose they raise the interesting point that the particularly simple form of p(fix) = 2s is due to the assumption that variance in offspring is equal to 1.

In addition, I raised some concerns about the analysis of empirical results on reproductive variance in my original review, and I don't believe that the authors responded to it at all. I'm not super worried about that analysis, but I think that the authors should probably respond to me.

Overall, I feel like I now have a better understanding of this manuscript. However, I think it still presents its results too strongly: Paradox 1 contains important words of caution that reflect what I am confident is an under appreciated possibility, and Paradox 3 is, as far as I'm concerned, not a paradox at all. I have not addressed Paradox 2 very much because I think that another reviewer had solid and interesting comments on that front and I am leaving it to them. That being said, I do think Paradox 2 actually presents a deep problem in the literature and that the authors' argument may actually represent a path toward a solution.

This manuscript can be a useful contribution to the literature, but as it's presented at the moment, I think most of it is worded too strongly and it continues to not engage appropriately with the literature. Theoretical advances are undoubtedly important, and I think the manuscript presents some interesting things to think about but ultimately needs to be better situated and several of the claims strongly toned down.

References:<br /> Kaj, I., & Krone, S. M. (2003). The coalescent process in a population with stochastically varying size. Journal of Applied Probability, 40(1), 33-48.

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.

Comments on revisions:

The author has addressed some of the concerns in my review, and I think the revised manuscript is more clear. I like the discussion about the caveats of the WFH model.

I hope the authors could also discuss the conditions needed for V(K)/Ne to be a reasonable approximation. It is currently unclear how the framework should be adopted in general.

The idea about estimating male-female V(K) ratios from population genetic data is interesting. Unfortunately, the results fell short. The accuracy of their estimators (derived using approximation Ne/V(K) approximation, and certain choice of theta, and then theta estimated with Watterson's estimator) should be tested with simulated results before applying to real data. The reliability of their estimator and their results from real data are unclear.

Arguments made in this paper sometimes lack precision (perhaps the authors want to emphasize intuition, but it seems more confusing than otherwise). For example: The authors stated that "This independence from N seems intuitively obvious: when an advantageous mutation increases to say, 100 copies in determining a population (depending mainly on s), its fixation would be almost certain, regardless of N.". Assuming large Ne, and with approximation, one could assume the probability of loss is e^(-2sn), but the writing about "100 copies" and "almost certain" is very imprecise, in fact, a mutation with s=0.001 segregating at 100 copies in a large Ne population is most probably lost. Whereas in a small population, it will be fixed. Yet the following sentence states "regardless of N. This may be a most direct argument against equating genetic drift, certainly no less important than 1/ N . with N, or Ne (which is supposed to be a function of N's)." I find this new paragraph misleading.

Some of the statements/wordings in this paper still seem too strong to me.

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:

- The theoretical results are well-described and easy to follow.<br /> - The analyses of different variances in offspring number (both experimentally and analyzing public data) are convincing that non-Poissonian offspring distributions are the norm.<br /> - 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.<br /> - I enjoyed the Density-Dependent Haldane model. It was a nice example of the decoupling of census size and effective size.<br /> - Equation (10) is a nice result (but see below)

Weaknesses:

- 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. As a concrete example, Mohle and Sagitov 2001 show that a "coalescent Ne" for the WF model should be (N-1)/Var(K). This resolves the exponentially growing bacteria "paradox" raised in the present paper --- when the bacteria are growing Var(K) ~ 0, and hence there should be very little drift. This exactly resolves the "paradox" raised by the authors. Instead, it merely underscores that Ne does not need to be equal to (or even positively correlated!) with N. I absolutely do not see this as a failure of the WF model. Whether one finds branching processes or the WF model more biologically intuitive is a matter of taste, but to say that WF models cannot explain this "paradox" is false, when a well-known paper from more than 20 years ago does just that.<br /> - Along these lines, the result that Ne in the Wright-Fisher process might not be related to N in any straightforward (or even positively correlated) 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...)<br /> - 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.<br /> - Given that Cannings' exchangeable models decouple N and Ne, it would not surprise me if something like equation (10) could be derived under such a model. I have not seen such a derivation, and the authors' result is nice, but I do not see it as proof that WF-type models (i.e., Cannings' models) are irreparably broken.

Author response:

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

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.

The prior literature the reviewers referred to are all "modified WF models". In the original submission, we lumped the standard and modified WF models together as the "generalized WF models". As the lumping causes confusions, their distinctions are now made clear.  That said, the Haldane model in our proposal is not a modification of the standard WF model because, conceptually, the two models are very different. WF is based on sampling whereas the Haldane model is based on gene transmission.

While the "modified WF models" often incorporate V(K) [variance in progeny number], the modification is still based on the WF model of population sampling. The modification is mathematically feasible but biologically untenable, as explained explicitly in the revised text. Most important, all four paradoxes are as incompatible with the modified WF models as with the standard model. Note that the Haldane model does not have the sampling step, which is absorbed into the V(K) term. In the integrated WF-Haldane model, these paradoxes are resolved (see the new sections of Discussion, quoted below).

If readers do not have time to ponder on all four paradoxes, they may simply read the first one, as follows. When the population size (N) is growing exponentially, such as in a bacteria culture, drift is nearly absent when N is small and becomes stronger as N increases, especially when approaching the carrying capacity.  Such common observations are exactly opposite of the WF model's central prediction. Any model based on sampling cannot escape the constraint of "greater drift, smaller N".

Revision - The following text is a reproduction of the last 7 paragraphs of Discussion.

“The standard WF model has been extended in several directions (overlapping generations, multiple alleles, ploidy, etc.). The modification most relevant to our studies here is the introduction of V(K) into the model, thus permitting V(K) ≠ E(K). While the modifications are mathematically valid, they are often biologically untenable. Kimura and Crow (1963) may be the first to offer a biological mechanism for V(K) ≠ E(K), effectively imposing the Haldane model on the WF model. Other models (Kimura and Crow 1963; Lynch, et al. 1995; Sjodin, et al. 2005; Der, et al. 2011; Cannings 2016) indeed model mathematically the imposition of the branching process on the population, followed by the WF sampling. The constructions of such models are biologically dubious but, more importantly, still unable to resolve the paradoxes. It would seem more logical to use the Haldane model in the first place by having two parameters, E(K) and V(K). 

Even if we permit V(K) ≠ E(K) under the WF sampling, the models would face other difficulties. For example, a field biologist needs to delineate a Mendelian population and determine its size, N or Ne. In all WF models, one cannot know what the actual population being studied is. Is it the fly population in an orchard being sampled, in the geographical region, or in the entire species range? It is unsatisfactory when a population biologist cannot identify the population being studied. The Haldane model is an individual-output model (Chen, et al. 2017), which does not require the delineation of a Mendelian population.

We shall now review the paradoxes specifically in relation to the modified WF models, starting with the multi-copy gene systems such as viruses and rRNA genes covered in the companion study (Wang, et al. 2024). These systems evolve both within and between hosts. Given the small number of virions transmitted between hosts, drift is strong in both stages as shown by the Haldane model (Ruan, Luo, et al. 2021; Ruan, Wen, et al. 2021; Hou, et al. 2023). Therefore, it does not seem possible to have a single effective population size in the WF models to account for the genetic drift in two stages. The inability to deal with multi-copy gene systems may explain the difficulties in accounting for the SARS-CoV-2 evolution (Deng, et al. 2022; Pan, Liu, et al. 2022; Ruan, Wen, et al. 2022; Hou, et al. 2023; Ruan, et al. 2023).

We now discuss the first paradox of this study, which is about the regulation of N. In the general WF models, N is imposed from outside of the model, rather than self-generating within the 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. As far as we know, no modifications of the WF model can account for this phenomenon that is opposite of its central tenet. In the general WF models, N is really the carrying capacity, not population size. 

The second paradox of sex chromosomes is rooted in V(K) ≠ E(K). As E(K) is the same between sexes but V(K) is different, clearly V(K) = E(K) would not be feasible. The mathematical solution of defining separate Ne's for males and females (Kimura and Crow 1963; Lynch, et al. 1995; Sjodin, et al. 2005; Der, et al. 2011; Cannings 2016) unfortunately obscures the interesting biology. As shown in Wang et al. (2024; MBE), the kurtosis of the distribution of K indicates the presence of super-breeder males. While the Haldane model can incorporate the kurtosis, the modified WF models are able to absorb only up to the variance term, i.e., the second moment of the distribution. The third paradox of genetic drift is manifested in the fixation probability of an advantageous mutation, 2_s_/V(K). As explained above, the fixation probability is determined by the probability of reaching a low threshold that is independent of N itself. Hence, the key parameter of drift in the WF model, N (or Ne), is missing. This paradox supports the assertion that genetic drift is fundamentally about V(K) with N being a scaling factor. 

As the domain of evolutionary biology expands, many new systems do not fit into the WF models, resulting in the lack of a genetic drift component in their evolutionary trajectories. Multi-copy gene systems are obvious examples. Others include domestications of animals and plants that are processes of rapid evolution  (Diamond 2002; Larson and Fuller 2014; Purugganan 2019; Chen, Yang, et al. 2022; Pan, Zhang, et al. 2022; Wang, et al. 2022). Due to the very large V(K) in domestication, drift must have played a large role. Somatic cell evolution is another example with “undefinable” genetic drift (Wu, et al. 2016; Chen, et al. 2017; Chen, et al. 2019; Ruan, et al. 2020; Chen, Wu, et al. 2022). The Haldane (or WFH) model, as an "individual output" model, can handle these general cases of genetic drift.

The Haldane model and the WF model are fundamentally different approaches to random forces of evolution. While the WF models encounter many biological contradictions, they have provided approximate mathematical solutions to more realistic scenarios. In systems such as in viral evolution (Ruan, Hou, et al. 2022; Hou, et al. 2023) or somatic cell evolution (Chen, Wu, et al. 2022; Zhai, et al. 2022) whereby the WF solution is absent, further development of the WFH model will be necessary.”

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

This point is addressed in the responses to reviewers' comments. Since they are quite technical, they do not fit in the overview here.

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.

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 reviewer's summary and weakness statement reflects the general criticism summarized by the editors. The reply and revision to these criticisms have been presented in the long reply to elife assessment above.

We hence re-emphasize only the key points here.

(1) The literature that the reviewers fault us for not citing is about the modifications of the standard WF model. We now cite them as well as a few others in that vein. However, the WF-Haldane model we propose is conceptually very different from the modified WF models. This WFH model is in essence the Haldane model which may use the results of the WF models as the starting point to find the exact solutions.

(2) The check of the power of the modified WF models is whether they can resolve the paradoxes. None of them can. The arguments apply to neutral cases as well as selection effects. Hence, our central point is that the modifications of the standard WF model [e.g., by incorporating V(K)] do not help the WF model in resolving the paradoxes.  Besides, the incorporation of V(K) is mathematically feasible but biologically untenable as presented in the new sections of Discussion.

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

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, …… - 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. [Since the comments above are highly technical and fairly long, they are not copied verbatim.]

We thank this reviewer for the detailed comments with respect to the potential confusion in the discussion of the Density Dependent Haldane (DDH) model.

First, the reviewer appears to ask how Eqs (5-6) are derived. We should clarify that both Eq (5) and (6) are assumptions rather than derived results. Both equations are assumptions based on population ecology. Eq (7) is then derived by substituting the assumptions in Eq (5) and (6) into Eq (3).

The definition in Equation (5) allows the growth rate of the population size to be dependent on N itself, such that growth rate E(K) (average offspring number per generation) is greater than 1 when N < Ck and less than 1 when N > Ck. The parameter z is introduced to adjust the sensitivity of E(K) to changes in population size (as shown in Fig. 3a).

Second, we appreciate the comments regarding the use of individual-based simulations and the apparent lack of interaction between individuals. In our simulations, there is indeed an interaction among individuals, which is represented by Eq (5). This equation reflects how the competition between two alleles affects the expected growth rate 𝐸(𝐾), which decreases as the population size increases. Furthermore, once 𝐸(𝐾) for the entire population is determined, the offspring numbers of the alleles are independent.

We believe that the primary purpose of our simulations was not clearly stated. This lack of clarity may be the root of the criticisms. We now note that the simulations are aimed at testing the accuracy of Equation (10).

Note that Eq. (10) is a textbook result and quite important in our study. This equation shows that the strength of genetic drift, as given by Pf (the fixation probability of an advantageous mutation), is not a function of N at all. This approximate solution has been obtained using the WF model by Kimura.  The Haldane model solution that can explain Paradox 1 is based on Equation (7) as shown below

Since the fixation probability of Equation (10) cannot be easily obtained using Eq. (7), we conducted simulations to confirm the accuracy of Eq. (10) when applied to the Haldane model.

We have revised the relevant sections of the manuscript to clarify these points and to better distinguish between assumptions and results. 

Revision - Details of the DDH model are given in the Supplementary Information. A synopsis is given here: We consider a non-overlapping haploid population with two neutral alleles. The population size at time t is Nt. We assume that expected growth rate E(K) is greater than 1 when N < Ck and less than 1 when N > Ck, as defined by Eq. (5) below:

The slope of E(K) vs. N (i.e., the sensitive of growth rate to changes in population size), as shown in Fig 3a, depends on z. To determine the variance V(K), we assume that K follows the negative binomial distribution whereby parents would suffer reproduction-arresting injury with a probability of pt at each birthing (Supplementary Information). Accordingly, V(K) can then be expressed as

By Eq. (6), the ratio of V(K)/E(K) could be constant, decrease or increase with the increase of population size. With E(K) and V(K) defined, we could obtain the effective population size by substituting Eq. (5) and Eq. (6) into Eq. (3).

Eq. (7) presents the relationship between effective population size (Ne) and the population size (N) as shown in Fig. 3. The density-dependent E(K) could regulate N with different strength (Fig. 3a). The steeper the slope in Fig. 3a, the stronger the regulation.

Simulation of genetic drift in the Haldane model and the Wright-Fisher (WF) model. In both models, interactions between individuals are implicitly included through the dependency of the average number of offspring on population size, as defined by Eq. (5). This dependency leads to the logistic population growth, reflecting the density-dependent interactions.

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 omitted

The comments are the summary of previous ones, which have been addressed in detail in the preceding sections.

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 [Ne]

Thanks.  The issue of Ne will be addressed below where the reviewer returns to this issue. The strength of the integrated WFH model is that N (or Ne) is generated by the model itself, rather than externally imposed as in WF models.

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.

Thanks. 

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 Haldane model treats N’s very differently from the WF models.  In the WF models, N’s are imposed externally (say, constant N, exponentially growing N, temporally fluctuating N’s and so on; all provided from outside of the model). Ne and coalescence are all derived from these given N’s.  In order to account for the first paradox (see the next paragraph), N needs to be regulated but the WF models cannot regulate N’s. The density-dependent Haldane model that Reviewer 1 inquired above is a model that regulates N internally. It can thus account for the paradox.

Paradox 1 -  When the population size (N) is growing exponentially, such as in a bacteria culture, drift is nearly absent when N is small and is much stronger as N increases, especially when approaching the carrying capacity.  Such a pattern is a common observation and is exactly opposite of the WF model's central prediction. In short, a model that does not regulate N cannot explain the paradox

Ne is a fix of the WF model in order to account for the missing components of genetic drift. The paradoxes presented in this one and the companion study show that the fix is rather inadequate.  In contrast, by the WFH model, N is regulated within the model itself as E(K) and V(K) are both functions of N.

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".

We appreciate greatly the illuminating advice.  Nevertheless, we should explain, or should have explained, more clearly that these four paradoxes presented are central to this pair of eLife papers. The WF and Haldane models are very different conceptual ideas altogether. The choice should not be based on mathematical grounds but on how they help us understand biological evolution. We are using four paradoxes to highlight the differences.  We have said in the papers that the origin and evolution of COVID-19 caused a lot of confusions partly because the WF models cannot handle multi-copy gene systems, including viruses that evolve both within- and between- hosts.

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.

We appreciate the question which has two aspects.  First, why do we think the WF models are insufficient? After all, for each assumption of the WF model (as given in the reviewer’s examples), there is often a solution by modifying Ne which relaxes the assumption. In this sense, there is only one grand assumption made by the WF models. That is, however complex the biology is, it is possible to find Ne that can make the WF model work. Our argument is that Ne is a cumbersome fix of the WF model and it does not work in many situations. That is how we replied about the importance of the paradoxes above.  We shall again use the first paradox as an example whereby drift is stronger as N becomes larger, the fix has to make Ne negatively correlated with N. In reality, it does not appear possible to resolve this paradox. Another paradox is the evolution of multi-copy gene systems. In short, it seems clear that Ne is not a useful or usable fix.

The second aspect is that “why, among the many modifications the WF models make, do we only emphasize the inclusion of V(K)?” This is the essence of the two papers of ours.  Although V(K) is a modification of the WF models, it does not enable the WF models to resolve the paradoxes. In contrast, the Haldane model has incorporate E(K) and V(K) in the model. In presenting paradox 3, it was stated that

This equation shows that the strength of genetic drift, as given by Pf (the fixation probability of an advantageous mutation), is not a function of N at all. It supports the view that the essence of genetic drift is V(K) with N as a scaling factor. Note that, if V(K) = 0, there is no genetic drift regardless of N. As V(K) is not an add-on to the Haldane model (unlike in WF models), the Haldane model can resolve the paradoxes.

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.

a' can be negative for the same reason that a (the male/female ratio in mutation rate) can be negative (Miyata, et al. 1987; Li, et al. 2002; Makova and Li 2002). Clearly, this has not been a problem in the large literature on a becoming negative.  In fact, in many reports, a is negative, which is read as a approaching infinity.  Imagine that our equation is a'^2 = 0.25, then a' can be 0.5 or -0.5, although the latter solution is not biologically meaningful.

As for theta, the reviewer asked why we do not use the pairwise genetic diversity (or theta[pi]) as the first-principle approach to estimating theta. While theta(pi) is the first estimator of theta used, the general principle is that every bin of the frequency spectrum can be used for estimating theta since the expected value is theta/i where i is the occurrence of the mutation in the sample.  (If the sample size is 100, then i is between 1 and 99.)  Hence, the issue is which part of the spectrum has the best statistical properties for the questions at hand.  The pairwise measure is theta(pi) [which the reviewer recommends]. While theta(pi) and theta(w) are most commonly used, there are in fact numerous ways to estimate theta.  ((Fu 2022) presents an excellent review.) For our purpose, we need a theta estimate least affected by selection and we choose the lowest frequency bin of the spectrum, which is theta(1) based on the singletons. Theta(1), least affected by selection, is the basis of the Fu and Li test. 

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.

Thanks.

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.

Please allow us to refer to, again, two of the four paradoxes.  We believe that that no modification of the WF model can resolve the paradoxes.

(1) When the population size (N) is growing exponentially, such as in a bacteria culture, drift is nearly absent when N is small and is much stronger as N increases, especially when approaching the carrying capacity.  Such common observations are exactly opposite of the WF model's key prediction. It is not possible for a model that does not regulate N to explain the paradox.

(2) There is no way the WF models can formulate Ne for, say viruses or ribosomal RNA genes that have two levels of populations – the within-host populations as well as the host population itself.

The fact that there are numerous Ne's suggests that Ne is a collection of cumbersome fixes of the WF model. By the WF-Haldane model, all factors are absorbed into V(K) resulting in a simpler model in the end. V(K) is often a measurable quantity. Note that, even if V(K) is incorporated into the WF model, the paradoxes remain unresolvable.

(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.

The criticism of lack of engagement with well-established literature has been responded extensively above.  Briefly, the literature is about modifications of the WF model which share the same feature of population sampling. With that feature, the paradoxes are unresolvable.  For example, however Ne is defined, the fixation probability of an advantageous mutation does not depend on N or Ne. This is the third paradox of the WF models.

(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.

We admire the learned understanding of the literature expressed by the review, which raise two points.  First, our model may not be able to handle the heavy-tailed progeny distribution (i.e., the kurtosis of the distribution of k). Second, the Xi coalescence models (cited above) can do that.  Below are our clarifications.

First, the WFH model is based on the general distribution of K, which includes flexible and realistic representations of offspring number distributions. In fact, we have used various forms of K distribution in our publications on the evolution of SARS-CoV-2 (see the Ruan et al publications in the bibliography). Power-law distribution is particularly useful as the K-distribution in viral transmission is highly kurtotic. This is reflected in the super-spreader hypothesis. In short, the branching process on which the WFH model is based in is mainly about the distribution of K. Nevertheless, the variance V(K) can often yield good approximations when the kurtosis is modest.

Second, we would like to comment on the models of Eldon and Wakely 2006. or Mohle and Sagitov 2001 and subsequent papers. These papers are based on the Moran model by considering a highly skewed distribution of offspring numbers. Fundamentally, the Moran models generally behave like WF models (standard or modified) and hence have the same problems with the paradoxes that are central to our studies. In fact, the reservations about introducing V(K) into the WF models apply as well to the Moran models.  The introduction of V(K) is mathematically valid but biologically untenable. Essentially, the WF models incorporate the Haldane model as a first step in the generation transition. The introduction of V(K) into the Moran model is even less biologically sensible. Furthermore, the model allows K to take only three discrete values: 0, 2, and Nψ (see Eq. (7) in Eldon and Wakely). Their model also assumes a constant population size, which contrasts with our model's flexibility in handling varying population sizes and more complex distributions for K.

In short, the modifications of the WF (and Moran) models are unnecessarily complicated, biologically untenable but still fail to account for the paradoxes. The WFH model can rectify these problems. 

(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...)

The reviewer is correct in pointing out the inexact correlation between N and Ne. Nevertheless, it should still be true that the WF models predict qualitatively weaker drift as N increases. The first paradox is as stated:

When the population size (N) is growing exponentially, such as in a bacteria culture, drift is nearly absent when N is small and is much stronger as N increases, especially when approaching the carrying capacity.  Such common observations are exactly opposite of the WF model's key prediction.

(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.

We thank the reviewer for this important comment. The main difference is that N is imposed from outside the WF models but can be generated from within the Haldane model (see the density-dependent Haldane model). In nature, N of the next generation is the sum of K’s among members of the population. It is how the Haldane model determines N(t+1) from N(t). In the WF models, N is imposed from outside the model and, hence the given N determines the distribution of K.  For this reason, N regulation is not possible in the WF models, thus resulting in the paradoxes.

(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.

We thank the reviewer and have used this more convincing case as suggested by the reviewer.

Recommendations for the authors:

General replies - We thank the editors and reviewers again.  The points below are re-iterations of the comments received above and have since been replied in detail. Specific instructions about wording and notations have also been rectified. Again, we are grateful for the inputs from which we learned a great deal.

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.

We have addressed this issue in the beginning of Introduction and Discussion, pointing to the long section in the new second half of Discussion. The essence is that the literature is all about the modified WF models.  The WF-Haldane model is conceptually and operationally distinct from the WF models, either standard or modified ones,

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.

This criticism is addressed together with the next one as they make the same point.

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.

Many thanks.  We have carefully reframed and presented the WF-Haldane model to make it clear and logically consistent. Whether a new model (i.e., the WF-Haldane model) deserves to be introduced depends on whether it makes any contribution for understanding nature. That is why we emphasize the four paradoxes. 

A most important disagreement between the reviewers and the authors is about the nature of the paradoxes. While the reviewers suggest that they "may" be resolvable by the conventional WF model (standard or modified), they did not offer the possible resolutions.  To use the analogy in our provisional response: the WF vs. Haldane models are compared to gas cars vs electric vehicles.  We can say confidently that the internal combustion engine cannot resolve the conflicting demands of transportation and zero emission. Its design has limited its capability. 

Reviewer #2 (Recommendations For The Authors):

Many thanks.  We have incorporated all these suggestions.  When the incorporation is not straightforward, we have carefully revised the text to minimize mis-communications.

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.

We change the word “simply” to “essentially”. This wording is supported by the fixation probability of advantageous mutations, 2s/(V(k). We have shown in the text that N does not matter here because the fixation is nearly deterministic when the copy number reaches, say, 100, regardless of whether N is 10^4 or 10^8,

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.

Thank you for your careful reading. Regarding the use of i_k and j_k  as probabilities, we initially considered using 𝑝 or 𝑞 to represent probabilities. However, since 𝑝 and 𝑞 are already used in the main text, we opted for 𝑖 and 𝑗 to avoid potential confusion potential confusion. As for your recommendation to use

E[X] and V[X] for expectation and variance, we would like to clarify that we follow the standard practice of italicizing these symbols to represent variables.

Eq A6, A7, While I manage to follow, P_{10}(t) and P_{10} are not defined anywhere in the text.<br /> Supplement page 7, the term "probability of fixation" is confusing in a branching model.

Thank you for your observation. We have carefully revised the supplement to provide clarity on these points.<br /> Revision - In population genetics, the fixation of M allele means that the population consist entirely of the M allele, with no W alleles remaining. We define the fixation probability of M allele by generation t as follows:

Given that M and W allele reproduce independently, this can be factored as:

As t approaches infinity, the ultimate fixation probability of M allele can be derived as follows:

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

We appreciate your careful review, and we will ensure this connection between the two equations is made clearer in the supplement. 

Revision - Note we would like to clarify that Eq. (A1) and Eq. (A28) are essentially the same, with the only difference being the subscript 𝑡, which indicates the time dependence in the dynamic process.

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.

Thank you for raising this concern. We have addressed this issue earlier.

🔥🔥🔥 can reproduce classic learnings sequences. plus some variety.

