Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors set out to understand how animals respond to visible light in an animal without eyes. To do so, they used the C. elegans model, which lacks eyes, but nonetheless exhibits robust responses to visible light at several wavelengths. Here, the authors report a promoter that is activated by visible light and independent of known pathways of light responses.

Strengths:

The authors convincingly demonstrate that visible light activates the expression of the cyp-14A5 promoter-driven gene expression in a variety of contexts and report the finding that this pathway is activated via the ZIP-2 transcriptionally regulated signaling pathway.

Weaknesses:

Because the ZIP-2 pathway has been reported to be activated predominantly by changes in the bacterial food source of C. elegans -- or exposure of animals to pathogens -- it remains unclear if visible light activates a pathway in C. elegans (animals) or if visible light potentially is sensed by the bacteria on the plate, which also lack eyes. Specifically, it is possible that the plates are seeded with excess E. coli, that E. coli is altered by light in some way, and in this context, alters its behavior in such a way that activates a known bacterially responsive pathway in the animals. This weakness would not affect the ability to use this novel discovery as a tool, which would still be useful to the field, but it does leave some questions about the applicability to the original question of how animals sense light in the absence of eyes.

Thank you for the insightful questions and suggestions. We have now performed a key experiment requested. Interesting new data (Fig. S1I) show that light induction of cyp-14A5p::GFP requires live bacteria that maintain a non-starved physiological state. Neither plates without food nor plates with heat-killed OP50 support robust induction. We now include this interesting new result in the paper and revised discussion on the bacteria-modulated mechanism but note that this bacterial requirement does not alter the central conclusions of the study. Rather, it reveals an intriguing mechanistic layer, namely, that bacterial metabolic activity likely influences the animal’s sensitivity to environmental light. We are pursuing this host–microbe interaction in a separate study. In the present work, we focus on the intrinsic regulation and functional significance of cyp-14A5 under standard laboratory conditions with live OP50. Accordingly, we have revised the Results and Discussion to reflect the appropriate scope.

Reviewer #2 (Public review):

Summary:

Ji, Ma, and colleagues report the discovery of a mechanism in C. elegans that mediates transcriptional responses to low-intensity light stimuli. They find that light-induced transcription requires a pair of bZIP transcription factors and induces expression of a cytochrome P450 effector. This unexpected light-sensing mechanism is required for physiologically relevant gene expression that controls behavioral plasticity. The authors further show that this mechanism can be co-opted to create light-inducible transgenes.

Strengths:

The authors rigorously demonstrate that ambient light stimuli regulate gene expression via a mechanism that requires the bZIP factors ZIP-2 and CEBP-2. Transcriptional responses to light stimuli are measured using transgenes and using measurements of endogenous transcripts. The study shows proper genetic controls for these effects. The study shows that this light-response does not require known photoreceptors, is tuned to specific wavelengths, and is highly unlikely to be an artifact of temperature-sensing. The study further shows that the function of ZIP-2 and CEBP-2 in light-sensing can be distinguished from their previously reported role in mediating transcriptional responses to pathogenic bacteria. The study includes experiments that demonstrate that regulatory motifs from a known light-response gene can be used to confer light-regulated gene expression, demonstrating sufficiency and suggesting an application of these discoveries in engineering inducible transgenes. Finally, the study shows that ambient light and the transcription factors that transduce it into gene expression changes are required to stabilize a learned olfactory behavior, suggesting a physiological function for this mechanism.

Weaknesses:

The study implies but does not show that the effects of ambient light on stabilizing a learned olfactory behavior are through the described pathway. To show this clearly, the authors should determine whether ambient light has any effect on mutants lacking CYP-14A5, ZIP-2, or CEBP-2. Other minor edits to the text and figures are suggested.

We appreciate the reviewer’s comment. Our study indeed implies that ambient light stabilizes learned olfactory behavior through effects on the described pathway. Importantly, the existing data already address this point. Mutants lacking CYP-14A5, ZIP-2, or CEBP-2 display impaired olfactory memory even when exposed to ambient light, indicating that these genes are required for the behavioral effect of light. Consistent with this, ambient light robustly induces cyp-14A5p::GFP in wild-type animals but fails to do so in zip-2 and cebp-2 mutants, demonstrating that light-dependent transcriptional activation is blocked upstream in these pathway mutants. Together, these results support the conclusion that ambient light acts through the ZIP-2 → CEBP-2 → CYP-14A5 pathway to stabilize memory. Minor textual and figure revisions have been made where helpful to clarify this point.

Reviewer #3 (Public review):

Ji et al. report a novel and interesting light-induced transcriptional response pathway in the eyeless roundworm Caenorhabditis elegans that involves a cytochrome P450 family protein (CYP-14A5) and functions independently from previously established photosensory mechanisms. Although the exact mechanisms underlying photoactivation of this pathway remain unclear, light-dependent induction of CYP-14A5 requires bZIP transcription factors ZIP-2 and CEBP-2 that have been previously implicated in worm responses to pathogens. The authors then suggest that light-induced CYP-14A5 activity in the C. elegans hypoderm can unexpectedly and cell-non-autonomously contribute to retention of an olfactory memory. Finally, the authors demonstrate the potential for this pathway to enable robust light-induced control of gene expression and behavior, albeit with some restrictions. Overall, the evidence supporting the claims of the authors is convincing, and the authors' work suggests numerous interesting lines of future inquiry.

(1) The authors determine that light, but not several other stressors tested (temperature, hypoxia, and food deprivation), can induce transcription of cyp-15A5. The authors use these experiments to suggest the potential specificity of the induction of CYP-14A5 by light. Given the established relationship between light and oxidative stress and the authors' later identification of ZIP-2, testing the effect of an oxidative stressor or pathogen exposure on transcription of cyp-14A5 would further strengthen the validity of this statement and potentially shed some insight into the underlying mechanisms.

We appreciate the reviewer’s thoughtful suggestion. We would like to clarify that the “specificity” we refer to is the strong and preferential induction of cyp-14A5 by light among pathogen or detoxification-related genes, rather than an assertion that cyp-14A5 is exclusively light-responsive. This does not preclude the possibility that cyp-14A5 can also be activated under other conditions. Indeed, prior work from the Troemel laboratory has identified cyp-14A5 as one of many pathogen-inducible genes, consistent with its role in stress physiology. Our data show that classical pathogen-responsive genes (e.g., irg-1) are not induced by light, whereas cyp-14A5 is strongly induced, highlighting the selective engagement of this cytochrome P450 by light under the conditions tested. We have revised the text to clarify this point.

(2) The authors suggest that short-wavelength light more robustly increases transcription of cyp-14A5 compared to equally intense longer wavelengths (Figure 2F and 2G). Here, however, the authors report intensities in lux of wavelengths tested. Measurements of and reporting the specific spectra of the incident lights and their corresponding irradiances (ideally, in some form of mW/mm2 - see Ward et al., 2008, Edwards et al., 2008, Bhatla and Horvitz, 2015, De Magalhaes Filho et al., 2018, Ghosh et al., 2021, among others, for examples) is critical for appropriate comparisons across wavelengths and facilitates cross-checking with previous studies of C. elegans light responses. On a related and more minor note, the authors place an ultraviolet shield in front of a visible light LED to test potential effects of ultraviolet light on transcription of cyp-14A5. A measurement of the spectrum of the visible light LED would help confirm if such an experiment was required. Regardless, the principal conclusions the authors made from these experiments will likely remain unchanged.

Thank you. We have revised the text to clarify this point. “Using controlled light versus dark conditions, we confirmed the finding from an integrated cyp-14A5p::GFP reporter and observed its robust widespread GFP expression in many tissues induced by moderate-intensity (500-3000 Lux, 16-48 hr duration) LED light exposure (Fig. 1A). The photometric Lux range is approximately 0.1–0.60 mW/cm² in radiometric (total radiant power) metric given the spectrum of the LED light source.”

(3) The authors report an interesting observation that animals exposed to ambient light (~600 lux) exhibit significantly increased memory retention compared to those maintained in darkness (Figure 4). Furthermore, light deprivation within the first 2-4 hours after learning appears to eliminate the effect of light on memory retention. These processes depend on CYP-14A5, loss of which can be rescued by re-expression of cyp-14A5 in mutant animals using a hypoderm-specific- and non-light-inducible- promoter. Taken together, the authors argue convincingly that hypodermal expression of cyp-14A5 can contribute to the retention of the olfactory memory. More broadly, these experiments suggest that cell-non-autonomous signaling can enhance retention of olfactory memory. How retention of the olfactory memory is enhanced by light generally remains unclear. In addition, the authors' experiments in Figure 1B demonstrate - at least by use of the transcriptional reporter - that light-dependent induction of cyp-14A5 transcription at 500 - 1000 lux is minimal and especially so at short duration exposures. Additional experiments, including verification of light-dependent changes in CYP-14A5 levels in the olfactory memory behavioral setup, would help further interpret these otherwise interesting results.

We thank the reviewer for these thoughtful comments. We agree that understanding how light enhances memory retention at a mechanistic level is an important direction for future work. Regarding the light intensities used in Figure 1B, we would like to clarify that 500–1000 lux does produce a measurable and statistically significant induction of cyp-14A5p::GFP, although the magnitude is lower than that observed at higher intensities. We interpret this modest induction as physiologically relevant: intermediate light levels appear sufficient to engage the CYP-14A5–dependent program required for memory stabilization, whereas stronger light intensities are detrimental to learning and reduce behavioral performance. Thus, the behavioral paradigm uses a light regime that activates the pathway without introducing stress-associated confounders.

(4) The experiments in Figure 4 nicely validate the usage of the cyp-14A5 promoter as a potential tool for light-dependent induction of gene expression. Despite the limitations of this tool, including those presented by the authors, it could prove useful for the community.

Thank you and we agree. In addition, we have included in the revised manuscript the single-copy integration strains based on UAS-GAL4 that produced similar results as transgenic strains and will be even more flexible and useful for the community.

Recommendations for the authors:

Reviewing Editor Comments:

While appreciating the quality and presentation of this important study, we had two major concerns that the authors need to address.

(1) Bacteria-versus-worm origin:

To rule out a bacterially derived stimulus, we suggest testing whether cyp-14A5p::GFP is inducible without bacteria (or killed bacteria). Checking whether the canonical immune reporters irg-5p::GFP and gst-4p::GFP are also light-inducible will further clarify this point.

We have now performed the key experiment requested by the reviewers. Interesting new data (Fig. S1I) show that light induction of cyp-14A5p::GFP requires live bacteria that maintain a non-starved physiological state. Neither plates without food nor plates with heat-killed OP50 support robust induction. Importantly, this requirement does not alter any of the central conclusions of the study. Rather, it reveals an intriguing mechanistic layer, namely, that bacterial metabolic activity influences the animal’s sensitivity to environmental light. We are pursuing this host–microbe interaction in a separate study. In the present work, we focus on the regulation and functional significance of cyp-14A5 under standard laboratory conditions with live OP50.

We included the data (Fig. 2D) to show that the canonical immune reporter irg-1p::GFP is not induced by the light condition that robustly induced cyp-14A5p::GFP, and gst-4p::GFP is only very mildly induced (Fig. S1J).

(2) Pathway-behaviour link:

The behavioural relevance of the newly described pathway is intriguing, but it needs direct support. Ideally, this would require comparing memory in WT, zip-2-/-, cebp-2-/-, and cyp-14A5-/- under both dark and light conditions. But at the very least, it would require testing if constitutive CYP-14A5 rescue in the dark bypasses the requirement of light.

We respectfully submit that additional experiments are not required to support the behavioral conclusions. Our model posits that cyp-14A5 is required but not sufficient for memory stabilization, one component within a broader set of light-induced genes. Thus, constitutive hypodermal expression of cyp-14A5 would not be expected to bypass the requirement for ambient light. The existing data are fully consistent with this framework and conclusions of the paper.

Reviewer #1 (Recommendations for the authors):

Overall, I think this paper is interesting to the field of C. elegans researchers at a minimum, as a light-inducible gene expression system might have a variety of uses throughout the diverse research paradigms that use this model system. With that said, I have a couple of suggestions that I think would substantially impact the ability to interpret these findings, which might be useful for broader implications of the study.

(1) Most importantly, the supplemental table of RNA-seq data should likely be updated and discussed further beyond the cyp-14A5 findings. First, the authors report 7,902 genes are differentially expressed in response to light and then break these into upregulated and downregulated genes. But there are only 1,785 upregulated genes and 3,632 downregulated genes. This adds up to 5417 genes, but doesn't match the 7,902 genes reported to change, and I could not find in the text if some other filters were applied that might explain this not adding up.

Thank you for this helpful comment. We agree that the exact numbers depend on statistical thresholds and are therefore somewhat arbitrary. To avoid implying unwarranted precision, we have revised the text to state that “thousands of genes are differentially regulated by light.”

(2) Among the upregulated genes in response to light are irg-5, irg-4, irg-6, irg-8, and gst-4. Indeed, all of these well-studied genes (or most) show even more induction by light than cyp-14A5. It is my opinion that this result needs further criticism as there are existing GFP reporters for gst-4 and irg-5 that are similarly well studied to irg-1, which is in the paper (and is not upregulated). In my opinion, the authors should test if they see activation of the irg-4 and gst-4 GFP reporters by light as well. This would not only validate their RNA-seq but might provide more important evidence for the field, as these other reporters are not considered light-inducible previously. If they are, several major studies might be impacted by this.

Thank you for the comments. We have irg-1p::GFP and gst-4p::GFP in the lab but did not find other reporters for the genes mentioned from CGC. Neither of the two reporters showed light induction (Figs. 2D and S1J) as strongly as cyp-14A5p::GFP. It is possible that irg-1 and gst-4 RNA levels are up-regulated but not reflected in our transgenic reporters that used their promoters to drive GFP expression. Stronger light induction of cyp-14A5p::GFP is unlikely caused by the multi-copy nature of the transgene since newly generated single-copy integration strains based on the UAS-GAL4 system produced similar robust results for light induction (Fig. S1I and see Method).

(3) Along the same lines, if at least 4 (and likely more) well characterized immune response genes are activated by light and these genes are known to mostly respond to differences in C. elegans bacterial food source/diet, then it stands to reason that maybe in this experimental context the light is not acting on "animals" at all, but rather triggering changes in E. coli (i.e. changing E. coli metabolism or pathogenicity like properties). If true, then perhaps the light affects bacteria in such a way that it activates a previously known bacterial pathogen response mechanism. This should be easy to test by seeing if this reporter is still activated by light in the presence of diverse bacterial diets, which are available from the CGC (CeMBio collection, for example). This is likely very important to the conclusions of the manuscript as it relates to animals sensing light, but might not be as important to the use of this system as a tool.

Thank you for the insightful questions and suggestions. Interesting new data (Fig. S1I) show that light induction of cyp-14A5p::GFP requires live bacteria that maintain a non-starved physiological state. Neither plates without food nor plates with heat-killed OP50 support robust induction. Importantly, this requirement does not alter any of the central conclusions of the study. Rather, it reveals an intriguing mechanistic layer, namely, that bacterial metabolic activity influences the animal’s sensitivity to environmental light. We are pursuing this host–microbe interaction in a separate study. In the present work, we focus on the regulation and functional significance of cyp-14A5 under standard laboratory conditions with live OP50. We have revised the Results and Discussion to reflect the appropriate scope of our study and implications of the new findings.

(4) Lastly, it seems unlikely that nearly half the C. elegans genome is transcriptionally regulated by light (or nearly half of the detected genes in the RNA-seq results). It seems likely that this list of 7,902 genes contains false positives. I would suggest upping some sort of filter, like moving to padj < 0.01 instead of 0.05, or adding a 4-fold change filter (2-fold and 0.01 still results in near 5000+ genes changing, which might explain the difference in up and down genes just being due to different padj filters. Along these lines, it is worth noting that the padj is generated using DESeq2 it appears and one of the first assumptions of DESeq2 is that the median expressed genes do not change, and there is a normalization. However, if MOST genes do change in expression, then one of the fundamental assumptions of DESeq2 is not valid, and thus would mean it might not be an appropriate analysis tool - perhaps there is some other normalization that could be done before running DESeq2 due to some other noise present in the RNA-seq runs?

Thank you for this helpful comment. We agree that the exact numbers depend on statistical thresholds and are therefore somewhat arbitrary. To avoid implying unwarranted precision, we have revised the text to state that “thousands of genes are differentially regulated by light.”

(5) Minor point - I would delete the reference to ER in line 92. While most CYPs do localize to the ER, the images shown are not clearly ER and probably do not have enough resolution to make claims about subcellular localization. To me, it would be easier to just delete this claim as it is not required for the main claims of the manuscript.

Reference deleted.

Reviewer #2 (Recommendations for the authors):

I have one request for clarification that likely requires additional data. Figure 3 shows that ambient light stabilizes learned changes to chemotaxis and further shows that CYP-14A5 has a similar function. The implication is that light promotes CYP-14A5 expression, which somehow promotes memory consolidation. The authors should test whether memory consolidation in cyp-15A5, zip-2, or cebp-2 mutants is no longer affected by ambient light.

It is also possible to test whether forced expression of CYP14A5 can bypass the effect of 'no light' conditions on memory consolidation.

Thank you for the comments. We respectfully submit that additional experiments are not required to support the behavioral conclusions. Our model posits that cyp-14A5 is required but not sufficient for memory stabilization, one component within a broader set of light-induced genes. Thus, constitutive hypodermal expression of cyp-14A5 would not be expected to bypass the requirement for ambient light. The existing data are fully consistent with this framework and conclusions of the paper.

I have several minor suggestions relating to the text and figures.

(1) In the introduction, the authors assert that little is known about non-visual light sensing and then list many examples of molecular mechanisms of non-visual light-sensing. They should emphasize that non-visual light sensing is important and accomplished by diverse molecular mechanisms.

Agree and revised accordingly.

(2) Check spacing between gene names (line 109).

Corrected.

(3) There should be a new paragraph break when the uORF experiments are described (line 146).

Corrected.

(4) 'Phenoptosis' is an esoteric word. Please define it (line 206).

Corrected.

(5) 'p' in the transgene name cyp-14A5p::nlp-22 is in italics, unlike the rest of the manuscript.

Corrected.

(6) 'Acknowledgment' should be 'Acknowledgments' (line 384).

Corrected.

(7) The color map in panel 1B should have units.

It was arbitrary unit (now added) to highlight relative not absolute differences.

(8) In panel 1E, it is confusing to have 'DARK' denoted by reddish bars and 'LIGHT' denoted by bluish bars. Perhaps 'DARK' is black/dark grey and 'LIGHT' is white?

Corrected.

(9) In panel 1D, it takes a minute to find the purple diamond. Please mark up the volcano plot to make it easier.

Corrected.

Reviewer #3 (Recommendations for the authors):

The authors generally present convincing experiments detailing interesting results in a well-written manuscript.

One quick note: the same Bhatla and Horvitz (2015) papers appear to be cited twice [line 52].

Corrected.

Author response:

The following is the authors’ response to the latest reviews:

"One remaining question is the interpretation of matching variants with very low stable posterior probabilities (~0), which the authors have analyzed in detail but without fully conclusive findings. I agree with the authors that this event is relatively rare and the current sample size is limited but this might be something to keep in mind for future studies."

Fine-mapping stability – on matching variants with very low stable posterior probability

We thank Reviewer 2 for encouraging us to think more about how low stable posterior probability matching variants can be interpreted. We describe a few plausible interpretations, even though – as Reviewer 2 and we have both acknowledged – our present experiments do not point to a clear and conclusive account.