In a third phase, the learner begins to experiment with actions toward zones of its environment where the external observer knows there are objects (the robot is not provided with a representation of the concept of “object”), but in a non-affordant manner (e.g., it vocalizes at the non-responding elephant or tries to bash the teacher robot which is too far to be touched). 4. In a fourth phase, the learner now explores the affordances of different objects in the environment: typically focusing first on grasping movements with the elephant, then shifting to bashing movements with the hanging toy, and finally shifting to explorations of vocalizing toward the imitating teacher. 5. In the end, the learner has learned sensorimotor affordances with several objects, as well as social affordances, and has mastered multiple skills. None of these specific objectives were pre-programmed. Instead, they self-organized through the dynamic interaction between curiosity-driven exploration, statistical inference, the properties of the body, and the properties of the environment

diagram with rough setup

Basic primitives which are just numbers to the robot.

for - fascism, polarization and climate crisis - interventions - love and listening strategy for climate crisis - Roger Hallam - Trump winning US election - is an opportunity - Roger Hallam - perspectival knowing - Deep Humanity - mini assemblies - Roger Hallam - listening - fascism - social intervention - from - Illuminem article - Proximity: The antidote to fascism - Kasper Benjamin Reimer Bjorkskov - on horizontal and vertical decision-making

Summary - Roger Hallam advocates for a new strategy for the rise of fascism, populism, polarization and the climate crisis - love - He believes that we need a new social strategy based on love, on reaching out to the other side with compassion and listening to them - He cites numerous research studies that show that this can be transformative, for instance, citing pyschologist Carl Rogers - SRG complexity mapping tool, Deep Humanity and Indyweb could be synergistic to this program because both depend on: - diversity and - perspectival knowing

from - Illuminem article - Proximity: The antidote to fascism - Kasper Benjamin Reimer Bjorkskov on horizontal and vertical decision-making - https://hyp.is/0Tv_Rqr3Ee-_-X8fKkCfpg/illuminem.com/illuminemvoices/proximity-the-antidote-to-fascism - Medium article - An Emerging Third Option: Reclaiming Democracy from Dark Money & Dark Tech Seven Observations On 2024 and What’s Next - Otto Scharmer - cutting across political lines / https://hyp.is/exS8dKtNEe-pfz-IhQFiZA/medium.com/presencing-institute-blog/an-emerging-third-option-reclaiming-democracy-from-dark-money-dark-tech-3886bcd0469b

for - key insight - quote - Why Harris lost US election - no perceived genuine alternative to BAU - Roger Hallam - from - Medium article - An Emerging Third Option: Reclaiming Democracy from Dark Money & Dark Tech Seven Observations On 2024 and What’s Next - Otto Scharmer - terminology - Status Quoism

key insight - quote - Why Harris lost US election - no perceived genuine alternative to BAU - Roger Hallam - (see below) - What the defeat of Harris shows about the US is this. People like everywhere else want a real alternative and - If there is no authentic left option, people vote for a fascist instead. - It happens again and again in history

from - Medium article - An Emerging Third Option: Reclaiming Democracy from Dark Money & Dark Tech Seven Observations On 2024 and What’s Next - Otto Scharmer - terminology - Status Quoism - https://hyp.is/Mxp1GqtFEe-pKzNGX6BrhQ/medium.com/presencing-institute-blog/an-emerging-third-option-reclaiming-democracy-from-dark-money-dark-tech-3886bcd0469b

for - love-based strategy for addressing fascism and polarization - listen and befriend - Roger Hallam - adjacency - Deep Humanity - Common Human Denominators - the intrinsic sacred - love-based strategy for addressing fascism and polarization - Roger Hallam - question - love-based strategy to address fascism - what about FAFO? - F around and Find Out - in which liberal women are separating from their Maga partners?

It might be useful to distinguish between pleasure and joy. But maybe everybody does this very easily, all the time, and only I am confused. A lot of people seem to feel that joy is only the most intense version of pleasure, arrived at by the same road—you simply have to go a little further down the track. That has not been my experience. And if you asked me if I wanted more joyful experiences in my life, I wouldn’t be at all sure I did, exactly because it proves such a difficult emotion to manage. It’s not at all obvious to me how we should make an accommodation between joy and the rest of our everyday lives. smith_1-011013.jpg

Butler Institute of American Art, Youngstown, Ohio

George Bellows: Geraldine Lee, No. 2, 1914; on view in the Metropolitan Museum of Art’s ‘George Bellows’ exhibition until February 18, 2013

Perhaps the first thing to say is that I experience at least a little pleasure every day. I wonder if this is more than the usual amount? It was the same even in childhood when most people are miserable. I don’t think this is because so many wonderful things happen to me but rather that the small things go a long way. I seem to get more than the ordinary satisfaction out of food, for example—any old food. An egg sandwich from one of these grimy food vans on Washington Square has the genuine power to turn my day around. Whatever is put in front of me, foodwise, will usually get a five-star review.

Wrong el

Estas ações incentivam os alunos a participarem ativamente no processo de aprendizagem, em vez de serem apenas recetores passivos de informação.

Nesta entrada do documento reside um ponto fundamental de interpretação ou diria má interpretação que nos leva para o contexto das e-atividades. Estou certo que o documento se baseia nestas atividades construídas de forma digital com todas as tecnologias disponíveis em linha (ou no suporte) ao desenvolvimento do ensino a distância, no entanto podem perfeitamente ser usadas (até devem na minha opinião) em qualquer contexto de aprendizagem ou formação. As e-atividades não podem pois ser um elemento que distingue os vários sistemas de aprendizagem e li em muitas anotações opiniões que se baseiam precisamente neste ponto. Sou um professor que utiliza (e desde sempre) a tecnologia como um meio importante no processo de ensino e aprendizagem, mas nunca em circunstancia alguma se poderá considerar que o ensino apoiado apenas em atividades ou recurso digitais seja uma optimização do processo. Um exemplo clássico deste paradigma é o professor que preparou cuidadosamente um conjunto de recursos com uma base tecnológica extensa e quando se prepara para dar a aula h á uma simples falha no sistema elétrico. Tenho a certeza que a aula será dada de qualquer forma.

sampling rate not over 10K

Briefing Doc : Approche scientifique et droits des enfants

Ce document synthétise les principaux thèmes et idées abordés lors de l'intervention de Gabrielle Chouin et Marie-Pierre Mackiewicz sur le sujet de l'approche scientifique et des droits des enfants, notamment en lien avec la protection de l'enfance.

Thèmes principaux :

La scolarité des enfants protégés : * Difficultés rencontrées par ces enfants (redoublement, orientation spécialisée, accès limité aux études supérieures...) * Manque d'information et de coordination entre les institutions (protection de l'enfance et éducation nationale) * Importance de la continuité scolaire et de l'accès aux mêmes droits que les autres élèves * Exemples de disparités : bonification Parcoursup, accès aux stages en voie professionnelle... * Nécessité d'une meilleure collaboration entre professionnels (mise en place d'alliances éducatives) * La recherche participative avec des mineurs protégés : * Importance de la participation des personnes concernées pour une meilleure compréhension des problématiques * Difficultés et enjeux éthiques de ce type de recherche (gestion des rapports de pouvoir, protection des enfants, fiabilité des résultats...) * Nécessité de protocoles spécifiques et d'une adaptation des méthodes de recherche (rythme, durée, lieu, accompagnement, convivialité...) * Exemples de recherches menées : étude sur la sortie des jeunes de la protection de l'enfance, étude sur le vécu d'enfants pris en charge * Le droit à la participation des enfants : * Importance de donner la parole aux enfants et de prendre en compte leur point de vue * Différents niveaux de participation (instances scolaires, politique, recherche...) * Difficultés de faire entendre la voix des enfants et de dépasser le stade des anecdotes

Idées/faits importants :

• Citation : "Il y avait des jeunes [...] qui était un peu perdu sur des logiques d'orientation qui étaient parfois complètement opposées et qui pouvait pas s'associer en mettant l'acteur le le jeune en en capacité de choisir son orientation." (Gabrielle Chouin) - Souligne le manque d'information et de coordination entre les institutions concernant l'orientation des jeunes protégés.
• Citation : "C'est comment une population qui subit des inégalités et qui est assignée au niveau identitaire [...] peut prendre parti sans être assigné au niveau identitaire." (Marie-Pierre Mackiewicz) - Met en avant l'enjeu des rapports de pouvoir et de l'identité dans la recherche participative.
• Citation : "Le chercheur va toujours être le représentant des normes sociales de de par ce statutl et comment peut-il de ce fait accéder à la parole de de groupes stigmatisés." (Marie-Pierre Mackiewicz) - Pose la question de la légitimité du chercheur et de sa capacité à comprendre les réalités des groupes marginalisés.
• Statistiques : 35% des jeunes sortant de l'aide sociale à l'enfance n'ont aucun diplôme ou seulement le brevet contre 16% pour les jeunes de même milieu. (France Stratégie, 2024) - Illustre les inégalités en matière de réussite scolaire pour les enfants protégés.

Recommandations de lecture :

Revue de littérature "Scolarité des enfants protégés" d'Aurélie Pico (2020) Note d'analyse "Scolarisation des enfants protégés" par France Stratégie (septembre 2024) Fiche "La participation collective des enfants protégés" par l'Observatoire national de la protection de l'enfance (ONPE)

Conclusion :

L'intervention met en lumière les défis et les opportunités liés à l'inclusion des enfants protégés, tant au niveau de leur scolarité que de leur participation à la société.

La recherche participative apparaît comme un outil prometteur pour mieux comprendre leurs réalités et faire évoluer les politiques publiques en leur faveur.

Remarques :

Ce briefing doc est basé sur une transcription orale et peut contenir des erreurs de transcription. Il est important de se référer aux sources originales pour une analyse plus approfondie.

Chronologie des événements

• Malheureusement, les sources fournies ne permettent pas d’établir une chronologie précise des événements. Le texte est une transcription d’une intervention orale, sans marqueurs temporels clairs.
• Cependant, on peut extraire quelques éléments pour une chronologie partielle:

Avant 2011:

• Marie-Pierre Mackiewicz travaille comme assistante sociale en Picardie et participe à la protection de l’enfance.
• Elle entreprend des études en sciences de l’éducation et rédige un mémoire sur la scolarité des élèves protégés.
• Manque de recherches et de ressources sur la scolarité des élèves protégés en France.

2011-2014:

• Gabrielle Chouin, alors étudiante, participe aux premières concertations nationales sur le droit à l'écoute et à la participation des enfants protégés.
• Elle rencontre Marie-Pierre Mackiewicz lors de ces concertations.
• Début des recherches universitaires sur la scolarité des élèves protégés.

2014:

Marie-Pierre Mackiewicz obtient un poste de maître de conférence à l’IUFM du Nord-Pas-de-Calais.

Entre 2014 et 2023:

• Marie-Pierre Mackiewicz travaille à l’Observatoire national de l’enfance en danger (devenu Observatoire national de l’enfance).
• Elle obtient un poste de maître de conférence à l’université de Créteil.
• Elle mène plusieurs recherches, dont une recherche majeure avec des jeunes majeurs passés par la protection de l’enfance, qui donne lieu à une pièce de théâtre.
• Une autre recherche est menée avec des enfants encore pris en charge, mais rencontre des difficultés et est moins valorisée.
• Une recherche sur la scolarité des enfants protégés est menée sans la participation de jeunes.
• 4 avril 2023:

Victoire pour les élèves placés : ils bénéficient désormais de la bonification lors de leurs vœux sur Parcoursup. 2020:

La Cour des comptes pointe des défaillances dans le suivi de la scolarité des enfants protégés. La Stratégie nationale de prévention concernant la protection de l’enfance définit des mesures pour améliorer la scolarité et l’accompagnement scolaire de ces enfants.

Personnages principaux

• Marie-Pierre Mackiewicz: Maître de conférence et chercheuse en sciences de l’éducation, spécialisée dans la protection de l’enfance. Elle a travaillé comme assistante sociale avant de se consacrer à la recherche. Elle a mené plusieurs recherches participatives avec des jeunes passés par la protection de l’enfance et s’intéresse particulièrement à la scolarité de ces enfants.
• Gabrielle Chouin: Ancienne enfant placée, elle est devenue une militante pour les droits des enfants et des jeunes. Elle s'est impliquée dans des concertations nationales et a participé à des recherches sur la protection de l'enfance. Elle travaille avec des associations d’anciens enfants placés.
• Laurence Rossignol: Ministre des Familles, de l'Enfance et des Droits des femmes de 2014 à 2016. Elle a mis en place les premières concertations nationales sur le droit à l'écoute et à la participation des enfants protégés.
• Adrien Taquet: Secrétaire d'État chargé de l'Enfance et des Familles de 2019 à 2022. Il a présidé un groupe de travail sur l'ambition scolaire des enfants protégés.
• Valérie Cabuil: Rectrice d’académie. Elle a co-présidé avec Adrien Taquet le groupe de travail sur l'ambition scolaire des enfants protégés.
• Pierine Robin: Chercheuse en sciences de l’éducation, elle a collaboré avec Marie-Pierre Mackiewicz sur plusieurs recherches, notamment celles menées avec des jeunes majeurs et des enfants encore pris en charge.
• Régine Sirota: Sociologue française, elle a contribué à l’établissement de la sociologie de l’enfance en France. Ses travaux interrogent la participation des enfants dans la recherche et les enjeux de la représentation de leur parole.

Remarques

• La transcription est parfois difficile à suivre et il manque des informations contextuelles pour une analyse plus approfondie.
• La chronologie est partielle et il est possible que certains événements n’aient pas été relevés.
• La liste des personnages n’est pas exhaustive et ne mentionne que les personnes les plus importantes.
• J'espère que ces informations vous seront utiles.

Table des Matières: Approche Scientifique et Droits des Enfants

Introduction

Cette table des matières structure l'intervention de Gabrielle Chouin et Marie-Pierre Mackiewicz lors d'une conférence portant sur l'approche scientifique et les droits des enfants.

I. Intervention de Gabrielle Chouin: Témoignage et Plaidoyer pour les Droits des Enfants Protégés

A. Contexte personnel et engagement initial (2011-2014)

Gabrielle Chouin évoque son premier mémoire universitaire portant sur la scolarité des élèves protégés et l'influence du partenariat entre la protection de l'enfance et l'éducation nationale. Elle souligne le manque d'information et de communication entre ces deux institutions, conduisant à des orientations scolaires parfois contradictoires et à un manque de choix pour les jeunes. Son engagement l'amène à participer à des concertations nationales, où elle défend le droit à l'écoute et à la participation effective des jeunes.

B. Importance des associations d'anciens protégés et l'évolution de la recherche

Gabrielle Chouin insiste sur le rôle crucial des associations d'anciens protégés (ADPEP) dans l'information et la défense des droits des jeunes. Elle note un développement significatif de la recherche sur la scolarité des élèves protégés au cours des dix dernières années.

C. Exemples de disparités et plaidoyer pour une continuité scolaire

Des exemples concrets de disparités entre élèves protégés et autres élèves sont présentés, notamment concernant l'accès aux bourses et aux stages (PFMP). Gabrielle Chouin plaide pour une meilleure collaboration entre protection de l'enfance et éducation nationale, afin d'assurer une continuité scolaire et des droits égaux pour tous.

D. L'importance du droit à la participation et la collaboration entre acteurs

Le droit à la participation ne doit pas être réservé aux élèves protégés, mais s'étendre à tous, via des instances comme les Conseils de Vie Collégienne et Lycéenne.

L'intervention souligne la nécessité d'une collaboration entre différents acteurs (professionnels, chercheurs, associations) pour faire progresser les droits des enfants. II. Intervention de Marie-Pierre Mackiewicz: La Recherche Participative avec des Mineurs Protégés

A. Introduction et parcours personnel

Marie-Pierre Mackiewicz présente son parcours professionnel et ses recherches en sciences de l'éducation, notamment sur la coéducation et la protection de l'enfance.

Elle introduit le concept de "coéducation professionnalisée", où la communication concernant la situation d'un enfant se fait entre professionnels de l'école et de la protection de l'enfance.

B. La recherche par les pairs: contexte et conditions

Présentation d'une recherche par les pairs menée avec 14 jeunes passés par la protection de l'enfance, en partenariat avec l'ONE, des conseils départementaux et des institutions.

Importance de créer des conditions de travail spécifiques pour ce type de recherche, incluant des espaces et des horaires flexibles, afin de favoriser la collaboration et la confiance.

C. Mise en perspective des recherches participatives

Historique des recherches participatives, apparues dans les années 70, et leur objectif de donner la parole aux populations marginalisées et de questionner les rapports de pouvoir.

Lien avec la protection de l'enfance et la nécessité de sortir d'une vision de l'enfant "symptôme" ou "à problème".

D. Questionnements et enjeux des recherches participatives avec des mineurs protégés

Différents questionnements sont abordés: la place des jeunes au-delà de la simple participation à l'enquête, la fiabilité et l'intérêt des résultats, le risque d'émancipation ou de réassignation identitaire.

Discussion sur les ambiguïtés de "faire entendre la voix de l'enfant": comment parler pour l'enfant, comment structurer les anecdotes, comment tenir compte du contexte et des "gatekeepers" institutionnels.

E. Protocoles de protection et adaptation des méthodes de recherche

Nécessité de mettre en place des protocoles spécifiques pour protéger les enfants impliqués dans la recherche, tout en assurant la validité des données.

Adaptation des méthodes de recherche en fonction de l'âge des enfants: la capacité d'élaborer un discours sur le passé diffère entre adolescents et jeunes enfants.

F. Difficultés et défis de la recherche participative

La dissymétrie de pouvoir entre chercheurs et populations marginalisées, la nécessité pour le chercheur de se décentrer et de reconnaître les pratiques "illégitimes" des personnes étudiées.

Risque d'exposition et de mise à mal des participants, obligation de respecter leurs limites et d'accepter d'être remis en question.

G. Exemples de recherches menées et leçons apprises

Présentation de deux recherches participatives: l'une sur la sortie des jeunes de la protection de l'enfance, ayant donné lieu à une pièce de théâtre et à la création d'une association; l'autre sur le vécu d'enfants encore pris en charge.

Différences observées en fonction de l'âge des participants et de l'implication des chercheurs pairs dans l'analyse et l'écriture.

H. L'importance des partenariats et la résistance aux pressions institutionnelles

Difficultés liées aux partenariats avec les institutions: imposition de priorités, sélection des participants, filtrage des résultats, enjeux d'image de marque et de financement.

Nécessité pour les chercheurs de résister à ces pressions et de préserver l'intégrité de leurs travaux.

III. La Scolarisation des Enfants de la Protection de l'Enfance: Un Enjeu Majeur

A. Évolution du paysage et prise de conscience des difficultés

Changements récents dans la prise en charge de la scolarité des enfants protégés, suite à des rapports de la Cour des comptes et à la stratégie nationale de prévention.

Mesures mises en place pour améliorer l'accompagnement scolaire et lutter contre les inégalités.

B. Données chiffrées et constats alarmants

Présentation de statistiques alarmantes sur la scolarité des enfants protégés: taux d'obtention de diplômes, redoublements, orientations spécialisées, accès aux études supérieures, emplois disqualifiés.

Comparaison avec les enfants de milieux sociaux similaires, soulignant l'impact spécifique de la protection de l'enfance.

C. Obstacles à la réussite scolaire et facteurs de risque

Identification des obstacles liés à la trajectoire familiale des enfants avant et pendant la prise en charge: placements multiples, instabilité, précarité, etc.

Impact de la durée, du type de placement et de l'âge de l'enfant sur ses chances de réussite scolaire.

D. Appel à l'engagement et à la confiance en la capacité de réussite de ces enfants

Importance de croire en la possibilité pour les enfants protégés de réussir leur scolarité et d'avoir des parcours positifs.

Responsabilité collective de tous les acteurs (professionnels, institutions, chercheurs, etc.) pour soutenir ces enfants et lutter contre les inégalités.

Conclusion

L'intervention de Gabrielle Chouin et Marie-Pierre Mackiewicz met en lumière les défis et les enjeux de la recherche participative avec des mineurs protégés, en soulignant l'importance de l'écoute, de la collaboration et de la confiance pour promouvoir leurs droits et améliorer leur scolarité.

Guide d'Étude: Approche Scientifique et Droits des Enfants

Quiz

Questions à réponse courte:

• Quel est le point commun entre les discours de Gabrielle Chouin lors des premières concertations nationales et ceux tenus lors de la deuxième concertation sur l'ambition scolaire ? (2-3 phrases)
• Expliquez la notion de "coéducation professionnalisée" dans le contexte de la protection de l'enfance. (2-3 phrases)
• Quels sont les objectifs principaux des recherches participatives, en particulier lorsqu'elles impliquent des mineurs protégés ? (2-3 phrases)
• Selon Régine Sirota, quelles sont les ambiguïtés liées à l'objectif de "faire entendre la voix de l'enfant" ? (2-3 phrases)
• Quels sont les défis spécifiques posés par l'implication de jeunes enfants dans des recherches participatives, par rapport aux adolescents ? (2-3 phrases)
• En quoi la situation de dissymétrie entre les chercheurs et les populations marginalisées pose-t-elle un défi dans le cadre de recherches participatives ? (2-3 phrases)
• Expliquez l'importance de la création d'un sentiment de "collectif" dans les recherches impliquant des personnes en situation de disqualification. (2-3 phrases)
• Quels sont les aspects positifs et négatifs de l'implication des chercheurs-pairs dans l'analyse et l'écriture des résultats de recherche ? (2-3 phrases)
• Quels sont les principaux obstacles à la réussite scolaire des enfants placés, liés à la fois à leur trajectoire familiale et aux prises en charge ? (2-3 phrases)
• Outre les rapports de recherche traditionnels, quelles sont les autres formes de restitution des résultats qui peuvent être envisagées dans le cadre de recherches participatives ? (2-3 phrases)

Corrigé du Quiz

Le point commun est l'importance de la collaboration entre le monde de la protection de l'enfance et celui de l'éducation nationale. Chouin a plaidé pour une meilleure communication, une formation commune et des partenariats concrets pour garantir aux élèves protégés l'accès aux mêmes droits et opportunités que les autres élèves.

La "coéducation professionnalisée" se réfère à la communication et à la collaboration entre professionnels de différents secteurs, comme l'école et la protection de l'enfance, concernant la situation d'un enfant. Dans ce contexte, les professionnels, plutôt que les parents, échangent des informations et travaillent ensemble pour élaborer des stratégies d'intervention.

Les recherches participatives visent à donner du pouvoir aux populations marginalisées en les impliquant activement dans le processus de recherche. Elles cherchent à rapprocher les savoirs experts et les savoirs profanes, à questionner les rapports de pouvoir et à promouvoir l'émancipation des participants. Sirota souligne la difficulté de parler "pour" l'enfant et de garantir une réelle prise en compte de son point de vue. Elle interroge la représentativité des témoignages individuels, la difficulté de situer les anecdotes dans un contexte plus large et les obstacles à l'accès aux enfants, souvent contrôlé par les institutions.

Les jeunes enfants ont un accès au langage et une capacité d'élaboration du discours moins développés que les adolescents. Leur participation à la recherche peut se traduire par une multitude d'anecdotes difficiles à structurer et à analyser. L'implication des chercheurs-pairs dans l'analyse et l'écriture peut être plus limitée.

La dissymétrie entre chercheurs et populations marginalisées peut créer des rapports de domination et influencer la parole des participants. Le chercheur, de par son statut, incarne les normes sociales et peut avoir du mal à accéder à la parole authentique des groupes stigmatisés.

Le sentiment de "collectif" permet de créer un espace de confiance et d'égalité entre chercheurs et participants. Il favorise l'expression libre et authentique des expériences et des points de vue, en minimisant les rapports de pouvoir et de domination.

L'implication des chercheurs-pairs enrichit l'analyse et l'écriture des résultats en apportant des perspectives vécues et une expertise issue de l'expérience. Cependant, elle peut aussi complexifier le processus de recherche et limiter la diffusion des résultats, en raison des difficultés de coordination et de l'impact émotionnel sur les participants.

Les obstacles liés à la trajectoire familiale incluent des antécédents d'échec scolaire et des difficultés socio-économiques. Les obstacles liés aux prises en charge sont la multiplicité des placements, l'instabilité des parcours et le manque de continuité dans l'accompagnement scolaire.

Au-delà des rapports, la restitution des résultats peut prendre la forme de pièces de théâtre, de films, d'expositions ou d'ateliers participatifs. Ces formes alternatives permettent de diffuser les résultats à un public plus large et de donner une voix aux participants, en valorisant leurs expériences et leurs points de vue.

Questions de dissertation

Analysez les enjeux éthiques liés à la participation de mineurs protégés à des recherches en sciences sociales. Discutez des mesures à prendre pour garantir leur protection et leur bien-être tout au long du processus de recherche.

Comparez et contrastez les approches traditionnelles et les approches participatives en recherche. En vous appuyant sur les exemples donnés dans les extraits, discutez des avantages et des limites de chaque approche dans le contexte de la recherche sur la protection de l'enfance.

Expliquez comment les recherches participatives peuvent contribuer à l'émancipation des populations marginalisées. Discutez du rôle des chercheurs-pairs dans ce processus et des défis liés à la reconnaissance de leur expertise et à la diffusion de leurs savoirs.

En vous appuyant sur les données présentées sur la scolarité des enfants placés, analysez les facteurs de risque et de protection qui influencent leur réussite éducative. Proposez des pistes d'action pour améliorer leur parcours scolaire et leur insertion sociale.

Discutez de l'importance de la collaboration entre les différents acteurs impliqués dans la protection de l'enfance, notamment l'éducation nationale, les services sociaux et les associations. En quoi cette collaboration peut-elle contribuer à garantir le respect des droits des enfants et à favoriser leur épanouissement ?

Glossaire

Termes clés:

• Recherche participative: Méthode de recherche qui implique activement les personnes concernées par le sujet d'étude dans toutes les étapes du processus de recherche, de la définition des questions de recherche à l'analyse des données et à la diffusion des résultats.
• Mineurs protégés: Enfants et adolescents bénéficiant de mesures de protection de l'enfance, suite à une situation de danger ou de risque pour leur sécurité, leur santé ou leur développement.
• Chercheurs-pairs: Personnes ayant une expérience vécue du sujet d'étude et qui participent à la recherche en tant que co-chercheurs, aux côtés des chercheurs académiques.
• Dissymétrie: Situation d'inégalité de pouvoir et de statut social entre les chercheurs et les populations marginalisées, pouvant influencer la parole des participants et la validité des résultats.
• Collectif: Sentiment d'appartenance à un groupe uni par un objectif commun, permettant de créer un espace de confiance et d'égalité entre chercheurs et participants.
• Gatekeepers: Personnes ou institutions contrôlant l'accès aux populations cibles de la recherche, pouvant influencer la sélection des participants et le déroulement de l'étude.
• Stigmatisation: Processus social par lequel un individu ou un groupe est marqué d'une étiquette négative, conduisant à la discrimination et à l'exclusion.
• Émancipation: Processus par lequel un individu ou un groupe acquiert le pouvoir de prendre des décisions et de contrôler sa propre vie, en se libérant des rapports de domination.
• Restitution des résultats: Action de partager les résultats de la recherche avec les participants et les autres parties prenantes, en utilisant des formats adaptés et accessibles.
• Trajectoire familiale: Ensemble des expériences vécues par un individu au sein de sa famille, pouvant influencer son parcours scolaire, son développement et son intégration sociale.

FAQ : Approche scientifique et droits des enfants 1. Quel est le lien entre la protection de l'enfance et la scolarité des élèves protégés ?

La protection de l'enfance a un impact majeur sur la scolarité des élèves protégés. Les recherches montrent que le partenariat entre les services de protection de l'enfance et l'Éducation nationale est crucial pour que ces élèves puissent réussir leur scolarité et obtenir un diplôme. Le manque de communication et d'information entre ces deux institutions peut entraîner des difficultés d'orientation et un manque d'accès aux droits communs pour ces jeunes.

2. Quels sont les obstacles à la réussite scolaire des enfants placés ?

Les obstacles à la réussite scolaire des enfants placés sont multiples. Ils peuvent être liés à leur trajectoire familiale antérieure, souvent marquée par l'échec scolaire, mais aussi aux caractéristiques de leur prise en charge : placements multiples, instables, tardifs, en institution plutôt qu'en famille d'accueil. La complexité de ces situations peut entraîner des redoublements, des retards, des orientations spécialisées et un accès limité aux études supérieures.

3. Quelles sont les initiatives prises pour améliorer la scolarité des enfants protégés ?

La prise de conscience de l'importance de la scolarité pour les enfants protégés a mené à la mise en place de différentes initiatives. La Cour des comptes a pointé les défaillances du système en matière de suivi scolaire, et la stratégie nationale de prévention de la protection de l'enfance a défini des mesures pour améliorer l'accompagnement scolaire de ces enfants. De plus, des dispositifs comme les alliances éducatives visent à renforcer la collaboration entre les différents acteurs impliqués dans la scolarité de ces élèves.

4. En quoi consiste la recherche participative avec des mineurs protégés ?

La recherche participative vise à impliquer les personnes concernées par une problématique, en l'occurrence les mineurs protégés, dans le processus de recherche. Il s'agit de reconnaître leurs savoirs et expériences, et de leur donner une voix pour contribuer à la production de connaissances. Cette approche permet de mieux comprendre les réalités vécues par ces jeunes, et de proposer des solutions adaptées à leurs besoins.

5. Quelles sont les difficultés rencontrées dans la mise en place de recherches participatives avec des enfants ?

La recherche participative avec des enfants présente des défis particuliers. Il est important de mettre en place des protocoles spécifiques pour protéger les enfants et respecter leur rythme et leur durée d'attention. La collaboration avec les institutions qui accueillent les enfants peut être complexe, car elles peuvent imposer leurs priorités et filtrer les résultats. De plus, l'implication des enfants dans l'analyse et l'écriture des résultats peut être difficile à mettre en œuvre.

6. Quel est l'impact de la participation des jeunes à la recherche sur leur identité ?

La participation des jeunes à la recherche peut avoir un impact positif sur leur identité. En s'engageant dans le processus de recherche, ils peuvent sortir d'une identité stigmatisée et expérimenter d'autres rôles sociaux, comme celui de "jeune chercheur". Cela peut contribuer à leur émancipation et à la construction d'une image positive d'eux-mêmes.

7. Comment les résultats de ces recherches sont-ils diffusés et utilisés ?

Les résultats des recherches participatives avec des mineurs protégés peuvent être diffusés sous différentes formes : rapports, publications scientifiques, pièces de théâtre, films.

L'objectif est de sensibiliser le public et les décideurs aux réalités vécues par ces jeunes, et de promouvoir des politiques publiques plus efficaces pour les soutenir.

8. Quel rôle peuvent jouer les professionnels de l'éducation dans l'amélioration de la scolarité des enfants protégés ?

Les professionnels de l'éducation ont un rôle essentiel à jouer dans l'amélioration de la scolarité des enfants protégés.

Ils doivent être sensibilisés aux difficultés spécifiques rencontrées par ces élèves, et adopter une posture bienveillante et encourageante.

Il est important de croire en leur potentiel de réussite et de mettre en place des dispositifs d'accompagnement adaptés à leurs besoins.

La collaboration avec les services de protection de l'enfance est également cruciale pour assurer la continuité de leur parcours scolaire.

Points forts de la vidéo "Approche scientifique et droits des enfants"

Cette vidéo présente une intervention de Gabrielle Chouin et Marie-Pierre Mackiewicz lors d'une journée d'étude à l'INSPÉ de Lille. Les deux intervenantes, expertes en protection de l'enfance et en recherche participative, abordent les thèmes suivants :

1. La scolarité des enfants protégés (0:00 - 8:30):

• Gabrielle Chouin partage son expérience personnelle et professionnelle en tant que CPE et ancienne enfant placée.
• Elle souligne les difficultés rencontrées par ces élèves en matière d'orientation, d'accès à l'information et aux droits communs.
• Elle met en avant l'importance de la collaboration entre l'Éducation Nationale et la Protection de l'enfance pour améliorer la scolarité et l'insertion des jeunes.
• Elle donne des exemples concrets de droits non effectifs pour les élèves protégés (Parcoursup, stages).
• Elle insiste sur le rôle des associations d'anciens pupilles de l'État et la nécessité de les faire connaître aux jeunes.

2. La recherche participative avec des mineurs protégés (8:30 - 25:00):

• Marie-Pierre Mackiewicz, chercheuse en sciences de l'éducation, présente les principes et les enjeux de la recherche participative.
• Elle explique la spécificité de ce type de recherche lorsqu'il s'agit de travailler avec des mineurs protégés :
  • Nécessité de prendre en compte les rapports de pouvoir et de créer un climat de confiance.
  • Importance de l'éthique et de la protection des participants.
  • Difficulté de concilier les exigences de la recherche académique avec les besoins et les contraintes des jeunes et des institutions.
• Elle illustre son propos avec des exemples de recherches menées avec des jeunes :
  • Une recherche sur la sortie des jeunes de la protection de l'enfance, qui a donné lieu à une pièce de théâtre.
  • Une recherche sur le vécu des enfants encore pris en charge.
  • Une recherche sur la scolarité, où l'absence de participation des jeunes a été préjudiciable.

3. Les obstacles à la réussite scolaire des enfants protégés (25:00 - 28:00):

• Marie-Pierre Mackiewicz dresse un état des lieux de la scolarité des enfants protégés en France, en s'appuyant sur des données récentes.
• Elle met en évidence les inégalités et les difficultés rencontrées par ces élèves.
• Elle souligne les obstacles liés à la fois à la trajectoire familiale des enfants et aux modalités des prises en charge.
• Elle appelle à la mobilisation de tous les acteurs pour favoriser la réussite scolaire de ces enfants.

Conclusion :

La vidéo met en lumière les défis et les perspectives de la scolarisation des enfants protégés. Elle souligne l'importance de la recherche participative pour donner la parole à ces jeunes et mieux comprendre leurs besoins. Elle invite à une collaboration étroite entre les différents acteurs pour garantir l'accès à une éducation de qualité pour tous les enfants, quel que soit leur parcours.

eLife Assessment

This study presents useful albeit preliminary findings on transcriptome changes in cardiac lymphatic cells after myocardial infarction in mice. Despite revision, the conclusions of the authors remain uncertain as sample sizes in general are very low, and even sometimes too low to allow for valid statistical comparisons. Accordingly, there are concerns regarding statistical robustness, raised by both the editors and the reviewers. While the single-cell transcriptomic data were analyzed using solid advanced methodology, too few cells were included in the scRNA-seq data set and the spatial transcriptomics analyses. Thus, this study rather represents more a collection of preliminary transcriptomic data than a full scientific report that would definitively advance the field.

Reviewer #1 (Public review):

Summary:

Assessment of cardiac LEC transcriptomes post-MI may yield new targets to improve lymphatic function. scRNAseq is a valid approach as cardiac LECs are rare compared to blood vessel endothelial cells.

Strengths:

Extensive bioinformatics approaches employed by the group

Weaknesses:

Too few cells included in scRNAseq data set and the spatial transcriptomics data that was exploited has little relevance, or rather specificity, for cardiac lymphatics. This study seems more a collection of preliminary transcriptomic data than a true scientific report to help advance the field.

Comments on revisions:

Thank you for the revision that helps clarify some outstanding questions.

(1) I still have questions relating to the relevance of the spatial maps generated and shown in fig 3C. They are supposedly generated using a 'molecular finger print' specific to each sub-cluster of LECs. However, given that at early stages postMI most populations are exceedingly rare in your analyses, could you please explain or comment on the relevance of the spatial maps?

(2) Fig 3 s1 would indicate that the population CaII is the majoritarian one in healthy hearts, while quantifications in Fig 3A show that rather the LEC Co subpopulation is majoritarian. Further, in mouse hearts histological analyses have demonstrated that cardiac lymphatics are restricted to the outer layers of the heart. This is not seen in your spatial maps. This seems to be the case only for the LEc Co population in healthy hearts, but not for other subpopulation signatures. Please explain.

(3) Further, the population of CaI, with 1 cell analysed in d3, but appears very prevalent in the spatial maps at d3. Please explain.

(4) In your list of 12 genes used as matrix anchors to identify LEC subpopulations in your screens, it is not apparent how LEC CaI, II and III differ so much as to allow selective detection of subpopulations. This similitude of profiles is supported by Fig 2F, and further explanations are needed to explain how the spatial maps of LEC ca subpopulations appear as distinct as shown in fig 3 S1 and Fig 3C.

Reviewer #2 (Public review):

Summary:

This study integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different time points post-MI. They identified four transcriptionally distinct subtypes of lymphatic endothelial cells and localized them in space. They observed that LECs subgroups are localized in different zones of infarcted heart with functions. Specifically, they demonstrated that LEC ca III may be involved in directly regulating myocardial injuries in the infarcted zone concerning metabolic stress, while LEC ca II may be related to the rapid immune inflammatory responses of the border zone in the early stage of MI. LEC ca I and LEC collection mainly participate in regulating myocardial tissue edema resolution in the middle and late stages post-MI. Finally, cell trajectory and Cell-Chat analyses further identified that LECs may regulate myocardial edema through Aqp1, and likely affect macrophage infiltration through the galectin9-CD44 pathway. The authors concluded that their study revealed the dynamic transcriptional heterogeneity distribution of LECs in different regions of the infarcted heart and that LECs formed different functional subgroups that may exert different bioeffects in myocardial tissue post-MI.

Strengths:

The study addresses a significant clinical challenge, and the results are of great translational value. All experiments were carefully performed, and their data support the conclusion.

Editors' comments (Public review):

Weaknesses:

(1) Figure 7C, 7E, 7I, and "Figure7-figure supplement 1 ": All data in these data panels are based on only n=3, which is insufficient. Sample sizes of n=3 are too low to correctly assess normality of distribution and, as a consequence, do not allow to select the appropriate parametric/non-parametric tests. Accordingly, no statistical comparison can be performed and all p values and symbols currently indicating statistically significant differences between groups must be removed.

(2) Figure 3A, 3B, or 3C: No information about n numbers per group. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

(3) Figure 4 E and 4F: No information about n numbers per group. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

(4) Figure 5: No information about n numbers per group is provided. Should n numbers per group be n=4 or less, no statistical comparison can be performed and all p values and symbols indicating statistically significant differences between groups must be removed.

Author response:

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

Reviewer #1 (Recommendations for The Authors):

Q1: In response to reviewers you noted totally 292 sequenced LECs, however in reviewer figure 3 B the numbers seem to add up to 221. Please include mention of the total number of LEC sequences. Please mention line 119, page 4 the total number of explored LEC transcriptomes

Thank you for your carefully review. We have updated Fig 2A, 2C and 2E. It was 242 (not 292) LECs included in our initial analysis, which contains the sample of d5 post MI in raw data (E-MTAB-7895). We dropped d5 in our subsequent analysis because the change in d5 did not significant differ from d3. Therefore, we included 221 LECs in our final analysis as we updated in Fig2A, 2C and 2E.

Q2-1: Figure 3A supposedly shows % of LEC subpopulations relative to their numbers found in day 0 samples. However, there seem to be some errors, because for example the subpop LEC Cap I include 13 cells day 1 and 6 cells day 1, which corresponds to 46% of initial numbers. However, from your graph 3B the blue population seems to occupy 10%. Please revise or explain how these relative % were calculated.

Thank you for your question. In the Figure 3A, each column was calculated by dn/d0*100%, that is d0=57/57*100%=100%, and d1= 21/57*100%=36.84%, d3=9/57*100%=15.79%, d7, d14, d28...Therefor, Cap I in d0 (13 cells) is 13/57*100%=22.81%, and Cap I in d1(6 cells) is 6/57*100%= 10.53%.

Q2-2: Further, based on the relative % of LEC subpopulations, using the numbers mentioned in Fig 3B, it would appear that the relative frequency LEC cap II population is actually stable at around 20-30% of all LECs per time point throughout the study (except day 1 drop). This contrasts with line 136 p. 4 statement. I would also urge caution for interpreting too much into the variation of relative levels of LEC co, as these represent exceeding rare cells in your samples, and could reflect technical issues rather than true biological variation (total LEC co numbers analyzed ranging from 1-24 cells/ time point). The same could be said of LEC cap II and cap III.

We strongly agree with your comment on the proportion of LEC cell subtypes post MI. As you pointed out, we have revised the result description on Page 4, line 137-143 as followed.

“In the early stages of myocardial infarction (D1 and D3), the quantity of LECs decreased sharply. The number of LECs gradually increasing from day 7 and returning to normal levels by day 14 after MI. Moreover, from day 14 onwards, the number and proportion of Ca I type LECs significantly increased.”

Q3: Please list in supplement the gene features used to identify in spatial transcriptomics the different LEC subpopulations, as their profiles (notably for capillary LECs) don't appear to be very different based on data in Fig 2F.

We have supplied gene features in supplementary materials.

Q4: In section 2.7 you refer to Gal9 secretion. Please replace with expression as no measure of protein levels from LECs has been described in your study.

Thank you for your suggestion, we have replaced secretion with expression.

Q5: The updated method to exclude non-lymphatic cells from lymphatic vessel analyses by incorporating pdpn as an additional marker ('present costained areas wherever possible' line 350 p 10)

Thank you for your correction. We have updated the description as follows and lighted them in the manuscript: rabbit anti-Lyve1 (1:300, ab14917, Abcam, UK), [Syrian hamster anti-Podoplanin (1:100, 53-5381-82, Thermo, USA), rabbit anti-Prox1(1:300, ab199359, Abcam, UK), both anti-podoplain and anti-prox1 are additional markers co-stained with Lyve1 to exclude non-lymphatic cells from lymphatic vessel].

Q6: Fig 1B, it is highly surprising to see the lymphatic density in the BZ go from 25 um² at day 3 to more than 1000 um² only four days later (day 7). Is it possible that your day 3 measurements were in the infarct area, and not BZ area? The H&E image shown in Fig1a for d3 sample would seem to indicate the analysis was done in a dead area, rather than BZ. Please revise (perhaps select similar zone as shown for d1 in fig 6D, adjusted for subepicardial region and not mid-myocardial as seems to be the case currently), and also provide lymphatic area measures in healthy myocardium for day 0 samples. The unit used (um²) also would depend on the size of the area examined. Is this unit per image? If so please report total imaged area as a reference.

A6: Thank you for your reminding and advises. We have labeled each zone on H&E and IF images in Fig1-supplementary Fig2B, and updated a clearer histological photo taken at 3 days post MI in Fig1A. Furthermore, we recalculated the lymphatic vessel area ratio as you suggested by calculating the ratio of LEC co-stained area to total imaged area under 100-fold magnification.

Q7: The mention that CD68 antibody isn't compatible with lyve1 antibody could easily have been bridged by using other macrophage markers, such as F4/80, which is readily available and often used marker for macs in mice and comes notably as a rat anti-mouse F4-80. It would have added much more relevant information to exclude Lyve1-/F4/80+ cells as compared to the current analysis, which may indeed include in area measures Lyve1+ /Pdpn- single cells erroneously spotted as 'lymphatic vessels'

Thank you for your excellent suggestion. We co-stained the sample with F4/80 and LYVE1 and supplied in the Fig1-supplementary Figure 1E, as shown in Author response image 1.

Author response image 1.

Immunofluorescence (IF) co-staining of tissue section with F4/80 and LYVE1 in sham and MI mice model at d3, d7, d14, and d28 post-MI. LYVE1: lymphatic vessel endothelial hyaluronan receptor 1; DAPI: 4’6-diamidino-2-phenylindole; scale bar in 10×-100 μm, 40×-25μm.

Reviewer 2 (Recommendations for The Authors):

Q1: Language expression must be improved. Many incomplete sentences exist throughout the manuscript. A few examples: Line 70-71: In order to further elucidate the effects and regulatory mechanisms of the lymphatic vessels in the repair process of myocardial injury following MI. Line 71-73. This study, integrated single-cell sequencing and spatial transcriptome data from mouse heart tissue at different timepoints after MI from publicly available data (E-MTAB-7895, GSE214611) in the ArrayExpress and gene expression omnibus (GEO) databases. Line 88-89: Since the membrane protein LYVE1 can present lymphatic vessel morphology more clearly than PROX1.

Thank you for your correction. We have carefully inspected and corrected the whole manuscript.

Q2: The type of animal models (i.e., permanent MI or MI plus reperfusion) included in Array Express and gene expression omnibus (GEO) databases must be clearly defined as these two models may have completely different effects on lymphatic vessel development during post-MI remodeling.

Thank you for your excellent suggestion. The animal models used in both E-MTAB-7895 and GSE214611 are permanent MI. We have modified the model information in the methodology section (page 12, line 400-401).

Q3: Line 119-120: Caution must be taken regarding Cav1 as a lymphocyte marker because Cav1 is expressed in all endothelial cells, not limited to LEC.

Thanks for your reminding. Cav 1 used in our clustering is one of the marker gene for its different expression in sub-types of LECs, referred in article PMID: 31402260

Q4: Figure 1 legend needs to be improved. RZ, BZ, and IZ need to be labeled in all IF images. Day 0 images suggest that RZ is the tissue section from the right ventricle.

Thank you for your suggestion. We have labeled and updated the regions of RZ, BZ, and IZ in H&E and IF image in Figure1-Figure supplement 2B.

Q5: The discussion section needs to be improved and better focused on the findings from the current study.

Thank you for your good comment. Based on your suggestion, we have revised the first paragraph of the discussion from lines 250-256 (Page 7) as followed:

Cardiac lymphatics play an important role in myocardial edema and inflammation. This study, for the first time, integrated single-cell sequencing data and spatial transcriptome data from mouse heart tissue at different time points of post-MI, and identified four transcriptionally distinct subtypes of LECs and their dynamic transcriptional heterogeneity distribution in different regions of myocardial tissue post-MI. These subgroups of LECs were shown to form different function involved in the inflammation, apoptosis, ferroptosis, and water absorption related regulation of vasopressin during the process of myocardial repair after MI.

for - adjacency - awakening the sleeping giant - of the commons - bridging Cosmo with local - Michel Bauwens - the Indyweb - from - Medium article - An Emerging Third Option: Reclaiming Democracy from Dark Money & Dark Tech Seven Observations On 2024 and What’s Next - Otto Scharmer - organizing around shared intentions

adjacency - between - awakening the sleeping giant - of the commons - bridging Cosmo with local - Michel Bauwens - the Indyweb - adjacency relationship - Using the web to enable cooperation at global scale between localities all over the world is Cosmo local - This lays at the heart of the Indyweb - To awaken the sleeping giant of the commons - requires completing the incomplete capacity of local communities

from - Medium article - An Emerging Third Option: Reclaiming Democracy from Dark Money & Dark Tech Seven Observations On 2024 and What’s Next - Otto Scharmer - organizing around shared intentions - https://hyp.is/Mxp1GqtFEe-pKzNGX6BrhQ/medium.com/presencing-institute-blog/an-emerging-third-option-reclaiming-democracy-from-dark-money-dark-tech-3886bcd0469b

for - Substack article - A global history of societal regulation - metacrisis, polycrisis - role of the commons and cosmolocal coordination - Michel Bauwens - from - Illuminem article - Proximity: The antidote to fascism - Kasper Benjamin Reimer Bjorkskov - on horizontal and vertical decision-making

article details - title: A global history of societal regulation - publisher: Substack - date_ 2024, Nov 20 - author: Michel Bauwens

Summary - Michel presents a history of economic and societal coordination and makes the claim that the commons has an important role to play in maintaining a wellbeing species that balances: - human activity - health of the natural environment - peace between different human groups - In particular, he observes the important role that cosmolocal coordination may play - Michel takes us to a journey through history to explore the various different systems that different cultures used in the past - It's very interesting that in modernity, we have a system which is seen as absolute but a study of history shows how relative it is - That raises the question of why the current system feels so intractable? What gives it its entrenchment - Perhaps it's that the global spread of neocapitalism around the globe has made it "too big to fail"? - and it will actually require failing before a new phoenix can emerge from the ashes? I hope not!

from - Illuminem article - Proximity: The antidote to fascism - Kasper Benjamin Reimer Bjorkskov on horizontal and vertical decision-making - https://hyp.is/0Tv_Rqr3Ee-_-X8fKkCfpg/illuminem.com/illuminemvoices/proximity-the-antidote-to-fascism

for - definition inflating the commons - Geoffrey West - superlinear relationship - of cosmolocalization - via digital cooperation - Michel Bauwens - adjacency relationship - inflating the commons - indyweb

adjacency - between - inflating the commons - indyweb - adjacency relationship - Indyweb could be one way to inflate the commons by weaving together cosmolocal groups around the group into a mycelial network

for - Technofeudalism: What killed capitalism - Yanis Varoufakis - from - interview - 2008 was the West's 1991 moment - Yanis Varoufakis - from - Medium article - An Emerging Third Option: Reclaiming Democracy from Dark Money & Dark Tech Seven Observations On 2024 and What’s Next - Otto Scharmer - neo feudalism

from - interview - 2008 was the West's 1991 moment - Yanis Varoufakis - https://hyp.is/BZ88pKj5Ee-k86snmHsbnQ/www.youtube.com/watch?v=8nTBWf4JgYQ - Medium article - An Emerging Third Option: Reclaiming Democracy from Dark Money & Dark Tech Seven Observations On 2024 and What’s Next - Otto Scharmer - neo feudalism - https://hyp.is/cVix6KtFEe-zA8PBZvgw8w/medium.com/presencing-institute-blog/an-emerging-third-option-reclaiming-democracy-from-dark-money-dark-tech-3886bcd0469b

Briefing Doc : Point de vue institutionnel des droits de l'enfant

Intervenante : Marie Derain de Vaucresson, ancienne Défenseure des enfants (2011-2014)

Contexte : Journée de réflexion organisée par le Rectorat du Nord sur la scolarisation des enfants placés et accompagnés.

Thèmes principaux:

Histoire de la protection de l'enfance et émergence des droits de l'enfant Principes fondamentaux de la Convention internationale des droits de l'enfant (CIDE) Rôle et évolution du Défenseur des enfants et du Défenseur des droits Défis et perspectives de la scolarisation des enfants placés

Idées et faits importants:

L'approche de la protection de l'enfant est apparue au 17e siècle, initialement axée sur la charité. L'approche par les droits de l'enfant est beaucoup plus récente, impulsée par la CIDE adoptée en 1989. La CIDE s'adresse aux États et non aux enfants, les obligeant à la transposer en droit national et à la rendre accessible aux enfants. La France a ratifié la CIDE en 1990 et a créé le Défenseur des enfants en 2000. Le Défenseur des enfants a fusionné avec d'autres institutions en 2011 pour former le Défenseur des droits, renforçant ses pouvoirs d'intervention. La loi de 2007 sur la protection de l'enfance a structuré le dispositif mais a été critiquée pour son "profilialisme". La loi de 2016 a opéré un changement de paradigme en plaçant l'enfant au centre du dispositif. La scolarisation des enfants placés présente des défis importants, notamment en termes de continuité du parcours et d'adaptation aux besoins spécifiques. La pluridisciplinarité, l'articulation des acteurs et la concertation sont essentielles pour garantir les droits de l'enfant.

Citations clés:

• "Tous les enfants sont protégés et sont à protéger et il n'y a pas des enfants plus protégés que d'autres."
• "L'approche par les droits n'a jamais été acquise en protection de l'enfance et elle est encore un combat à défendre."
• "Les droits de l'enfance c'est ici et maintenant dans tous les lieux où sont où c'est où sont les enfants y compris quand ils sont placés bien évidemment."
• "La question des droits de l'enfant c'est ni plus ni moins qu'une manière de répondre aux besoins des enfants."
• "Les solutions elles sont jamais évidentes elles sont très propres à des contextes et en particulier à des territoires."
• Recommandations:
• Poursuivre et renforcer les efforts de concertation entre les acteurs de la protection de l'enfance, y compris l'Éducation nationale.
• Développer des solutions innovantes pour garantir la continuité du parcours scolaire des enfants placés, en s'adaptant aux moments clés de leur scolarité.
• Sensibiliser l'ensemble de la communauté éducative aux droits de l'enfant et aux besoins spécifiques des enfants placés.

Conclusion:

Le discours de Marie Derain de Vaucresson souligne l'importance cruciale de l'approche par les droits de l'enfant dans le contexte de la protection de l'enfance.

La scolarisation, en tant que droit fondamental, joue un rôle majeur dans le développement et l'épanouissement de tous les enfants, y compris ceux en situation de placement.

L'engagement collectif et la recherche de solutions concertées sont indispensables pour garantir le respect de leurs droits et leur offrir les meilleures chances de réussite.

Chronologie des événements clés XVIIe siècle:

Emergence de la protection des enfants avec Vincent de Paul qui met en place un système d'accueil pour les enfants abandonnés. XIXe siècle:

• 1842: Loi sur le travail des enfants. Mise en place d'un âge minimum (8 ans) pour travailler dans les mines et limitation du temps de travail.
• 1882: Loi sur l'obligation d'instruction de 6 à 13 ans.
• XXe siècle:
• 1924: Janusz Korczak, médecin polonais, développe une approche pédagogique centrée sur la participation des enfants et rédige une première déclaration des droits de l'enfant.
• 1959: La Déclaration des droits de l'enfant de 1924 est renforcée.
• 1979: Année internationale de l'enfant, impulsée par l'ONU.
• 20 novembre 1989: Adoption de la Convention internationale des droits de l'enfant (CIDE) à l'unanimité.
• XXIe siècle:
• Août 1990: Ratification de la CIDE par la France.
• 2000: Création du Défenseur des enfants en France.
• 2007: Loi relative à la protection de l'enfance. Organisation de la protection de l'enfance autour des conseils départementaux et des cellules de recueil d'informations préoccupantes (CRIP).
• 2011: Création du Défenseur des droits, intégrant le Défenseur des enfants. Renforcement des pouvoirs d'intervention en matière de défense des droits des enfants.
• 2012: Interdiction du mariage avant 18 ans en France.
• 2016: Loi relative à la protection de l'enfant. Changement de paradigme : l'approche par les droits de l'enfant et la satisfaction de ses besoins sont placées au centre du dispositif.
• 2022: Loi relative à la protection des enfants (loi Taquet). Remobilisation de l'État dans la protection de l'enfance et consolidation du dispositif.