One explanation is that the locus captured by the variant might not be well-resolved, in the sense that many correlated variants exist around the locus. Thus, the variant itself is unlikely causal, but the set of variants in high LD with it may contain the true causal variant, or it's possible that the causal variant itself was not sequenced but lies in that locus. A comparison of LD patterns across ancestries at the locus would be helpful here.

Another explanation rests on the following observation. For a variant to be matching between top and stable PICS and to also have very small stable PP, it has to have the largest PP after residualization on the ALL slice but also have positive PP with gene expression on many other slices. In other words, failing to control for potential confounders shrinks the PP. If one assumes that the matching variant is truly causal, then our observation points to an example of negative confounding (aka suppressor effect). This can occur when the confounders (PCs) are correlated with allele dosage at the causal variant in a different direction than their correlation with gene expression, so that the crude association between unresidualized gene expression and causal variant allele dosage is biased toward 0.

Although our present study does not allow us to systematically confirm either interpretation – since we found that matching variants were depleted in causal variants in our simulations, violating the second argument, but we also found functional enrichment in analyses of GEUVADIS data though only 17 matching variants with low stable PP were reported – we believe a larger-scale study using larger cohort sizes (at least 1000 individuals per ancestry) and many more simulations (to increase yield of such cases) would be insightful.

———

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

Reviewer #1:

Major comments:

(1) It would be interesting to see how much fine-mapping stability can improve the fine-mapping results in cross-population. One can simulate data using true genotype data and quantify the amount the fine-mapping methods improve utilizing the stability idea.

We agree, and have performed simulation studies where we assume that causal variants are shared across populations. Specifically, by mirroring the simulation approach described in Wang et al. (2020), we generated 2,400 synthetic gene expression phenotypes across 22 autosomes, using GEUVADIS gene expression metadata (i.e., gene transcription start site) to ensure largely cis expression phenotypes were simulated. We additionally generated 1,440 synthetic gene expression phenotypes that incorporate environmental heterogeneity, to motivate our pursuit of fine-mapping stability in the first place (see Response to Reviewer 2, Comment 6). These are described in Results section “Simulation study”:

We evaluated the performance of the PICS algorithm, specifically comparing the approach incorporating stability guidance against the residualization approach that is more commonly used — similar to our application to the real GEUVADIS data. We additionally investigated two ways of “combining” the residualization and stability guidance approaches: (1) running stability-guided PICS on residualized phenotypes; (2) prioritizing matching variants returned by both approaches. See Response to Reviewer 2, Comment 5.

(2) I would be very interested to see how other fine-mapping methods (FINEMAP, SuSiE, and CAVIAR) perform via the stability idea.

Thank you for this valuable comment. We ran SuSiE on the same set of simulated datasets. Specifically, we ran a version that uses residualized phenotypes (supposedly removing the effects of population structure), and also a version that incorporates stability. The second version is similar to how we incorporate stability in PICS. We investigated the performance of Stable SuSiE in a similar manner to our investigation of PICS. First we compared the performance relative to SuSiE that was run on residualized phenotypes. Motivated by our finding in PICS that prioritizing matching variants improves causal variant recovery, we did the same analysis for SuSiE. This analysis is described in Results section “Stability guidance improves causal variant recovery in SuSiE.”

We reported overall matching frequencies and causal variant recovery rates of top and stable variants for SuSiE in Figures 2C&D.

Frequencies with which Stable and Top SuSiE variants match, stratified by the simulation parameters, are summarized in Supplementary File 2C (reproduced for convenience in Response to Reviewer 2, Comment 3). Causal variant recovery rates split by the number of causal variants simulated, and stratified by both signal-to-noise ratio and the number of credible sets included, are reported in Figure 2—figure supplements 16-18. We reproduce Figure 2—figure supplement 18 (three causal variants scenario) below for convenience. Analogous recovery rates for matching versus non-matching top or stable variants are reported in Figure 2—figure supplements 19, 21 and 23.

(3) I am a little bit concerned about the PICS's assumption about one causal variant. The authors mentioned this assumption as one of their method limitations. However, given the utility of existing fine-mapping methods (FINEMAP and SuSiE), it is worth exploring this domain.

Thank you for raising this fair concern. We explored this domain, by considering simulations that include two and three causal variants (see Response to Reviewer 2, Comment 3). We looked at how well PICS recovers causal variants, and found that each potential set largely does not contain more than one causal variant (Figure 2—figure supplements 20 and 22). This can be explained by the fact that PICS potential sets are constructed from variants with a minimum linkage disequilibrium to a focal variant. On the other hand, in SuSiE, we observed multiple causal variants appearing in lower credible sets when applying stability guidance (Figure 2—figure supplements 21 and 23). A more extensive study involving more fine-mapping methods and metrics specific to violation of the one causal variant assumption could be pursued in future work.

Reviewer #2:

Aw et al. presents a new stability-guided fine-mapping method by extending the previously proposed PICS method. They applied their stability-based method to fine-map cis-eQTLs in the GEUVADIS dataset and compared it against what they call residualization-based method. They evaluated the performance of the proposed method using publicly available functional annotations and claimed the variants identified by their proposed stability-based method are more enriched for these functional annotations.

While the reviewer acknowledges the contribution of the present work, there are a couple of major concerns as described below.

Major:

(1) It is critical to evaluate the proposed method in simulation settings, where we know which variants are truly causal. While I acknowledge their empirical approach using the functional annotations, a more unbiased, comprehensive evaluation in simulations would be necessary to assess its performance against the existing methods.

Thank you for this point. We agree. We have performed a simulation study where we assume that causal variants are shared across populations (see response to Reviewer 1, Comment 1). Specifically, by mirroring the simulation approach described in Wang et al. (2020), we generated 2,400 synthetic gene expression phenotypes across 22 autosomes, using GEUVADIS gene expression metadata (i.e., gene transcription start site) to ensure cis expression phenotypes were simulated.

(2) Also, simulations would be required to assess how the method is sensitive to different parameters, e.g., LD threshold, resampling number, or number of potential sets.

Thank you for raising this point. The underlying PICS algorithm was not proposed by us, so we followed the default parameters set (LD threshold, r² \= 0.5; see Taylor et al., 2021 Bioinformatics) to focus on how stability considerations will impact the existing fine-mapping algorithm. We attempted to derive the asymptotic joint distribution of the p-values, but it was too difficult. Hence, we used 500 permutations because such a large number would allow large-sample asymptotics to kick in. However, following your critical suggestion we varied the number of potential sets in our analyses of simulated data. We briefly mention this in the Results.

“In the Supplement, we also describe findings from investigations into the impact of including more potential sets on matching frequency and causal variant recovery…”

A detailed write-up is provided in Supplementary File 1 Section S2 (p.2):

“The number of credible or potential sets is a parameter in many fine-mapping algorithms. Focusing on stability-guided approaches, we consider how including more potential sets for stable fine-mapping algorithms affects both causal variant recovery and matching frequency in simulations…

Causal variant recovery. We investigate both Stable PICS and Stable SuSiE. Focusing first on simulations with one causal variant, we observe a modest gain in causal variant recovery for both Stable PICS and Stable SuSiE, most noticeably when the number of sets was increased from 1 to 2 under the lowest signal-to-noise ratio setting…”

We observed that increasing the number of potential sets helps with recovering causal variants for Stable PICS (Figure 2—figure supplements 13-15). This observation also accounts for the comparable power that Stable PICS has with SuSiE in simulations with low signal-to-noise ratio (SNR), when we increase the number of credible sets or potential sets (Figure 2—figure supplements 10-12).

(3) Given the previous studies have identified multiple putative causal variants in both GWAS and eQTL, I think it's better to model multiple causal variants in any modern fine-mapping methods. At least, a simulation to assess its impact would be appreciated.

We agree. In our simulations we considered up to three causal variants in cis, and evaluated how well the top three Potential Sets recovered all causal variants (Figure 2—figure supplements 13-15; Figure 2—figure supplement 15). We also reported the frequency of variant matches between Top and Stable PICS stratified by the number of causal variants simulated in Supplementary File 2B and 2C. Note Supplementary File 2C is for results from SuSiE fine-mapping; see Response to Reviewer 1, Comment 2.

Supplementary File 2B. Frequencies with which Stable and Top PICS have matching variants for the same potential set. For each SNR/ “No. Causal Variants” scenario, the number of matching variants is reported in parentheses.

Supplementary File 2C. Frequencies with which Stable and Top SuSiE have matching variants for the same credible set. For each SNR/ “No. Causal Variants” scenario, the number of matching variants is reported in parentheses.

(4) Relatedly, I wonder what fraction of non-matching variants are due to the lack of multiple causal variant modeling.

PICS handles multiple causal variants by including more potential sets to return, owing to the important caveat that causal variants in high LD cannot be statistically distinguished. For example, if one believes there are three causal variants that are not too tightly linked, one could make PICS return three potential sets rather than just one. To answer the question using our simulation study, we subsetted our results to just scenarios where the top and stable variants do not match. This mimics the exact scenario of having modeled multiple causal variants but still not yielding matching variants, so we can investigate whether these non-matching variants are in fact enriched in the true causal variants.

Because we expect causal variants to appear in some potential set, we specifically considered whether these non-matching causal variants might match along different potential sets across the different methods. In other words, we compared the stable variant with the top variant from another potential set for the other approach (e.g., Stable PICS Potential Set 1 variant vs Top PICS Potential Set 2 variant). First, we computed the frequency with which such pairs of variants match. A high frequency would demonstrate that, even if the corresponding potential sets do not have a variant match, there could still be a match between non-corresponding potential sets across the two approaches, which shows that multiple causal variant modeling boosts identification of matching variants between both approaches — regardless of whether the matching variant is in fact causal.

Low frequencies were observed. For example, when restricting to simulations where Top and Stable PICS Potential Set 1 variants did not match, about 2-3% of variants matched between the Potential Set 1 variant in Stable PICS and Potential Sets 2 and 3 variants in Top PICS; or between the Potential Set 1 variant in Top PICS and Potential Sets 2 and 3 variants in Stable PICS (Supplementary File 2D). When looking at non-matching Potential Set 2 or Potential Set 3 variants, we do see an increase in matching frequencies (between 10-20%) between Potential Set 2 variants and other potential set variants between the different approaches. However, these percentages are still small compared to the matching frequencies we observed between corresponding potential sets (e.g., for simulations with one causal variant this was 70-90% between Top and Stable PICS Potential Set 1, and for simulations with two and three causal variants this was 55-78% and 57-79% respectively).

We next checked whether these “off-diagonal” matching variants corresponded to the true causal variants simulated. Here we find that the causal variant recovery rate is mostly less than the corresponding rate for diagonally matching variants, which together with the low matching frequency suggests that the enrichment of causal variants of “off-diagonal” matching variants is much weaker than in the diagonally matching approach. In other words, the fraction of non-matching (causal) variants due to the lack of multiple causal variant modeling is low.

We discuss these findings in Supplementary File 1 Section S2 (bottom of p.2).

(5) I wonder if you can combine the stability-based and the residualization-based approach, i.e., using the residualized phenotypes for the stability-based approach. Would that further improve the accuracy or not?

This is a good idea, thank you for suggesting it. We pursued this combined approach on simulated gene expression phenotypes, but did not observe significant gains in causal variant recovery (Figure 2B; Figure 2—figure supplements 2, 13 and 15). We reported this Results “Searching for matching variants between Top PICS and Stable PICS improves causal variant Recovery.”

“We thus explore ways to combine the residualization and stability-driven approaches, by considering (i) combining them into a single fine-mapping algorithm (we call the resulting procedure Combined PICS); and (ii) prioritizing matching variants between the two algorithms. Comparing the performance of Combined PICS against both Top and Stable PICS, however, we find no significant difference in its ability to recover causal variants (Figure 2B)...”

However, we also confirmed in our simulations that prioritizing matching variants between the two approaches led to gains in causal variant recovery (Figure 2D; Figure 2—figure supplements 4, 19, 20 and 22). We reported this Results “Searching for matching variants between Top PICS and Stable PICS improves causal variant Recovery.”

“On the other hand, matching variants between Top and Stable PICS are significantly more likely to be causal. Across all simulations, a matching variant in Potential Set 1 is 2.5X as likely to be causal than either a non-matching top or stable variant (Figure 2D) — a result that was qualitatively consistent even when we stratified simulations by SNR and number of causal variants simulated (Figure 2—figure supplements 19, 20 and 22)...”

This finding is consistent with our analysis of real GEUVADIS gene expression data, where we reported larger functional significance of matching variants relative to non-matching variants returned by either Top of Stable PICS.

(6) The authors state that confounding in cohorts with diverse ancestries poses potential difficulties in identifying the correct causal variants. However, I don't see that they directly address whether the stability approach is mitigating this. It is hard to say whether the stability approach is helping beyond what simpler post-hoc QC (e.g., thresholding) can do.

Thank you for raising this fair point. Here is a model we have in mind. Gene expression phenotypes (Y) can be explained by both genotypic effects (G, as in genotypic allelic dosage) and the environment (E): Y = G + E. However, both G and E depend on ancestry (A), so that Y = G|A+E|A. Suppose that the causal variants are shared across ancestries, so that (G|A=a)=G for all ancestries a. Suppose however that environments are heterogeneous by ancestry: (E|A=a) = e(a) for some function e that depends non-trivially on a. This would violate the exchangeability of exogenous E in the full sample, but by performing fine-mapping on each ancestry stratum, the exchangeability of exogenous E is preserved. This provides theoretical justification for the stability approach.

We next turned to simulations, where we investigated 1,440 simulated gene expression phenotypes capturing various ways in which ancestry induces heterogeneity in the exogenous E variable (simulation details in Lines 576-610 of Materials and Methods). We ran Stable PICS, as well as a version of PICS that did not residualize phenotypes or apply the stability principle. We observed that (i) causal variant recovery performance was not significantly different between the two approaches (Figure 2—figure supplements 24-32); but (ii) disagreement between the approaches can be considerable, especially when the signal-to-noise ratio is low (Supplementary File 2A). For example, in a set of simulations with three causal variants, with SNR = 0.11 and E heterogeneous by ancestry by letting E be drawn from N(2σ,σ²) for only GBR individuals (rest are N(0,σ²)), there was disagreement between Potential Set 1 and 2 variants in 25% of simulations — though recovery rates were similar (Probability of recovering at least one causal variant: 75% for Plain PICS and 80% for Stable PICS). These points suggest that confounding in cohorts can reduce power in methods not adjusting or accounting for ancestral heterogeneity, but can be remedied by approaches that do so. We report this analysis in Results “Simulations justify exploration of stability guidance”

In the current version of our work, we have evaluated, using both simulations and empirical evidence, different ways to combine approaches to boost causal variant recovery. Our simulation study shows that prioritizing matching variants across multiple methods improves causal variant recovery. On GEUVADIS data, where we might not know which variants are causal, we already demonstrated that matching variants are enriched for functional annotations. Therefore, our analyses justify that the adverse consequence of confounding on reducing fine-mapping accuracy can be mitigated by prioritizing matching variants between algorithms including those that account for stability.

(7) For non-matching variants, I wonder what the difference of posterior probabilities is between the stable and top variants in each method. If the difference is small, maybe it is due to noise rather than signal.

We have reported differences in posterior probabilities returned by Stable and Top PICS for GEUVADIS data; see Figure 3—figure supplement 1. For completeness, we compute the differences in posterior probabilities and summarize these differences both as histograms and as numerical summary statistics.

Potential Set 1

- Number of non-matching variants = 9,921

- Table of Summary Statistics of (Stable Posterior Probability – Top Posterior Probability)

Author response table 1.

- Histogram of (Stable Posterior Probability – Top Posterior Probability)

Author response image 1.

Potential Set 2

- Number of non-matching variants = 14,454

- Table of Summary Statistics of (Stable Posterior Probability – Top Posterior Probability)

Author response table 2.

- Histogram of (Stable Posterior Probability – Top Posterior Probability)

Author response image 2.

Potential Set 3

- Number of non-matching variants = 16,814

- Table of Summary Statistics of (Stable Posterior Probability – Top Posterior Probability)

Author response table 3.

- Histogram of (Stable Posterior Probability – Top Posterior Probability)

Author response image 3.

We also compared the difference in posterior probabilities between non-matching variants returned by Stable PICS and Top PICS for our 2,400 simulated gene expression phenotypes. Focusing on just Potential Set 1 variants, we find two equally likely scenarios, as demonstrated by two distinct clusters of points in a “posterior probability-posterior probability” plot. The first is, as pointed out, a small difference in posterior probability (points lying close to y=x). The second, however, reveals stable variants with very small posterior probability (of order 4 x 10^–5 to 0.05) but with a non-matching top variant taking on posterior probability well distributed along [0,1]. Moving down to Potential Sets 2 and 3, the distribution of pairs of posterior probabilities appears less clustered, indicating less tendency for posterior probability differences to be small ( Figure 2—figure supplement 8).

Here are the histograms and numerical summary statistics.

Potential Set 1

- Number of non-matching variants = 663 (out of 2,400)

- Table of Summary Statistics of (Stable Posterior Probability – Top Posterior Probability)

Author response table 4.

- Histogram of (Stable Posterior Probability – Top Posterior Probability)

Author response image 4.

Potential Set 2

Number of non-matching variants = 1,429 (out of 2,400)

- Table of Summary Statistics of (Stable Posterior Probability – Top Posterior Probability)

Author response table 5.

- Histogram of (Stable Posterior Probability – Top Posterior Probability)

Author response image 5.

Potential Set 3

- Number of non-matching variants = 1,810 (out of 2,400)

- Table of Summary Statistics of (Stable Posterior Probability – Top Posterior Probability)

Author response table 6.

- Histogram of (Stable Posterior Probability – Top Posterior Probability)

Author response image 6.

(8) It's a bit surprising that you observed matching variants with (stable) posterior probability ~ 0 (SFig. 1). What are the interpretations for these variants? Do you observe functional enrichment even for low posterior probability matching variants?

Thank you for this question. We have performed a thorough analysis of matching variants with very low stable posterior probability, which we define as having a posterior probability < 0.01 (Supplementary File 1 Section S11). Here, we briefly summarize the analysis and key findings.

Analysis

First, such variants occur very rarely — only 8 across all three potential sets in simulations, and 17 across all three potential sets for GEUVADIS (the latter variants are listed in Supplementary 2E). We begin interpreting these variants by looking at allele frequency heterogeneity by ancestry, support size — defined as the number of variants with positive posterior probability in the ALL slice* — and the number of slices including the stable variant (i.e., the stable variant reported positive posterior probability for the slice).

*Note that the stable variant posterior probability need not be at least 1/(Support Size). This is because the algorithm may have picked a SNP that has a lower posterior probability in the ALL slice (i.e., not the top variant) but happens to appear in the most number of other slices (i.e., a stable variant).

For variants arising from simulations, because we know the true causal variants, we check if these variants are causal. For GEUVADIS fine-mapped variants, we rely on functional annotations to compare their relative enrichment against other matching variants that did not have very low stable posterior probability.

Findings

While we caution against generalizing from observations reported here, which are based on very small sample sizes, we noticed the following. In simulations, matching variants with very low stable posterior probability are largely depleted in causal variants, although factors such as the number of slices including the stable variant may still be useful. In GEUVADIS, however, these variants can still be functionally enriched. We reported three examples in Supplementary File 1 Section S11 (pp. 8-9 of Supplement), where the variants were enriched in either VEP or biologically interpretable functional annotations, and were also reported in earlier studies. We partially reproduce our report below for convenience.

“However, we occasionally found variants that stand out for having large functional annotation scores. We list one below for each potential set.

- Potential Set 1 reported the variant rs12224894 from fine-mapping ENSG00000255284.1 (accession code AP006621.3) in Chromosome 11. This variant stood out for lying in the promoter flanking region of multiple cell types and being relatively enriched for GC content with a 75bp flanking region. This variant has been reported as a cis eQTL for AP006632 (using whole blood gene expression, rather than lymphoblastoid cell line gene expression in this study) in a clinical trial study of patients with systemic lupus erythematosus (Davenport et al., 2018). Its nearest gene is GATD1, a ubiquitously expressed gene that codes for a protein and is predicted to regulate enzymatic and catabolic activity. This variant appeared in all 6 slices, with a moderate support size of 23.

- Potential Set 2 reported the variant rs9912201 from fine-mapping ENSG00000108592.9 (mapped to FTSJ3) in Chromosome 17. Its FIRE score is 0.976, which is close to the maximum FIRE score reported across all Potential Set 2 matching variants. This variant has been reported as a SNP in high LD to a GWAS hit SNP rs7223966 in a pan-cancer study (Gong et al., 2018). This variant appeared in all 6 slices, with a moderate support size of 32.

- Potential Set 3 reported the variant rs625750 from fine-mapping ENSG00000254614.1 (mapped to CAPN1-AS1, an RNA gene) in Chromosome 11. Its FIRE score is 0.971 and its B statistic is 0.405 (region under selection), which lie at the extreme quantiles of the distributions of these scores for Potential Set 3 matching variants with stable posterior probability at least 0.01. Its associated mutation has been predicted to affect transcription factor binding, as computed using several position weight matrices (Kheradpour and Kellis, 2014). This variant appeared in just 3 slices, possibly owing to the considerable allele frequency difference between ancestries (maximum AF difference = 0.22). However, it has a small support size of 4 and a moderately high Top PICS posterior probability of 0.64.

To summarize, our analysis of GEUVADIS fine-mapped variants demonstrates that matching variants with very low stable posterior probability could still be functionally important, even for lower potential sets, conditional on supportive scores in interpretable features such as the number of slices containing the stable variant and the posterior probability support size…”

May need changing if Emma cannot present - JB to confirm

is just getting started.

Ide ezt tenném be : A szerződés (a továbbiakban: Contract) a Letétkezelési üzletágnak a Client részére nyújtott szolgáltatását leíró, a CNXT-ben létező technikai entitás, nem igényli tényleges (fizikai) dokumentum meglétét, azt (ha létezik ilyen) nem rögzítjük a CNXT-ben.

Jelleg szempontjából háromféle Contractot különböztetünk meg CNXT-ben, ezek az alábbiak:

Account jellegű Contract, amely értékpapírszámlavezetési és kapcsolódó szolgáltatás nyújtására vonatkozik (pl. értékpapír számlavezetés és letétkezelés).

Framework jellegű Contract, amely már létező számlához további addicionális szolgáltatás nyújtására vonatkozik (pl. portfóliókezelés)

Classification jellegű Contract, amely a Client típusa szerinti addicionális szolgáltatás nyújtására vonatkozik (pl. privátbank)

A fentiek közül az ERSTE esetén csak az Account jellegű Contract lesz használva. Erre tekintettel a továbbiakban leírtak is erre a jellegre vonatkoznak.

EGészítsük ki még, ha itt a kötelező töltendő mezők vannak akkor még ide Custom value-kat is fel kell sorolni mert ezekből is van 3 ami nélkül nem menthető az ügyfél : C2 ID - C2 ID BEVA insured vagy BEVA Exception érték Fatca status

Átgondolom még ezt a részt

Margó szerintem ez nem lett nyelvesítve . ÍRok ki erre JIRA jegyet . Ha a magyar felületen Törzsadat szerepel, akkor itt is annak kéne.

EGészítsük ki még, ha itt a kötelező töltendő mezők vannak akkor még ide Custom value-kat is fel kell sorolni mert ezekből is van 3 ami nélkül nem menthető az ügyfél : C2 ID - C2 ID BEVA insured vagy BEVA Exeption érték Fatca status

Reviewer #1 (Public review):

While the structure of the melibiose permease in both outward and inward-facing forms has been solved previously, there remains unanswered questions regarding its mechanism. Hariharan et al set out to address this with further crystallographic studies complemented with ITC and hydrogen deuterium exchange (HDX) mass spectrometry. They first report 4 different crystal structures of galactose derivatives to explore molecular recognition showing that the galactose moiety itself is the main source of specificity. Interestingly, they observe a water-mediated hydrogen bonding interaction with the protein and suggest that this water molecule may be important in binding.

The results from the crystallography appear sensible, though the resolution of the data is low with only the structure with NPG better than 3Å. Support for the conclusion of the water molecule in the binding site, as interpreted from the density, is given by MD studies.

The HDX also appears to be well done and is explained reasonably well in the revision.

Reviewer #3 (Public review):

Summary:

The melibiose permease from Salmonella enterica serovar Typhimurium (MelBSt) is a member of the Major Facilitator Superfamily (MFS). It catalyzes the symport of a galactopyranoside with Na⁺, H⁺, or Li⁺, and serves as a prototype model system for investigating cation-coupled transport mechanisms. In cation-coupled symporters, a coupling cation typically moves down its electrochemical gradient to drive the uphill transport of a primary substrate; however, the precise role and molecular contribution of the cation in substrate binding and translocation remain unclear. In a prior study, the authors showed that the binding affinity for melibiose is increased in the presence of Na+ by about 8-fold, but the molecular basis for the cooperative mechanism remains unclear. The objective of this study was to better understand the allosteric coupling between the Na+ and melibiose binding sites. To verify the sugar-recognition specific determinants, the authors solved the outward-facing crystal structures of a uniport mutant D59C with four sugar ligands containing different numbers of monosaccharide units (α-NPG, melibiose, raffinose, or α-MG). The structure with α-NPG bound has improved resolution (2.7 Å) compared to a previously published structure and to those with other sugars. These structures show that the specificity is clearly directed toward the galactosyl moiety. However, the increased affinity for α-NPG involves its hydrophobic phenyl group, positioned at 4 Å-distance from the phenyl group of Tyr26 forms a strong stacking interaction. Moreover, a water molecule bound to OH-4 in the structure with α-NPG was proposed to contribute to the sugar recognition and appears on the pathway between the two specificity-determining pockets. Next, the authors analyzed by hydrogen-to-deuterium exchange coupled to mass spectrometry (HDX-MS) the changes in structural dynamics of the transporter induced by melibiose, Na+, or both. The data support the conclusion that the binding of the coupling cation at a remote location stabilizes the sugar-binding residues to switch to a higher-affinity state. Therefore, the coupling cation in this symporter was proposed to be an allosteric activator.

Strengths:

(1) The manuscript is generally well written.

(2) This study builds on the authors' accumulated knowledge of the melibiose permease and integrates structural and HDX-MS analyses to better understand the communication between the sodium ion and sugar binding sites. A high sequence coverage was obtained for the HDX-MS data (86-87%), which is high for a membrane protein.

The revised manuscript shows clear improvement, and the authors have addressed my concerns in a satisfactory manner. Of note, I noticed two mistakes that should be corrected:

- page 11. Unless I am mistaken, the sentence "In contrast, Na+ alone or with melibiose primarily caused deprotections" should be corrected with "protections". The authors may wish to verify this sentence and also the previous one in the main text.

- Figure 8 displays two cytoplasmic gates (one of them should be periplasmic)

Author response:

The following is the authors’ response to the original reviews

eLife Assessment

This manuscript presents useful insights into the molecular basis underlying the positive cooperativity between the co-transported substrates (galactoside sugar and sodium ion) in the melibiose transporter MelB. Building on years of previous studies, this work improves on the resolution of previously published structures and reports the presence of a water molecule in the sugar binding site that would appear to be key for its recognition, introduces further structures bound to different substrates, and utilizes HDX-MS to further understand the positive cooperativity between sugar and the co-transported sodium cation. Although the experimental work is solid, the presentation of the data lacks clarity, and in particular, the HDX-MS data interpretation requires further explanation in both methodology and discussion, as well as a clearer description of the new insight that is obtained in relation to previous studies. The work will be of interest to biologists and biochemists working on cation-coupled symporters, which mediate the transport of a wide range of solutes across cell membranes.

We express our gratitude to the associate editor, review editor, and reviewers for their favorable evaluation of this manuscript, as well as their constructive comments and encouragement. Their feedback has been integrated to fortify the evidence, refine the data analysis, and elevate the presentation of the results, thereby enhancing the overall quality and clarity of the manuscript.

A brief summary of the modifications in this revision:

(a) We performed four new experiments: 1) intact cell [³H]raffinose transport assay; 2) intact cell p-nitrophenol detection to demonstrate α-NPG transport; 3) ITC binding assay for the D59C mutant; and 4) molecular dynamics to simulate the water-1 in sugar-binding site and the dynamics of side chains in the Na⁺- and melibiose-binding pockets. All data consistently support the conclusion draw in this article.

(b) We have added a new figure to show the apo state dynamics (the new Fig. 5a,b) and annotated the amino acid residue positions and marked positions in sugar- or Na⁺-binding pockets.

(c) As suggested by reviewer-3, we have moved the individual mapping of ligand effects on HDX data to the main figure, combined with the residual plots, and marked the amino-acid residue positions.

(d) We have added more deuterium uptake plots to cover all residues in the sugar- or Na⁺-binding pockets in the current figure 7 (previously figure 6).

(e) We have added a new figure 8 showing the positions at the well-studied cytoplasmic gating salt-bridge network and other loops likely important for conformational changes, along with a membrane topology marked with the HDX data. We have added a new figure 9 from MD simulations.

Reviewer #1:

While the structure of the melibiose permease in both outward and inward-facing forms has been solved previously, there remain unanswered questions regarding its mechanism. Hariharan et al set out to address this with further crystallographic studies complemented with ITC and hydrogen-deuterium exchange (HDX) mass spectrometry.

(1) They first report 4 different crystal structures of galactose derivatives to explore molecular recognition, showing that the galactose moiety itself is the main source of specificity. Interestingly, they observe a water-mediated hydrogen bonding interaction with the protein and suggest that this water molecule may be important in binding.

We thank you for understanding what we've presented in this manuscript.

(2) The results from the crystallography appear sensible, though the resolution of the data is low, with only the structure with NPG better than 3Å. However, it is a bit difficult to understand what novel information is being brought out here and what is known about the ligands. For instance, are these molecules transported by the protein or do they just bind? They measure the affinity by ITC, but draw very few conclusions about how the affinity correlates with the binding modes. Can the protein transport the trisaccharide raffinose?

The four structures with bound sugars of different sizes were used to identify the binding motif on both the primary substrate (sugar) and the transporter (MelB_St). Although the resolutions of the structures complexed with melibiose, raffinose, or a-MG are relatively low, the size and shape of the densities at each structure are consistent with the corresponding sugar molecules, which provide valuable data for confirming the pose of the bound sugar proposed previously. In this revision, we further refine the α-NPG-bound structure to 2.60 Å. The identified water-1 in this study further confirms the orientation of C4-OH. Notably, this transporter does not recognize or transport glucosides in which the orientation of the C4-OH at the glucopyranosyl ring is opposite. To verify the water in the sugar-binding site, we initiated a new collaborative study using MD simulations. Results showed that Wat-1 exhibited nearly full occupancy when melibiose was present, regardless of whether Na⁺ was bound at the cation-binding site.

As detailed in the Summary, we added two additional sets of transport assays and confirmed that raffinose and α-NPG are transportable substrates of MelB_St. For α-NPG transport, we measured the end products of the process—enzyme hydrolysis and membrane diffusion of p-nitrophenol released from intracellular α-NPG.

As a bonus, based on the WT-like downhill α-NPG transport activity by the D59C uniporter mutant that failed in active transport against a sugar concentration gradient, we further emphasized that the sugar translocation pathway is isolated from the cation-binding site. The new data strongly support the allosteric effects of cation binding on sugar-binding affinity. Thank you for this helpful suggestion.

A meaningful analysis of ITC data heavily depends on the quality of the data. My laboratory has extensive experience with ITC and has gained rich, insightful mechanistic knowledge of MelB_St. Because of the low affinity in raffinose and a-MG, unfortunately, no further information can be convincingly obtained. Therefore, we did not dissect the enthalpic and entropic contributions but focused on the Kd value and binding stoichiometry.

(3) The HDX also appears to be well done; however, in the manuscript as written, it is difficult to understand how this relates to the overall mechanism of the protein and the conformational changes that the protein undergoes.

We are sorry for not presenting our data clearly in the initial submission. In this revised manuscript, we have made numerous improvements, as described in the Summary. These enhancements in the HDX data analysis provided new mechanistic insights into the allosteric effects, leading us to conclude that protein dynamics and conformational transitions are coupled with sugar-binding affinity. Na⁺ binding restricts protein conformational flexibility, thereby increasing sugar-binding affinity. The HDX study revealed that the major dynamic region includes a sugar-binding residue, Arg149, which also plays a gating role. Structurally, this dual-function residue undergoes significant displacement during the sugar-affinity-coupled conformational transition, thereby coupling the sugar binding and structural dynamics.

Reviewer #2:

This manuscript from Hariharan, Shi, Viner, and Guan presents x-ray crystallographic structures of membrane protein MelB and HDX-MS analysis of ligand-induced dynamics. This work improves on the resolution of previously published structures, introduces further sugar-bound structures, and utilises HDX to explore in further depth the previously observed positive cooperatively to cotransported cation Na⁺. The work presented here builds on years of previous study and adds substantial new details into how Na⁺ binding facilitates melibiose binding and deepens the fundamental understanding of the molecular basis underlying the symport mechanism of cation-coupled transporters. However, the presentation of the data lacks clarity, and in particular, the HDX-MS data interpretation requires further explanation in both methodology and discussion.

We appreciate this reviewer's time in reading our previous articles related to this manuscript.

Comments on Crystallography and biochemical work:

(1) It is not clear what Figure 2 is comparing. The text suggests this figure is a comparison of the lower resolution structure to the structure presented in this work; however, the figure legend does not mention which is which, and both images include a modelled water molecule that was not assigned due to poor resolution previously, as stated by the authors, in the previously generated structure. This figure should be more clearly explained.

This figure is a stereo view of a density map created in cross-eye style. In this revision, we changed this figure to Fig. 3 and showed only the density for sugar and water-1. 

(2) It is slightly unclear what the ITC measurements add to this current manuscript. The authors comment that raffinose exhibiting poor binding affinity despite having more sugar units is surprising, but it is not surprising to me. No additional interactions can be mapped to these units on their structure, and while it fits into the substrate binding cavity, the extra bulk of additional sugar units is likely to reduce affinity. In fact, from their listed ITC measurements, this appears to be the trend. Additionally, the D59C mutant utilised here in structural determination is deficient in sodium/cation binding. The reported allostery of sodium-sugar binding will likely influence the sugar binding motif as represented by these structures. This is clearly represented by the authors' own ITC work. The ITC included in this work was carried out on the WT protein in the presence of Na⁺. The authors could benefit from clarifying how this work fits with the structural work or carrying out ITC with the D59C mutant, or additionally, in the absence of sodium.

Thank this reviewer for your helpful suggestions. We have performed the suggested ITC measurements with the D59C mutant. The purpose of the ITC experiments was to demonstrate that MelB_St can bind raffinose and α-MG to support the crystal structures.

Comments on HDX-MS work:

While the use of HDX-MS to deepen the understanding of ligand allostery is an elegant use of the technique, this reviewer advises the authors to refer to the Masson et al. (2019) recommendations for the HDX-MS article (https://doi.org/10.1038/s41592-019-0459-y) on how to best present this data. For example:

All authors value this reviewer's comments and suggestions, which have been included in this revision.

(1) The Methodology includes a lipid removal step. Based on other included methods, I assumed that the HDX-MS was being carried out in detergent-solubilised protein samples. I therefore do not see the need for a lipid removal step that is usually included for bilayer reconstituted samples. I note that this methodology is the same as previously used for MelB. It should be clarified why this step was included, if it was in fact used, aka, further details on the sample preparation should be included.

Yes, a lipid/detergent removal step was included in this study and previous ones, and this information was clearly described in the Methods.

(2) A summary of HDX conditions and results should be given as recommended, including the mean peptide length and average redundancy per state alongside other included information such as reaction temperature, sequence coverage, etc., as prepared for previous publications from the authors, i.e., Hariharan et al., 2024.

We have updated the Table S2 and addressed the reviewer’ request for the details of HDX experiments.

(3) Uptake plots per peptide for the HDX-MS data should be included as supporting information outside of the few examples given in Figure 6.

We have prepared and presented deuterium uptake time-course plots for any peptides with ΔD > threshold in Fig. S5a-c.

(4) A reference should be given to the hybrid significance testing method utilised. Additionally, as stated by Hageman and Weis (2019) (doi:10.1021/acs.analchem.9b01325), the use of P < 0.05 greatly increases the likelihood of false positive ΔD identifications. While the authors include multiple levels of significance, what they refer to as high and lower significant results, this reviewer understands that working with dynamic transporters can lead to increased data variation; a statement of why certain statistical criteria were chosen should be included, and possibly accompanied by volcano plots. The legend of Figure 6 should include what P value is meant by * and ** rather than statistically significant and highly statistically significant.

We appreciate this comment and have cited the suggested article on the hybrid significance method. We fully acknowledge that using a cutoff of P < 0.05 can increase the likelihood of false-positive identifications. By applying multiple levels of statistical testing, we determined that P < 0.05 is an appropriate threshold for this study. The threshold values were presented in the residual plots and explained in the text. For the previous Fig. 6 (renamed Fig. S4b in the current version), we have reported the P value. *, < 0.05; **, < 0.01. (The text for 0.01 was not visible in the previous version. Sorry for the confusion.)

(5) Line 316 states a significant difference in seen in dynamics, how is significance measured here? There is no S.D. given in Table S4. Can the authors further comment on the potential involvement in solvent accessibility and buried helices that might influence the overall dynamics outside of their role in sugar vs sodium binding? An expected low rate of exchange suggests that dynamics are likely influenced by solvent accessibility or peptide hydrophobicity. The increased dynamics at peptides covering the Na binding site on overall more dynamic helices suggests that there is no difference between the dynamics of each site.

The current Table S3 (combined from previous Tables S3 and S4 as suggested) was prepared to provide an overall view of the dynamic regions with SD values provided. For other questions, if we understand correctly, this reviewer asked us to comment on the effects of solvent accessibility or hydrophobic regions on the overall dynamics outside the binding residues of the peptides that cover them. Since HDX rates are influenced by two linked factors: solvent accessibility and hydrogen-bonding interactions that reflect structural dynamics, poor solvent accessibility in buried regions should result in low deuterium uptakes. The peptides in our dataset that include the Na⁺-binding site showed lower HDX, likely due to limited solvent accessibility and lower structural stability. It is unclear what this reviewer meant by "increased dynamics at peptides covering the Na binding site on overall more dynamic helices." We did not observe increased dynamics in peptides covering the Na⁺-binding site; instead, all Na⁺-binding residues and nearby sugar-binding residues have lower degrees of deuteriation.

(6) Previously stated HDX-MS results of MelB (Hariharan et al., 2024) state that the transmembrane helices are less dynamic than polypeptide termini and loops with similar distributions across all transmembrane bundles. The previous data was obtained in the presence of sodium. Does this remove the difference in dynamics in the sugar-binding helices and the cation-binding helices? Including this comparison would support the statement that the sodium-bound MelB is more stable than the Apo state, along with the lack of deprotection observed in the differential analysis.