Personnages principaux

• Vincent de Paul (1581 - 1660): Prêtre catholique français connu pour ses œuvres de charité. Il a mis en place au XVIIe siècle un système d'accueil pour les enfants abandonnés, marquant une étape importante dans l'histoire de la protection de l'enfance.
• Janusz Korczak (1878 - 1942): Médecin et pédagogue polonais. Précurseur dans l'approche pédagogique centrée sur la participation des enfants. Il a rédigé une première déclaration des droits de l'enfant en 1924. Il a été déporté et assassiné à Treblinka avec les enfants de son orphelinat.
• Claire Brisset (née en 1948): Première Défenseure des enfants en France de 2000 à 2006. Elle a joué un rôle important dans la promotion et la défense des droits de l'enfant, notamment en obtenant l'interdiction du mariage avant 18 ans.
• Dominique Bodis: Défenseur des droits de 2011 à 2014. A succédé à Claire Brisset et a poursuivi son action en matière de défense des droits des enfants.
• Marie Derain de Vaucresson: Défenseure des enfants adjointe de Dominique Bodis de 2011 à 2014. Experte en droits de l'enfant et de la protection de l'enfance. Elle a notamment travaillé sur la question de la scolarisation des enfants placés et des mineurs non accompagnés.
• Adrien Taquet: Secrétaire d'État chargé de l'Enfance et des Familles de 2019 à 2022. Il a porté la loi relative à la protection des enfants de 2022, visant à remobiliser l'État dans la protection de l'enfance.
• Agès Jean duuc: Directrice de la recherche à l'ENPJJ. A collaboré avec Marie Derain de Vaucresson sur des concertations concernant la protection de l'enfance et la scolarisation des enfants placés.
• Laurence Rossignol: Ministre des Familles, de l'Enfance et des Droits des femmes de 2016 à 2017. A conduit des travaux de concertation sur la protection de l'enfance en 2016.
• Jean-Pierre Rosenczveig: Magistrat et expert en justice des mineurs. Membre de la "bande des quatre" avec les anciennes Défenseures des enfants, il s'engage pour la défense des droits des enfants.
• Dominique Houf: Philosophe du droit et éducateur, il a critiqué l'éloignement de la protection de l'enfance d'une philosophie des droits de l'enfant.
• Gabrielle: Personne non identifiée dans le texte, mais qui semble intervenir après Marie Derain de Vaucresson et aborder la question de la concertation et de la co-construction dans la protection de l'enfance.

L'enfant au cœur du système: Droits de l'enfant et protection de l'enfance

Résumé du contenu

Ce document retranscrit une intervention de Marie Derain de Vaucresson, ancienne Défenseure des enfants, lors d'une journée de réflexion sur les droits de l'enfant et la protection de l'enfance.

Elle y aborde l'histoire de la protection de l'enfance, l'évolution de l'approche par les droits, les grands principes de la Convention internationale des droits de l'enfant (CIDE) et son application en France, et les défis de la scolarisation des enfants placés.

L'intervention met en lumière l'importance de la CIDE, un traité international qui impose aux États des obligations en matière de promotion et de protection des droits de l'enfant.

En France, la création du Défenseur des enfants puis du Défenseur des droits a permis de renforcer l'accès au droit des enfants et de leur garantir une meilleure protection.

La loi de 2007, qui structure la protection de l'enfance, est analysée avec ses forces et ses faiblesses.

L'accent est mis sur l'importance d'une prise en compte des besoins de l'enfant, de la stabilité de son parcours et de l'adaptation du dispositif à sa situation.

Enfin, Marie Derain de Vaucresson souligne les défis spécifiques de la scolarisation des enfants placés, notamment la continuité du parcours scolaire, les ruptures liées aux placements successifs, et l'importance de l'apprentissage de la lecture et de la socialisation.

Elle appelle à une pluridisciplinarité, une articulation des acteurs et une concertation au plus près des réalités des territoires pour répondre au mieux aux besoins des enfants.

Quiz

Instructions: Répondez aux questions suivantes en 2-3 phrases.

• Pourquoi Marie Derain de Vaucresson s'oppose-t-elle à l'expression "enfants protégés"?
• Quelles sont les deux grandes étapes de l'évolution de la protection des enfants au XIXème siècle ?
• Qui est Janusz Korczak et quel est son rôle dans l'histoire des droits de l'enfant ?
• Pourquoi la Convention internationale des droits de l'enfant (CIDE) est-elle importante ?
• Quelle est la différence entre une déclaration et une convention en droit international ?
• Quels sont les deux grands principes de la CIDE mis en avant par Marie Derain de Vaucresson ?
• Quel est le rôle du Défenseur des enfants / du Défenseur des droits en France ?
• Quelles sont les principales critiques adressées à la loi de 2007 sur la protection de l'enfance ?
• En quoi la loi de 2016 marque-t-elle un changement de paradigme dans l'approche de la protection de l'enfance ?
• Quels sont les défis spécifiques de la scolarisation des enfants placés ?

Corrigé du quiz

• Marie Derain de Vaucresson s'oppose à l'expression "enfants protégés" car elle sous-entend qu'il existe des catégories d'enfants plus protégés que d'autres, ce qui est contraire à l'esprit de la Convention des droits de l'enfant qui affirme que tous les enfants doivent être protégés.
• Les deux grandes étapes de l'évolution de la protection des enfants au XIXème siècle sont la loi de 1842 sur le travail des enfants qui fixe un âge minimum pour travailler dans les mines et limite le temps de travail, et la loi de 1882 qui rend l'instruction obligatoire pour les enfants de 6 à 13 ans.
• Janusz Korczak est un médecin et pédagogue polonais qui a développé une approche novatrice de l'éducation des enfants, basée sur la participation et l'autonomie. Il est considéré comme un pionnier des droits de l'enfant et a joué un rôle important dans la rédaction de la première déclaration des droits de l'enfant en 1924.
• La CIDE est importante car c'est un traité international juridiquement contraignant qui impose aux États des obligations en matière de promotion et de protection des droits de l'enfant. Elle garantit un socle commun de droits à tous les enfants du monde et constitue un cadre de référence pour les politiques publiques en faveur de l'enfance.
• En droit international, une déclaration est un texte qui exprime des intentions ou des principes, mais qui n'a pas force obligatoire pour les États. Une convention, en revanche, est un traité international qui crée des obligations juridiques pour les États qui l'ont ratifié.
• Les deux grands principes de la CIDE mis en avant par Marie Derain de Vaucresson sont le meilleur intérêt de l'enfant et la non-discrimination.
• Le Défenseur des enfants / du Défenseur des droits est une institution indépendante qui a pour mission de promouvoir et de défendre les droits de l'enfant en France. Il peut intervenir auprès des pouvoirs publics et des institutions pour faire respecter les droits des enfants et proposer des solutions aux problèmes qu'ils rencontrent.
• La loi de 2007 est critiquée pour son manque de prise en compte des besoins spécifiques des enfants placés, pour l'absence de garanties de sorties sécurisées du dispositif de protection de l'enfance, et pour les parcours chaotiques des enfants qui en découlent.
• La loi de 2016 marque un changement de paradigme dans l'approche de la protection de l'enfance en plaçant l'enfant au cœur du système. Elle met l'accent sur la satisfaction des besoins de l'enfant, la reconnaissance de ses droits, et la recherche de son meilleur intérêt dans toutes les décisions qui le concernent.
• Les défis spécifiques de la scolarisation des enfants placés sont la continuité du parcours scolaire souvent interrompue par les placements successifs, le risque de retard scolaire lié aux ruptures et aux absences, et la nécessité d'une prise en charge adaptée aux besoins spécifiques de ces enfants.
• Questions pour une dissertation
• En quoi l'histoire de la protection de l'enfance en France reflète-t-elle l'évolution de la place de l'enfant dans la société ?
• Comment concilier le principe de protection de l'enfant avec celui de sa participation dans la mise en œuvre de ses droits ?
• Quels sont les obstacles à la pleine application de la Convention internationale des droits de l'enfant en France ?
• Comment l'école peut-elle mieux répondre aux besoins spécifiques des enfants placés et contribuer à leur réussite scolaire ?
• Quel est le rôle de la société civile dans la promotion et la protection des droits de l'enfant ?

Glossaire des termes clés

• Convention internationale des droits de l'enfant (CIDE): Traité international adopté en 1989 qui définit l'ensemble des droits civils, politiques, économiques, sociaux et culturels de tous les enfants.
• Protection de l'enfance: Ensemble des mesures prises par les pouvoirs publics pour garantir la sécurité, le bien-être et le développement des enfants en danger ou en risque de l'être.
• Placement d'enfant: Décision judiciaire ou administrative qui confie la garde d'un enfant à une personne ou à un service spécialisé, lorsque sa famille n'est pas en mesure d'assurer sa protection.
• Aide sociale à l'enfance (ASE): Service du Conseil départemental chargé de mettre en œuvre la politique de protection de l'enfance au niveau local.
• Cellule de recueil des informations préoccupantes (CRIP): Service chargé de recueillir, d'analyser et de transmettre aux autorités compétentes les informations préoccupantes concernant des enfants en danger ou en risque de l'être.
• Défenseur des enfants / Défenseur des droits: Institution indépendante chargée de promouvoir et de défendre les droits de l'enfant en France.
• Intérêt supérieur de l'enfant / Meilleur intérêt de l'enfant: Principe fondamental de la CIDE qui impose de prendre en considération l'intérêt de l'enfant dans toutes les décisions qui le concernent.
• Non-discrimination: Principe qui interdit toute distinction, exclusion ou restriction fondée sur des motifs tels que la race, la couleur, le sexe, la langue, la religion, l'opinion politique ou toute autre opinion, l'origine nationale ou sociale, la fortune, la naissance ou toute autre situation.
• Participation de l'enfant: Droit de l'enfant à exprimer son opinion sur les décisions qui le concernent et à être entendu.
• Transversalité: Principe qui impose de prendre en compte les droits de l'enfant dans tous les domaines de la vie sociale et dans toutes les politiques publiques.
• FAQ : Droits de l'enfant et protection de l'enfance

1. Quelle est la différence entre l'approche traditionnelle de la protection de l'enfance et l'approche par les droits de l'enfant ?

L'approche traditionnelle de la protection de l'enfance, apparue au 17ème siècle, se focalisait sur une vision sanitaire et sociale, visant à assurer une force de travail saine. L'approche par les droits de l'enfant, issue de la Convention internationale des droits de l'enfant de 1989, place l'enfant au centre et reconnaît ses droits fondamentaux, notamment le droit à la participation, à la non-discrimination et à vivre dans un environnement familial stable.

2. Pourquoi la terminologie "enfant protégé" est-elle critiquée ?

L'expression "enfant protégé" peut laisser entendre qu'il existe des catégories d'enfants plus protégés que d'autres, ce qui est contraire à l'esprit de la Convention des droits de l'enfant. Tous les enfants, sans exception, doivent être protégés et avoir leurs droits respectés.

3. Quels sont les principaux apports de la Convention internationale des droits de l'enfant ?

La Convention de 1989 a introduit des principes fondamentaux pour la protection de l'enfance, notamment :

Le meilleur intérêt de l'enfant : toute décision concernant un enfant doit prendre en compte son meilleur intérêt. La non-discrimination : tous les enfants doivent avoir les mêmes droits, sans distinction de sexe, d'origine, de religion, de handicap, etc. Le droit à la participation : les enfants doivent pouvoir exprimer leur opinion et être associés aux décisions qui les concernent. Le droit de vivre dans une famille : l'enfant doit pouvoir grandir dans un environnement familial stable et aimant.

4. Quel est le rôle du Défenseur des droits en matière de protection de l'enfance ?

Le Défenseur des droits, institution indépendante créée en 2011, a pour mission de promouvoir et de défendre les droits de l'enfant. Il peut intervenir pour signaler des manquements au respect de ces droits, enquêter sur des situations problématiques et formuler des recommandations aux autorités compétentes. Il dispose de pouvoirs renforcés d'intervention, comme l'accès aux centres de rétention administrative.

5. Quels sont les principaux défis de la scolarisation des enfants placés ?

Les enfants placés sont confrontés à des difficultés accrues en matière de scolarisation :

Ruptures de parcours : les changements fréquents de lieu de placement entrainent des interruptions de scolarité et des retards d'apprentissage.

Manque de continuité et de stabilité : l'adaptation à un nouvel environnement scolaire et la reconstruction de liens avec les enseignants et les camarades de classe peuvent être difficiles. Absence de suivi individualisé : les besoins spécifiques des enfants placés en matière d'apprentissage ne sont pas toujours pris en compte.

6. Comment améliorer la prise en charge scolaire des enfants placés ?

Plusieurs pistes peuvent être envisagées pour améliorer la scolarisation des enfants placés :

Assurer la continuité du parcours scolaire : faciliter les transferts de dossiers scolaires, organiser des accompagnements individualisés lors des changements d'établissement, mettre en place des dispositifs de soutien pédagogique adaptés.

Favoriser la stabilité des placements : limiter les changements de lieu de placement, privilégier les solutions d'accueil durables. Sensibiliser les professionnels de l'éducation : former les enseignants aux besoins spécifiques des enfants placés, développer des partenariats entre l'Éducation nationale et les services de protection de l'enfance.

Encourager la participation des enfants : les associer aux décisions concernant leur scolarité, leur donner la parole pour exprimer leurs difficultés et leurs besoins.

7. Quelle est l'importance de la concertation entre les différents acteurs de la protection de l'enfance ?

La protection de l'enfance nécessite une approche pluridisciplinaire et une collaboration étroite entre les différents acteurs concernés (Éducation nationale, santé, justice, services sociaux, associations...). La concertation permet d'échanger des informations, de coordonner les actions et de construire des solutions adaptées aux besoins des enfants sur chaque territoire.

8. Quel est le rôle de la société civile dans la protection de l'enfance ?

La protection de l'enfance est une responsabilité collective qui engage l'ensemble de la société. Les citoyens, les associations et les organisations non gouvernementales peuvent jouer un rôle important en :

• Sensibilisant l'opinion publique aux droits de l'enfant.
• Soutenant les familles en difficulté.
• Accompagnant les enfants placés.
• Participant à la mise en œuvre des politiques de protection de l'enfance.
• L'engagement de tous est essentiel pour garantir le respect des droits de l'enfant et son épanouissement dans un environnement protecteur et bienveillant.

https://www.youtube.com/watch?v=vDwVm2l8AbY

Points forts de la vidéo avec timestamps

• Introduction et contexte (0:00-2:00): La vidéo commence par une introduction de Marie Derain de Vaucresson, ancienne Défenseure des enfants, qui souligne l'importance de la protection de l'enfance et rappelle que tous les enfants doivent être protégés. Elle insiste sur le fait que le terme "enfants protégés" est une erreur stratégique car il laisse entendre qu'il existe des enfants qui ne méritent pas d'être protégés.
• Histoire de la protection de l'enfance (2:00-10:00): La vidéo retrace l'histoire de la protection de l'enfance en France, en commençant par l'apparition des premières mesures au 17ème siècle.
  • Travail des enfants (2:00-4:00): Marie Derain de Vaucresson évoque la loi de 1842 qui fixe un âge minimum pour le travail des enfants dans les mines et limite leur temps de travail.
  • Obligation d'instruction (4:00-5:00): La loi de 1882 sur l'obligation d'instruction est ensuite abordée, marquant une étape importante dans la prise en compte des besoins éducatifs des enfants.
  • Janusz Korczak et l'approche pédagogique (5:00-10:00): La vidéo met en lumière le rôle de Janusz Korczak, un médecin polonais qui a développé une approche pédagogique centrée sur la participation des enfants. Korczak a mis en place un système éducatif où les enfants étaient encouragés à prendre des responsabilités et à participer à la vie de l'orphelinat. Il a notamment créé un journal des enfants, un tribunal des enfants pour gérer les conflits et a impliqué les enfants dans l'aménagement des lieux et la création des règles de vie. Son destin tragique pendant la Seconde Guerre mondiale est également évoqué.
• La Convention internationale des droits de l'enfant (CIDE) (10:00-16:00): La vidéo s'attarde ensuite sur la CIDE adoptée en 1989, un texte fondamental qui reconnaît les droits spécifiques des enfants et impose aux États des obligations pour les garantir.
  • Principes clés de la CIDE (10:00-12:00): Les principes clés de la CIDE sont expliqués, notamment la non-discrimination, le meilleur intérêt de l'enfant et l'obligation pour les États de mettre en place un mécanisme de défense des droits de l'enfant.
  • Création du Défenseur des enfants en France (12:00-16:00): La création du Défenseur des enfants en France en 2000 est présentée comme une conséquence directe de la CIDE. Le rôle et les missions du Défenseur des enfants sont expliqués, ainsi que l'évolution de l'institution avec la création du Défenseur des droits en 2011, qui a élargi ses pouvoirs d'intervention. L'importance des délégués du Défenseur des enfants pour faciliter l'accès au droit est également soulignée.
• Evolution de la protection de l'enfance en France (16:00-23:00): La vidéo aborde les différentes lois qui ont marqué l'évolution de la protection de l'enfance en France, notamment la loi de 2007, la loi de 2016 et la loi de 2022.
  • Loi de 2007 (18:00-20:00): La loi de 2007 est analysée avec ses points forts, comme la structuration de la protection de l'enfance et la mise en place de projets pour les enfants placés, mais aussi ses limites, notamment la tendance au "profilialisme" et l'absence de garantie de sorties sécurisées du dispositif de protection.
  • Loi de 2016 (20:00-21:00): La loi de 2016 marque un changement de paradigme en plaçant l'enfant au centre du dispositif de protection et en affirmant l'importance de ses besoins.
  • Travaux du Défenseur des enfants (21:00-23:00): La vidéo présente les travaux menés par Marie Derain de Vaucresson lorsqu'elle était Défenseure des enfants, notamment son rapport de 2011 sur les enfants placés. Ce rapport met en lumière les difficultés rencontrées par ces enfants en matière de scolarité, notamment les ruptures dans leur parcours et l'importance de la continuité éducative.
• Conclusion (23:00-24:00): La vidéo conclut en soulignant l'importance de la pluridisciplinarité, de la concertation et de la co-construction pour répondre aux besoins des enfants et garantir leurs droits. L'initiative du rectorat d'organiser une journée de réflexion sur ces questions est saluée et présentée comme un exemple à suivre.

J'espère que cette liste des points forts de la vidéo avec timestamps vous sera utile. N'hésitez pas à me poser d'autres questions si vous souhaitez plus de précisions sur un point en particulier.

for - commons - new definition - pathological conservatism - new definition - benign conservatism - new definition - beneficial conservatism - adjacency - citizen assemblies - cosmolocal - community organization - horizontal and vertical decision-making as cosmolocal - Fair Share Commons - FSC - pathological conservatism - hypocrisy of modern conservatism that cannot acknowledge first nations - TPF as a vehicle for citizen assembly in each ward and district of a city - to - Youtube - Trump won, now what? - Roger Hallam - to - Substack article - - A global history of societal regulation - metacrisis, polycrisis - role of the commons and cosmolocal coordination - Michel Bauwens

adjacency - between - citizen assemblies - cosmolocal - community organization - citizen assemblies - horizontal and vertical - Fair Share Commons - FSC - town anywhere - TPF - one per city ward or district - progress traps - wicked problem - pathological conservatism - deep conservatism - ECOnomy is a subset of ECOlogy - Modernity has many forms of shallow, pathological conservatism - Indigenous and first nations peoples practice deep, beneficial conservatism - adjacency relationship - One of the biggest progress traps is pathological conservatism when - a technology has become popular and ubiquitous but an unintended consequence becomes exposed - In that case, incumbents who profit from the established supply chain will defend it at great cost, even if the harm it causes becomes increasingly obvious. - They will do this until it reaches a point that the harm is so great that it can no longer be defended. - Often, great harm is done before that point is reached, if it is reached. - Misinformation, gaslighting and fascism can emerge as a form of pathological conservatism in an attempt to preserve the harmful aspect of the status quo. - Fossil fuels, internal combustion engines and the climate change they cause are an example of this, creating a wicked problem in which those trying to solve the problem are also contributing to it - Citizen assemblies are a bottom up response and counterweight to centralized power that is driving pathological conservatism - In contrast to the pathological conservatism, environmental awareness is a practice of benign and beneficial conservatism - the conservation of our natural environment - In fact, many who call themselves conservatives and nationalists are hypocritical because - if they went further in their conservativism logic, they would have to acknowledge the first nations people who came before them - The natural resources that were part of indigenous peoples lives for millenia that colonialists have built their entire fortune on represents even greater degree of conservatism, yet the hypocrisy is that - modern conservatives often cannot acknowledge this reality of a deeper form of conservatism as it threatens their false entitlement - This brings into question their claim of practicing conservatism - pathological conservatives act as if the ECOlogy is subordinate to the ECOnomy when in fact, the ECOnomy cannot exist without a functioning ECOlogy - citizen assemblies can be implemented in each ward and district of a large city - On top of these, Fair Share Commons and community cooperatives can be built as formal structures to drive specific projects - In order for participatory democracy to work effectively requires education on Deep Humanity and conflict resolution, otherwise risks low resiliency due to internal conflicts and derailment of vision - In order to scale, it requires both horizontal and vertical components or organization. This implies a cosmolocal strategy: - horizontal decision-making with local group is local, whilst - vertical decision-making with non-local groups based on broader issues is cosmo - A global Tipping Point Festival that employs social tipping point theory to emerge a global network of citizen assemblies / commons assemblies / people's assemblies in each ward and district of a city to relocate healthy power back to the people

to - Youtube - Trump won, now what? - a love-based approach to replace power-based approach for dealing with fascism and polarization - Roger Hallam - https://hyp.is/wUDpaKsAEe-DM9fteMUtzw/www.youtube.com/watch?v=AiKWCHAcS7E - Substack article - A global history of societal regulation - metacrisis, polycrisis - role of the commons and cosmolocal coordination - Michel Bauwens - https://hyp.is/wlywbqkTEe-ROXfhSmA3bA/4thgenerationcivilization.substack.com/p/a-global-history-of-societal-regulation

:)

Variation with Latitude:

Earth is not a perfect sphere; it is an oblate spheroid, meaning it is slightly flattened at the poles and bulging at the equator. Gravity is stronger at the poles and weaker at the equator because: The poles are closer to the Earth's center (smaller radius, stronger gravitational pull). The equator experiences a greater outward centrifugal force due to Earth's rotation.

Constants :

Incorporate Earth's flattening and the effects of rotation.

SI system is absolute (gravitationally independent) as: Mass - in relation with Planck constant Time - in terms of vibration of isotope of Cesium Length - in terms of distance covered by Light All of which are independent of effect of gravity

Soit sur la doc du site ePoc ou ici, mais il faudrait dans cette partie expliquer ce que chaque terme veut dire lorsque qu'on paramètre les critères d'obtention d'un badge. Ça peut se faire sous forme de tableau car c'est assez long.

il faut fixer un terme " activités", "exercice" ou "questions" ?

dans le "panneau d'édition"

on a appelé ça "zone d'édition" au chapitre 1

on a appelé ça "panneau d'édition" au chapitre 1

idem remplacer par "activités"

"un ensemble d'activité" (c'est pas forcément une question)

je remplacerais par "apprenant"

remplacer "projet" par "parcours" car on explique que c'est pour créer un "parcours pédagogique tous ces éléments

Pour améliorer la lisibilité, mettre en gras "barre d'outils", "panneau de configuration" et "barre des composants"

Il faudrait aussi préciser qu'il s'affiche lorsque l'on clique sur un composant

Pourquoi ne pas remplacer par "espaces d'interaction" au lieu de "zones" ? Étant donné qu'un espace s'appelle "zone d'édition", le mot zone est déjà employé et ça peut créer de la confusion.

j'aurais appelé ça "une activité"

Module 1 - page 1 : "les zones d'interractions" : enlever un r et le s --> "les zones d'interaction". Idem page 2 "les zones d'interaction" avec 1 seul r.

Dans le titre de l'ePoc, j'aurais changé le verbe "produire" par "créer" par ce que produire me fait penser à de la fabrication. --> Donc ça deviendrait "Créer mon 1er ePoc" ou "Créer son 1er ePoc".

Partie "résumé" de la page de présentation : ajouter un retour à la ligne dès l'annonce des pré requis, à "Il est conseillé de télécharger..."

Local variables' declaration always occurs before it is used, the VM can resolve them at compile time, even in a simple single-pass compiler.

eLife Assessment

This important study presents a series of results aimed at uncovering the involvement of the endosomal sorting protein SNX4 in neurotransmitter release. While the evidence supporting the conclusions is solid, the molecular mechanisms remain unclear. This paper will be of interest to cell biologists and neurobiologists.

Reviewer #1 (Public review):

Summary:

In the work Josse Poppinga and collaborators addressed the synaptic function of Sortin-Nexin 4 (SNX4). Employing a newly-developed in vitro KO model, with live imaging experiments, electrophysiological recordings and ultrastructural analysis, the authors evaluate modifications in synaptic morphology and function upon loss of SNX4. The data demonstrate increased neurotransmitter release and alteration in synapse ultrastructure with higher number of docked vesicles and shorter AZ. The evaluation of presynaptic function of SNX4 is of relevance and tackles an open and yet unresolved question in the field of presynaptic physiology.

Strengths:

The sequential characterization of the cellular model is nicely conducted, and the different techniques employed are appropriate for the morpho-functional analysis of the synaptic phenotype and the derived conclusions on SNX4 function at presynaptic site. The authors succeeded in presenting a novel in vitro model that results in chronic deletion of SNX4 in neurons. A convincing sequence of experimental techniques are applied to the model to unravel the role of SNX4, whose functions in neuronal cells and at synapses are largely unknown. The understanding of the role of endosomal sorting at presynaptic site is relevant and of high interest in the field of synaptic physiology and on the pathophysiology of the many described synaptopathies that broadly result in loss of synaptic fidelity and quality control at release sites.

Weaknesses:

The flow of the data presentation is mostly descriptive with several consistent morphological and functional modifications upon SNX loss. The paper would benefit from a wider characterization that would allow to address the physiological roles of SNX4 at synaptic site and speculate on the underlying molecular mechanisms. The novel experiments on autophagy progression as well as spontaneous neurotransmission are well conducted, although do not assist for the explanation of the molecular mechanism underneath.

Comments on revisions:

Other implementations in the revised version are quite limited and would benefit from a more detailed presentation and description. i.e.: Sholl analysis in the new figure 1h, is presented with no definition of number of cells employed and standard deviations of the replication. The "simil" Sholl analysis performed on VAMP2 is still puzzling and some explanations on the reason for the constant value of VAMP2 fluorescent signal from less than 0 to 160 µm from the cell body is to be added. How is the increased number of active synapses explained? How is this related to shorter AZ and higher number of docked vesicles?

Reviewer #2 (Public review):

Summary:

SNX4 is thought to mediate recycling from endosomes back to the plasma membrane in cells. In this study, the authors demonstrate the increases in the amounts of transmitter release and the number of docked vesicles by combining genetics, electrophysiology and EM. They failed to find evidence for its role in synaptic vesicle cycling and endocytosis, which may be intuitively closer to the endosome function.

Strengths:

The electrophysiological data and EM data are in principle, convincing, though there are several issues in the study.

Weaknesses:

It is unclear why the increase in the amounts of transmitter release and docked vesicles happened in the SNX4 KO mice. In other words, it is unclear how the endosomal sorting proteins in the end regulate or are connected to presynaptic, particularly the active zone function.

Comments on revisions:

I am fine with revision in principle. the authors have addressed my concerns.

Reviewer #3 (Public review):

Summary:

The study aims to determine whether the endosomal protein SNX4 performs a role in neurotransmitter release and synaptic vesicle recycling. The authors exploited a newly generated conditional knockout mouse to allow them to interrogate SNX4 function. A series of basic parameters were assessed, with an observed impact on neurotransmitter release and active zone morphology. The work is interesting, however as things currently stand, the work is descriptive with little mechanistic insight. There are a number of places where some of the conclusions require further validation.

Strengths:

The strengths of the work are the state-of-the-art methods to monitor presynaptic function.

Weaknesses:

The weaknesses are the fact that the work is largely descriptive, with no mechanistic insight into the role of SNX4.

Comments on revisions:

The authors have addressed a couple of the more major concerns with the manuscript, however many of the original weaknesses remain. The primary weakness being the lack of mechanism. It is disappointing that real-time VAMP2 trafficking was not investigated, and the authors justification as to why the experiment was not performed was not convincing (especially since this is the approach that all other groups employ to examine SV cargo trafficking). In a number of instances "contractual constraints" are referred to as an explanation for not performing additional experiments. It was unclear whether this refers to licencing issues with the mouse line or the lack of personnel to perform the work. Regardless it still leaves this work as somewhat incomplete.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In the work: "Endosomal sorting protein SNX4 limits synaptic vesicle docking and release" Josse Poppinga and collaborators addressed the synaptic function of Sortin-Nexin 4 (SNX4). Employing a newly developed in vitro KO model, with live imaging experiments, electrophysiological recordings, and ultrastructural analysis, the authors evaluate modifications in synaptic morphology and function upon loss of SNX4. The data demonstrate increased neurotransmitter release and alteration in synapse ultrastructure with a higher number of docked vesicles and shorter AZ. The evaluation of the presynaptic function of SNX4 is of relevance and tackles an open and yet unresolved question in the field of presynaptic physiology.

Strengths:

The sequential characterization of the cellular model is nicely conducted and the different techniques employed are appropriate for the morpho-functional analysis of the synaptic phenotype and the derived conclusions on SNX4 function at presynaptic site. The authors succeeded in presenting a novel in vitro model that resulted in chronical deletion of SNX4 in neurons. A convincing sequence of experimental techniques is applied to the model to unravel the role of SNX4, whose functions in neuronal cells and at synapses are largely unknown. The understanding of the role of endosomal sorting at the presynaptic site is relevant and of high interest in the field of synaptic physiology and in the pathophysiology of the many described synaptopathies that broadly result in loss of synaptic fidelity and quality control at release sites.

We thank the reviewer for their positive evaluation of our manuscript.

Weaknesses:

The flow of the data presentation is mostly descriptive with several consistent morphological and functional modifications upon SNX loss. The paper would benefit from a wider characterization that would allow us to address the physiological roles of SNX4 at the synaptic site and speculate on the underlying molecular mechanisms. In addition, due to the described role of SNX4 in autophagy and the high interest in the regulation of synaptic autophagy in the field of synaptic physiology, an initial evaluation of the autophagy phenotype in the neuronal SNX4KO model is important, and not to be only restricted to the discussion section.

We thank the reviewer for their suggestions and agree that broader characterization would help us speculate on the underlying mechanism. To address this, we have conducted additional independent experiments investigating the role of SNX4 in neuronal autophagy, as suggested by this reviewer. These experiments are now included in the main figures and are no longer limited to the discussion section. Please see the detailed responses to this reviewer's recommendations below.

Reviewer #2 (Public Review):

Summary:

SNX4 is thought to mediate recycling from endosomes back to the plasma membrane in cells. In this study, the authors demonstrate the increases in the amounts of transmitter release and the number of docked vesicles by combining genetics, electrophysiology, and EM. They failed to find evidence for its role in synaptic vesicle cycling and endocytosis, which may be intuitively closer to the endosome function.

Strengths:

The electrophysiological data and EM data are in principle, convincing, though there are several issues in the study.

We thank the reviewer for their positive evaluation of our manuscript.

Weaknesses:

It is unclear why the increase in the amounts of transmitter release and docked vesicles happened in the SNX4 KO mice. In other words, it is unclear how the endosomal sorting proteins in the end regulate or are connected to presynaptic, particularly the active zone function.

We thank the reviewer for their suggestions and agree that further characterization would help to understand how endosomal sorting proteins regulate presynaptic neurotransmission. We have now added extra data on electrophysiological recordings clarifying SNX4’s role in the synapse. Please see the detailed responses to this reviewer's recommendations below.

Reviewer #3 (Public Review):

Summary:

The study aims to determine whether the endosomal protein SNX4 performs a role in neurotransmitter release and synaptic vesicle recycling. The authors exploited a newly generated conditional knockout mouse to allow them to interrogate the SNX4 function. A series of basic parameters were assessed, with an observed impact on neurotransmitter release and active zone morphology. The work is interesting, however as things currently stand, the work is descriptive with little mechanistic insight. There are a number of places where the data appear to be a little preliminary, and some of the conclusions require further validation.

Strengths:

The strengths of the work are the state-of-the-art methods to monitor presynaptic function.

We thank the reviewers for their positive evaluation of our manuscript.

Weaknesses:

The weaknesses are the fact that the work is largely descriptive, with no mechanistic insight into the role of SNX4. Further weaknesses are the absence of controls in some experiments and the design of specific experiments.

We thank the reviewer for their suggestions and agree that addition of extra control groups and experiments would strengthen interpretation of the observed phenotype. To address this, we have now performed experiments to investigate the miniature excitatory postsynaptic currents and added extra control groups such as overexpression of SNX4 on control background. In addition, we assessed SNX4-mediated neuronal autophagy as a potential molecular mechanism by which SNX4 affects synaptic output. Please see the detailed responses to this reviewers’ recommendations below.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) The characterization of the neurite outgrowth presented in Figure 1 is a necessary starting point for the characterization of the model and the interpretation of the following data. Being the analysis conducted at 21 DIV, a significant portion of the neurite tree is out of the analyzed field. Adding sholl analysis will better indicate the complexity of the that appears to be influenced by SNX4 loss in the representative images shown in Figure 1f.

We fully agree and have now performed a Sholl analysis of dendrite branches to investigate dendritic complexity. (Figure 1(i), page 2-3, line 86-88). SNX4 depletion does not affect dendrite length or dendrite branching.

(2) Analogously, the characterization of synapse number is of relevance for the interpretation of the data. For a better flow of the data, Figure 4 might be presented as Figure 2 (without the repetition of panel h in Figure 1). An explanation of how VAMP2 puncta are processed is necessary in the method section. A double labelling with a postsynaptic marker would allow trafficking organelles to be distinguished from mature synaptic contacts. Indeed, the analysis of VAMP2 intensity along neurite in mature 21DIV neurons should reveal peaks in the intensity profile that represent synaptic contacts. For unexplained reasons, the profile is rather flat in the two experimental groups. Focusing on axonal branches will surely result in a peaked profile for VAMP2 labelling.

We fully agree that the characterization of synapses is relevant for the interpretation of the data. We have now added a section in our Material and Methods how the VAMP2 puncta are processed (p14 line 517-520). Instead of labeling mature synapses using double labeling of VAMP2 and PSD95, we analyzed the number of active synapses in live neurons using SypHy (Fig. 3g). The reviewer is correct that the VAMP2 data presented in Fig 1I and Fig 4 is part of the same dataset and we have clarified this in the figure legend. In Fig 1I only the total number of VAMP2 puncta is plotted as a marker for synapse number, while in Fig 4 we assess VAMP2 as potential SNX4 sorting cargo (Ma et al., 2017). Because of these different aims, we prefer to keep the figures separate. The analysis of VAMP2 intensity along the distance of the soma is a Sholl analysis (Fig. 4d), represents the average VAMP2 intensity over distance from the soma of 35-41 neurons per group. In contrast to a line scan of a single neurite, this average profile lacks the peaks of individual synapses.

(3) Miniature excitatory postsynaptic currents recordings would strengthen the synaptic characterization and complement the electrophysiological recordings shown in Figure 2. Analyzing frequency and amplitude parameters would complement the data on the number of synaptic connections defined by the pre and postsynaptic colocalization puncta as suggested above and may support the data shown in Figure 3 g that suggests a decreased number of active synapses in SNX4-KO cells.

We fully agree that the characterization of miniature excitatory postsynaptic currents would strengthen the synaptic characterization and complement the other electrophysiological data. Therefore, we have now added additional experiments showing the mEPSCs (Fig. 2k-m, page 4) in SNX4 cKO neurons versus control. This data shows that the amplitude and frequency of spontaneous miniature EPSCs (mEPSCs) were not affected upon SNX4 depletion, consistent with a normal first evoked EPSC and RRP estimate. Furthermore, these data suggest that it is unlikely that the observed increase in neurotransmission is due to post-synaptic effects.

(4) Recordings on the first evoked response shown in Figure 2 b and quantified in Figures c and d suggest that SNX4 overexpression per se exerts some effect on the Amplitude and the Charge of the first evoked response. This is also evident in the supplementary Figure 2 with lower frequency trains. An additional experimental group, namely control+SNX4 is needed for the correct interpretation of the observed phenotype. The possibility that SNX4 per se exerts an effect on evoked transmission could be discussed in terms of putative mechanisms and interactions.

We thank the reviewer for their suggestion and agree that an additional experimental group (control + SNX4) would strengthen interpretation of the observed phenotype. We have now added a new experimental condition with overexpression of SNX4 on a control background (Supplementary Fig. 3, page 20). This data shows that the amplitude and charge of the first evoked response were not affected in control + SNX4 neurons compared to control, and no differences were detected in the response to the 40 Hz stimulation train (Supplementary Fig. 3a-e).  Together, these data suggest that SNX4 overexpression in itself does not affect the neurotransmission protocols studied in SNX4 cKO experiments.

(5) To correctly interpret the SyPhy experiments and exclude an effect of SNX silencing on SV recycling, it is suggested to repeat the experiments shown in Figure 3 in the absence and in the presence of bafilomycin. Indeed, the quantifications shown in Figure 3 d and f do not represent "release fraction" as stated (lines 139/140) but they rather refer to an average difference between release fraction and recovered fraction. With the use of bafilomycin, the comparison of the deltaFmax/deltaFNH4Cl with and without bafilomycin would enable the release fraction to be correctly evaluated and compared.

We appreciate the reviewer’s suggestion and agree on the importance of considering the impact of SV recycling when evaluating the released fraction. We agree that the presence of bafilomycin is critical to isolate the released component during stimulation. We have now rephrased this conclusion. To assess synaptic recycling in these assays, bafilomycin in not critically required and we show by multiple independent experiments, including SypHy and FM64 dye assays, that SV recycling is either not affected or the effect is too small to be detected by these methods.

(6) In the ultrastructural analysis, additional quantifications are needed to exclude the accumulation of endosome-like structures. It is not clear if, in the evaluation of total SV number (Figure 5e), the authors counted all vesicles or vesicles < 50nm. This has to be explained and additional quantification of # of SV < 50nm and # SV > 50nm is informative, taking into account the endosomal nature of SNX4. Indeed, although the average size of SV is not changed (fig. 5 d), the density of "bigger vesicle" may result from endosomal-like structure accumulation. An additional suggested quantification is on vesicle # SV > 80nm as previously reported in the cited references dealing with endosomal proteins and presynaptic morphology.

We fully agree that the characterization of vesicle size is important and that it was not clearly stated which vesicles were included in the total number of SV (Fig. 5e). We have now added this to the figure description. We have also added a histogram that contains the vesicle numbers of different bin sizes for SNX4 cKO synapses and control synapses (Supplementary Fig. 4, page 21) including # SVs > 80nm. (Whilst it seems that there are more “bigger” vesicles in the KO, further analysis revealed that this is mostly driven by one experiment and this effect is not consistent.)

(7) Due to the high scientific interest in presynaptic autophagy for SV recycling and degradation, and the paucity of experimental work assessing the proteins involved, an initial evaluation of the neuronal autophagy process (by western blot analysis and immunocytochemistry) for the characterization of the model will better support the paragraph in the discussion (lines 314-322) and contribute to future work in the field. Although very rare, autophagosomes quantification at presynaptic sites can also be performed from the already acquired images. A double membrane structure with the material inside is evident in the representative control image presented!

We appreciate the reviewer’s suggestion and agree that presynaptic autophagy is an interesting potential mechanism that would elaborate our current working model. To address the reviewers’ suggestion, we added multiple independent experiments to investigate basal autophagy markers such as ATG5 using western blot analysis, characterization of p62 levels using immunohistochemistry and performed additional morphometric analysis on the electron microscopy data (Supplementary Fig. 5). In SNX4 cKO neurons, there was no significant difference in P62 puncta numbers or P62 somatic intensity under basal conditions or after blocking autophagic P62 degradation by bafilomycin treatment, suggesting that autophagic flux remains normal. Also, no changes in total ATG5 protein levels were observed and ultrastructural analysis revealed no differences in the total number of autophagosomes. Collectively, these data indicate that SNX4 depletion does not impact the basal autophagic flux, ATG5 protein levels, or the number of autophagosomes.

Minor points:

(1) Dorrbaun et al. 2018 is missing from the reference list. In the legend to figure 1 there is an incorrect reference to Figure 6, rather than Figure 4.

We have now adjusted the figure legend and added the reference (page 16, line 604).

(2) Information on the construct employed for the rescue is missing. Is it a fluorescent tag construct? Representative images of the three autaptic neurons (control, KO, KO+SNX4) would nicely complement data presentation in Figure 2. 

We have now elaborated on this in material and methods section (p12, line 418-421). Unfortunately, we did not obtain pictures of autaptic neurons used for electrophysiology experiments.

Reviewer #2 (Recommendations For The Authors):

(1) Figure 2d and f are somewhat inconsistent. Total charges for the 1st EPSCs differ almost 2-fold in the same condition.

We appreciate the reviewer’s concern. The average EPSCs charge of the first evoked was 89, 122 and 57 pC for control, KO and rescued neurons respectfully. The average charge of the first pulse of 40Hz train was 41,58 and 32 pC for control, KO and rescued neurons respectfully, which is roughly 50% of the naïve response of the same cells. These trains were recorded after 2 or 3 other stimulation paradigms, which can have affected the total charge released in the 40Hz train. That said, the proportional difference between groups is high comparable, with a 37% increased average charge released in SNX4 cKO compared to control in the naïve response and 41% increased response in the first response of the 40 Hz train, and rescued cells show a 53% reduction in average released charge compared to control in the naïve response compared to a 44% reduction in the first response of the 40 Hz train. Although the absolute values differ between these readouts, we conclude that the biological comparison between groups is consistent.

(2) Figure 2h. This type of analysis has a drawback. See Neher (2015) for the problems associated with this analysis.

We fully agree with the reviewer’s comment. As noted in our discussion (page 9 line 285), while this analysis has its limitations, it can still provide an indication of the ready releasable pool.   

(3) The EPSC phenotype may be due to postsynaptic effects. This should be excluded by additional experiments (mEPSC analysis) or further clarification.

We fully agree that the characterization of miniature excitatory postsynaptic currents recording would strengthen the synaptic characterization and complement the electrophysiological recordings. Therefore, we have now added additional experiments showing the mEPSCs (Fig. 2k-m) in SNX4 cKO neurons versus control. This data shows that the amplitude and frequency of spontaneous miniature EPSCs (mEPSCs) were not affected upon SNX4 depletion, suggesting that it is unlikely that the observed increase in neurotransmission is due to post-synaptic effects.

(4) The increased number of docked vesicles observed in EM and the increased slope (vesicle recruitment, Figure 2h) are not consistent with each other. Maybe the definition of docked vesicles is unclear in this version of the manuscript.

As noted in our material & methods (page 15, line 547-548), SVs were defined as docked if there was no distance visible between the SV membrane and the active zone membrane. We have added the pixel size for clarification. Indeed, we do not observe an increase in release probability or first evoked response, which would correspond with an increased docked pool. However, we think that the increase in docked vesicles might contribute to an enhanced SV recruitment (see discussion).

(5) Figure 3: Vesicle cycling was monitored in only a limited condition. It is known that there are multiple pathways of vesicle cycling. Ideally, these pathways should be dissected. At least, the authors mention the possibility that they have missed some "positive" conditions.

We fully agree with the reviewer’s comment that vesicle recycling is complex with several parallel pathways involved. While we did not study individual endocytosis pathways, we used different assays covering various recycling pathways. The SypHy assay (Fig. 3c & f) combined with the 100 AP stimulation paradigm at room temperature predominantly addresses clathrin-mediated endocytosis. Additionally, the FM-64 dye assay at 37 degrees Celsius covers ultrafast endocytosis pathways as well as bulk endocytosis routes. Since neither assay showed major effects, we decided not to pursue further experiments focusing on different endocytosis pathways.

Reviewer #3 (Recommendations For The Authors):

Major points:

(1) Since all of the work here is culture-focussed, the in vivo phenotype is not as relevant, however the in vitro properties are. The incomplete Cre-dependent removal of SNX4 is concerning (especially axonal SNX4 levels identified via immunofluorescence), however, the main concern is that there was no profiling of the other molecular changes within these cultures. This is important, since there may be considerable alterations in the expression of a number of presynaptic proteins which may explain the observed phenotypes. Ideally, these cultures could have been profiled in an unbiased manner via mass spectrometry to identify potential changes in the presynaptic proteome, or at the very least the levels of key fusion molecules would have been assessed via Western blotting.

We thank the reviewer for their suggestion and agree that mass spectrometry would strengthen the interpretation of the observed phenotype. However, due to contractual constraints, we are unable to pursue a mass spectrometry follow-up experiment. We agree that characterizing key fusion molecules is of potential interest. Therefore, based on literature, we selected a likely candidate, VAMP2, which did not show any alterations in expression levels when knocking out SNX4. Given the previously described role of SNX4 in the degradation pathway, one would expect increased degradation of key fusion molecules if they are recycled by SNX4. Other literature indicates that reduced levels of key fusion molecules, such as synaptotagmin or SNAP-25 (Broadie et al., 1994; Washbourne et al., 2001) , do not mimic our phenotype.

(2) The experiments reported in Figure 2, in particular those in 2c and 2d, suggest that overexpression of SNX4 has a dominant-negative effect on neurotransmitter release. This is strongly supported by the supplementary data during a stimulus train (particularly the start point of the 5 Hz train in Supplementary Figure 2). Therefore, the perceived rescue of EPSC charge in Figure 2f, 2g may be a result of SNX4 inhibiting neurotransmitter release. A determination of the impact of SNX4 overexpression (and level of overexpression) in WT neurons is essential to show that this is a bonefide rescue, rather than a direct inhibition by SNX4 overexpression.

We thank the reviewer for their suggestion and agree that an additional experimental group (control + SNX4) would strengthen interpretation of the observed phenotype. We have now added a new experiment with an extra experimental condition with overexpression of SNX4 on a control background (Supplementary Fig. 3 page 21). This data shows that the amplitude and charge of the first evoked response were not affected in control + SNX4 neurons compared to control, and no differences were detected in the response to the 40 Hz stimulation train (Supplementary Fig. 3a-e).  Together, these data suggest that SNX4 overexpression in itself does not affect the neurotransmission protocols studied in SNX4 cKO experiments.

(3) The experiments in Figure 3 clearly reveal a lack of effect of SNX4 depletion on synaptic vesicle endocytosis. However, the assumption that synaptic vesicle recycling is unaffected is a little premature. The fact that the second evoked SypHy peak is significantly larger than the first (Figures 3c-e) suggests that more vesicles may be recycling in KO neurons. Furthermore, the FM dye experiments do not aid interpretation, since there may be insufficient time (10 min) for new vesicles to be generated from endosomal intermediates experiments. Therefore, to confirm an absence of effect on recycling, the authors could either 1) perform the same experiment as 3c, but with 4 stimulation trains (to drive the system harder to reveal any phenotype) or 2) repeat the FM dye experiment but increase the time between loading and unloading to 30 min.

We fully agree with the reviewers' comment that vesicle recycling is an important component to consider and is complex with several parallel pathways involved. We conducted multiple independent experiments covering the most significant recycling pathways. The SypHy assay (Fig. 3c & f) combined with the 100 AP stimulation paradigm at room temperature predominantly addresses clathrin-mediated endocytosis. Additionally, the FM-64 dye assay at 37 degrees Celsius covers ultrafast endocytosis pathways as well as bulk endocytosis routes. To further challenge the system and reveal recycling phenotypes, we included a second 100 AP stimulation in our SypHy assay. While only the increase of the second SypHy peak is significant, the absolute numbers do not differ much from the first peak (0,17 for control and 0,21 for KO second peak and 0,19 for control and 0,22 for KO first peak, Supplementary table1). We nevertheless do not see any effects on recycling after the second peak (mean decay time is 27 for control and 26 for KO Supplementary Table 1). A single 100 AP 40 Hz train depletes all the synchronous release (not shown) and most of the evoked charge (see Fig 2f), hence two of these trains with one minute recovery is already a very demanding protocol. Although increasing the time between loading and unloading to 30 minutes might uncover other recycling components, it has been shown that ultrafast endocytosis occurs within 30 seconds (Watanabe et al., 2013), suggesting that 10 minutes should provide enough time for synaptic vesicle recycling. This is also evident from the fact that we can significantly destain synapses loaded with FM dye by electrical stimulation (Fig 3j), indicating that synaptic vesicle recycling took place. Since neither assay showed major effects, we concluded that under these circumstances, synaptic recycling is not significantly affected. However, we cannot exclude the possibility that recycling deficits in SNX4 cKO neurons could be detected in other paradigms,

(4) There is no obvious effect on VAMP2 levels or location in SNX4 KO neurons (Figure 4). However, when one considers that SNX4 is proposed to have a role in VAMP2 trafficking, it is surprising that an experiment examining the live trafficking of VAMP2-SypHy was not performed. This would have revealed activity-dependent alterations that would have been missed by simply measuring VAMP2 expression and localization, and potentially provided a molecular explanation for the enhanced neurotransmitter release during a stimulus train.

We appreciate the reviewer’s suggestion and agree that it could be a valuable experiment However, overexpressing a VAMP2-pHluorin construct might obscure potential phenotypes related to VAMP2 trafficking. SNX4 is expected to be involved in VAMP2 recycling, even with activity-dependent changes. Mis-sorted VAMP2 would accumulate in acidic vesicles, which could be masked by the VAMP2-pHluorin construct. Similarly, mis-sorting of other SNX4 cargo, such as the transferrin receptor, has been identified through lysosomal degradation, as shown by Western blot analysis of expression levels of the endogenous protein. We did not detect any differences in endogenous levels of VAMP2 within 21 days of SNX4 deletion (Fig 4), indicating that SNX4-dependent endosome sorting is not essential for VAMP2 recycling.

(5) The morphological data in Figure 5 report a series of small changes in docked vesicles and active zone length. In many cases, significance is obtained due to synapses being used as the experimental n, and thus inflating the statistical power. When one considers that no significant effect was observed on evoked release (apart from during a stimulus train), it suggests that the number of docked vesicles does not alter release probability in this system (which the authors point out). Instead, they suggest that an increased supply of vesicles is responsible, via increased recruitment to RRP/releasable pool (but not via increased recycling). If this is the case, it should have been reflected as an increase in the evoked SypHy response in Fig 2c,d (which is borderline significant). What may help is to determine the morphological landscape immediately after a stimulus strain, since this is the only condition where enhanced release is observed, and thus provide a morphological correlate to the physiological data.

We fully agree with the reviewer’s suggestion that an ultrastructural characterization immediately after a stimulus train would be informative. Unfortunately, contract constraints prevent us from performing this experiment. For our ultrastructural morphological data, we treated synapses as individual experimental n since it is not possible to determine whether synapses in a micronetwork on one sapphire originate from the same neuron. We used 18 independent sapphires from 3 independent pups to ensure the technical and biological replication of our data and measuring independent neurons. We fully agree with the reviewers comment to be careful with ‘inflating the statistical power’ due to potential nesting effects when using synapses as experimental n. To mitigate the potential nesting effect of analyzing multiple synapses per neuron, the intracluster correlation (ICC) is calculated per variable and per nesting effect. If ICC was close to 0.1, indicating that a considerable portion of the total variance can be attributed to e.g. synapse or sapphire, multilevel analysis was performed to accommodate nested data (Aarts et al., 2014).