Thanks for this suggestion. The previous datasets were collected in the presence of Na⁺. In the current study, we also have two Na⁺-containing datasets. Both showed similar results: the multiple overlapping peptides covering the sugar-binding residues on helices I and V have higher HDX rates than those peptides covering the Na⁺-binding residues, even when Na⁺ was present.

(7) Have the authors considered carrying out an HDX-MS comparison between the WT and the D59C mutant? This may provide some further information on the WT structure (particularly a comparison with sugar-bound). This could be tied into a nice discussion of their structural data.

Thank you for this suggestion. Comparing HDX-MS between the WT and the D59C mutant is certainly interesting, especially with the increasing amount of structural, biochemical, and biophysical data now available for this mutant. However, due to limited resources, we might consider it later.

(8) Have the authors considered utilising Li⁺ to infer how cation selectivity impacts the allostery? Do they expect similar stabilisation of a higher-affinity sugar binding state with all cations?

We have shown that Li⁺ also works positively with melibiose. Li⁺ binds to MelB_St with a higher affinity than Na⁺ and modifies MelB_St differently. It is important to study this thoroughly and separately. To answer the second question, H⁺ is a weak coupling cation with little effect on melibiose binding. Since its pKa is around 6.5, only a small population of MelB_St is protonated at pH 7.5. The order of sugar-binding cooperativity is highest with Na⁺, then Li⁺, and finally H⁺.

(9) MD of MelB suggests all transmembrane helices are reorientated during substrate translocation, yet substrate and cotransporter ligand binding only significantly impacts a small number of helices. Can the authors comment on the ensemble of states expected from each HDX experiment? The data presented here instead shows overall stabilisation of the transporter. This data can be compared to that of HDX on MFS sugar cation symporter XylE, where substrate binding induces a transition to the OF state. There is no discussion of how this HDX data compares to previous MFS sugar transporter HDX. The manuscript could benefit from this comparison rather than a comparison to LacY. It is unlikely that there are universal mechanisms that can be inferred even from these model proteins. Highlighting differences between these transport systems provides broader insights into this protein class. Doi: 10.1021/jacs.2c06148 and 10.1038/s41467-018-06704-1.

The sugar translocation free-energy landscape simulations showed that both helix bundles move relative to the membrane plane. This analysis aimed to clarify a hypothesis in the field—that the MFS transporter can use an asymmetric mode to perform the conformational transition between inward- and outward-facing states. In the case of MelB_St, we clearly demonstrated that both domains move and each helix bundle moves as a unit. So only a small number of helices and loops showed labeling changes. Thanks for the suggestion about comparing with XylE. We have included that in the discussion.

(10) Additionally, the recent publication of SMFS data (by the authors: doi:10.1016/j.str.2022.11.011) states the following: "In the presence of either melibiose or a coupling Na⁺-cation, however, MelB increasingly populates the mechanically less stable state which shows a destabilized middle-loop C3." And "In the presence of both substrate and co-substrate, this mechanically less stable state of MelB is predominant.". It would benefit the authors to comment on these data in contrast to the HDX obtained here. Additionally, is the C3 loop covered, and does it show the destabilization suggested by these studies? HDX can provide a plethora of results that are missing from the current analysis on ligand allostery. The authors instead chose to reference CD and thermal denaturation methods as comparisons.

Thank this reviewer for reading the single-molecule force spectroscopy (SMFS) study on MelB_St.  The C3 loop mentioned in this SMFS article is partially covered in the dataset Mel or Mel plus Na⁺ vs. apo, and there is more coverage in the Na⁺ vs. apo dataset. In either condition, no deprotection was detected. The labeling time point might not be long enough to detect it.

Reviewer #3:

Summary:

The melibiose permease from Salmonella enterica serovar Typhimurium (MelB_St) is a member of the Major Facilitator Superfamily (MFS). It catalyzes the symport of a galactopyranoside with Na⁺, H⁺, or Li⁺, and serves as a prototype model system for investigating cation-coupled transport mechanisms. In cation-coupled symporters, a coupling cation typically moves down its electrochemical gradient to drive the uphill transport of a primary substrate; however, the precise role and molecular contribution of the cation in substrate binding and translocation remain unclear. In a prior study, the authors showed that the binding affinity for melibiose is increased in the presence of Na⁺ by about 8-fold, but the molecular basis for the cooperative mechanism remains unclear. The objective of this study was to better understand the allosteric coupling between the Na⁺ and melibiose binding sites. To verify the sugar-recognition specific determinants, the authors solved the outward-facing crystal structures of a uniport mutant D59C with four sugar ligands containing different numbers of monosaccharide units (α-NPG, melibiose, raffinose, or α-MG). The structure with α-NPG bound has improved resolution (2.7 Å) compared to a previously published structure and to those with other sugars. These structures show that the specificity is clearly directed toward the galactosyl moiety. However, the increased affinity for α-NPG involves its hydrophobic phenyl group, positioned at 4 Å-distance from the phenyl group of Tyr26, which forms a strong stacking interaction. Moreover, a water molecule bound to OH-4 in the structure with α-NPG was proposed to contribute to the sugar recognition and appears on the pathway between the two specificity-determining pockets. Next, the authors analyzed by hydrogen-to-deuterium exchange coupled to mass spectrometry (HDX-MS) the changes in structural dynamics of the transporter induced by melibiose, Na⁺, or both. The data support the conclusion that the binding of the coupling cation at a remote location stabilizes the sugar-binding residues to switch to a higher-affinity state. Therefore, the coupling cation in this symporter was proposed to be an allosteric activator.

Strengths:

(1) The manuscript is generally well written.

(2) This study builds on the authors' accumulated knowledge of the melibiose permease and integrates structural and HDX-MS analyses to better understand the communication between the sodium ion and sugar binding sites. A high sequence coverage was obtained for the HDX-MS data (86-87%), which is high for a membrane protein.

Thank this reviewer for your positive comments.

Weaknesses:

(1) I am not sure that the resolution of the structure (2.7 Å) is sufficiently high to unambiguously establish the presence of a water molecule bound to OH-4 of the α-NPG sugar. In Figure 2, the density for water 1 is not obvious to me, although it is indeed plausible that water mediates the interaction between OH4/OH6 and the residues Q372 and T373.

A water molecule can be modeled at a resolution ranging from 2.4 to 3.2 Å, and the quality of the model depends on the map quality and water location. In this revision, we refined the resolution to 2.6 Å using the same dataset and also performed all-atom MD simulations. All results support the occupancy of water-1 in the sugar-bound MelB_St.

(2) Site-directed mutagenesis could help strengthen the conclusions of the authors. Would the mutation(s) of Q372 and/or T373 support the water hypothesis by decreasing the affinity for sugars? Mutations of Thr121, Arg 295, combined with functional and/or HDX-MS analyses, may also help support some of the claims of the authors regarding the allosteric communication between the two substrate-binding sites.

The authors thank this reviewer for the thoughtful suggestions. MelB_St has been subjected to Cys-scanning mutagenesis (https://doi.org/10.1016/j.jbc.2021.101090). Placing a Cys residue at Gln372 significantly decreased the transport initial rate, accumulation, and melibiose fermentation, with minimal effect on protein expression, as shown in Figure 2 of this JBC article, which could support its role in the binding pocket. The T373C mutant retained most of the WT's activities. Our previous studies showed that Thr121 is only responsible for Na⁺ binding in MelB_St, and mutations decreased protein stability; now, HDX reveals that this is the rigid position. Additionally, our previous studies indicated that Arg295 is another conformationally important residue. In this version, we have added more HDX analysis to explore the relationship between the two substrate-binding sites with conformational dynamics, especially focusing on the gating salt-bridge network including Arg295, which has provided meaningful new insights.

(3) The main conclusion of the authors is that the binding of the coupling cation stabilizes those dynamic sidechains in the sugar-binding pocket, leading to a high-affinity state. This is visible when comparing panels c and a from Figure S5. However, there is both increased protection (blue, near the sugar) and decreased protection in other areas (red). The latter was less commented, could the increased flexibility in these red regions facilitate the transition between inward- and outward-facing conformations? The HDX changes induced by the different ligands were compared to the apo form (see Figure S5). It might be worth it for data presentation to also analyze the deuterium uptake difference by comparing the conditions sodium ion+melibiose vs melibiose alone. It would make the effect of Na⁺ on the structural dynamics of the melibiose-bound transporter more visible. Similarly, the deuterium uptake difference between sodium ion+melibiose vs sodium ion alone could be analyzed too, in order to plot the effect of melibiose on the Na⁺-bound transporter.

Thanks for this important question. We have added more discussion of the deprotected data and prepared a new Fig. 8b to highlight the melibiose-binding-induced flexibility in several loops, especially the gating area on both sides of the membrane. We also proposed that these changes might facilitate the formation of the transition-competent state. The overall effects induced by substrate binding are relatively small, and the datasets for apo and Na were collected separately, so comparing melibiose&Na⁺ versus Na⁺ might not be as precise. In fact, the Na⁺ effects on the sugar-binding site can be clearly seen in the deuterium uptake plots shown in Figures 7-8, by comparing the first and last panels.

(4) For non-specialists, it would be beneficial to better introduce and explain the choice of using D59C for the structural analyses.

Asp59 is the only site that responds to the binding of all coupling cations: Na⁺, Li⁺, or H⁺. Notably, this thermostable mutant D59C selectively abolishes all cation binding and associated cotransport activities, but it maintains intact sugar binding and exhibits conformational transition as the WT, as demonstrated by electroneutral transport reactions including α-NPG transport showed in this articles, and melibiose exchange and fermentation showed previously. Therefore, the structural data derived from this mutant are significant and offer important mechanistic insights into sugar transport, which supports the conclusion that the Na⁺ functions as allosteric activator.

(5) In Figure 5a, deuterium changes are plotted as a function of peptide ID number. It is hardly informative without making it clearer which regions it corresponds to. Only one peptide is indicated (213-226). I would recommend indicating more of them in areas where deuterium changes are substantial.

We appreciate this comment and have modified the plots by marking the residue position as well as labeled several peptides of significant HDX in the Fig 5b. We also provided a deuteriation map based on peptide coverage (Fig. 5a).

(6) From prior work of the authors, melibiose binding also substantially increases the affinity of the sodium ion. Can the authors interpret this observation based on the HDX data?

This is an intriguing mechanistic question. In this HDX study, we found that the cation-binding pocket and nearby sugar-binding residues are conformationally rigid, while some sugar-binding residues farther from the cation-binding pocket are flexible. We concluded that conformational dynamics regulate sugar-binding affinity, but the increase in Na-binding affinity caused by melibiose is not related to protein dynamics. Our previous interpretation based on structural data remains our preferred explanation; therefore, the bound melibiose physically prevents the release of Na⁺ or Li⁺ from the cation-binding pocket. We also proposed the mechanism of intracellular NA⁺ release in the 2024 JBC paper (https://doi.org/10.1016/j.jbc.2024.107427); after sugar release, the rotamer change of Asp55 will help NA⁺ exit the cation pocket into the empty sugar pocket, and the negative membrane potential inside the cell will further facilitate movement from MelB_St to the cytosol.

Recommendations for the authors:

Reviewing Editor Comments:

(1) It would help the reader if the previous work were introduced more clearly, and if the results of the experiments reported in this manuscript were put into the context of the previous work. Lines 283-296 discuss observations that are similar to previous reported structures as well as novel interpretations. It would help the reader to be clearer about what the new observations are.

Thank you for the important comment. We have revised accordingly by adding related citations and words “as showed previously” when we stated our previous observations.

(2) The affinity by ITC is measured for various ligands, but very few conclusions are drawn about how the affinity correlates with the binding modes. Are the other ligands that are investigated in this study transported by the protein, or do they just bind? Can the protein transport the trisaccharide raffinose? The authors comment that raffinose exhibiting poor binding affinity despite having more sugar units is surprising, but this is not surprising to me. No additional interactions can be mapped to these units on their structure, and while it fits into the substrate binding cavity, the extra bulk of additional sugar units is likely to reduce affinity. In fact, from their listed ITC measurements, this appears to be the trend.

Additionally, the D59C mutant utilized here in structural determination is deficient in sodium/cation binding. The reported allostery of sodium-sugar binding will likely influence the sugar binding motif as represented by these structures. This is clearly represented by the authors' own ITC work. The ITC included in this work was carried out on the WT protein in the presence of Na⁺. The authors could benefit from clarifying how this work fits with the structural work or carrying out ITC with the D59C mutant, or additionally, in the absence of sodium. For non-specialists, please better introduce and explain the choice of using D59C for the structural analyses.

Thank you for the meaningful comments. We have comprehensively addressed all the concerns and suggestions as listed in the summary of this revision. Notably, the D59C mutant does not catalyze any electrogenic melibiose transport involved in a cation transduction but catalyze downhill transport location of the galactosides, as shown by the downhill α-NPG transport assay in Fig. 1a. The intact downhill transport results from D59C mutant further supports the allosteric coupling between the cation- and sugar-binding sites.

The binding isotherm and poor affinity of the ITC measurements do not support to further analyze the binding mode since none showed sigmoidal curve, so the enthalpy change cannot be accurately determined. But authors thank this comment.

(3) It is not clear what Figure 2 is comparing. The text suggests this figure is a comparison of the lower resolution structure to the structure presented in this work; however, the figure legend does not mention which is which, and both images include a modelled water molecule that was not assigned due to poor resolution previously, as stated by the authors, in the previously generated structure. This figure should be more clearly explained.

We have addressed these concerns in the response to the Public Reviews at reviewer-2 #1.

(4) I am not sure that the resolution of the structure (2.7 Å) is sufficiently high to unambiguously establish the presence of a water molecule bound to OH-4 of the α-NPG sugar. In Figure 2, the density for water 1 is not obvious to me, although it is indeed plausible that water mediates the interaction between OH4/OH6 and the residues Q372 and T373. Please change line 278 to state "this OH-4 water molecule is likely part of sugar binding".

We have addressed these concerns in the response to the Public Reviews at reviewer-3 #1.

(5) Line 290-296: The Thr121 is not represented in any figures, while the Lys377 is. Their relative positioning between sugar water and sodium is not made clear by any figure.

Thanks for this comment. This information has been clearly presented in the Figs. 7-8. Lys377 is closer to the cation site and related far from the sugar-binding site.

(6) Methodology includes a lipid removal step. Based on other included methods, I assumed that the HDX-MS was being carried out in detergent-solubilized protein samples. I therefore do not see the need for a lipid removal step that is usually included for bilayer reconstituted samples. I note that this methodology is the same as previously used for MelB. It should be clarified why this step was included, if it was in fact used, aka, further details on the sample preparation should be included.

(7) A summary of HDX conditions and results should be given as recommended, including the mean peptide length and average redundancy per state alongside other included information such as reaction temperature, sequence coverage, etc., as prepared for previous publications from the authors, i.e., Hariharan et al., 2024.

We have addressed these concerns in the response to the Public Reviews at reviewer-2 #4.

(8) Uptake plots per peptide for the HDX-MS data should be included as supporting information outside of the few examples given in Figure 6.

We have addressed these concerns in the response to the Public Reviews at reviewer-2 #4.

(9) A reference should be given to the hybrid significance testing method utilised. Additionally, as stated by Hageman and Weis (2019) (doi:10.1021/acs.analchem.9b01325), the use of P < 0.05 greatly increases the likelihood of false positive ΔD identifications. While the authors include multiple levels of significance, what they refer to as high and lower significant results, and this reviewer understands that working with dynamic transporters can lead to increased data variation, a statement of why certain statistical criteria were chosen should be included, and possibly accompanied by volcano plots. The legend of Figure 6 should include what P value is meant by * and ** rather than statistically significant and highly statistically significant.

We have addressed these concerns in the response to the Public Reviews at reviewer-2 #4.

(10) The table (S3) and figure (S4) showing uncovered residues is an unclear interpretation of the data; this would be better given as a peptide sequence coverage heat map. This would also be more informative for the redundancy in covered regions, too. In this way, S3 and S4 can be combined.

We have addressed these concerns in the response to the Public Reviews at reviewer-2 #4.

(11) Residual plots in Figure 5 could be improved by a topological map to indicate how peptide number resembles the protein amino acid sequence.

Thanks for the request, due to the figure 6 is big so that we add a transmembrane topology plot colored with the HDX results in Fig. 8c.

(12) The presentation of data in S5 could be clarified. Does the number of results given in the brackets indicate overlapping peptides? What are the lengths of each of these peptides? Classical HDX data presentation utilizes blue for protection and red for deprotection. The use of yellow ribbons to show protection in non-sugar binding residues takes some interpretation and could be clarified by also depicting in a different blue. I also don't see the need to include ribbon and cartoon representation when also using colors to depict protection and deprotection. The authors should change or clarify this choice.

We have moved this figure into the current Fig. 6b as suggested by Reviewer-3. To address your questions listed in the figure legend, the number of results shown in brackets indeed indicates overlapping peptides. What are the lengths of each of these peptides? The sequences of each peptide are shown in Figures 7-8 and are also included in Supplemental Figure S5. Regarding the use of color, both blue and green were used to distinguish peptides protecting the substrate-binding site from other regions. The ribbon and cartoon representations are provided for clarity, as the cartoon style hides many helices.

(13) In Table S5, the difference between valid points and protection is unclear. And what is indicated by numbers in brackets or slashes? Additionally, it should be highlighted again here that single-residue information is inferred from peptide-level data. By value, are the authors referring to peptide-level differential data?

Please review our responses in the Public Reviews at reviewer-2 #5.

(14) Line 316 states a significant difference in seen in dynamics, how is significance measured here? There is no S.D. given in Table S4. Can the authors further comment on the potential involvement in solvent accessibility and buried helices that might influence the overall dynamics outside of their role in sugar vs sodium binding? An expected low rate of exchange suggests that dynamics are likely influenced by solvent accessibility or peptide hydrophobicity? The increased dynamics at peptides covering the Na binding site on overall more dynamic helices suggests that there isn't a difference between the dynamics of each site.

Please review our responses in the Public Reviews at reviewer-2 #5.

(15) Previously stated HDX-MS results of MelB (Hariharan et al., 2024) state that the transmembrane helices are less dynamic than polypeptide termini and loops with similar distributions across all transmembrane bundles. The previous data was obtained in the presence of sodium. Does this remove the difference in dynamics in the sugar-binding helices and the cation-binding helices? Including this comparison would support the statement that the sodium-bound MelB is more stable than the Apo state, along with the lack of deprotection observed in the differential analysis.

Please review our responses in the Public Reviews.

(16) MD of MelB suggests all transmembrane helices are reorientated during substrate translocation, yet substrate and cotransporter ligand binding only significantly impacts a small number of helices. Can the authors comment on the ensemble of states expected from each HDX experiment? The data presented here instead shows overall stabilisation of the transporter. This data can be compared to that of HDX on MFS sugar cation symporter XylE, where substrate binding induces a transition to the OF state. There is no discussion of how this HDX data compares to previous MFS sugar transporter HDX. The manuscript could benefit from this comparison rather than a comparison to LacY. It is unlikely that there are universal mechanisms that can be inferred even from these model proteins. Highlighting differences instead between these transport systems provides broader insights into this protein class. Doi: 10.1021/jacs.2c06148 and 10.1038/s41467-018-06704-1.

Please review our responses in the Public Reviews.