Minor points

(1) When a new mouse model is generated, it is usually accompanied by a thorough characterization of its properties. However, in this case, there was no information provided about the conditional SNX4 knockout mouse. This is surprising and at a minimum, the following should be provided a) the background strain, b) method of generation, c) the number of animals used to establish the colony, d) breeding strategy, e) backcrossing strategy, f) genotyping protocol.

We apologize that a thorough characterization of our novel mouse model was lacking and therefore added this to our material & methods section (page 11, line 377-391).

(2) There is a noticeable difference between WT and KO neurons during train stimulation in Figure 2f, however, this appears to be due to the fact that there is a far higher EPSC charge to begin with in KO neurons. Why is there such a disparity when there is no difference in response to single pulses (Figures 2b-d) or presynaptic plasticity (Figure 2e)?

We understand the reviewer’s concern. We excluded an outlier (3x SD) in the KO dataset that drove the initial far higher EPSC charge in the graph (was already excluded for the statistics, Supplementary table 1). The average charge of the first pulse of 40Hz train is 41 pC and for KO neurons 58 pC, which did not differ significantly.  These trains of Fig. 2f were recorded after 2 or 3 other stimulation paradigms, which can have affected the total charge released in the 40Hz train. That said, the proportional difference between groups is high comparable between Fig 2b-d and 2f, with a 37% increased average charge released in SNX4 cKO compared to control in the naïve response (Fig. 2d) and 41% increased response in the first response of the 40 Hz train (Fig. 2f), and rescued cells show a 53% reduction in average released charge compared to control in the naïve response compared to a 44% reduction in the first response of the 40 Hz train. Although the absolute values differ between these readouts, we conclude that the biological comparison between groups is consistent.

(3) Line 343-344 - "(Supplementary Figure 1a)" should be "(Figure 1a)".

We thank the reviewer for this comment and adjusted this in the manuscript.

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La vidéo "Meetup Loïc Donot" de Toulouse DataViz aborde plusieurs points clés. Voici un résumé détaillé avec les minutages :

0:00 - 5:00

Introduction et contexte : - La vidéo commence par une introduction sur Loïc Donot et son expérience en visualisation de données et cartographie à la Direction départementale des territoires (DDT31). - Loïc Donot explique l'importance de la visualisation de données dans la prise de décision et la gestion des territoires.

5:01 - 10:00

Projets de visualisation de données : - Loïc Donot présente plusieurs projets de visualisation de données qu'il a réalisés à la DDT31. - Il explique comment ces projets ont aidé à améliorer la compréhension des données et à faciliter la prise de décision.

10:01 - 15:00

Techniques et outils utilisés : - La vidéo explore les différentes techniques et outils de visualisation de données utilisés par Loïc Donot. - Il mentionne des logiciels comme QGIS et des langages de programmation comme Python pour créer des visualisations interactives.

15:01 - 20:00

Défis rencontrés et solutions : - Loïc Donot discute des défis qu'il a rencontrés lors de la réalisation de ses projets de visualisation de données. - Il partage des solutions et des bonnes pratiques pour surmonter ces défis et réussir des projets de visualisation de données.

20:01 - 23:22

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Pour plus de détails, vous pouvez regarder la vidéo sur YouTube.

La vidéo "L’abc du X,Y : rendre accessibles les données aux non experts grâce à la dataviz" de Toulouse DataViz aborde plusieurs points clés. Voici un résumé détaillé avec les minutages :

0:00 - 10:00 Introduction et contexte :

La vidéo commence par une introduction sur l'importance de la datavisualisation pour rendre les données accessibles aux non-experts.

Les présentateurs expliquent comment la datavisualisation peut transformer des données brutes en informations compréhensibles et exploitables.

10:01 - 20:00 Principes de base de la datavisualisation :

Les intervenants discutent des principes fondamentaux de la datavisualisation, tels que la clarté, la précision et l'efficacité.

Ils expliquent comment choisir les bons types de graphiques et de visualisations en fonction des données et du message à transmettre.

20:01 - 30:00 Outils et techniques de datavisualisation :

La vidéo présente différents outils et logiciels de datavisualisation, comme Tableau, Power BI et D3.js.

Les intervenants montrent comment utiliser ces outils pour créer des visualisations interactives et engageantes.

30:01 - 40:00 Études de cas et exemples concrets :

Des études de cas et des exemples concrets de datavisualisation sont présentés pour illustrer les bonnes pratiques.

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40:01 - 49:06 Conclusion et perspectives d'avenir :

La vidéo se termine par une réflexion sur l'avenir de la datavisualisation et son rôle croissant dans la prise de décision basée sur les données.

Les présentateurs encouragent les spectateurs à continuer à explorer et à expérimenter avec la datavisualisation pour améliorer la compréhension et l'utilisation des données.

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Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The study by McKim et al seeks to provide a comprehensive description of the connectivity of neurosecretory cells (NSCs) using a high-resolution electron microscopy dataset of the fly brain and several single-cell RNA seq transcriptomic datasets from the brain and peripheral tissues of the fly. They use connectomic analyses to identify discrete functional subgroups of NSCs and describe both the broad architecture of the synaptic inputs to these subgroups as well as some of the specific inputs including from chemosensory pathways. They then demonstrate that NSCs have very few traditional presynapses consistent with their known function as providing paracrine release of neuropeptides. Acknowledging that EM datasets can't account for paracrine release, the authors use several scRNAseq datasets to explore signaling between NSCs and characterize widespread patterns of neuropeptide receptor expression across the brain and several body tissues. The thoroughness of this study allows it to largely achieve it's goal and provides a useful resource for anyone studying neurohormonal signaling.

Strengths:

The strengths of this study are the thorough nature of the approach and the integration of several large-scale datasets to address short-comings of individual datasets. The study also acknowledges the limitations that are inherent to studying hormonal signaling and provides interpretations within the the context of these limitations.

Weaknesses:

Overall, the framing of this paper needs to be shifted from statements of what was done to what was found. Each subsection, and the narrative within each, is framed on topics such as "synaptic output pathways from NSC" when there are clear and impactful findings such as "NSCs have sparse synaptic output". Framing the manuscript in this way allows the reader to identify broad takeaways that are applicable to other model system. Otherwise, the manuscript risks being encyclopedic in nature. An overall synthesis of the results would help provide the larger context within which this study falls.

We agree with the reviewer and will replace all the subsection titles as suggested.

The cartoon schematic in Figure 5A (which is adapted from a 2020 review) has an error. This schematic depicts uniglomerular projection neurons of the antennal lobe projecting directly to the lateral horn (without synapsing in the mushroom bodies) and multiglomerular projection neurons projecting to the mushroom bodies and then lateral horn. This should be reversed (uniglomerular PNs synapse in the calyx and then further project to the LH and multiglomerular PNs project along the mlACT directly to the LH) and is nicely depicted in a Strutz et al 2014 publication in eLife.

We thank the reviewer for spotting this error. We will modify the schematic as suggested.

Reviewer #2 (Public review):

Summary:

The authors aim to provide a comprehensive description of the neurosecretory network in the adult Drosophila brain. They sought to assign and verify the types of 80 neurosecretory cells (NSCs) found in the publicly available FlyWire female brain connectome. They then describe the organization of synaptic inputs and outputs across NSC types and outline circuits by which olfaction may regulate NSCs, and by which Corazon-producing NSCs may regulate flight behavior. Leveraging existing transcriptomic data, they also describe the hormone and receptor expressions in the NSCs and suggest putative paracrine signaling between NSCs. Taken together, these analyses provide a framework for future experiments, which may demonstrate whether and how NSCs, and the circuits to which they belong, may shape physiological function or animal behavior.

Strengths:

This study uses the FlyWire female brain connectome (Dorkenwald et al. 2023) to assign putative cell types to the 80 neurosecretory cells (NSCs) based on clustering of synaptic connectivity and morphological features. The authors then verify type assignments for selected populations by matching cluster sizes to anatomical localization and cell counts using immunohistochemistry of neuropeptide expression and markers with known co-expression.

The authors compare their findings to previous work describing the synaptic connectivity of the neurosecretory network in larval Drosophila (Huckesfeld et al., 2021), finding that there are some differences between these developmental stages. Direct comparisons between adults and larvae are made possible through direct comparison in Table 1, as well as the authors' choice to adopt similar (or equivalent) analyses and data visualizations in the present paper's figures.

The authors extract core themes in NSC synaptic connectivity that speak to their function: different NSC types are downstream of shared presynaptic outputs, suggesting the possibility of joint or coordinated activation, depending on upstream activity. NSCs receive some but not all modalities of sensory input. NSCs have more synaptic inputs than outputs, suggesting they predominantly influence neuronal and whole-body physiology through paracrine and endocrine signaling.

The authors outline synaptic pathways by which olfactory inputs may influence NSC activity and by which Corazon-releasing NSCs may regulate flight. These analyses provide a basis for future experiments, which may demonstrate whether and how such circuits shape physiological function or animal behavior.

The authors extract expression patterns of neuropeptides and receptors across NSC cell types from existing transcriptomic data (Davie et al., 2018) and present the hypothesis that NSCs could be interconnected via paracrine signaling. The authors also catalog hormone receptor expression across tissues, drawing from the Fly Cell Atlas (Li et al., 2022).

Weaknesses:

The clustering of NSCs by their presynaptic inputs and morphological features, along with corroboration with their anatomical locations, distinguished some, but not all cell types. The authors attempt to distinguish cell types using additional methodologies: immunohistochemistry (Figure 2), retrograde trans-synaptic labeling, and characterization of dense core vesicle characteristics in the FlyWire dataset (Figure 1, Supplement 1). However, these corroborating experiments often lacked experimental replicates, were not rigorously quantified, and/or were presented as singular images from individual animals or even individual cells of interest. The assignments of DH44 and DMS types remain particularly unconvincing.

We thank the reviewer for this comment. We would like to clarify that the images presented in Figure 2 and Figure 1 Supplement 1 are representative images based on at least 5 independent samples. We will clarify this in the figure caption and methods. The electron micrographs showing dense core vesicle (DCV) characteristics (Figure 1 Supplement E-G) are also representative images based on examination of multiple neurons. However, we agree with the reviewer that a rigorous quantification would be useful to showcase the differences between DCVs from NSC subtypes. Therefore, we have now performed a quantitative analysis of the DCVs in putative m-NSC^DH44 (n=6), putative m-NSC^DMS (n=6) and descending neurons (n=4) known to express DMS. For consistency, we examined the cross section of each cell where the diameter of nuclei was the largest. We quantified the mean gray value of at least 50 DCV per cell. Our analysis shows that mean gray values of putative m-NSC^DMS and DMS descending neurons are not significantly different, whereas the mean gray values of m-NSC^DH44 are significantly larger. This analysis is in agreement with our initial conclusion.

Author response image 1.

The authors present connectivity diagrams for visualization of putative paracrine signaling between NSCs based on their peptide and receptor expression patterns. These transcriptomic data alone are inadequate for drawing these conclusions, and these connectivity diagrams are untested hypotheses rather than results. The authors do discuss this in the Discussion section.

We fully agree with the reviewer and will further elaborate on the limitations of our approach in the revised manuscript. However, there is a very high-likelihood that a given NSC subtype can signal to another NSC subtype using a neuropeptide if its receptor is expressed in the target NSC. This is due to the fact that all NSC axons are part of the same nerve bundle (nervi corpora cardiaca) which exits the brain. The axons of different NSCs form release sites that are extremely close to each other. Neuropeptides from these release sites can easily diffuse via the hemolymph to peripheral tissues that (e.g. fat body and ovaries) that are much further away from the release sites on neighboring NSCs. We believe that neuropeptide receptors are expressed in NSCs near these release sites where they can receive inputs not just from the adjacent NSCs but also from other sources such as the gut enteroendocrine cells. Hence, neuropeptide diffusion is not a limiting factor preventing paracrine signaling between NSCs and receptor expression is a good indicator for putative paracrine signaling.

Reviewer #3 (Public review):

Summary:

The manuscript presents an ambitious and comprehensive synaptic connectome of neurosecretory cells (NSC) in the Drosophila brain, which highlights the neural circuits underlying hormonal regulation of physiology and behaviour. The authors use EM-based connectomics, retrograde tracing, and previously characterised single-cell transcriptomic data. The goal was to map the inputs to and outputs from NSCs, revealing novel interactions between sensory, motor, and neurosecretory systems. The results are of great value for the field of neuroendocrinology, with implications for understanding how hormonal signals integrate with brain function to coordinate physiology.

The manuscript is well-written and provides novel insights into the neurosecretory connectome in the adult Drosophila brain. Some, additional behavioural experiments will significantly strengthen the conclusions.

Strengths:

(1) Rigorous anatomical analysis

(2) Novel insights on the wiring logic of the neurosecretory cells.

Weaknesses:

(1) Functional validation of findings would greatly improve the manuscript.

We agree with this reviewer that assessing the functional output from NSCs would improve the manuscript. Given that we currently lack genetic tools to measure hormone levels and that behaviors and physiology are modulated by NSCs on slow timescales, it is difficult to assess the immediate functional impact of the sensory inputs to NSC using approaches such as optogenetics. However, since l-NSC^CRZ are the only known cell type that provide output to descending neurons, we will functionally test this output pathway using different behavioral assays recommended by this reviewer.

The way Lizzie thinks is also an example of dissociating/derealization. She knows there's a bigger problem with the world but can't truly face what's going on without attempting to remove her personal emotions about it. This can be seen as a reflection of how some people view the climate change topic.

Lizzie's way of thinking helps her perceive the world on a larger scale. If humans as a whole develop this type of thinking, will we be able to fully understand the complexity of climate change?

eLife Assessment

This manuscript provides fundamental studies to help us better understand the effects of mutations in the presenilin-1 (PSEN1) gene on proteolytic processing of the amyloid precursor protein (APP). The authors provide compelling evidence using mutations in PSEN to understand what drives alternative substrate turnover with conclusive data and rigorous analysis. This deep mechanistic study provides a framework towards the development of small molecule inhibitors to treat Alzheimer's disease.

Reviewer #1 (Public review):

Summary:

Arafi et al. present results of studies designed to better understand the effects of mutations in the presenilin-1 (PSEN1) gene on proteolytic processing of the amyloid precursor protein (APP). This is important because APP processing can result in the production of the amyloid β-protein (Aβ), a key pathologic protein in Alzheimer's disease (AD). Aβ exists in various forms that differ in amino acid sequence and assembly state. The predominant forms of Aβ are Aβ40 and Aβ42, which are 40 and 42 amino acids in length, respectively. Shorter and longer forms derive from processive proteolysis of the Aβ region of APP by the heterotetramer β-secretase, within which presenilin 1 possesses the active site of the enzyme. Each form may become toxic if it assembles into non-natively folded, oligomeric, or fibrillar structures. A deep mechanistic understanding of enzyme-substrate interactions is a first step toward the design and successful use of small-molecule therapeutics for AD.

The key finding of Arafi et al. is that three PSEN mutations display unusual profiles of effects on Aβ production that have novel implications for the stalled E-S complex hypothesis. PSEN1 F386S is unique in that initial ε cleavage is not reduced compared with WT PSEN1; only certain trimming steps are deficient, results consistent with FLIM experiments that reveal stabilized E-S complexes only in Aβ-rich regions in the cell. In contrast, PSEN1 A431E and A434T display very little ε cleavage and therefore very little overall Aβ production, suggesting a limited role of Aβ in the pathogenesis of these two mutants and pointing to stalled E-S complexes as the common factor. For the biochemist, this may not be surprising, but in the context of understanding and treating AD, it is immense because it shifts the paradigm from targeting the results of γ-secretase action, viz., Aβ oligomers and fibrils, to targeting initial Aβ production at the molecular level. It is the equivalent of taking cancer treatment from simple removal of tumorous tissue to prevention of tumor formation and growth. Arafi et al. have provided us with a blueprint for the design of small-molecule inhibitors of γ-secretase. The significance of this achievement cannot be overstated.

Strengths and weaknesses:

The comprehensiveness and rigor of the study are notable. Rarely have I reviewed a manuscript reporting the results of so many orthogonal experiments, all of which support the authors' hypotheses, and of so many excellent controls. In addition, as found in clinical trial reports, the limitations of the study were discussed explicitly. None of these significantly affected the conclusions of the study.

Reviewer #2 (Public review):

Summary:

The work by Arafi et al. shows the effect of Familial Alzheimer's Disease presenilin-1 mutants on endoproteinase and carboxylase activity. They have elegantly demonstrated how some mutants alter each step of processing. Together with FLIM experiments, this study provides additional evidence to support their 'stalled complex hypotheses'.

Strengths:

This is a beautiful biochemical work. The approach is comprehensive.

Weaknesses:

(1) It appears that the purified g-secretase complex generates the same amount of Ab40 and Ab42, which is quite different in cellular and biochemical studies. Is there any explanation for this?

(2) It has been reported the Ab production lines from Ab49 and Ab48 can be crossed with various combinations (PMID: 23291095 and PMID: 38843321). How does the production line crossing impact the interpretation of this work?

(3) In Figure 5, did the authors look at the protein levels of PS1 mutations and C99-720, as well as secreted Ab species? Do the different amounts of PS1 full-length and PS1-NTF/CTF influence FILM results?

(4) It is interesting that both Ab40 and Ab42 Elisa kits detect Ab43. Have the authors tested other kits in the market? It might change the interpretation of some published work.

Author response:

Reviewer 2:

(1) It appears that the purified γ-secretase complex generates the same amount of Aβ40 and Aβ42, which is quite different in cellular and biochemical studies. Is there any explanation for this?

Roughly equal production of Aβ40 and Aβ42 is a phenomenon seen with purified enzyme assays, and the reason for this has not been identified. However, we suggest that what is meaningful in our studies is the relative difference between the effects of FAD-mutant vs. WT PSEN1 on each proteolytic processing step. All FAD mutations are deficient in multiple cleavage steps in γ-secretase processing of APP substrate, and these deficiencies correlate with stabilization of E-S complexes.

(2) It has been reported the Aβ production lines from Aβ49 and Aβ48 can be crossed with various combinations (PMID: 23291095 and PMID: 38843321). How does the production line crossing impact the interpretation of this work?

In the cited reports, such crossover was observed when using synthetic Aβ intermediates as substrate. In PMID 2391095 (Okochi M et al, Cell Rep, 2013), Aβ43 is primarily converted to Aβ40, but also to some extent to Aβ38. In PMID: 38843321 (Guo X et al, Science, 2024), Aβ48 is ultimately converted to Aβ42, but also to a minor degree to Aβ40. We have likewise reported such product line “crossover” with synthetic Aβ intermediates (PMID: 25239621; Fernandez MA et al, JBC, 2014). However, when using APP C99-based substrate, we did not detect any noncanonical tri- and tetrapeptide co-products of Aβ trimming events in the LC-MS/MS analyses (PMID: 33450230; Devkota S et al, JBC, 2021). In the original report on identification of the small peptide coproducts for C99 processing by γ-secretase using LC-MS/MS (PMID: 19828817; Takami M et al, J Neurosci, 2009), only very low levels of noncanonical peptides were observed. In the present study, we did not search for such noncanonical trimming coproducts, so we cannot rule out some degree of product line crossover.

(3) In Figure 5, did the authors look at the protein levels of PS1 mutations and C99-720, as well as secreted Aβ species? Do the different amounts of PS1 full-length and PS1-NTF/CTF influence FILM results?

This is a good question. Our preliminary investigation by Western Blot shows no correlation between C99 and PSEN1 expressions and FLIM results, but we will fully address the concern in our point-by-point responses submitted with a revised manuscript. 

(4) It is interesting that both Aβ40 and Aβ42 Elisa kits detect Aβ43. Have the authors tested other kits in the market? It might change the interpretation of some published work.

We have not tested other ELISA kits. In light of our findings, it would be a good idea for other investigators to test whatever ELISAs they use for specificity vis-à-vis Aβ43.

拜伦与希腊独立战争：一位诗人的革命情怀

拜伦，这位19世纪英国最伟大的浪漫主义诗人之一，不仅以其才华横溢的诗歌闻名于世，更因其对希腊独立战争的热忱参与而成为一个传奇人物。

为什么是希腊？

• 古典文化的传承： 古希腊文明是西方文明的摇篮，其民主思想、哲学艺术对欧洲产生了深远影响。拜伦作为一位饱学之士，深受希腊古典文化的熏陶，对希腊有着特殊的感情。
• 民族解放的呼声： 19世纪的欧洲，民族主义思潮高涨。希腊人在奥斯曼帝国的长期压迫下，掀起了争取独立的斗争，这与拜伦心中对自由、平等的追求不谋而合。
• 浪漫主义情怀： 拜伦的诗歌充满浪漫主义色彩，他歌颂自由、爱情、反抗精神。希腊独立战争正是这种浪漫主义精神的体现，自然深深吸引了拜伦。

拜伦在希腊做了什么？

• 提供物资和资金： 拜伦不仅用自己的诗歌为希腊独立战争宣传，还倾尽家产，购买武器、粮食等物资，资助希腊的反抗军。
• 亲自参战： 拜伦甚至亲自前往希腊，加入了希腊的军队，与希腊人民并肩作战。
• 提升国际影响力： 拜伦在欧洲享有盛名，他的参与使得更多的欧洲人关注到希腊的独立战争，并给予了希腊革命者道义上的支持。

拜伦的逝世

不幸的是，拜伦在希腊因病去世。但他短暂而辉煌的一生，为希腊的独立事业做出了巨大的贡献。他的死讯传遍欧洲，激起了人们对希腊革命的同情和支持，加速了希腊独立进程。

拜伦成为希腊民族英雄的原因

• 无私奉献： 拜伦不计个人安危，为希腊独立事业倾尽所有。
• 国际影响力： 拜伦的参与，提升了希腊独立战争的国际知名度。
• 浪漫主义形象： 拜伦的诗歌和他在希腊的经历，将他塑造成了一位浪漫主义英雄的形象。

历史影响

拜伦的参与，不仅加速了希腊的独立，也激发了其他被压迫民族的反抗精神。他的故事，成为了一段流传至今的传奇，激励着一代又一代的人为自由而奋斗。

总结

拜伦参与希腊独立战争，不仅仅是一个历史事件，更体现了人类对自由、平等的追求。他的故事，是浪漫主义精神的一次伟大实践，也是人道主义的一次胜利。

想了解更多关于拜伦和希腊独立战争，可以参考以下关键词：

• 拜伦
• 希腊独立战争
• 浪漫主义
• 民族英雄
• 欧洲历史

您想了解更多关于拜伦的诗歌、他的生活经历，还是希腊独立战争的历史背景呢？

eLife Assessment

This valuable study provides a novel method to detect sleep cycles based on variations in the slope of the power spectrum from electroencephalography signals. The method, dispensing with time-consuming and potentially subjective manual identification of sleep cycles, is supported by solid evidence and analyses. This study will be of interest to researchers and clinicians working on sleep and brain dynamics.

Reviewer #1 (Public review):

In this study, Rosenblum et al introduce a novel and automatic way of calculating sleep cycles from human EEG. Previous results have shown that the slope of the non-oscillatory component of the power spectrum (called the aperiodic or fractal component) changes with sleep stage. Building on this, the authors present an algorithm that extracts the continuous-time fluctuations in the fractal slope and propose that peaks in this variable can be used to identify sleep cycle limits. Cycles defined in this way are termed "fractal cycles". The main focus of the article is a comparison of "fractal" and "classical" (ie defined manually based on the hypnogram) sleep cycles in numerous datasets.

The manuscript amply illustrates through examples the strong overlap between fractal and classical cycle identification. Accordingly, a high percentage (81%) can be matched one-to-one between methods and sleep cycle duration is well correlated (around R = 0.5). Moreover, the methods track certain global changes in sleep structure in different populations: shorter cycles in children and longer cycles in patients medicated with REM-suppressing anti-depressants. Finally, a major strength of the results is that they show similar agreement between fractal and classical sleep cycle length in 5 different data sets, showing that it is robust to changes in recording settings and methods.

The match between fractal and classical cycles is not one-to-one. For example, the fractal method identifies a correlation between age and cycle duration in adults that is not apparent with the classical method.<br /> The difference between the fractal and classical methods appear to be linked to the uncertain definition of sleep cycles since they are tied to when exactly the cycle begins/ends and whether or not to count cycles during fractured sleep architecture at sleep onset. Moreover, the discrepancies between the two are on the order of that found between classical cycles defined manually or via an automatic algorithm.

Overall the fractal cycle is an attractive method to study sleep architecture since it dispenses with time-consuming and potentially subjective manual identification of sleep cycles. However, given its difference with the classical method, it is unlikely that fractal scoring will be able to replace classical scoring directly. By providing a complementary quantification, it will likely contribute to refining the definition of sleep cycles that is currently ambiguous in certain cases. Moreover, it has the potential to be applied on animal studies which rarely deal with sleep cycle structure.

Reviewer #2 (Public review):

Summary:

This study focused on using strictly the slope of the power spectral density (PSD) to perform automated sleep scoring and evaluation of the durations of sleep cycles. The method appears to work well because the slope of the PSD is highest during slow-wave sleep, and lowest during waking and REM sleep. Therefore, when smoothed and analyzed across time, there are cyclical variations in the slope of the PSD, fit using an IRASA (Irregularly resampled auto-spectral analysis) algorithm proposed by Wen & Liu (2016).

Strengths:

The main novelty of the study is that the non-fractal (oscillatory) components of the PSD that are more typically used during sleep scoring can be essentially ignored because the key information is already contained within the fractal (slope) component. The authors show that for the most part, results are fairly consistent between this and conventional sleep scoring, but in some cases show disagreements that may be scientifically interesting.

Weaknesses:

The previous weaknesses were well-addressed by the authors in the revised manuscript. I will note that from the fractal cycle perspective, waking and REM sleep are not very dissimilar. Combining these states underlies some of the key results of this study.

Author response:

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

Reviewer 1:

Weaknesses:

The match between fractal and classical cycles is not one-to-one. For example, the fractal method identifies a correlation between age and cycle duration in adults that is not apparent with the classical method. This raises the question as to whether differences are due to one method being more reliable than another or whether they are also identifying different underlying biological differences. It is not clear for example whether the agreement between the two methods is better or worse than between two human scorers, which generally serve as a gold standard to validate novel methods. The authors provide some insight into differences between the methods that could account for differences in results. However, given that the fractal method is automatic it would be important to clearly identify criteria for recordings in which it will produce similar results to the classical method.

We thank the reviewer for the insightful suggestions. In the revised Manuscript, we have added a number of additional analyses that provide a quantitative comparison between the classical and fractal cycle approaches aiming to identify the source of the discrepancies between classical and fractal cycle durations. Likewise, we assessed the intra-fractal and intra-classical method reliability.

Reviewer 2:

One weakness of the study, from my perspective, was that the IRASA fits to the data (e.g. the PSD, such as in Figure 1B), were not illustrated. One cannot get a sense of whether or not the algorithm is based entirely on the fractal component or whether the oscillatory component of the PSD also influences the slope calculations. This should be better illustrated, but I assume the fits are quite good.

Thank you for this suggestion. In the revised Manuscript, we have added a new figure (Fig.S1 E, Supplementary Material 2), illustrating the goodness of fit of the data as assessed by the IRASA method.

The cycles detected using IRASA are called fractal cycles. I appreciate the use of a simple term for this, but I am also concerned whether it could be potentially misleading? The term suggests there is something fractal about the cycle, whereas it's really just that the fractal component of the PSD is used to detect the cycle. A more appropriate term could be "fractal-detected cycles" or "fractal-based cycle" perhaps?

We agree that these cycles are not fractal per se. In the Introduction, when we mention them for the first time, we name them “fractal activity-based cycles of sleep” and immediately after that add “or fractal cycles for short”. In the revised version, we renewed this abbreviation with each new major section and in Abstract. Nevertheless, given that the term “fractal cycles” is used 88 times, after those “reminders”, we used the short name again to facilitate readability. We hope that this will highlight that the cycles are not fractal per se and thus reduce the possible confusion while keeping the manuscript short.

The study performs various comparisons of the durations of sleep cycles evaluated by the IRASA-based algorithm vs. conventional sleep scoring. One concern I had was that it appears cycles were simply identified by their order (first, second, etc.) but were not otherwise matched. This is problematic because, as evident from examples such as Figure 3B, sometimes one cycle conventionally scored is matched onto two fractal-based cycles. In the case of the Figure 3B example, it would be more appropriate to compare the duration of conventional cycle 5 vs. fractal cycle 7, rather than 5 vs. 5, as it appears is currently being performed.

In cases where the number of fractal cycles differed from the number of classical cycles (from 34 to 55% in different datasets as in the case of Fig.3B), we did not perform one-to-one matching of cycles. Instead, we averaged the duration of the fractal and classical cycles over each participant and only then correlated between them (Fig.2C). For a subset of the participants (45 – 66% of the participants in different datasets) with a one-to-one match between the fractal and classical cycles, we performed an additional correlation without averaging, i.e., we correlated the durations of individual fractal and classical cycles (Fig.4S of Supplementary Material 2). This is stated in the Methods, section Statistical analysis, paragraph 2.

There are a few statements in the discussion that I felt were either not well-supported. L629: about the "little biological foundation" of categorical definitions, e.g. for REM sleep or wake? I cannot agree with this statement as written. Also about "the gradual nature of typical biological processes". Surely the action potential is not gradual and there are many other examples of all-or-none biological events.

In the revised Manuscript, we have removed these statements from both Introduction and Discussion.

The authors appear to acknowledge a key point, which is that their methods do not discriminate between awake and REM periods. Thus their algorithm essentially detected cycles of slow-wave sleep alternating with wake/REM. Judging by the examples provided this appears to account for both the correspondence between fractal-based and conventional cycles, as well as their disagreements during the early part of the sleep cycle. While this point is acknowledged in the discussion section around L686. I am surprised that the authors then argue against this correspondence on L695. I did not find the "not-a-number" controls to be convincing. No examples were provided of such cycles, and it's hard to understand how positive z-values of the slopes are possible without the presence of some wake unless N1 stages are sufficient to provide a detected cycle (in which case, then the argument still holds except that its alterations between slow-wave sleep and N1 that could be what drives the detection).

In the revised Manuscript, we have removed the “NaN analysis” from both Results and Discussion. We have replaced it with the correlation between the difference between the durations of the classical and fractal cycles and proportion of wake after sleep onset. The finding is as follows:

“A larger difference between the durations of the classical and fractal cycles was associated with a higher proportion of wake after sleep onset in 3/5 datasets as well as in the merged dataset (Supplementary Material 2, Table S10).” Results, section “Fractal cycles and wake after sleep onset”, last two sentences. This is also discussed in Discussion, section “Fractal cycles and age”, paragraph 1, last sentence. 

To me, it seems important to make clear whether the paper is proposing a different definition of cycles that could be easily detected without considering fractals or spectral slopes, but simply adjusting what one calls the onset/offset of a cycle, or whether there is something fundamentally important about measuring the PSD slope. The paper seems to be suggesting the latter but my sense from the results is that it's rather the former.

Thank you for this important comment. Overall, our paper suggests that the fractal approach might reflect the cycling nature of sleep in a more precise and sensitive way than classical hypnograms. Importantly, neither fractal nor classical methods can shed light on the mechanism underlying sleep cycle generation due to their correlational approach. Despite this, the advantages of fractal over classical methods mentioned in our Manuscript are as follows:

(1) Fractal cycles are based on a real-valued metric with known neurophysiological functional significance, which introduces a biological foundation and a more gradual impression of nocturnal changes compared to the abrupt changes that are inherent to hypnograms that use a rather arbitrary assigned categorical value (e.g., wake=0, REM=-1, N1=-2, N2=-3 and SWS=-4, Fig.2 A).

(2) Fractal cycle computation is automatic and thus objective, whereas classical sleep cycle detection is usually based on the visual inspection of hypnograms, which is time-consuming, subjective and error-prone. Few automatic algorithms are available for sleep cycle detection, which only moderately correlated with classical cycles detected by human raters (r’s = 0.3 – 0.7 in different datasets here).

(3) Defining the precise end of a classical sleep cycle with skipped REM sleep that is common in children, adolescents and young adults using a hypnogram is often difficult and arbitrary.   The fractal cycle algorithm could detect such cycles in 93% of cases while the hypnogram-based agreement on the presence/absence of skipped cycles between two independent human raters was 61% only; thus, 32% lower.

(4) The fractal analysis showed a stronger effect size, higher F-value and R-squared than the classical analysis for the cycle duration comparison in children and adolescents vs young adults. The first and second fractal cycles were significantly shorter in the pediatric compared to the adult group, whereas the classical approach could not detect this difference.

(5) Fractal – but not classical – cycle durations correlated with the age of adult participants.

These bullets are now summarized in Table 5 that has been added to the Discussion of the revised manuscript.

Reviewer #1 (Recommendations for the authors):

The authors have added a lot of quantifications to provide a more complete comparison of classical and fractal cycles that address the points I raised.

Regarding, the question of skipped REM cycles: I am not sure the comparison of skipped cycle accuracies between fractal and manual methods makes sense. To make a fair comparison fractal and 2nd scorer classifications should be compared to the same baseline dataset which doesn't seem to be the case since the number of skipped cycles is not the same. Moreover, it's not indicated whether the fractal method identifies any false positive skipped cycles.

Thank you for this comment. In the revised Manuscript, we have reported the number of false positive skipped cycles identified by the fractal algorithm. Likewise, we have added the comparison between the fractal algorithm and the second scorer detection of cycles with skipped REM sleep (Results, the section “Skipped cycles”, last paragraph). The text has been revised as follows:

“Visual inspection of the hypnograms from Datasets 1 – 6 was performed by two independent researchers. Scorer 1 and Scorer 2 detected that out of 226 first sleep cycles 58 (26%) and 64 (28%), respectively, lacked REM episodes. The agreement on the presence of skipped cycles between two human raters equaled 91% (58 cycles detected by both raters out of 64 cycles detected by either one or two scorers). The fractal cycle algorithm detected skipped cycles in 57 out of 58 (98%) cases detected by Scorer 1 with one false positive (which, however, was tagged as a skipped cycle by Scorer2), and in 58 out of 64 (91%) cases detected by Scorer 2 with no false positives.”

Minor points

I suggest reporting the values of inter-method / inter-scorer correlations with the classical method in the main text since otherwise interpreting the value for fractal vs classical is impossible.

Thank you for this comment. In the revised Manuscript, we have moved this section to the main text (Table 3).

Table 5 + text of discussion: cycle identification based on hypnograms is claimed to be. "based on arbitrary assigned categorical values" the categories are not arbitrary since they correspond to well-validate sleep states, only the number associated it and this does not seem to be very important since it's only for visualization purposes.

Thank you for this comment. In the revised Manuscript, we have removed the phrase “arbitrary assigned“.

Letting go of feelings of ill will without reconciling with the other person.

Forgiving someone and reconciling with them

from play list

Crockford on JavaScript

**Volume 1: The Early Years **

self.link

eLife Assessment

This important study investigates how working memory load influences the Stroop effect from a temporal dynamics perspective. Convincing evidence is provided that the working memory load influences the Stroop effect in the late-stage stimulus-response mapping instead of the early sensory stage. This study will be of interest to both neuroscientists and psychologists who work on cognitive control.

Reviewer #1 (Public review):

Summary:

This study investigates an intriguing question in cognitive control from a temporal dynamics perspective: why does concurrent verbal working memory load eliminate the color-word Stroop effect? Through a series of thorough data analyses, the authors propose that verbal working memory load occupies the stimulus-response mapping resources represented by theta-band activity, thereby disrupting the mapping process for task-irrelevant distractors. This reduces the response tendency to the distractors, ultimately leading to the elimination of the Stroop effect.

Strengths:

The behavioral and neural evidence presented in the manuscript is solid, and the findings have valuable theoretical implications for research on Stroop conflict processing.

Comments on revisions:

The authors have addressed all concerns

Reviewer #2 (Public review):

Summary

Li et al. explored which stage of Stroop conflict processing was influenced by working memory loads. Participants completed a single task (Stroop task) and a dual task (the Sternberg working memory task combined with the Stroop task) while their EEG data was recorded. They adopted the event-related potential (ERP), and multivariate pattern analyses (MVPA) to investigate the interaction effect of task (single/dual) and congruency (congruent/incongruent). The results showed that the interaction effect was significant on the sustained potential (SP; 650-950 ms), the late theta (740-820 ms), and beta (920-1040 ms) power but not significant on the early P1 potential (110-150 ms). They used the representational similarity analyses (RSA) method to explore the correlation between behavioral and neural data, and the results revealed a significant contribution of late theta activity.

Strength

The experiment is well designed.<br /> The data were analyzed in depth from both time and frequency domain perspectives by combining several methods.

Comments on revisions:

All my concerns have been properly addressed, no further comments.

Author response:

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

Reviewer #1 (Public review):

Comment 1: In the Results section, the rationale behind selecting the beta band for the central (C3, CP3, Cz, CP4, C4) regions and the theta band for the fronto-central (Fz, FCz, Cz) regions is not clearly explained in the main text. This information is only mentioned in the figure captions. Additionally, why was the beta band chosen for the S-ROI central region and the theta band for the S-ROI fronto-central region? Was this choice influenced by the MVPA results?

We thank the reviewer for the question regarding the rationale for the S-ROI selection in our study. The beta band was chosen for the central region due to its established relevance in motor control (Engel & Fries, 2010), movement planning (Little et al., 2019) and motor inhibition (Duque et al., 2017). The fronto-central theta band (or frontal midline theta) was a widely recognized indicator in cognitive control research (Cavanagh & Frank, 2014), associated with conflict detection and resolution processes. Moreover, recent empirical evidence suggested that the fronto-central theta reflected the coordination and integration between stimuli and responses (Senoussi et al., 2022). Although we have described the cognitive processes linked to these different frequencies in the introduction and discussion sections, along with the potential patterns of results observed in Stroop-related studies, we did not specify the involved cortical areas. Therefore, we have specified these areas in the introduction to enhance the clarity of the revised version (in the fourth paragraph of the Introduction section).

Regarding whether the selection of S-ROIs was influenced by the MVPA results, we would like to clarify here that we selected the S-ROIs based on prior research and then conducted the decoding analysis. Specifically, we first extracted the data representing different frequency indicators (three F-ROIs and three S-ROIs) as features, followed by decoding to obtain the MVPA results. Subsequently, the time-frequency analysis, combined with the specific time windows during which each frequency was decoded, provided detailed interaction patterns among the variables for each indicator. The specifics of feature selection are described in the revised version (in the first paragraph of the Multivariate Pattern Analysis section).

Comment 2: In the Data Analysis section, line 424 states: “Only trials that were correct in both the memory task and the Stroop task were included in all subsequent analyses. In addition, trials in which response times (RTs) deviated by more than three standard deviations from the condition mean were excluded from behavioral analyses.” The percentage of excluded trials should be reported. Also, for the EEG-related analyses, were the same trials excluded, or were different criteria applied?

We thank the reviewer for this suggestion. Beyond the behavioral exclusion criteria, trials with EEG artifacts were also excluded from the data for the EEG-related analyses. We have now reported the percentage of excluded trials for both behavioral and EEG data analyses in the revised version (in the second paragraph of the EEG Recording and Preprocessing section and the first paragraph of the Behavioral Analysis section).

Comment 3: In the Methods section, line 493 mentions: “A 400-200 ms pre-stimulus time window was selected as the baseline time window.” What is the justification in the literature for choosing the 400-200 ms pre-stimulus window as the baseline? Why was the 200-0 ms pre-stimulus period not considered?

We thank the reviewer for this question and would like to provide the following justification. First, although a baseline ending at 0 ms is common in ERP analyses, it may not be suitable for time-frequency analysis. Due to the inherent temporal smoothing characteristic of wavelet convolution in time-frequency decomposition, task-related early activities can leak into the pre-stimulus period (before 0 ms) (Cohen, 2014). This means that extending the baseline to 0 ms will include some post-stimulus activity in the baseline window, thereby increasing baseline power and compromising the accuracy of the results. Second, an ideal baseline duration is recommended to be around 10-20% of the entire trial of interest (Morales & Bowers, 2022). In our study, the epoch duration was 2000 ms, making 200-400 ms an appropriate baseline length. Third, given that the minimum duration of the fixation point before the stimulus in our experiment was 400 ms, we chose the 400 ms before the stimulus as the baseline point to ensure its purity. In summary, considering edge effects, duration requirements, and the need to exclude other influences, we selected a baseline correction window of -400 to -200 ms. To enhance the clarity of the revised version, we have provided the rationale for the selected time windows along with relevant references (in the first paragraph of the Time-frequency analysis section).

Comment 4: Is the primary innovation of this study limited to the methodology, such as employing MVPA and RSA to establish the relationship between late theta activity and behavior?

We thank the reviewer for this insightful question and would like to clarify that our research extends beyond mere methodological innovation; rather, it utilized new methods to explore novel theoretical perspectives. Specifically, our research presents three levels of innovation: methodological, empirical, and theoretical. First, methodologically, MVPA overcame the drawbacks of traditional EEG analyses based on specific averaged voltage intensities, providing new perspectives on how the brain dynamically encoded particular neural representations over time. Furthermore, RSA aimed to identify which indicators among the decoded were directly related to behavioral representation patterns. Second, in terms of empirical results, using these two methods, we have identified for the first time three EEG markers that modulate the Stroop effect under verbal working memory load: SP, late theta, and beta, with late theta being directly linked to the elimination of the behavioral Stroop effect. Lastly, from a theoretical perspective, we proposed the novel idea that working memory played a crucial role in the late stages of conflict processing, specifically in the stimulus-response mapping stage (the specific theoretical contributions are detailed in the second-to-last paragraph of the Discussion section).

Comment 5: On page 14, lines 280-287, the authors discuss a specific pattern observed in the alpha band. However, the manuscript does not provide the corresponding results to substantiate this discussion. It is recommended to include these results as supplementary material.

We thank the reviewer for this suggestion. We added a new figure along with the corresponding statistical results that displayed the specific result patterns for the alpha band (Supplementary Figure 1).

Comment 6: On page 16, lines 323-328, the authors provide a generalized explanation of the findings. According to load theory, stimuli compete for resources only when represented in the same form. Since the pre-memorized Chinese characters are represented semantically in working memory, this explanation lacks a critical premise: that semantic-response mapping is also represented semantically during processing.

We thank the reviewer for this insightful suggestion. We fully agree with the reviewer’s perspective. As stated in our revised version, load theory suggests that cognitive resources are limited and dependent on a specific type (in the second paragraph of the Discussion section). The previously memorized Chinese characters are stored in working memory in the form of semantic representations; meanwhile the stimulus-response mapping should also be represented semantically, leading to resource occupancy. We have included this logical premise in the revised version (in the third-to-last paragraph of the Discussion section).

Comment 7: The classic Stroop task includes both a manual and a vocal version. Since stimulus-response mapping in the vocal version is more automatic than in the manual version, it is unclear whether the findings of this study would generalize to the impact of working memory load on the Stroop effect in the vocal version.

We fully agree with the reviewer’s point that the verbal version of the Stroop task differs from the manual version in terms of the degree of automation in the stimulus-response mapping. Specifically, the verbal version relies on mappings that are established through daily language use, while the manual version involves arbitrary mappings created in the laboratory. Therefore, the stimulus-response mapping in the verbal response version is more automated and less likely to be suppressed. However, our previous research indicated that the degree of automation in the stimulus-response mapping was influenced by practice (Chen et al., 2013). After approximately 128 practice trials, semantic conflict almost disappears, suggesting that the level of automation in stimulus-response mapping for the verbal Stroop task is comparable to that of the manual version (Chen et al., 2010). Given that participants in our study completed 144 practice trials (in the Procedure section), we believe these findings can be generalized to the verbal version.

Comment 8: While the discussion section provides a comprehensive analysis of the study’s results, the authors could further elaborate on the theoretical and practical contributions of this work.

We thank the reviewer for the constructive suggestions. We recognize that the theoretical and practical contributions of the study were not thoroughly elaborated in the original manuscript. Therefore, we have now provided a more detailed discussion. Specifically, the theoretical contributions focus on advancing load theory and highlighting the critical role of working memory in conflict processing. The practical contributions emphasize the application of load theory and the development of intervention strategies for enhancing inhibitory control. A more detailed discussion can be found in the revised version (in the second-to-last paragraph of the Discussion section).

Reviewer #2 (Public review):

Comment 1: As the researchers mentioned, a previous study reported a diminished Stroop effect with concurrent working memory tasks to memorize meaningless visual shapes rather than memorize Chinese characters as in the study. My main concern is that lower-level graphic processing when memorizing visual shapes also influences the Stroop effect. The stage of Stroop conflict processing affected by the working memory load may depend on the specific content of the concurrent working memory task. If that’s the case, I sense that the generalization of this finding may be limited.

We thank the reviewer for this insightful concern. As mentioned in the manuscript, this may be attributed to the inherent characteristics of Chinese characters. In contrast to English words, the processing of Chinese characters relies more on graphemic encoding and memory (Chen, 1993). Therefore, the processing of line patterns essentially occupies some of the resources needed for character processing, which aligns with our study’s hypothesis based on dimensional overlap. Additionally, regarding the results, even though the previous study presents lower-level line patterns, the results still showed that the working memory load modulated the later theta band. We hypothesize that, regardless of the specific content of the pre-presented working memory load, once the stimulus disappears from view, these loads are maintained as representations in the working memory platform. Therefore, they do not influence early perceptual processing, and resource competition only occurs once the distractors reach the working memory platform. Lastly, previous study has shown that spatial loads, which do not overlap with either the target or distractor dimensions, do not influence conflict effect (Zhao et al., 2010). Taken together, we believe that regardless of the specific content of the concurrent working memory tasks, as long as they occupy resources related to irrelevant stimulus dimensions, they can influence the late-stage processing of conflict effect. Perhaps our original manuscript did not convey this clearly, so we have rephrased it in a more straightforward manner (in the second paragraph of the Discussion section).

Comment 2: The P1 and N450 components are sensitive to congruency in previous studies as mentioned by the researchers, but the results in the present study did not replicate them. This raised concerns about data quality and needs to be explained.

We thank the reviewer for this insightful concern. For P1, we aimed to convey that the early perceptual processing represented by P1 is part of the conflict processing process. Therefore, we included it in our analysis. Additionally, as mentioned in the discussion, most studies find P1 to be insensitive to congruency. However, we inappropriately cited a study in the introduction that suggested P1 shows differences in congruency, which is among the few studies that hold this perspective. To prevent confusion for readers, we have removed this citation from the introduction.

As for N450, most studies have indeed found it to be influenced by congruency. In our manuscript, we did not observe a congruency effect at our chosen electrodes and time window. However, significant congruency effects were detected at other central-parietal electrodes (CP3, CP4, P5, P6) during the 350-500 ms interval. The interaction between task type and consistency remained non-significant, consistent with previous results. Furthermore, with respect to the location of the electrodes chosen, existing studies on N450 vary widely, including central-parietal electrodes and frontal-central electrodes (for a review, see Heidlmayr et al., 2020). We speculate that this phenomenon may be related to the extent of practice. With fewer total trials, the task may involve more stimulus conflicts, engaging more frontal brain areas. On the other hand, with more total trials, the task may involve more response conflicts, engaging more central-parietal brain areas (Chen et al., 2013; van Veen & Carter, 2005). Due to the extensive practice required in our study, we identified a congruency N450 effect in the central-parietal region. We apologize for not thoroughly exploring other potential electrodes in the previous manuscript, and we have revised the results and interpretations regarding N450 accordingly in the revised version (in the N450 section of the ERP results and the third paragraph of the Discussion section).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Comment 1: In the Introduction, line 108 states: “Second, alpha oscillations (8-13 Hz) can serve as a neural inverse index of mental activity or alertness, while a decrease in alpha power reflects increased alertness or enhanced attentional inhibition of distractors (Arakaki et al., 2022; Tafuro et al., 2019; Zhou et al., 2023; Zhu et al., 2023).” Please clarify which specific psychological process related to conflict processing is reflected by alpha oscillations.

We appreciate your suggestion and we have clearly highlighted the role of alpha oscillations in attentional engagement during conflict processing in the revised version (in the third-to-last paragraph of the introduction).

Comment 2: In Figures 3C and 3E, a space is needed between “amplitude” and the preceding parenthesis. Similar adjustments are required in Figures 4A, 4B, 4C, 5C, and 6C. Additionally, in Figures 3B and 3D, a space should be added between the numbers and “ms.” This issue also appears in Figure 8. Please review all figures for these formatting inconsistencies.

We apologize for the inconsistency in formatting and have corrected them throughout the revised version.

Comment 3: There are some clerical errors in the manuscript that need correction. For instance, on page 19, line 403: “Participants were asked to answer by pressing one of two response buttons (“S with the left ring finger and “L” with the left ring finger).” This should be corrected to: “L” with the right ring finger. I recommend that the authors carefully proofread the manuscript to identify and correct such errors.

We sincerely apologize for the errors present in the manuscript and have now carefully proofread it (in the Procedure section).

Comment 4: On page 13, line 254, the elimination of the Stroop effect should not be interpreted as an improvement in processing.

We greatly appreciate your suggestion. We agree that the elimination of the Stroop effect should not be confused with improvements in processing. We have corrected this in the revised version (the second paragraph of the Discussion section).

Reviewer #3 (Recommendations for the authors):

Comment 1: In the introduction section, the N450 was introduced as “a frontal-central negative deflection”, but in the methods part the N450 was computed using central-parietal electrodes. This inconsistency is confusing and needs to be clarified.

We apologize for this confusion. We have provided a detailed explanation regarding the differences in electrodes and the rationale behind choosing central-parietal electrodes in our response to Reviewer 2’s second comment. To clarify, we have updated the introduction to consistently label them as central-parietal deflections (in the third paragraph of the Introduction section).

Comment 2: I speculate the “beta” was mistakenly written as “theta” in line 212.

We sincerely apologize for this mistake. We have corrected this error (in the RSA results section).

Comment 3: The speculation that “changes in beta bands may be influenced by theta bands, thereby indirectly influencing the behavioral Stroop effect” needs to be rationalized.

We appreciate your suggestion. What we intended to convey is that we found an interaction effect in the beta bands; however, the RSA results did not show a correlation with the behavioral interaction effect. We speculate that beta activity might be influenced by the theta bands. On the one hand, we realize that the idea of beta bands indirectly influencing the behavioral Stroop effect was inappropriate, and we have removed this point in the revised version. On the other hand, we have provided rational evidence for the idea that beta bands may be influenced by theta bands. This is based on the biological properties of theta oscillations, which support communication between different cortical neural signals, and their functional role in integrating and transmitting task-relevant information to response execution (in the third-to-last paragraph of the Discussion section).

Comment 4: Typo in line 479: [10,10].

We sincerely apologize for this mistake. We have corrected this error: [-10,10] (in the Multivariate pattern analysis section).

Reference

Cavanagh, J. F., & Frank, M. J. (2014). Frontal theta as a mechanism for cognitive control. Trends in Cognitive Sciences, 18(8), 414–421. https://doi.org/10.1016/j.tics.2014.04.012

Chen, M. J. (1993). A Comparison of Chinese and English Language Processing. In Advances in Psychology (Vol. 103, pp. 97–117). North-Holland. https://doi.org/10.1016/S0166-4115(08)61659-3

Chen, X. F., Jiang, J., Zhao, X., & Chen, A. (2010). Effects of practice on semantic conflict and response conflict in the Stroop task. Psychol. Sci., 33, 869–871.