(17) Additionally, the recent publication of SMFS data (by the authors: doi:10.1016/j.str.2022.11.011) states the following: "In the presence of either melibiose or a coupling Na⁺-cation, however, MelB increasingly populates the mechanically less stable state which shows a destabilized middle-loop C3." And "In the presence of both substrate and co-substrate this mechanically less stable state of MelB is predominant.". It would benefit the authors to comment on these data in contrast to the HDX obtained here. Additionally, is the C3 loop covered, and does it show the destabilization suggested by these studies? HDX can provide a plethora of results that are missing from the current analysis on ligand allostery. The authors instead chose to reference CD and thermal denaturation methods as comparisons.

Please review our responses in the Public Reviews.

(18) The main conclusion of the authors is that the binding of the coupling cation stabilizes those dynamic sidechains in the sugar-binding pocket, leading to a high-affinity state. This is visible when comparing panels c and a from Figure S5. However, there is both increased protection (blue, near the sugar) and decreased protection in other areas (red). The latter was less commented, could the increased flexibility in these red regions facilitate the transition between inward- and outward-facing conformations? The HDX changes induced by the different ligands were compared to the apo form (see Figure S5). It might be worth it for data presentation more visible to also analyze the deuterium uptake difference by comparing the conditions sodium ion+melibiose vs melibiose alone. You would make the effect of Na⁺ on the structural dynamics of the melibiose-bound transporter. Similarly, the deuterium uptake difference between sodium ion+melibiose vs sodium ion alone could be analyzed too, in order to plot the effect of melibiose on the Na⁺-bound transporter.

Please review our responses in the Public Reviews.

(19) In Figure 5a, deuterium changes are plotted as a function of peptide ID number. It is hardly informative without making it clearer which regions it corresponds to. Only one peptide is indicated (213-226); I would recommend indicating more of them, in areas where deuterium changes are substantial.

Please review our responses in the Public Reviews.

(20) Figure 6, please indicate in the legend what the black and blue lines are (I assume black is for the apo?)

We are sorry that we did not make it clear. Yes, the black was used for apo state and blue was used for all bound states

(21) From prior work of the authors, melibiose binding also substantially increases the affinity of the sodium ion. Can the authors interpret this observation based on the HDX data?

Please review our responses in the Public Reviews.

Addressing the following three points would strengthen the manuscript, but also involve a significant amount of additional experimental work. If the authors decide not to carry out the experiments described below, they can still improve the assessment by focusing on points (1-21) described above.

(22) Have the authors considered carrying out an HDX-MS comparison between the WT and the D59C mutant? This may provide some further information on the WT structure (particularly a comparison with sugar-bound). This could be tied into a nice discussion of their structural data.

Please review our responses in the Public Reviews.

(23) Have the authors considered utilising Li⁺ to infer how cation selectivity impacts the allostery? Do they expect similar stabilisation of a higher-affinity sugar binding state with all cations?

Please review our responses in the Public Reviews.

(24) Site-directed mutagenesis could help strengthen the conclusions. Would the mutation(s) of Q372 and/or T373 support the water hypothesis by decreasing the affinity for sugars? Mutations of Thr 121 and Arg 295, combined with functional and/or HDX-MS analyses, may also help support some of the authors' claims regarding allosteric communication between the two substrate-binding sites.

Please review our responses in the Public Reviews.

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

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Reply to the reviewers

Reviewer #1

Evidence, reproducibility and clarity

__Summary

Köver et al. examine the genetic and environmental underpinnings of multicellular-like phenotypes (MLPs) in fission yeast, studying 57 natural isolates of Schizosaccharomyces pombe. They uncover that a noteworthy subset of these isolates can develop MLPs, with the extent of these phenotypes varying according to growth media. Among these, two strains demonstrate pronounced MLP across a range of conditions. By genetically manipulating one strain with an MLP phenotype (distinct from the previously mentioned two strains), they provide evidence that genes such as MBX2 and SRB11 play a direct role in MLP formation, strengthening their genetic mapping findings. The study also reveals that while some key genes and their phenotypic effects are strikingly similar between budding and fission yeast, other aspects of MLP formation are not conserved, which is an intriguing finding.

Overall, the manuscript is well-written, dense yet logically structured, and the figures are well presented. The combination of phenotypic, genetic, and bioinformatics analyses, particularly from wet lab experiments, is commendable. The study addresses a significant gap in our understanding, primarily explored in budding yeast, by providing comprehensive data on MLP diversity in fission yeast and the interplay of genetic and environmental factors.

In summary, I enjoyed reading the manuscript and have only a few minor suggestions to strengthen the paper:

Minor revisions:

1. Although this may seem like a minor revision, but it is a crucial point. Please make sure that all raw data used to generate figures, run stats, sequence data, and scripts used to run data analysis are made publicly available. Provide relevant accession numbers and links to public data repositories. It is important that others can download the various types of data that went into the major conclusions of this paper in order to replicate your analysis or expand upon the scope of this work. I am not sure if the journal has a policy regarding this, but it should be followed to allow for transparency and reproducibility of the research.__

Reply: We very much agree with the reviewer that sharing raw data and scripts is an essential part of open science. All code and data are deposited to Github (https://github.com/BKover99/S.-Pombe-MLPs) and Figshare (https://figshare.com/articles/software/S_-Pombe-MLPs/25750980), which have now been updated to reflect our revisions. Additionally, the sequenced genomes have been deposited to ENA (PRJEB69522). Where external data was used, it was properly referenced and specifically included in Supplementary Table 3.

Two out of 57 strains exhibit strong and consistent MLP across multiple environments. Providing more information on these strains (JB914 and JB953), such as their natural habitats and distinct appearances of their MLP phenotypes under varying conditions, would provide valuable insights.

First, a brief discussion highlighting what differentiates these two strains from the rest would be helpful for readers (e.g. insight into their unique genetic and environmental background that might be linked to the MLP phenotype).

Additionally, culture tube and microscopy images of these strains, similar to those presented for JB759 in Figure 2A, can be included in the supplementary materials. My reasoning is that these images could help illustrate variation or lack thereof in aggregative group size across different media.

Reply: We thank the reviewer for highlighting this issue. Our further investigation into these strains has added additional interesting insights. JB914 and JB953 were isolated from molasses in Jamaica and the exudate of Eucalyptus in Australia, respectively, though it remains unclear whether these environments are related or even selective for the ability of these strains to form MLPs. We note that the environment from which a strain is isolated is an incomplete way of assessing its ecology. Indeed, recent research suggests that the primary habitat of S. pombe is honeybee honey and suggests that bees, which may be attracted to a number of sugary substances, may be a vector by which fission yeast are transported (1). Therefore, isolation from a particular nectar or food production environment might not reflect significant ecological differences. We now refer to the location of strain isolation in the manuscript text (lines 208-209).

However, there is more to learn from the genetic backgrounds of these two strains. We found that JB914 possesses the same variant in srb11 causally related to MLPs as JB759, the MLP-forming parental strain for our QTL analysis. To understand whether the appearance of this variant in these two strains derived from a single mutation event or was a case of convergent evolution, we analysed homology between the genomes of JB759 and JB914, focusing specifically on that variant. We found an approximately 20kb region of homology between JB759 and JB914 surrounding the srb11 truncation variant, in contrast to the majority of the genome, which does not share homology between those two strains (New Supplementary Figure 9A, B)). This result suggests that, while the two strains are largely unrelated, that specific region shares a recent common ancestor and is likely a result of interbreeding across strains.

Importantly, this analysis further emphasizes the point that the srb11 variant segregates with the MLP-forming phenotype. We conclude this because none of the other strains similar to JB759 (either across the whole genome, or specifically in the region surrounding srb11) exhibit MLPs (New Supplementary Figure 9C). This thereby further complements our QTL analysis on the significance of this variant. We have added this analysis to the manuscript text (lines 337-349).

Furthermore, we searched other strains which exhibited MLPs in our experiments (e.g. JB953) for frame shifts, insertions or deletions in any other genes in the CKM module or in the genes that were identified in our deletion library screen as adhesive, and did not identify any severe mutations falling into coding regions (other than the srb11 truncation in JB914 and JB759). This indicates that MLPs in these other strains may be caused by differences in regulatory regions surrounding these genes, or variants in other genes that were not identified in our screen. We have added this analysis to our manuscript (lines 424-425) and Supplementary Table 13.

We agree that microscopy and culture tube images of JB914 and JB953 may give insight into the nature of the MLPs exhibited by those strains. We have included such images of cultures grown in YES, EMM and EMM-Phosphate media in our revision (Lines 207-208, Supplementary Figures 4 and 5). These images are consistent with our adhesion assay screen and show that JB914 and JB953 are adhesive at the microscopic level in the relevant conditions (EMM or EMM-Phosphate).

The phenotypic outcome of overexpressing MXB2 is striking, as shown in Supplementary Figure 4C. Incorporating at least one of the culture tube images depicting large flocs into the main text, perhaps adjacent to Figure 3 panel D, would improve the visual appeal and highlight this key finding (at the moment those images are only shown in the supplementary materials).

Reply: We thank the reviewer for this suggestion. In response to Reviewer 2's suggestion to overexpress mbx2 in YES, we created new mbx2 overexpression strains that could overexpress mbx2 in YES, which was not possible in our previous strain in which mbx2 overexpression was triggered by removal of thymine from the media. We have replaced our original data from Figure 3D with data from the new mbx2 overexpression experiment, including flask images.

I know that the authors discuss the knowledge gap in the intro and results, but the abstract does not mention this critical gap. Please stress this critical gap (i.e., MLPs understudied in fission yeast) with a brief sentence in the abstract. Similarly, please consider writing a brief concluding sentence summarizing the paper's most significant finding referring to the knowledge gap would provide a clearer takeaway message for the reader - the abstract ends abruptly without any conclusion.

Reply: We agree and have now emphasized the critical gap in our abstract:

"As MLP formation remains understudied in fission yeast compared to budding yeast, we aimed to narrow this gap." at lines 18-19.

Additionally, we added the following final sentence to give the reader a clearer takeaway message:

"Our findings provide a comprehensive genetic survey of MLP formation in fission yeast, and a functional description of a causal mutation that drives MLP formation in nature." at lines 31-32.

1. The observation that strains with adhesive phenotypes have a lower growth rate compared to non-adhesive strains is a noteworthy point (lines 532-535). This represents yet another example of this classical trade-off. This point could be emphasized in the Discussion or alongside the relevant result, with a brief speculative explanation for this phenomenon.

Reply: We agree that the nature of the trade-off between MLP formation is an interesting discussion point that could arise from our work. Understanding this trade-off is made more complicated by the fact that growth is always condition-dependent, and measuring growth in strains exhibiting MLPs is non-trivial, as adhesion to labware and thick clumps of cells separated by regions of cell-free media can add variability. Nonetheless, there has been some previous work on this problem. In S. cerevisiae, it was shown that larger group size correlates with slower growth rate (3), and that flocculating cells grow more slowly (4). In S. cerevisiae, cAMP, a signalling molecule heavily involved in regulating growth in response to nutrient availability, also regulates filamentation (5). However, the relationship between flocculation and slow growth is not consistent in the literature. In some settings overexpressing the flocculins FLO8, FLO5, and FLO10 results in slower growth (6), while in others it does not (7). In addition, ethanol production has been shown to improve for biofilms (7).

Furthermore, in S. cerevisiae, MLP-forming cells grow better in low sucrose concentrations (8) and under various stress conditions (4). Flocculating cells have also shown faster fermentation in media containing common industrial bioproduction inhibitors, despite slower fermentation than non-flocculating cells in non-inhibitory media (9). However, any consequence of this possible advantage on growth has not been characterised.

In S. pombe, there is less work on this topic; however, it has been shown that deletions of rpl3201 and rpl3202, which code for ribosomal proteins, cause flocculation and slow growth (10). In that case, it is not clear if there is any causal relationship between slow growth and flocculation or if they are both parallel consequences of the ribosomal pathway disruption. We have added some of these points to the portion of the discussion that discusses this tradeoff (Lines 477-499).

To get a better understanding of this tradeoff in our system, we took several approaches. First, we added a supporting analysis (New Supplementary Figure 12B), using published growth data based on measurements on agar plates for the S. pombe gene deletion library (11). There, the authors defined a set of deletion strains that grow more slowly on EMM than the wild-type lab strain. We found that our MLP hit strains were significantly enriched in this "EMM-slow" category. This information is now included in the manuscript (Lines 409-413, New Supplementary Figure 12B).

It is, however, possible that for the assays from that work, the appearance of slow growth on solid agar in adhesive cells could be partially artifactual. Indeed, we have observed that adhesive cells tend to stick to flasks and, when grown on agar plates, cells in the same colony can stick to one another rather than to inoculation loops or pin pads. Both of these dynamics can reduce initial inoculation densities. This is less of a concern for our adhesion assay and Figures 2E, 5B, and 5F, because our before-wash intensity was done with a 7x7 pinned square about 10x10 mm2. Nonetheless, as we wanted to make a point about srb10 and srb11 mutants growing faster than other deletion mutants that exhibit MLP-formation, we also conducted growth assays in liquid media (New Figure 5F).

We observed that srb10Δ and srb11Δ strains (which exhibit MLPs in EMM) show growth curves similar to wild-type cells in minimal (EMM) and rich media (YES). On the other hand, other strains that grow similarly to wild type cells in YES, such as tlg2Δ and rpa12Δ, grow much more slowly in EMM when they clump together. There are also some strains, mus7Δ and kgd2Δ, that grow more slowly in both YES and EMM but are only adhesive in EMM.

The text mentions two lab strains, JB22 and JB50, displaying strong adhesion under phosphate starvation (lines 525-526), yet the data point for JB22 in Figure 2C is not labeled.

Reply: We agree that highlighting JB22 on the figure is crucial, given that it was mentioned in the main text. JB22 is now highlighted in green on Fig 2C.

1. Although I generally avoid commenting on formatting, I found the manuscript to be dense. As mentioned above, I truly enjoyed reading it! But I couldn't help but think of ways to make the manuscript more concise for readers. The Results section spans nine pages (excluding figure captions), and the Discussion is five pages long. The main text contains 6 figures with approximately 27 panels and 32 plots and Venn diagrams, while the supplementary material has 11 figures with 22 panels and about 59 plots. Altogether, the manuscript comprises 17 figures, 49 panels, and roughly 91 plots and Venn diagrams! While I will not request any changes, I encourage the authors to consider streamlining the text/data where possible to focus on the core theme of the study.

We thank the reviewer for these suggestions and have reorganised some of our figures and text to appear less dense. We have also added several figures and panels in response to reviewer comments. While we endeavor to make our points clear and concise in the main figures, we believe that it is important to retain key supplementary figures so that an interested reader can evaluate the data in more detail:

A summary of our major changes to the figures is below, and we also provide a manuscript with changes tracked for the reviewers' convenience:

Fig 2:

Added Panel E in response to reviewer comments. Fig 3:

Removed axes for pfl3 and pfl7 from Fig 3C, as the point was made by the other genes displayed (mbx2, pfl8 and gsf2) Replaced Fig 3D with similar data from an improved experiment in response to reviewer comments. Added New Fig 3F from Original Supp Fig 5 Fig 5:

Moved Original Fig 5A to New Supp Fig 10A. Added New Fig 5F in response to reviewer comments. Original Supp Fig 4 / New Supp Fig 6:

Removed mbx2 overexpression images from Original Fig 4C, to be replaced by new overexpression data and images in New Fig 3D. Added flask images for srb10 and srb11 deletion mutants from Original Supp Fig 5A to New Supp Fig 6C. Added microscope image for srb11 deletion mutant from Ooriginal Supp Fig 5A to New Supp Fig 6C. Added adhesion assay results from Original Supp Fig 5C to New Supp Fig 6C. Added New Supp Fig 6D in response to review Original Supp Fig 5

Removed this figure. Original Supp Fig 5A and 5B were moved to New Supp Fig 6. Original Supp Fig 5B was removed to make the manuscript more concise. Original Supp Figs 6, 7 and 8 were combined into New Supp Fig 8.

Original Supp Fig 6A and 6B are now New Supp Fig 8A and 8B. Original Supp Fig 7 is now New Supp Fig 8C. Original Supp Fig 8A is now New Supp Fig 8D and 8E. Original Supp Fig 8B is now New Supp Fig 8F Original Supp Fig 9/New Supp Fig 10

Added Original Fig 5A as new Supp Fig 10A. Original Supp Fig 11/New Supp Fig 12

Removed Original Fig 11B and the relevant text to make the manuscript more concise. Added New Supp Fig 12B in response to reviewer comments. New Supplementary Figures added in response to reviewer comments:

New Supp Fig 4: Microscopy images of natural isolates. New Supp Fig 5: Flask images of natural isolates New Supp Fig 7: Microscopy and flask images of mbx2 overexpression strains. New Supp Fig 9: Genomic comparisons between JB759 and the MLP-forming wild isolate, JB914. Removed some less relevant points from our discussion, to reduce the length.

Added new Supplementary Tables:

Supplementary Table 13: Variants in candidate genes. Added in response to reviewer comments Supplementary Table 14: List of plasmids used in the study.

**Referees cross-commenting**

There are many useful recommendations from all the other reviewers that will help improve the final product. Once those points are revised, I think this will be a nice paper of interest to folks interested in natural variation in MLPs and its genetic background.

Significance

My expertise: evolutionary genetics, evolution of multicellularity, yeast genetics, experimental evolution

Overall, the manuscript is well-written, dense yet logically structured, and the figures are well presented. The combination of phenotypic, genetic, and bioinformatics analyses, particularly from wet lab experiments, is commendable. The study addresses a significant gap in our understanding, primarily explored in budding yeast, by providing comprehensive data on MLP diversity in fission yeast and the interplay of genetic and environmental factors.

In summary, I enjoyed reading the manuscript and have only a few minor suggestions to strengthen the paper.

Reviewer #2

Evidence, reproducibility and clarity

REVIEWER COMMENTS

Yeast species, including fission yeast and budding yeast, could form multicellular-like phenotypes (MLP). In this work, Kӧvér and colleagues found most proteins involved in MLP formation are not functionally conserved between S. pombe and budding yeast by bioinformatic analysis. The authors analyzed 57 natural S. pombe isolates and found MLP formation to widely vary across different nutrient and drug conditions. The authors demonstrate that MLP formation correlated with expression levels of the transcription factor gene mbx2 and several flocculins. The authors also show that Cdk8 kinase module and srub11 deletions also resulted in MLP formation. The experimental design is logic, the manuscript is well-written and organized. I have a few concerns that should be addressed before the publication.

Major points:

1) Line 61-62, how did the authors grow yeast cells in the liquid medium? Shaking or static? If shaking, the nutrient should be even distributed in the medium.

If static culture, most single yeast cells could precipitate on the bottom, how do you address the advantage of flocculation for increasing the sedimentation? In addition, under static culture, the bottom will have less air than the up medium, how to balance the air and nutrients?

Reply: In line 61-62 we stated that "Similarly, flocculation could increase sedimentation in liquid media, thereby assisting the search for more nutrient-rich or less stressful environments (4)".

Our intent was to speculate on the advantages of multicellular-like growth, and cited a review article which has mentioned sedimentation. After further consideration, we decided that this is a minor point and is rather speculative, and removed it altogether from the manuscript.

In response to the Reviewer's question about how cells were grown in liquid medium, throughout the paper we used shaking cultures for our flocculation assays and for pre-cultures. We have made this more clear in the text where it was ambiguous (e.g. line 189, throughout the methods section, and in the legend of Fig. 2A).