Chen, Z., Lei, X., Ding, C., Li, H., & Chen, A. (2013). The neural mechanisms of semantic and response conflicts: An fMRI study of practice-related effects in the Stroop task. NeuroImage, 66, 577–584. https://doi.org/10.1016/j.neuroimage.2012.10.028

Cohen, M. X. (2014). Analyzing Neural Time Series Data: Theory and Practice. The MIT Press. https://doi.org/10.7551/mitpress/9609.001.0001

Duprez, J., Gulbinaite, R., & Cohen, M. X. (2020). Midfrontal theta phase coordinates behaviorally relevant brain computations during cognitive control. NeuroImage, 207, 116340. https://doi.org/10.1016/j.neuroimage.2019.116340

Duque, J., Greenhouse, I., Labruna, L., & Ivry, R. B. (2017). Physiological Markers of Motor Inhibition during Human Behavior. Trends in Neurosciences, 40(4), 219–236. https://doi.org/10.1016/j.tins.2017.02.006

Engel, A. K., & Fries, P. (2010). Beta-band oscillations—Signalling the status quo? Current Opinion in Neurobiology, 20(2), 156–165. https://doi.org/10.1016/j.conb.2010.02.015

Heidlmayr, K., Kihlstedt, M., & Isel, F. (2020). A review on the electroencephalography markers of Stroop executive control processes. Brain and Cognition, 146, 105637. https://doi.org/10.1016/j.bandc.2020.105637

Little, S., Bonaiuto, J., Barnes, G., & Bestmann, S. (2019). Human motor cortical beta bursts relate to movement planning and response errors. PLOS Biology, 17(10), e3000479. https://doi.org/10.1371/journal.pbio.3000479

Morales, S., & Bowers, M. E. (2022). Time-frequency analysis methods and their application in developmental EEG data. Developmental Cognitive Neuroscience, 54, 101067. https://doi.org/10.1016/j.dcn.2022.101067

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van Veen, V., & Carter, C. S. (2005). Separating semantic conflict and response conflict in the Stroop task: A functional MRI study. NeuroImage, 27(3), 497–504. https://doi.org/10.1016/j.neuroimage.2005.04.042

Zhao, X., Chen, A., & West, R. (2010). The influence of working memory load on the Simon effect. Psychonomic Bulletin & Review, 17(5), 687–692. https://doi.org/10.3758/PBR.17.5.687

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by Hans Schenker

eLife Assessment

This study uses carefully designed experiments to generate a useful behavioural and neuroimaging dataset on visual cognition. The results provide solid evidence for the involvement of higher-order visual cortex in processing visual oddballs and asymmetry. However, the evidence provided for the very strong claims of homogeneity as a novel concept in vision science, separable from existing concepts such as target saliency, is incomplete. The authors and the reviewers do not agree on several points, which are explained in the reviews and author response.

Reviewer #1 (Public review):

Summary:

The authors define a new metric for visual displays, derived from psychophysical response times, called visual homogeneity (VH). They attempt to show that VH is explanatory of response times across multiple visual tasks. They use fMRI to find visual cortex regions with VH-correlated activity. On this basis, they declare a new visual region in human brain, area VH, whose purpose is to represent VH for the purpose of visual search and symmetry tasks.

Link to original review: https://elifesciences.org/reviewed-preprints/93033v2/reviews#peer-review-0

Comments on latest version:

Authors rebuttal: We agree that visual homogeneity is similar to existing concepts such as target saliency, memorability etc. We have proposed it as a separate concept because visual homogeneity has an independent empirical measure (the reciprocal of target-absent search time in oddball search, or the reciprocal of same response time in a same-different task, etc) that may or may not be the same as other empirical measures such as saliency and memorability. Investigating these possibilities is beyond the scope of our study but would be interesting for future work. We have now clarified this in the revised manuscript (Discussion, p. 42).

Reviewer response to rebuttal: Neither the original ms nor the comments on that ms pretended that "visual homogeneity" was entirely separate from target saliency etc. So this is a response to a criticism that was never made. What the authors do claim, and what the comments question, is that they have successfully subsumed long-recognized psychophysical concepts like target saliency etc. under a new, uber-concept, "visual homogeneity" that explains psychophysical experimental results in a more unified and satisfying way. This subsumption of several well-established psychophysical concepts under a new, unified category is what reviewers objected to.

Authors rebuttal: However, we'd like to emphasize that the question of whether visual homogeneity is novel or related to existing concepts misses entirely the key contribution of our study.

Reviewer response to rebuttal: Sorry, but the claim of a new uber-concept in psychophysics, "visual homogeneity", is a major claim of the paper. The fact that it is not the only claim made does not absolve the authors from having to prove it satisfactorily.

"Authors rebuttal: "In addition, the large regions of VH correlations identified in Experiments 1 and 2 vs. Experiments 3 and 4 are barely overlapping. This undermines the claim that VH is a universal quantity, represented in a newly discovered area of visual cortex, that underlies a wide variety of visual tasks and functions."<br /> • We respectfully disagree with your assertion. First of all, there is partial overlap between the VH regions, for which there are several other obvious explanations that must be considered first before dismissing VH outright as a flawed construct. We acknowledge these alternatives in the Results (p. 27), and the relevant text is reproduced below.

"We note that it is not straightforward to interpret the overlap between the VH regions identified in Experiments 2 & 4. The lack of overlap could be due to stimulus differences (natural images in Experiment 2 vs silhouettes in Experiment 4), visual field differences (items in the periphery in Experiment 2 vs items at the fovea in Experiment 4) and even due to different participants in the two experiments. There is evidence supporting all these possibilities: stimulus differences (Yue et al., 2014), visual field differences (Kravitz et al., 2013) as well as individual differences can all change the locus of neural activations in object-selective cortex (Weiner and Grill-Spector, 2012a; Glezer and Riesenhuber, 2013). We speculate that testing the same participants on search and symmetry tasks using similar stimuli and display properties would reveal even larger overlap in the VH regions that drive behavior."

Reviewer response to rebuttal: The authors are saying that their results merely look unconvincing (weak overlap between VH regions defined in different experiments) because there were confounding differences between their experiments, in subject population, stimuli, etc. That is possible, but in that case it is up to the authors to show that their definition of a new "area VH" is convincing when the confounding differences are resolved, e.g. by using the same stimuli in the different experiments they attempt to agglomerate here. That would require new experiments, and none are offered in this revision.

Authors rebuttal: • Thank you for carefully thinking through our logic. We agree that a distance-to-centre calculation is entirely unnecessary as an explanation for target-present visual search. The similarity between target and distractor, so there is nothing new to explain here. However, this is a narrow and selective interpretation of our findings because you are focusing only on our results on target-present searches, which are only half of all our data. The other half is the target-absent responses which previously have had no clear explanation. You are also missing the fact that we are explaining same-different and symmetry tasks as well using the same visual homogeneity computation. We urge you to think more deeply about the problem of how to decide whether an oddball is present or not in the first place. How do we actually solve this task?

Reviewer response to rebuttal: It is the role of the authors to think deeply about their paper and on that basis present a clear and compelling case that readers can understand quickly and agree with. That is not done here.

Authors rebuttal: There must be some underlying representation and decision process. Our study shows that a distance-to-centre computation can actually serve as a decision variable to solve disparate property-based visual tasks. These tasks pose a major challenge to standard models of decision-making because the underlying representation and decision variable have been unclear. Our study resolves this challenge by proposing a novel computation that can be used by the brain to solve all these disparate tasks, and bring these tasks into the ambit of standard theories of decision-making.

Reviewer response to rebuttal: There is only a "challenge" if you accept the authors' a priori assumption that all of these tasks must have a common explanation and rely on a single neural mechanism. I do not accept that assumption, and I don't think the authors provide evidence to support the assumption. There is nothing "unclear" about how search, oddball, etc. have been thoroughly explained, separately, in the psychophysical literature that spans more than a century.

Authors rebuttal: • You are indeed correct in noting that both Experiment 1 & 2 involve oddball search, and so at the superficial level, it looks circular that the oddball search data of Experiment 1 is being used to explain the oddball search data of Experiment 2.<br /> However a deeper scrutiny reveals more fundamental differences: Experiment 1 consisted of only oddball search with the target appearing on the left or right, whereas Experiment 2 consisted of oddball search with the target either present or completely absent. In fact, we were merely using the search dissimilarities from Experiment 1 to reconstruct the underlying object representation, because it is well-known that neural dissimilarities are predicted well by search dissimilarities (Sripati & Olson, 2009; Zhivago et al, 2014).

Reviewer response to rebuttal: Here again the authors cite differences between their multiple experiments as a virtue that supports their conclusions. Instead, the experiments should have been designed for maximum similarity if the authors intended to explain them with the same theory.

Authors rebuttal: To thoroughly refute any lingering concern about circularity, we reasoned that the model predictions for Experiment 2 could have been obtained by a distance-to-center computation on any brain like object representation. To this end, we used object representations from deep neural networks pretrained on object categorization, whose representations are known to match well with the brain, and asked if a distance-to-centre computation on these representations could predict the search data in Experiment 2. This was indeed the case, and these results are now included an additional section in Supplementary Material (Section S1).

Reviewer response to rebuttal: The authors' claims are about human performance and how it is based on the human brain. Their claims are not well supported by the human experiments that they performed. It serves no purpose to redo the same experiments in silico, which cannot provide stronger evidence that compensates for what was lacking in the human data.

Authors rebuttal: "Confirming the generality of visual homogeneity<br /> We performed several additional analyses to confirm the generality of our results, and to reject alternate explanations.

First, it could be argued that our results are circular because they involve taking oddball search times from Experiment 1 and using them to explain search response times in Experiment 2. This is a superficial concern since we are using the search dissimilarities from Experiment 1 only as a proxy for the underlying neural representation, based on previous reports that neural dissimilarities closely match oddball search dissimilarities (Sripati and Olson, 2010; Zhivago and Arun, 2014). Nonetheless, to thoroughly refute this possibility, we reasoned that we would get similar predictions of the target present/absent responses in Experiment using any other brain-like object representation. To confirm this, we replaced the object representations derived from Experiment 1 with object representations derived from deep neural networks pretrained for object categorization, and asked if distance-to-center computations could predict the target present/absent responses in Experiment 2. This was indeed the case (Section S1).

Second, we wondered whether the nonlinear optimization process of finding the best-fitting center could be yielding disparate optimal centres each time. To investigate this, we repeated the optimization procedure with many randomly initialized starting points, and obtained the same best-fitting center each time (see Methods).

Third, to confirm that the above model fits are not due to overfitting, we performed a leave-one-out cross validation analysis. We left out all target-present and target-absent searches involving a particular image, and then predicted these searches by calculating visual homogeneity estimated from all other images. This too yielded similar positive and negative correlations (r = 0.63, p < 0.0001 for target-present, r = -0.63, p < 0.001 for target-absent).

Fourth, if heterogeneous displays indeed elicit similar neural responses due to mixing, then their average distance to other objects must be related to their visual homogeneity. We confirmed that this was indeed the case, suggesting that the average distance of an object from all other objects in visual search can predict visual homogeneity (Section S1).

Fifth, the above results are based on taking the neural response to oddball arrays to be the average of the target and distractor responses. To confirm that averaging was indeed the optimal choice, we repeated the above analysis by assuming a range of relative weights between the target and distractor. The best correlation was obtained for almost equal weights in the lateral occipital (LO) region, consistent with averaging and its role in the underlying perceptual representation (Section S1).

Finally, we performed several additional experiments on a larger set of natural objects as well as on silhouette shapes. In all cases, present/absent responses were explained using visual homogeneity (Section S2)."

Reviewer response to rebuttal: The authors can experiment on side questions for as long as they please, but none of the results described above answer the concern about how center-fitting undercuts the evidentiary value of their main results.

Authors rebuttal: • While it is true that the optimal center needs to be found by fitting to the data, there no particular mystery to the algorithm: we are simply performing a standard gradient-descent to maximize the fit to the data. We have described the algorithm clearly and are making our codes public. We find the algorithm to yield stable optimal centers despite many randomly initialized starting points. We find the optimal center to be able to predict responses to entirely novel images that were excluded during model training. We are making no assumption about the location of centre with respect to individual points. Therefore, we see no cause for concern regarding the center-finding algorithm.

Reviewer response to rebuttal: The point of the original comment was that center-fitting should not be done in the first place because it introduces unknowable effects.

•Authors rebuttal: Most visual tasks, such as finding an animal, are thought to involve building a decision boundary on some underlying neural representation. Even visual search has been portrayed as a signal-detection problem where a particular target is to be discriminated from a distractor. However none of these formulations work in the case of property-based visual tasks, where there is no unique feature to look for.<br /> We are proposing that, when we view a search array, the neural response to the search array can be deduced from the neural responses to the individual elements using well-known rules, and that decisions about an oddball target being present or absent can be made by computing the distance of this neural response from some canonical mean firing rate of a population of neurons. This distance to center computation is what we denote as visual homogeneity. We have revised our manuscript throughout to make this clearer and we hope that this helps you understand the logic better.<br /> • You are absolutely correct that the stimulus complexity should matter, but there are no good empirically derived measures for stimulus complexity, other than subjective ratings which are complex on their own and could be based on any number of other cognitive and semantic factors. But considering what factors are correlated with target-absent response times is entirely different from asking what decision variable or template is being used by participants to solve the task.

Reviewer response to rebuttal: If stimulus complexity is what matters, as the authors agree here, then it is incumbent on them to measure stimulus complexity. The difficulty of measuring stimulus complexity does not justify avoiding the problem with an analysis that ignores complexity.

Authors rebuttal: • We have provided empirical proof for our claims, by showing that target-present response times in a visual search task are correlated with "different" responses in the same-different task, and that target-absent response times in the visual search task are correlated with "same" responses in the same-different task (Section S4).

Reviewer response to rebuttal: Sorry, but there is still no reason to think that same-different judgments are based on a mythical boundary halfway between the two. If there is a boundary, it will be close to the same end of the continuum, where subjects might conceivably miss some tiny difference between two stimuli. The vast majority of "different" stimuli will be entirely different from the same stimulus, producing no confusability, and certainly not a decision boundary halfway between two extremes.

Authors rebuttal: • Again, the opposite correlations between target present/absent search times with VH are the crucial empirical validation of our claims that a distance-to-center calculation explain how we perform these property-based tasks. The VH predictions do not fully explain the data. We have explicitly acknowledged this shortcoming, so we are hardly dismissing it as a problem.

Reviewer response to rebuttal: The authors' acknowledgement of flaws in the ms does not argue in favor of publication, but rather just the opposite.

Authors rebuttal: • Finding an oddball, deciding if two items are same or different and symmetry tasks are disparate visual tasks that do not fit neatly into standard models of decision-making. The key conceptual advance of our study is that we propose a plausible neural representation and decision variable that allows all three property-based visual tasks to be reconciled with standard models of decision-making.

Reviewer response to rebuttal: The original comment stands as written. Same/different will have a boundary very close to the "same" end of the continuum. The boundary is only halfway between two choices if the stimulus design forces the boundary to be there, as in the motion and cat/dog experiments.

Authors rebuttal: "There is no inherent middle point boundary between target present and target absent. Instead, in both types of trial, maximum information is present when target and distractors are most dissimilar, and minimum information is present when target and distractors are most similar. The point of greatest similarity occurs at then limit of any metric for similarity. Correspondingly, there is no middle point dip in information that would produce greater difficulty and higher response times. Instead, task difficulty and response times increase monotonically with similarity between targets and distractors, for both target present and target absent decisions. Thus, in Figs. 2F and 2G, response times appear to be highest for animals, which share the largest numbers of closely similar distractors."<br /> • Your alternative explanation rests on vague factors like "maximum information" which cannot be quantified. By contrast we are proposing a concrete, falsifiable model for three property-based tasks - same/different, oddball present/absent and object symmetry. Any argument based solely on item similarity to explain visual search or symmetry responses cannot explain systematic variations observed for target-absent arrays and for symmetric objects, for the reasons explained earlier.

Reviewer response to rebuttal: There is nothing vague about this comment. The authors use an analysis that assumes a decision boundary at the centerpoint of their arbitrarily defined stimulus space. This assumption is not supported, and it is unlikely, considering that subjects are likely to notice all but the smallest variations between same and different stimuli, putting the boundary nearly at the same end of the continuum, not the very middle.

Authors rebuttal: "(1) The area VH boundaries from different experiments are nearly completely non-overlapping.

In line with their theory that VH is a single continuum with a decision boundary somewhere in the middle, the authors use fMRI searchlight to find an area whose responses positively correlate with homogeneity, as calculated across all of their target present and target absent arrays. They report VH-correlated activity in regions anterior to LO. However, the VH defined by symmetry Experiments 3 and 4 (VHsymmetry) is substantially anterior to LO, while the VH defined by target detection Experiments 1 and 2 (VHdetection) is almost immediately adjacent to LO. Fig. S13 shows that VHsymmetry and VHdetection are nearly non-overlapping. This is a fundamental problem with the claim of discovering a new area that represents a new quantity that explains response times across multiple visual tasks. In addition, it is hard to understand why VHsymmetry does not show up in a straightforward subtraction between symmetric and asymmetric objects, which should show a clear difference in homogeneity."

• We respectfully disagree. The partial overlap between the VH regions identified in Experiments 1 & 2 can hardly be taken as evidence against the quantity VH itself, because there are several other obvious alternate explanations for this partial overlap, as summarized earlier as well. The VH region does show up in a straightforward subtraction between symmetric and asymmetric objects (Section S7), so we are not sure what the Reviewer is referring to here.

Reviewer response to rebuttal: In disagreeing with the comment quoted above, the authors are maintaining that a new functional area of cerebral cortex can be declared even if that area changes location on the cortical map from one experiment to another. That position is patently absurd.

Authors rebuttal: "(3) Definition of the boundaries and purpose of a new visual area in the brain requires circumspection, abundant and convergent evidence, and careful controls.

Even if the VH metric, as defined and calculated by the authors here, is a meaningful quantity, it is a bold claim that a large cortical area just anterior to LO is devoted to calculating this metric as its major task. Vision involves much more than target detection and symmetry detection. Cortex anterior to LO is bound to perform a much wider range of visual functionalities. If the reported correlations can be clarified and supported, it would be more circumspect to treat them as one byproduct of unknown visual processing in cortex anterior to LO, rather than treating them as the defining purpose for a large area of visual cortex."

• We totally agree with you that reporting a new brain region would require careful interpretation and abundant and converging evidence. However, this requires many studies worth of work, and historically category-selective regions like the FFA have achieved consensus only after they were replicated and confirmed across many studies. We believe our proposal for the computation of a quantity like visual homogeneity is conceptually novel, and our study represents a first step that provides some converging evidence (through replicable results across different experiments) for such a region. We have reworked our manuscript to make this point clearer (Discussion, p 32).

Reviewer response to rebuttal: Indeed, declaring a new brain area depends on much more work than is done here. Thus, the appropriate course here is to wait before claiming to have identified a new cortical area.

Reviewer #2 (Public review):

Summary:

This study proposes visual homogeneity as a novel visual property that enables observers perform to several seemingly disparate visual tasks, such as finding an odd item, deciding if two items are same, or judging if an object is symmetric. In Exp 1, the reaction times on several objects were measured in human subjects. In Exp 2, visual homogeneity of each object was calculated based on the reaction time data. The visual homogeneity scores predicted reaction times. This value was also correlated with the BOLD signals in a specific region anterior to LO. Similar methods were used to analyze reaction time and fMRI data in a symmetry detection task. It is concluded that visual homogeneity is an important feature that enables observers to solve these two tasks.

Strengths:

(1) The writing is very clear. The presentation of the study is informative.

(2) This study includes several behavioral and fMRI experiments. I appreciate the scientific rigor of the authors.

Weaknesses:

Before addressing the manuscript itself, I would like to comment the review process first. Having read the lasted revised manuscript, I shared many of the concerns raised by the two reviewers in the last two rounds of review. It appears that the authors have disagreed with the majority of comments made by the two reviewers. If so, I strongly recommend that the authors proceed to make this revision as a Version of Record and conclude this review process. According to eLife's policy that the authors have the right to make a Version of Record at any time during the review process, and I fully respect that right. However, I also ask that the authors respect the reviewer's right to retain the comments regarding this paper.

Beside that, I still have several further questions about this study.

(1) My main concern with this paper is the way visual homogeneity is computed. On page 10, lines 188-192, it says: "we then asked if there is any point in this multidimensional representation such that distances from this point to the target-present and target-absent response vectors can accurately predict the target-present and target-absent response times with a positive and negative correlation respectively (see Methods)". This is also true for the symmetry detection task. If I understand correctly, the reference point in this perceptual space was found by deliberating satisfying the negative and positive correlations in response times. And then on page 10, lines 200-205, it shows that the positive and negative correlations actually exist. This logic is confusing. The positive and negative correlations emerge only because this method is optimized to do so. It seems more reasonable to identify the reference point of this perceptual space independently, without using the reaction time data. Otherwise, the inference process sounds circular. A simple way is to just use the mean point of all objects in Exp 1, without any optimization towards reaction time data.<br /> I raised this question in my initial review. However, the authors did not address whether the positive and negative correlations still hold if the mean point is defined as the reference point without any optimization. The authors also argue that it is similar to a case of fitting a straight line. It is fine that the authors insist on the straight line (e.g., correlation). However, I would not call "straight line correlations" a "quantitative model" as a high-profile journals like eLife. Please remove all related arguments of a novel quantitative model.

(2) Visual homogeneity (at least given the current form) is an unnecessary term. It is similar to distractor heterogeneity/distractor variability/distractor saliency in literature. However, the authors attempt to claim it as a novel concept. Both R1 and me raised this question in the very first review. However, the authors refused to revise the manuscript. In the last review, I mentioned this and provided some example sentences claiming novelty. The authors only revised the last sentence of the abstract, and even did not bother to revise the last sentence of significance: "we show that these tasks can be solved using a simple property WE DEFINE as visual homogeneity". Also, lines 851 still shows "we have defined a NOVEL image property, visual homogeneity...". I am confused about whether the authors agree or disagree that "visual homogeneity is an unnecessary term". If the authors agree, they should completely remove the related phrase throughout the paper. If not, they should keep all these and state the reasons. I don't think this is a correct approach to revising a manuscript.

(3) If the authors agree that visual homogeneity is not new, I suggest a complete rewrite of the title, abstract, significance, and introduction. Let me ask a simple question, can we remove "visual homogeneity" and use some more well-established term like "image feature similarity"? If yes, visual homogeneity is unnecessary.

(4) If I understand it correctly, one of the key findings of this paper is "the response times for target-present searches were positively correlated with visual homogeneity. By contrast, the response times for target-absent searches were negatively correlated with visual homogeneity" (lines 204-207). I think the authors have already acknowledged that this positive correlation is not surprising at all because it reflects the classic target-distractor similarity effect. If this is the case, please completely remove the positive correlation as a novel prediction and finding.

(5) In my last review, I mentioned the seminal paper by Duncan and Humphreys (1989) has clearly stated that "difficulty increases with increased similarity of targets to nontargets and decreased similarity between nontargets" (the sentence in their abstract). Here, "similarity between nontargets" is the same as the visual homogeneity defined here. Similar effects have been shown in Duncan (1989) and Nagy, Neriani, and Young (2005). See also the inconsistent results in Nagy& Thomas, 2003, Vicent, Baddeley, Troscianko&Gilchrist, 2009. More recently, Wei Ji Ma has systematically investigated the effects of heterogeneous distractors in visual search. I think the introduction part of Wei Ji Ma's paper (2020) provides a nice summary of this line of research.

Thanks to the authors' revision, I now better understand the negative correlation. The between-distrator similarity mentioned above describes the heterogeneity of distractors WITHIN an image. However, if I understand it correctly, this study aims to address the negative correlation of reaction time and target-absent stimuli ACROSS images. In other words, why do humans show a shorter reaction time to an image of four pigeons than to an image of four dogs (as shown in Figure 2C), simply because the later image is closer to the reference point of the image space. In this sense, this negative correlation is indeed not the same as distractor heterogeneity. However, this is known as the saliency effect or oddball effects. For example, it seems quite natural to me that humans respond faster to a fish image if the image set contains many images of four-leg dogs that look very different from fish. If this is indeed a saliency effect, why should we define a new term "visual homogeneity"?

(6) The section "key predictions" is quite straightforward. I understand the logic of positive and negative correlations. However, what is the physical meaning of "decision boundary" (Fig. 1G) here? How does the "decision boundary" map on the image space?

(7) In my opinion, one of the advantages of this study is the fMRI dataset, which is valuable because previous studies did not collect fMRI data. The key contribution may be the novel brain region associated with display heterogeneity. If this is the case, I would suggest using a more parametric way to measure this region. For example, one can use Gabor stimuli and systematically manipulate the variations of multiple Gabor stimuli, the same logic also applies to motion direction. If this study uses static Gabor, random dot motion, object images that span from low-level to high-level visual stimuli, and consistently shows that the stimulus heterogeneity is encoded in one brain region, I would say this finding is valuable. But this sounds another experiment. In other words, it is insufficient to claim a new brain region given the current form of the manuscript.

References:

* Duncan, J., & Humphreys, G. W. (1989). Visual search and stimulus similarity. Psychological Review, 96(3), 433-458. doi: 10.1037/0033-295x.96.3.433<br /> * Duncan, J. (1989). Boundary conditions on parallel processing in human vision. Perception, 18(4), 457-469. doi: 10.1068/p180457<br /> * Nagy, A. L., Neriani, K. E., & Young, T. L. (2005). Effects of target and distractor heterogeneity on search for a color target. Vision Research, 45(14), 1885-1899. doi: 10.1016/j.visres.2005.01.007<br /> * Nagy, A. L., & Thomas, G. (2003). Distractor heterogeneity, attention, and color in visual search. Vision Research, 43(14), 1541-1552. doi: 10.1016/s0042-6989(03)00234-7<br /> * Vincent, B., Baddeley, R., Troscianko, T., & Gilchrist, I. (2009). Optimal feature integration in visual search. Journal of Vision, 9(5), 15-15. doi: 10.1167/9.5.15<br /> * Singh, A., Mihali, A., Chou, W. C., & Ma, W. J. (2023). A Computational Approach to Search in Visual Working Memory.<br /> * Mihali, A., & Ma, W. J. (2020). The psychophysics of visual search with heterogeneous distractors. BioRxiv, 2020-08.<br /> * Calder-Travis, J., & Ma, W. J. (2020). Explaining the effects of distractor statistics in visual search. Journal of Vision, 20(13), 11-11.

Reviewer #3 (Public review):

Summary of the review process from the Reviewing Editor:

The authors and the reviewers did not agree on several important points made in this paper. The reviewers were critical of the operationalisation of the concept of visual homogeneity (VH), and questioned its validity. For instance, they found it unsatisfying that VH was not calculated on the basis of images themselves, but on the basis of reaction times instead. The authors responded by providing further explanation and argumentation for the importance of this novel concept, but the reviewers were not persuaded. The reviewers also pointed out some data features that did not fit the theory (e.g., overlapping VH between present and absent stimuli), which the authors acknowledge as a point that needs further refining. Finally, the reviewers pointed out that the new so-called visual homogeneity brain region does not overlap very much in the two studies, to which the authors have responded that it is remarkable that there is even partial overlap, given the many confounding differences between the two studies. Altogether, the authors have greatly elaborated their case for VH as an important concept, but the reviewers were not persuaded, and we conclude that the current evidence does not yet meet the high bar for declaring that a novel image property, visual homogeneity, is computed in a localised brain region.