2) Line 555, it will be interesting to test whether overexpression of mbx2 could cause flocculation in YES medium. In Figure 3D, the authors use two control strains, but only one mbx2 OE strain, mbx2 OE should be tested in both strains. In addition, did the authors transform empty plasmid into the control strains, please indicate in the figure.

In this experiment, mbx2 was overexpressed using a thiamine-repressible nmt1 promoter, which is a standard construct in fission yeast studies. Assaying MLP formation was not feasible in YES with this strain, because YES is a rich media made up of yeast extract which contains thiamine. Thus, we could not remove thiamine from the media to trigger mbx2 overexpression.

In order to test the influence of mbx2 overexpression in YES, we constructed strains in which mbx2 was integrated into the genome and expression was driven by the rpl2102 promoter, which has been shown to provide constitutive moderate expression levels (12). We observed strong flocculation in both EMM and YES (Fig 3D, New Supplementary Figure 7) . We did not see strong flocculation in a control in which GFP was expressed under the rpl2102 promoter. The flocculation phenotype was so strong that our original adhesion assay protocol required modification for this experiment, including resuspension in 10 mM EDTA before repinning (Methods). We observed strong adhesion for the mbx2 overexpression strains (Fig 3D), but not for control strains in YES. We could not check adhesion in EMM for those strains because cells pinned on EMM did not survive resuspension in EDTA.

We performed these experiments in two backgrounds, 968 h90 (JB50), which is one of the parental strains of the segregant library analysed in Figure 3 and 972 h- (JB22), which is an appropriate background for the gene deletion collection.

We have replaced the data from the original Figure 3D with the new adhesion assay and added New Supplementary Figure 7 to the manuscript (Lines 236-244).

This result also helped us to further refine our model for the pathway. We can now say that the repression of MLPs in rich media must act via Mbx2, as overexpression of mbx2 is sufficient to abolish it, and is likely to act transcriptionally (if it acted on the protein level, the mild overexpression would likely not have led to the phenotype) (Figure 6, Lines 554-556 in the discussion)

3) Line 600-601, the authors may do the backcross of srb11Δ::Kan to exclude the possibility caused by other mutations.

Reply: We thank the reviewer for noticing our concern about suppressor mutations arising in the srb11Δ strain obtained from our deletion library. This initial concern arose following the observation that while qualitatively the srb11Δ::Kan and srb11Δ(CRISPR) strains were both strongly adhesive, there was a minor quantitative difference in their adhesion.

As we obtained this strain from an h+ deletion library strain backcrossed with a prototrophic h- strain (JB22) in order to restore auxotrophies (13), the chances for a suppressor mutation to arise are very low. We have therefore removed that language from our text. We now suspect that a more likely explanation for this small difference could be the strain background, as our CRISPR engineered strain was made in a JB50 background which has the h90 mating type, while the deletion library strains are h- without auxotrophic markers.

We would like to emphasize, however, that despite this quantitative difference in the adhesion phenotype between the two srb11Δ strains, they both have a large increase in the adhesion phenotype relative to the respective wild-type strains. To address this point, we have removed the unnecessary statistical comparison of these two deletion strains and focused on their qualitatively high levels of adhesion in the text (lines 267-269) and in our Revised Supplementary Figure 6D.

Minor points:

1) Line 506, what are the growth conditions of cells in Figure 2A? Did the authors use the liquid or solid medium? Please mention in the Methods or figure legends.

Reply: We have updated the manuscript to include the relevant details in the text (line 189), figure caption for Fig. 2A and in the methods section (lines 829-831).

2) Line 533-535, please explain why the strains exhibiting strong adhesion have a decreased growth rate. Is there any related research? Please add some references.

Reply: Please see reply to Reviewer 1, comment 5.

**Referees cross-commenting**

I agree with most of the comments from other reviewers. This publication may indeed be of interest to a minor area. But the results and the interpretations of the data are interesting and warranted, the findings are scientifically important.

Significance

The authors did many large-scale screens and bioinformatic analyses. The experiments in the manuscript are generally logical and sound. This study is useful for deciphering the mechanism of multicellular-like phenotype formation in the fission yeast, with some implications for some other organisms.

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

Summary: Using a variety of targeted and genome wide analyses, the authors investigate the basis for "multicellular-like phenotypes" in S. pombe. Authors developed several methodologies to detect and quantify "multicellular-like phenotypes" (flocculation, aggregation...) and defined genes involved in these processes in laboratory and wild S. pombe.

SECTION A - Evidence, reproducibility and clarity

This is a very solid manuscript that is well-written and supported by convincing data. While one can imagine many additional experiments, the manuscript stands on its own and presents a quite exhaustive analysis of the area. I commend the author for their rigorous work and clear presentation. They are only a few minor points that warrant comments or corrections: - Supplementary Figure 1 is a typical example of the "necessity" to have statistics and P-values everywhere. The data are convincing but what is the evidence that the Filtering assay and the Plate-reader assay values should be linearly related? Lets imagine that Plate-reader assay value is proportional to the square of the Filtering assay value. What would be the Pearson R and P-value in this case? What is most appropriate? Why would one use a linear correlation? What is the "real" significance?

Reply: We thank the reviewer for pointing out that the data in Supplementary Figure 1 does not appear to be linear and, therefore, reporting the Pearson correlation coefficient may not be the best way to represent the relationship between the two assays. The nonlinear nature of this data could indicate that

The filtering assay saturates before the plate reader assay, and is less able to distinguish between strains that flocculate strongly and The filtering assay may be more sensitive for strains that show lower levels of flocculation. In general, we observed fewer strains with intermediate phenotypes for both assays, making it difficult to ascertain the true relationship between them; however, we believe that the key result is that the strains with the highest level of flocculation have the highest values in both assays. To capture this aspect of the data, we now report the Spearman correlation which is non-parametric and indicates how similar the ranking of each strain is based on both assays. With the alternative hypothesis being that the correlation is > 0, we report a Spearman correlation coefficient of 0.24 and a P-value of 0.04 (lines 823-826)

• Minor points: * They are several "personal communications" in the manuscript (page 11, page 18, page 23). It should be checked whether this is accepted in the journal that publishes this manuscript.

Reply: We thank the reviewer for highlighting this issue. We had three instances of "personal communications" in our original submission.

The first instance was an acknowledgement for advice on our DNA extraction protocol from Dan Jeffares. We now include this in the Acknowledgements section instead.

The second communication with Angad Garg described that they observed flocculation while growing cells in phosphate starvation conditions, which was not reported in their publication (14). Though we appreciate their willingness to share unpublished data with us, we have removed this observation from our manuscript and instead rely only on our own observations and arguments based on their published RNA-seq data to make our point.

The third personal communication with Olivia Hillson supplements a minor hypothesis, namely that deletion of SPNCRNA.781 might cause MLP formation by affecting the promoter of hsr1, for which we had access to unpublished ChIP-seq data, showing its binding to flocculins. Recently published work from a different group (15) also suggests this link between hsr1 and flocculation and is now discussed in our manuscript instead of the result based on unpublished data obtained from personal communication at Lines 397-398.

* Page 4 check "a few regulators"

Reply: For clarity, this has now been changed to "several regulatory proteins" at Line 108. The specific proteins we are referring to are highlighted in Figure 1C.

* Page 19, line 567: "remaining 8 strains" may be confusing as Material and Methods states "remaining 10 strains".

Reply: Two of the 10 strains were found to be redundant after sequencing as explained in the Methods (Lines 930-934). Therefore, we only added 8 new strains to the analysis. We thank the reviewer for highlighting this as a potential source of misunderstanding, and clarified this point in the text (Lines 247-250 and in the methods).

**Referees cross-commenting**

I concur with most comments. Overall, the reviewers agree that this is a solid piece of work that could benefit from minor modifications and should be published. I reiterate that, for me, despite its quality, this publication will only be of interest to specialists.

Reviewer #3 (Significance (Required)):

A limited number of studies have investigated "multicellular-like phenotypes" in S. pombe. This manuscript brings therefore new and solid information. Yet, despite an impressive amount of work, our conceptual advance in understanding this process and its phylogenetic conservation remains limited. This is probably best illustrated in the figure 6 that summarize the study and contains 3 question marks and an additional unknown mechanism. (Most of the solid arrows in this figure correspond to interactions within the Mediator complex that were well known before this study.) In addition, while only few studies have been published in this area, the authors' findings are often only bringing additional support to already published observations. Overall, while this manuscript will be of interest to a restricted group of aficionados, it will most likely not attract the attention of a wide readership.

__ Reviewer #4 (Evidence, reproducibility and clarity (Required)):__

In this manuscript, the authors explore how multicellular-like phenotypes (MLPs) arise in the fission yeast S. pombe. Although yeasts are characterized as unicellular fungi, diverse species show MLPs, including filamentous growth on agar plates and flocculation in liquid media. MLPs may provide certain advantages in nutritionally poor conditions and protection against external challenges, upon which natural selection can then act. Previous work on MLPs has mostly been carried out in the budding yeasts S. cerevisiae and C. albicans, and little was known about these behaviors in S. pombe. The authors thus set out to investigate both genetic and environmental regulators of MLP formation.

First, their analysis of published data revealed a limited number of shared regulators of MLP between S. pombe, S. cerevisiae, and C. albicans, although the cell adhesion proteins themselves are largely not conserved. Next, the authors screened a set of non-clonal natural isolates using two high-throughput assays that they developed and found that MLPs vary in strains and depending on nutrient conditions. Focusing on a natural isolate that showed both adhesion on agar plates and flocculation in liquid medium, they then analyzed a segregant library generated from this and a laboratory strain using their assays. Using QTL analysis, they uncovered a frameshift in the srb11 gene, which encodes a subunit of the Mediator complex, as the likely causal inducer of MLP. This was confirmed by additional analyses of strains lacking srb11 or other members of Mediator. Furthermore, the authors showed that loss of srb11 function resulted in the upregulation of the Mbx2 transcription factor, which was both necessary and sufficient for MLP formation in this background. Finally, screening of two additional yeast strain collections (gene and long intergenic non-coding RNA deletion) identified both known and novel regulators representing different pathways that may be involved in MLP formation.

Altogether, this study provides new perspectives into our understanding of the diverse inputs that regulate multicellular-like phenotypes in yeast.

Major comments:

• The methods for screening for adhesion and flocculation are well described, with representative figures that show plates and flasks. However, there are few microscopy images of cells, and it would be interesting and helpful for the reader to have an idea of how cells look when they exhibit MLPs. For instance, are there any differences in cell shape or size when strains present different degrees of adhesion or flocculation? In addition, the authors mention that mutants with strong adhesion generally had lower colony density and are likely to be slower growing. Although their analyses suggest otherwise (page 22), this has a potential for introducing error in their observations, and including images of the adhesion/flocculation phenotypes may provide further support for their conclusions. I suggest that the authors present microscopy images 1) similar to what is shown for JB759 in Figure 2A and 2) of cells growing on agar in the adhesion assay. This could be included for the different Mediator subunit deletions that they tested, where there appear to be varying phenotypes. It could also be informative for a subset of the 31 high-confidence candidates that they identified in their screen.

Reply: We thank the reviewer for highlighting the need for further microscopic characterisation of MLP forming strains. We therefore now include images of JB914, JB953 (New Supplementary Figures 4, Figure 2E) in liquid media in EMM, EMM-Phosphate, and YES; an srb11 deletion strain (Figure 3F), and mbx2 overexpression strains (New Supplementary Figure 7).

• Upon identifying a frameshift in srb11 that is responsible for the MLP, the authors assessed whether deletion of other Mediator subunits would result in the same phenotype. They found that srb10 and srb11 deletions both flocculate and show adhesion, while other mutants had milder phenotypes. However, the authors also found that a new deletion of srb11 that they generated had a stronger adhesion phenotype than the srb11 deletion from the prototrophic deletion library, which was attributed this the accumulation of suppressor mutations in the strains of the deletion collection. As the authors make clear distinctions between the phenotypes of different Mediator mutants, I suggest generating and analyzing "clean" deletions of the 6 other subunits that they tested. This would strengthen their conclusion and help to rule out accumulated suppressors as the cause of the differences in the observed phenotypes.

Reply: We thank the reviewer for noticing our concern about suppressor mutations in the manuscript. As we describe above in response to a similar question from reviewer 2, as the prototrophic deletion library from which we extracted the Mediator deletion strains had been backcrossed during its construction (13), we no longer suspect that small difference between the srb11Δ::Kan strain from the deletion library and the newly created srb11Δ (CRISPR) strains is due to suppressor mutations. Rather, we think they may be a result of the difference in genetic background and possibly mating type between the two strains. We also want to emphasize that this difference is small compared to the difference between the adhesion ratios of the srb11Δ strains and their respective control strains.

Nevertheless, we made clean, independent Mediator mutants for 5 out of 6 Mediator genes tested (med10Δ, med13Δ, med19Δ, med27Δ, and srb10Δ) as well as an additional mutant that we didn't have in our library, med12Δ (Figure R9). When running the assay on these new strains we got an overall lower dynamic range, possibly due to variations in the water flow rate relative to the first assay. However, we saw a strong phenotype for both library and our own srb10Δ and CRISPR srb11Δ strains. We did not see a significant increase in adhesion for the other Mediator deletion mutants in EMM relative to wild type with the exception of for med10Δ in both the library strain and for our clean mutant, for which we did not observe a phenotype in our previous experiment. We included the experiment for the newly created mutants as New Supplementary Figure S6E and described them in lines 276-281 in our revised manuscript.

Minor comments:

• One point that recurs in the manuscript is the idea that mutations that give rise to strong MLPs also generally lead to slower growth, representing a potential trade-off. This idea could be reinforced with measurements of growth rate or generation time by optical density or cell number, for instance, rather than comparisons of colony density. Also, it would be interesting to mention if the slow growth phenotype is only observed in MLP-inducing conditions or also in rich medium.

Reply: As described above in response to item 5 from Reviewer 1, we have conducted growth assays in liquid media for srb10Δ, srb11Δ, and other mutants from our adhesion screen (tlg2Δ, rpa12Δ, mus7Δ and kgd2Δ) that showed a similar phenotype to those genes in both minimal (EMM) and rich (YES) media. We observe that in rich media, srb10Δ and srb11Δ cells grow similarly to control strains, and they exhibit a lower decrease in growth rate than the other similarly adhesive strains. Both mus7Δ and kgd2Δ cells grow more slowly, even in rich media.

We have also added data on the tradeoff between growth and adhesion based on growth on solid media from (11) for all mutants identified in our screen (New Supp Fig 12B)).

Thus, the relationship between slow growth and clumpiness depends on the mutation, and specifically, mutations of the Mediator, including those to srb11 and srb10, seem to decrease the impact of any tradeoff between growth and adhesion.

• The authors show that the MLPs of the srb10 and srb11 deletions occur through mbx2 upregulation. Do the varying strengths of the phenotypes of the strains lacking different Mediator subunits correlate with mbx2 levels in these backgrounds?

Reply: There is some evidence from previous work that the relationship between the strength of the MLPs and the expression of mbx2 may not be perfectly proportional. In (16), med12Δ had a higher (though qualitatively comparable) level of mbx2 upregulation than srb10Δ (New Supp Fig 8E), even though that paper reported a milder phenotype for med12Δ than for srb10Δ cells. We did not observe a significant increase in adhesion in our med12Δ strain (New Supp Fig 6D). This suggests that in the case of these mutants, it is not simply the level of mbx2 that controls MLP formation, but that there are likely additional regulatory mechanisms. We have added some discussion on this context in the manuscript (lines 545-547).

**Referees cross-commenting**

I agree overall with the comments and suggestions from the other reviewers. The revision would require only minor modifications. The paper is interesting both for the combination of methodologies used and its findings, and I believe that it would benefit a growing community of researchers.

Reviewer #4 (Significance (Required)):

This study employed a variety of methods that allowed the authors to uncover previously unknown regulators of MLPs. Taking advantage of the diversity of natural fission yeast isolates as well as the constructed gene and non-coding RNA deletion collections, the authors identified novel genetic determinants that give rise to MLPs, opening new avenues into this exciting area of research. The overall conclusions of the work are solid and supported by the reported results and analyses. This study will be appreciated by a broad audience of readers who are interested in understanding how organisms respond to environmental challenges as well as how MLPs may result in emergent properties that play key roles in these responses. Some of the limitations of the work are described above, with recommendations for addressing these points.

Keywords for my field of expertise: fission yeast, cell cycle, transcription, replication.

References for Response to Reviews

1. Brysch-Herzberg M, Jia GS, Seidel M, Assali I, Du LL. Insights into the ecology of Schizosaccharomyces species in natural and artificial habitats. Antonie Van Leeuwenhoek. 2022 May 1;115(5):661-95.
2. Jeffares DC, Rallis C, Rieux A, Speed D, Převorovský M, Mourier T, et al. The genomic and phenotypic diversity of Schizosaccharomyces pombe. Nat Genet. 2015 Mar;47(3):235-41.
3. Ratcliff WC, Denison RF, Borrello M, Travisano M. Experimental evolution of multicellularity. Proc Natl Acad Sci. 2012 Jan 31;109(5):1595-600.
4. Smukalla S, Caldara M, Pochet N, Beauvais A, Guadagnini S, Yan C, et al. FLO1 is a variable green beard gene that drives biofilm-like cooperation in budding yeast. Cell. 2008 Nov 14;135(4):726-37.
5. Lorenz MC, Heitman J. Yeast pseudohyphal growth is regulated by GPA2, a G protein alpha homolog. EMBO J. 1997 Dec 1;16(23):7008-18.
6. Ignacia DGL, Bennis NX, Wheeler C, Tu LCL, Keijzer J, Cardoso CC, et al. Functional analysis of Saccharomyces cerevisiae FLO genes through optogenetic control. FEMS Yeast Res. 2025 Sept 24;25:foaf057.
7. Wang Z, Xu W, Gao Y, Zha M, Zhang D, Peng X, et al. Engineering Saccharomyces cerevisiae for improved biofilm formation and ethanol production in continuous fermentation. Biotechnol Biofuels Bioprod. 2023 July 31;16(1):119.
8. Koschwanez JH, Foster KR, Murray AW. Improved use of a public good selects for the evolution of undifferentiated multicellularity. eLife. 2013 Apr 2;2:e00367.
9. Westman JO, Mapelli V, Taherzadeh MJ, Franzén CJ. Flocculation Causes Inhibitor Tolerance in Saccharomyces cerevisiae for Second-Generation Bioethanol Production. Appl Environ Microbiol. 2014 Nov;80(22):6908-18.
10. Li R, Li X, Sun L, Chen F, Liu Z, Gu Y, et al. Reduction of Ribosome Level Triggers Flocculation of Fission Yeast Cells. Eukaryot Cell. 2013 Mar;12(3):450-9.
11. Rodríguez-López M, Bordin N, Lees J, Scholes H, Hassan S, Saintain Q, et al. Broad functional profiling of fission yeast proteins using phenomics and machine learning. Marston AL, James DE, editors. eLife. 2023 Oct 3;12:RP88229.
12. Hebra T, Smrčková H, Elkatmis B, Převorovský M, Pluskal T. POMBOX: A Fission Yeast Cloning Toolkit for Molecular and Synthetic Biology. ACS Synth Biol. 2024 Feb 16;13(2):558-67.
13. Malecki M, Bähler J. Identifying genes required for respiratory growth of fission yeast. Wellcome Open Res. 2016 Nov 15;1:12.
14. Garg A, Sanchez AM, Miele M, Schwer B, Shuman S. Cellular responses to long-term phosphate starvation of fission yeast: Maf1 determines fate choice between quiescence and death associated with aberrant tRNA biogenesis. Nucleic Acids Res. 2023 Feb 16;51(7):3094-115.
15. Ohsawa S, Schwaiger M, Iesmantavicius V, Hashimoto R, Moriyama H, Matoba H, et al. Nitrogen signaling factor triggers a respiration-like gene expression program in fission yeast. EMBO J. 2024 Oct 15;43(20):4604-24.
16. Linder T, Rasmussen NN, Samuelsen CO, Chatzidaki E, Baraznenok V, Beve J, et al. Two conserved modules of Schizosaccharomyces pombe Mediator regulate distinct cellular pathways. Nucleic Acids Res. 2008 May;36(8):2489-504.

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Referee #4

Evidence, reproducibility and clarity

In this manuscript, the authors explore how multicellular-like phenotypes (MLPs) arise in the fission yeast S. pombe. Although yeasts are characterized as unicellular fungi, diverse species show MLPs, including filamentous growth on agar plates and flocculation in liquid media. MLPs may provide certain advantages in nutritionally poor conditions and protection against external challenges, upon which natural selection can then act. Previous work on MLPs has mostly been carried out in the budding yeasts S. cerevisiae and C. albicans, and little was known about these behaviors in S. pombe. The authors thus set out to investigate both genetic and environmental regulators of MLP formation.

First, their analysis of published data revealed a limited number of shared regulators of MLP between S. pombe, S. cerevisiae, and C. albicans, although the cell adhesion proteins themselves are largely not conserved. Next, the authors screened a set of non-clonal natural isolates using two high-throughput assays that they developed and found that MLPs vary in strains and depending on nutrient conditions. Focusing on a natural isolate that showed both adhesion on agar plates and flocculation in liquid medium, they then analyzed a segregant library generated from this and a laboratory strain using their assays. Using QTL analysis, they uncovered a frameshift in the srb11 gene, which encodes a subunit of the Mediator complex, as the likely causal inducer of MLP. This was confirmed by additional analyses of strains lacking srb11 or other members of Mediator. Furthermore, the authors showed that loss of srb11 function resulted in the upregulation of the Mbx2 transcription factor, which was both necessary and sufficient for MLP formation in this background. Finally, screening of two additional yeast strain collections (gene and long intergenic non-coding RNA deletion) identified both known and novel regulators representing different pathways that may be involved in MLP formation.

Altogether, this study provides new perspectives into our understanding of the diverse inputs that regulate multicellular-like phenotypes in yeast.

Major comments:

• The methods for screening for adhesion and flocculation are well described, with representative figures that show plates and flasks. However, there are few microscopy images of cells, and it would be interesting and helpful for the reader to have an idea of how cells look when they exhibit MLPs. For instance, are there any differences in cell shape or size when strains present different degrees of adhesion or flocculation? In addition, the authors mention that mutants with strong adhesion generally had lower colony density and are likely to be slower growing. Although their analyses suggest otherwise (page 22), this has a potential for introducing error in their observations, and including images of the adhesion/flocculation phenotypes may provide further support for their conclusions. I suggest that the authors present microscopy images 1) similar to what is shown for JB759 in Figure 2A and 2) of cells growing on agar in the adhesion assay. This could be included for the different Mediator subunit deletions that they tested, where there appear to be varying phenotypes. It could also be informative for a subset of the 31 high-confidence candidates that they identified in their screen.
• Upon identifying a frameshift in srb11 that is responsible for the MLP, the authors assessed whether deletion of other Mediator subunits would result in the same phenotype. They found that srb10 and srb11 deletions both flocculate and show adhesion, while other mutants had milder phenotypes. However, the authors also found that a new deletion of srb11 that they generated had a stronger adhesion phenotype than the srb11 deletion from the prototrophic deletion library, which was attributed this the accumulation of suppressor mutations in the strains of the deletion collection. As the authors make clear distinctions between the phenotypes of different Mediator mutants, I suggest generating and analyzing "clean" deletions of the 6 other subunits that they tested. This would strengthen their conclusion and help to rule out accumulated suppressors as the cause of the differences in the observed phenotypes.

Minor comments:

• One point that recurs in the manuscript is the idea that mutations that give rise to strong MLPs also generally lead to slower growth, representing a potential trade-off. This idea could be reinforced with measurements of growth rate or generation time by optical density or cell number, for instance, rather than comparisons of colony density. Also, it would be interesting to mention if the slow growth phenotype is only observed in MLP-inducing conditions or also in rich medium.
• The authors show that the MLPs of the srb10 and srb11 deletions occur through mbx2 upregulation. Do the varying strengths of the phenotypes of the strains lacking different Mediator subunits correlate with mbx2 levels in these backgrounds?

Referees cross-commenting

I agree overall with the comments and suggestions from the other reviewers. The revision would require only minor modifications. The paper is interesting both for the combination of methodologies used and its findings, and I believe that it would benefit a growing community of researchers.

Significance

This study employed a variety of methods that allowed the authors to uncover previously unknown regulators of MLPs. Taking advantage of the diversity of natural fission yeast isolates as well as the constructed gene and non-coding RNA deletion collections, the authors identified novel genetic determinants that give rise to MLPs, opening new avenues into this exciting area of research. The overall conclusions of the work are solid and supported by the reported results and analyses. This study will be appreciated by a broad audience of readers who are interested in understanding how organisms respond to environmental challenges as well as how MLPs may result in emergent properties that play key roles in these responses. Some of the limitations of the work are described above, with recommendations for addressing these points.

Keywords for my field of expertise: fission yeast, cell cycle, transcription, replication.

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Referee #2

Evidence, reproducibility and clarity

Yeast species, including fission yeast and budding yeast, could form multicellular-like phenotypes (MLP). In this work, Kӧvér and colleagues found most proteins involved in MLP formation are not functionally conserved between S. pombe and budding yeast by bioinformatic analysis. The authors analyzed 57 natural S. pombe isolates and found MLP formation to widely vary across different nutrient and drug conditions. The authors demonstrate that MLP formation correlated with expression levels of the transcription factor gene mbx2 and several flocculins. The authors also show that Cdk8 kinase module and srub11 deletions also resulted in MLP formation. The experimental design is logic, the manuscript is well-written and organized. I have a few concerns that should be addressed before the publication.

Major points:

1. Line 61-62, how did the authors grow yeast cells in the liquid medium? Shaking or static? If shaking, the nutrient should be even distributed in the medium. If static culture, most single yeast cells could precipitate on the bottom, how do you address the advantage of flocculation for increasing the sedimentation? In addition, under static culture, the bottom will have less air than the up medium, how to balance the air and nutrients?
2. Line 555, it will be interesting to test whether overexpression of mbx2 could cause flocculation in YES medium. In Figure 3D, the authors use two control strains, but only one mbx2 OE strain, mbx2 OE should be tested in both strains. In addition, did the authors transform empty plasmid into the control strains, please indicate in the figure.
3. Line 600-601, the authors may do the backcross of srb11Δ::Kan to exclude the possibility caused by other mutations.

Minor points:

1. Line 506, what are the growth conditions of cells in Figure 2A? Did the authors use the liquid or solid medium? Please mention in the Methods or figure legends.
2. Line 533-535, please explain why the strains exhibiting strong adhesion have a decreased growth rate. Is there any related research? Please add some references.

Referees cross-commenting

I agree with most of the comments from other reviewers. This publication may indeed be of interest to a minor area. But the results and the interpretations of the data are interesting and warranted, the findings are scientifically important.

Significance

The authors did many large-scale screens and bioinformatic analyses. The experiments in the manuscript are generally logical and sound. This study is useful for deciphering the mechanism of multicellular-like phenotype formation in the fission yeast, with some implications for some other organisms.

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Referee #1

Evidence, reproducibility and clarity

Summary

Köver et al. examine the genetic and environmental underpinnings of multicellular-like phenotypes (MLPs) in fission yeast, studying 57 natural isolates of Schizosaccharomyces pombe. They uncover that a noteworthy subset of these isolates can develop MLPs, with the extent of these phenotypes varying according to growth media. Among these, two strains demonstrate pronounced MLP across a range of conditions. By genetically manipulating one strain with an MLP phenotype (distinct from the previously mentioned two strains), they provide evidence that genes such as MBX2 and SRB11 play a direct role in MLP formation, strengthening their genetic mapping findings. The study also reveals that while some key genes and their phenotypic effects are strikingly similar between budding and fission yeast, other aspects of MLP formation are not conserved, which is an intriguing finding.

Overall, the manuscript is well-written, dense yet logically structured, and the figures are well presented. The combination of phenotypic, genetic, and bioinformatics analyses, particularly from wet lab experiments, is commendable. The study addresses a significant gap in our understanding, primarily explored in budding yeast, by providing comprehensive data on MLP diversity in fission yeast and the interplay of genetic and environmental factors.

In summary, I enjoyed reading the manuscript and have only a few minor suggestions to strengthen the paper:

Minor revisions:

1. Although this may seem like a minor revision, but it is a crucial point. Please make sure that all raw data used to generate figures, run stats, sequence data, and scripts used to run data analysis are made publicly available. Provide relevant accession numbers and links to public data repositories. It is important that others can download the various types of data that went into the major conclusions of this paper in order to replicate your analysis or expand upon the scope of this work. I am not sure if the journal has a policy regarding this, but it should be followed to allow for transparency and reproducibility of the research.
2. Two out of 57 strains exhibit strong and consistent MLP across multiple environments. Providing more information on these strains (JB914 and JB953), such as their natural habitats and distinct appearances of their MLP phenotypes under varying conditions, would provide valuable insights.

First, a brief discussion highlighting what differentiates these two strains from the rest would be helpful for readers (e.g. insight into their unique genetic and environmental background that might be linked to the MLP phenotype).

Additionally, culture tube and microscopy images of these strains, similar to those presented for JB759 in Figure 2A, can be included in the supplementary materials. My reasoning is that these images could help illustrate variation or lack thereof in aggregative group size across different media. 3. The phenotypic outcome of overexpressing MXB2 is striking, as shown in Supplementary Figure 4C. Incorporating at least one of the culture tube images depicting large flocs into the main text, perhaps adjacent to Figure 3 panel D, would improve the visual appeal and highlight this key finding (at the moment those images are only shown in the supplementary materials). 4. I know that the authors discuss the knowledge gap in the intro and results, but the abstract does not mention this critical gap. Please stress this critical gap (i.e., MLPs understudied in fission yeast) with a brief sentence in the abstract. Similarly, please consider writing a brief concluding sentence summarizing the paper's most significant finding referring to the knowledge gap would provide a clearer takeaway message for the reader - the abstract ends abruptly without any conclusion. 5. The observation that strains with adhesive phenotypes have a lower growth rate compared to non-adhesive strains is a noteworthy point (lines 532-535). This represents yet another example of this classical trade-off. This point could be emphasized in the Discussion or alongside the relevant result, with a brief speculative explanation for this phenomenon. 6. The text mentions two lab strains, JB22 and JB50, displaying strong adhesion under phosphate starvation (lines 525-526), yet the data point for JB22 in Figure 2C is not labeled. 7. Although I generally avoid commenting on formatting, I found the manuscript to be dense. As mentioned above, I truly enjoyed reading it! But I couldn't help but think of ways to make the manuscript more concise for readers. The Results section spans nine pages (excluding figure captions), and the Discussion is five pages long. The main text contains 6 figures with approximately 27 panels and 32 plots and Venn diagrams, while the supplementary material has 11 figures with 22 panels and about 59 plots. Altogether, the manuscript comprises 17 figures, 49 panels, and roughly 91 plots and Venn diagrams! While I will not request any changes, I encourage the authors to consider streamlining the text/data where possible to focus on the core theme of the study.

Referees cross-commenting

There are many useful recommendations from all the other reviewers that will help improve the final product. Once those points are revised, I think this will be a nice paper of interest to folks interested in natural variation in MLPs and its genetic background.

Significance

My expertise: evolutionary genetics, evolution of multicellularity, yeast genetics, experimental evolution

Overall, the manuscript is well-written, dense yet logically structured, and the figures are well presented. The combination of phenotypic, genetic, and bioinformatics analyses, particularly from wet lab experiments, is commendable. The study addresses a significant gap in our understanding, primarily explored in budding yeast, by providing comprehensive data on MLP diversity in fission yeast and the interplay of genetic and environmental factors.

In summary, I enjoyed reading the manuscript and have only a few minor suggestions to strengthen the paper.

Author response:

The following is the authors’ response to the original reviews

Reviewing Editor Comment:

The reviewers felt that the study could be improved by (1) better integrating the results with the existing literature in the field

(1) In the Introduction and Results section of the manuscript, we had made every attempt to cite the relevant literature. (Reviewer 1 stated that “The literature is appropriately cited”). We agree with the Reviewing Editor that rather than simply cite the relevant literature, we could have done a better job of integrating our findings with what has been previously discovered by others. We have attempted to do this in the revised manuscript. Also, we have included many additional citations in the Introduction and in the first section of the Results where work by others has provided a framework for interpreting our single-cell studies.

and (2) manipulating Trib expression and analyzing the expression of 1-2 HIX genes.

(2) We are grateful for this suggestion. As suggested by the Reviewing Editor we have attempted to increase and decrease trbl expression and assess the effect on expression of two genes, Swim and CG15784.

We increased trbl levels in the wing pouch using rn-Gal4, tub-Gal80^ts and UAS-trbl. By transferring larvae for 24 h from 18oC to 31oC, we were able to induce trbl expression in the wing pouch. When these larvae were irradiated at 4000 rad, we found reduced levels of apoptosis in the wing pouch of discs that overexpressed trbl (Figure 7-figure supplement 1). This indicated that upregulation of trbl is radioprotective. Consistent with our findings, others have previously shown that upregulation of trbl and stalling in the G2 phase of the cells cycle protects cells from JNK-induced apoptosis (Cosolo et al., 2019, PMID:30735120) or that downregulating the G2/M progression promoting factor string protects cells from X-ray radiation induced apoptosis (Ruiz-Losada et al., 2021, PMID:34824391).

As suggested by the Reviewing Editor, we also examined the effect of trbl overexpression on the induction of two “highly induced by X-ray irradiation (HIX)” gene, Swim and CG15784. Increasing trbl expression had no effect on the induction of Swim and only a modest decrease in the induction of CG15784 (Figure 7-figure supplement 2). Thus, increasing trbl expression, is in itself, insufficient to promote HIX gene expression indicating that other factors are necessary for HIX gene induction.

We also attempted to reduce trbl expression, using three different RNAi lines. While some of these lines have been used previously by others to reduce trbl expression under unirradiated conditions (Cosolo et al., 2019, PMID:30735120), we nevertheless wanted to check if they reduced trbl induction following irradiation. For each of the three lines, we observed no obvious reduction in trbl RNA following irradiation when visualized using HCR (Author response image 1). Thus, any effects on gene expression that we observe could not be attributed to a decrease in trbl expression. We have therefore included the images showing a lack of knockdown in this Response to Reviews document but not included these experiments in the revised manuscript.

Author response image 1.

RNA in situ hybridizations using the hybridization chain reaction performed using probes to trbl. In A-F, the RNAi is expressed using nubbin-Gal4. In G-I the RNAi is expressed using rn-Gal4, tub-Gal80^ts. white-RNAi was used as a control (A, B, G, H). Three different RNAi lines directed against trbl were tested: Vienna lines VDRC 106774 (C, D) and VDRC 22113 (E, F), and Bloomington line BL42523. In no case was a reduction in trbl RNA upregulation in the wing pouch following 4000 rad observed, except for one disc (n = 6) of VDRC 106774 crossed to nubbin-gal4.

Reviewer #1 (Public review):

Summary:

The authors analyze transcription in single cells before and after 4000 rads of ionizing radiation. They use Seuratv5 for their analyses, which allows them to show that most of the genes cluster along the proximal-distal axis. Due to the high heterogeneity in the transcripts, they use the Herfindahl-Hirschman index (HHI) from Economics, which measures market concentration. Using the HHI, they find that genes involved in several processes (like cell death, response to ROS, DNA damage response (DDR)) are relatively similar across clusters. However, ligands activating the JAK/STAT, Pvr, and JNK pathways and transcription factors Ets21C and dysf are upregulated regionally. The JAK/STAT ligands Upd1,2,3 require p53 for their upregulation after irradiation, but the normal expression of Upd1 in unirradiated discs is p53-independent. This analysis also identified a cluster of cells that expressed tribbles, encoding a factor that downregulates mitosis-promoting String and Twine, that appears to be G2/M arrested and expressed numerous genes involved in apoptosis, DDR, the aforementioned ligands, and TFs. As such, the tribbles-high cluster contains much of the heterogeneity.

Strengths:

(1) The authors have used robust methods for rearing Drosophila larvae, irradiating wing discs, and analyzing the data with Seurat v5 and HHI.

(2) These data will be informative for the field.

(3) Most of the data is well-presented

(4) The literature is appropriately cited.

We thank the reviewer for these comments.

Weaknesses:

(1) The data in Figure 1 are single-image representations. I assume that counting the number of nuclei that are positive for these markers is difficult, but it would be good to get a sense of how representative these images are and how many discs were analyzed for each condition in B-M.

For each condition at least 5 discs were imaged but we imaged up to 15 discs in some cases. We tried to choose a representative disc for each condition after looking at all of them. All discs imaged under each condition are shown below; the disc chosen for the figure is indicated with an asterisk. All scale bars are 100 mm.

Author response image 2.

Images for discs shown in Manuscript Figure 1panels B, C

Author response image 3.

Images for discs shown in Manuscript Figure 1panels D, E

Author response image 4.

Images used in Manuscript Figure 1, F, G

Author response image 5.

Images used in Manuscript Figure 1H, I

Author response image 6.

Images used in Manuscript Figure 1J, K

Author response image 7.

Images used in Manuscript Figure 1L, M

(2) Some of the figures are unclear.

It is unclear to us exactly which figures the Reviewer is referring to. Perhaps this is the same issue mentioned below in “Recommendations for the authors”. We address it below.

Reviewer #1 (Recommendations for the authors):

(1) Regarding Figure 1, what is stained in blue? Is it DAPI? If so, this should be added to the figure legend.

Thank you for pointing out this omission. This has been addressed in the revised manuscript.

It is very difficult to see blue on black, so could the authors please outline the discs?

Alternatively, they could show DAPI in green and the markers (pH2Av, etc) in magenta.

We used DAPI (blue) as a way of outlining the discs. While we appreciate the reviewer’s concern, after reviewing the images, we found that the blue is clearly visible when the document is viewed on the screen. It is less obvious if the document is printed on some kinds or printers. Since boosting this channel would make the signal from the channels more difficult to see, we left the images as they were.

(2) Figure 3, Figure Supplement 2, panel B. It is not possible to read the gene names in the panel's current form. Please break this up into 4 lines (as much as possible from the current 2).

Thank you for this suggestion. We have done this in the revised manuscript.

Reviewer #2 (Public review):

This manuscript investigates the question of cellular heterogeneity using the response of Drosophila wing imaginal discs to ionizing radiation as a model system. A key advance here is the focus on quantitatively expressing various measures of heterogeneity, leveraging single-cell RNAseq approaches. To achieve this goal, the manuscript creatively uses a metric from the social sciences called the HHI to quantify the spatial heterogeneity of expression of individual genes across the identified cell clusters. Inter- and intra-regional levels of heterogeneity are revealed. Some highlights include the identification of spatial heterogeneity in the expression of ligands and transcription factors after IR. Expression of some of these genes shows dependence on p53. An intriguing finding, made possible by using an alternative clustering method focusing on cell cycle progression, was the identification of a high-trbl subset of cells characterized by concordant expression of multiple apoptosis, DNA damage repair, ROS-related genes, certain ligands, and transcription factors, collectively representing HIX genes. This high-trbl set of cells may correspond to an IR-induced G2/M arrested cell state.

Overall, the data presented in the manuscript are of high quality but are largely descriptive. This study is therefore perceived as a resource that can serve as an inspiration for the field to carry out follow-up experiments.

Thank you for your assessment of the work.

Reviewer #2 (Recommendations for the authors):

I suggest two major points for improvement:

(1) It is important to test whether manipulation of trbl levels (i.e., overexpression, knockdown, mutation) would result in measurable biological outcomes after IR, such as altered HIX gene expression, altered cell cycle progression, or both. This may help disentangle the question of whether high trbl expression and correlated HIX gene expression are a cause or consequence of G2/M stalling.

We have described these experiments at the beginning of this Response to Reviews document when addressing the comments made by the Reviewing Editor. Please see Figure 7, figure supplements 1 and 2. These experiments suggest that upregulation of trbl offers some protection from radiation-induced death, yet it is itself insufficient to induce expression of two HIX genes tested. As we have also described earlier, three different RNAi lines tested did not reduce trbl upregulation after irradiation.

(2) A more extensive characterization of the high-trbl cell state would also be appropriate, particularly in terms of their relationship to the cell cycle.

We attempted to address this issue in two ways. First, we used the expression of a trbl-gfp transgene and RNA in-situ hybridization experiments to visualize the distribution of the high-trbl cells (shown in new manuscript figure, Figure 6-figure supplement 3). When examining trbl RNA in irradiated discs, there is no obvious demarcation between cells that express high levels of trbl and other cells. This is also apparent in the UMAP shown in Figure 6A and A’. Most cells seem to express trbl; cells in the “high trbl” cluster simply express more trbl than others. We observed cells expressing trbl and PCNA as well as cells expressing only one of those two genes at detectable levels. Thus, it was not possible to distinguish the “high trbl” cells from other cells by this approach.

We decided instead to focus on examining the expression of other cell-cycle genes in the high-trbl cluster. We have added a paragraph in the Results section that details our findings. Many transcriptional changes are indeed consistent with stalling in G2 such as high levels of trbl and low levels of string (stg). Additionally, that the cells are likely in G2 is consistent with reduced levels of genes that are normally expressed at other stages of the cell cycle: G1 genes such as E2f1 and Dp, S-phase genes such as several Mcm genes, PCNA and RnrS, and genes that encode mitotic proteins such as polo, Incenp and claspin. There are however, several anomalies such as slightly increased expression of the early-G1 cyclin, CycD, and the retinoblastoma ortholog Rbf. Thus, at least as assessed by the transcriptome, this cluster may not correspond to a cell state that is found under normal physiological conditions.

(3) Minor: p. 12, line 3. Figure 5A is mentioned, but it seems that it should be 4A instead.

Thank you for pointing this out. We have addressed this in our revisions.

Reviewer #3 (Public review):

Strengths:

Overall, the manuscript makes a compelling case for heterogeneity in gene expression changes that occur in response to uniform induction of damage by X-rays in a single-layer epithelium. This is an important finding that would be of interest to researchers in the field of DNA damage responses, regeneration, and development.

Weaknesses:

This work would be more useful to the field if the authors could provide a more comprehensive discussion of both the impact and the limitations of their findings, as explained below.

Propidium iodide staining was used as a quality control step to exclude cells with a compromised cell membrane. But this would exclude dead/dying cells that result from irradiation. What fraction of the total do these cells represent? Based on the literature, including works cited by the authors, up to 85% of cells die at 4000R, but this likely happens over a longer period than 4 hours after irradiation. Even if only half of the 85% are PI-positive by 4 hr, this still removes about 40% of the cell population from analysis. The remaining cells that manage to stay alive (excluding PI) at 4 hours and included in the analysis may or may not be representative of the whole disc. More relevant time points that anticipate apoptosis at 4 hr may be 2 hr after irradiation, at which time pro-apoptotic gene expression peaks (Wichmann 2006). Can the authors rule out the possibility that there is heterogeneity in apoptosis gene expression, but cells with higher expression are dead by 4 hours, and what is left behind (and analyzed in this study) may be the ones with more uniform, lower expression? I am not asking the authors to redo the study with a shorter time point, but to incorporate the known schedule of events into their data interpretation.

We thank the reviewer for these important comments. The generation of single-cell RNA-seq data from irradiated cells is tricky. Many cells have already died. Even those that do not incorporate propidium iodide are likely in early stages of apoptosis or are physiologically unhealthy and likely made it through our FACS filters. Indeed, in irradiated samples up to 57% of sequenced cells were not included in our analysis since their RNA content seemed to be of low quality. It is therefore likely that our data are biased towards cells that are less damaged. As advised by the reviewer, we will include a clearer discussion of these issues as well as the time course of events and how our analysis captures RNA levels only at a single time point.

If cluster 3 is G1/S, cluster 5 is late S/G2, and cluster 4 is G2/M, what are clusters 0, 1, and 2 that collectively account for more than half of the cells in the wing disc? Are the proportions of clusters 3, 4, and 5 in agreement with prior studies that used FACS to quantify wing disc cells according to cell cycle stage?

Work by others (Ruiz-Losada et al., 2021, PMID:34824391) has shown that almost 80% of cells have a 4C DNA content 4 h after 4,000 rad X-ray irradiation. The high-trbl cluster accounts for only 18% of cells and can therefore account for a minority of cells with a 4C DNA content.

Thus clusters 0, 1 and 2 could potentially contain other populations that also have a 4C DNA content. Importantly, similar proportions of cells in these clusters are also observed in unirradiated discs.

We expect that clusters 1 and 2 are largely comprised of cells in G2/M. Together, these clusters are marked by some genes previously found to be higher in FACS separated G2 cells compared to G1 cells (Liang et al., 2014, PMID: 24684830). These genes include Det, aurA, and ana1. Strangely, cluster 0 is not strongly marked by any of the 175 cell cycle genes used in our clustering (eff being the strongest marker) and has a lower-than-average expression of 165/175 cell cycle genes. Cluster 0 is however marked by the genes ac and sc, which are known to be expressed in proneuronal cell clusters interspersed throughout the disc that stall in G2 and form mitotically quiescent domains (Usui & Kimura 1992, Development, 116 (1992), pp. 601-610 (no PMID); Nègre et al., 2003, PMID: 12559497). Given these observations, we hypothesize that cluster 0 is largely comprised of stalled G2 cells like those found in ac/sc-expressing proneural clusters.

The EdU data in Figure 1 is very interesting, especially the persistence in the hinge. The authors speculate that this may be due to cells staying in S phase or performing a higher level of repair-related DNA synthesis. If so, wouldn't you expect 'High PCNA' cells to overlap with the hinge clusters in Figures 6G-G'? Again, no new experiments are needed. Just a more thorough discussion of the data.

We have found that the locations of elevated PCNA expression do not always correlate with the location of EdU incorporation either by examining scRNA-seq data or by using HCR to detect PCNA. PCNA expression is far more widespread as we now show in Figure 6-figure supplement 3.

Trbl/G2/M cluster shows Ets21C induction, while the pattern of Ets21C induction as detected by HCR in Figures 5H-I appears in localized clusters. I thought G2/M cells are not spatially confined. Are Ets21C+ cells in Figure 5 in G2/M? Can the overlap be confirmed, for example, by co-staining for Trbl or a G2/M marker with Ets21C?

The data show that the high-trbl cells are higher in Ets21C transcripts relative to other cell-cycle-based clusters after irradiation. This does not imply that high-trbl-cells in all regions of the disc upregulate Ets21C equally. Ets21C expression is likely heterogeneous in both ways – by location in the disc and by cell-cycle state.

Induction of dysf in some but not all discs is interesting. What were the proportions? Any possibility of a sex-linked induction that can be addressed by separating male and female larvae?

We can separate the cells in our dataset into male and female cells by expression of lncRNA:roX1/2. When we do this, we see X-ray induced dysf expressed similarly in both male and female cells. We think that it is therefore unlikely that this difference in expression can be attributed to cell sex. Another possibility is that dysf upregulation might be acutely sensitive to the developmental stage of the disc. This would require experiments with very precisely-staged larvae. We have not investigated this further as it is not a central issue in our paper.

Reviewer #3 (Recommendations for the authors):

Please check the color-coding in Figure 1A. The region marked as pouch appears to include hinge folds that express Zfh2 (a hinge marker) in Figure 2A (even after accounting for low Zfh2 expression in part of the pouch).

We have corrected this and have marked the pouch region based on the analysis of expression of different hinge and pouch markers by Ayala-Camargo et al. 2013 (PMID 2398534).

The statement 'Furthermore, within tissues, stem cells are most sensitive while differentiated cells are relatively radioresistant' needs to be qualified, as there are differences in radiosensitivity of adult versus embryonic stem cells (e.g., PMID: 30588339)

We thank the reviewer for bringing this point to our attention and for pointing us to an article that addresses this issue in detail. We appreciate that our statement was rather simplistic – we have modified it and added two additional references.

Synthèse du Webinaire : Aménagements d'Examens pour les Élèves à Besoins Éducatifs Particuliers

Résumé Exécutif

Ce document de synthèse résume les points clés du webinaire organisé par la FCPE nationale le 20 novembre 2025, animé par Guillaume Laffitte, conseiller technique académique pour l'École inclusive, et Laurence Noël, chef de la division des examens et concours (DEC) de l'académie de Montpellier.

L'objectif central était de clarifier les droits, les procédures et les délais concernant les aménagements d'examens.

Les aménagements ne sont pas des faveurs, mais un droit fondamental pour garantir l'égalité des chances et permettre une évaluation juste et adaptée aux besoins de chaque élève.

Le concept central est la cohérence du "parcours de l'élève" : les aménagements aux examens doivent être l'aboutissement logique des aides pédagogiques mises en place durant toute la scolarité.

Deux acteurs principaux collaborent : le Pôle École Inclusive, qui se concentre sur l'accompagnement pédagogique en amont, et la Division des Examens et Concours (DEC), qui gère le cadre réglementaire et logistique des épreuves.

La procédure de demande se divise en deux voies : une procédure simplifiée pour les élèves bénéficiant déjà d'un PAP, PAI ou PPS, et une procédure complète pour les autres cas ou les demandes nouvelles.

L'anticipation est cruciale : les démarches doivent être entamées dès la classe de quatrième pour le brevet et en seconde pour le baccalauréat.

Enfin, des outils pédagogiques innovants comme les "matrices pédagogiques" sont encouragés pour renforcer l'autonomie des élèves, illustrant une évolution vers une "école pour tous" où les adaptations bénéfiques pour certains le sont pour l'ensemble des élèves.

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1. Principes Fondamentaux et Philosophie

Le webinaire établit d'emblée que les démarches d'aménagement d'examen sont essentielles pour garantir l'égalité des chances. Elles constituent un parcours souvent lourd et mal compris pour les familles.

• Un Droit, Pas une Faveur : Il est rappelé que les aménagements sont un "droit indispensable pour que chaque élève soit évalué dans des conditions le plus juste et adaptée à leurs besoins".

• De l'École Inclusive à l'École pour Tous : Guillaume Laffitte propose de dépasser le terme "école inclusive" pour viser une "école pour tous", qui répond aux besoins de chacun sans étiqueter les élèves. La diversité est présentée comme normale et bénéfique.

• Le Parcours de l'Élève : L'idée centrale est que l'examen n'est pas une simple étape, mais l'aboutissement de toute la scolarité.

Il doit exister une cohérence systématique entre les aménagements pédagogiques fournis en classe tout au long du parcours et ceux accordés lors des épreuves. Cette continuité renforce l'autonomie de l'élève.

"Il faut vraiment qu'on puisse corréler systématiquement [...] le parcours de l'élève jusqu'aux épreuves pour le candidat, parce qu'il faut une cohérence et c'est comme ça qu'on peut renforcer finalement les élèves face à leur autonomie en situation d'apprentissage." - Guillaume Laffitte

2. Les Acteurs Clés et Leurs Rôles

La gestion des aménagements repose sur la collaboration de deux services principaux au sein du rectorat, ici illustrés par l'Académie de Montpellier.

Le Pôle Académique École Inclusive

Dirigé par Guillaume Laffitte, ce pôle se concentre sur l'accompagnement pédagogique de l'élève tout au long de sa scolarité.

• Coordination : Il pilote l'organisation de l'école inclusive au niveau académique, en s'appuyant sur les orientations nationales.

• Collaboration : Il travaille en lien étroit avec tous les services de l'académie, notamment la Division des Examens et Concours (DEC).

• Création de Ressources : Il produit des guides pour les familles et les équipes, comme le "guide académique pour les aménagements des examens, mais du parcours de l'élève jusqu'aux aménagements des examens".

• Priorités Académiques : L'une des priorités est l'utilisation des matrices pédagogiques comme réponse pédagogique cohérente.

La Division des Examens et Concours (DEC)

Dirigée par Laurence Noël, la DEC est le service administratif et logistique qui organise l'ensemble des épreuves et gère l'application réglementaire des aménagements.

Chaque rectorat possède une DEC (à Paris, il s'agit du SIEC).

Missions principales :

• Organisation Globale : Organisation de tous les examens (DNB, CAP, Baccalauréats, BTS, etc.) et des concours de recrutement de l'Éducation Nationale.

• Volet Sujets : Élaboration et adaptation des sujets d'examen (ex: dictée aménagée, sujets agrandis, sujets en braille).

• Volet Organisationnel : Gestion des inscriptions, élaboration des calendriers (en tenant compte des tiers temps qui allongent la durée des épreuves), répartition des candidats dans les centres, et communication des aménagements aux chefs de centre.

• Volet Logistique : Fourniture de matériel spécifique comme les copies spéciales (mais pas les ordinateurs ou le mobilier ergonomique).

• Volet Administratif :

◦ Notification : C'est la DEC qui envoie la décision officielle d'aménagement (la "notification") aux familles via l'application Cyclades.  

◦ Recours : Elle traite les recours des familles en cas de désaccord avec une décision.   

◦ Fraudes : Elle gère les commissions de discipline, y compris celles liées à un mauvais usage des aménagements (ex: aide humaine qui donne les réponses, ordinateur non vidé de son contenu).

3. Le Cadre des Aménagements d'Examens

Types d'Aménagements Possibles

Les aménagements peuvent porter sur divers aspects de l'épreuve pour répondre aux besoins spécifiques du candidat.

Catégorie

Exemples d'aménagements

Temps

- Temps majoré (ex: tiers temps) pour les épreuves écrites, orales ou pratiques.<br>- Temps compensatoire pour permettre des soins ou des pauses.<br>- Temps pour se lever et faire quelques pas.

Espace

- Composition en rez-de-chaussée.<br>- Placement spécifique dans la salle (près d'une fenêtre).<br>- Composition dans une salle isolée.

Aides Techniques

- Utilisation d'un ordinateur (personnel ou fourni par le centre).<br>- Matériel spécifique (tables ou chaises ergonomiques, non fournies par la DEC).<br>- Sujets adaptés : en braille, agrandis, sur support numérique.

Aides Humaines

- Secrétaire : Tâches d'exécution pure (lecteur, scripteur sous la dictée).<br>- Assistant : Marge d'autonomie (reformulation ou séquençage des consignes, recentrage de l'attention).<br>- AESH : Missions précises définies dans le cadre d'un PPS.

Adaptations & Dispenses

- Adaptation de l'épreuve : Dictée aménagée pour le DNB.<br>\

• Dispense d'épreuve : Très réglementée et spécifique à chaque examen (ex: dispense de langue vivante, non applicable à tous les diplômes).<br>\

• Étalement : Possibilité de passer les épreuves sur plusieurs sessions consécutives.<br>\

• Conservation des notes : Les notes obtenues peuvent être conservées durant cinq ans.

Correction

- Anonymat respecté : Le correcteur n'a pas connaissance du handicap.<br>\

• Non-pénalisation de l'orthographe : Si validé, un sigle sur la copie anonyme l'indique au correcteur.

Les "Matrices Pédagogiques" : Un Outil d'Avenir

Fortement mises en avant par Guillaume Laffitte, les matrices sont des outils méthodologiques qui aident l'élève à séquencer une tâche et à organiser sa pensée.

• Principe : Elles ne sont pas une antisèche, mais une fiche qui guide l'élève dans les étapes d'une tâche (ex: comment utiliser son brouillon, construire un fil conducteur, organiser son temps).

• Cohérence : Elles permettent à l'élève d'utiliser le jour de l'examen un outil qu'il maîtrise déjà pour l'avoir utilisé en classe.

• Autonomie : Elles visent à rendre l'élève plus autonome et à renforcer son estime de soi.

• Statut : L'utilisation de matrices est un aménagement réglementaire autorisé pour les examens.

_"Ce qui réussit à l'élève qui a le plus de besoins, il n'y a pas de raison que ce ne soit pas utile à tous.

C'est ce qu'on appelle la conception universelle des apprentissages."_ - Guillaume Laffitte

Distinction Cruciale : Dispense d'Enseignement et Aménagement d'Examen

Il est essentiel de ne pas confondre ces deux notions :

• Dispense d'enseignement : Décision très rare, prise uniquement par le recteur à la demande des parents, pour un élève en situation de handicap.

Elle a un impact majeur sur le parcours et l'orientation future de l'élève et doit être évaluée en cohérence avec les examens à venir.

• Dispense d'épreuve d'examen : Fait partie des aménagements possibles mais est strictement encadrée par la réglementation de chaque diplôme.

La DEC ne peut valider une dispense que si le règlement de l'examen le permet.

4. Procédures de Demande d'Aménagement

La procédure a été simplifiée en 2020 pour garantir la continuité entre le parcours scolaire et les examens. Elle s'articule en deux voies principales.

Pour Qui ?

Tout candidat présentant un handicap (reconnu par la MDPH), un trouble de santé invalidant (dans le cadre d'un PAP ou PAI) ou une limitation temporaire d'activité (ex: bras cassé avant l'épreuve) peut demander un aménagement, quel que soit son statut (scolarisé, candidat individuel, etc.).

Procédure Simplifiée

• Conditions : Réservée aux élèves scolarisés en établissement public ou privé sous contrat, disposant d'un PAP, PAI ou PPS valide, et dont les aménagements demandés pour l'examen sont identiques à ceux déjà mis en place durant leur scolarité.

• Processus : La demande ne nécessite pas l'avis d'un médecin. Le chef d'établissement signe le formulaire, qui est ensuite transmis à la DEC.

Procédure Complète

• Conditions : S'applique à tous les autres candidats (individuels, hors contrat), à ceux qui n'ont pas de plan formalisé (PAP, PAI, PPS), ou à ceux qui demandent des aménagements différents ou nouveaux par rapport à leur scolarité.

Elle est également requise en cas d'aggravation de l'état de santé ou pour une majoration de temps au-delà du tiers temps (mi-temps).

• Processus : Le dossier est examiné par l'équipe pédagogique et doit obligatoirement recevoir l'avis d'un médecin de l'Éducation Nationale avant d'être transmis à la DEC.

Calendrier et Délais Clés

L'anticipation est le maître-mot. L'interlocuteur principal pour les familles est le chef d'établissement.

Examen

Moment pour Entamer la Procédure

DNB / CFG

En classe de quatrième

Baccalauréats (général, techno, pro)

Fin du second trimestre de la classe de seconde

Autres examens (CAP, BTS, etc.)

Au cours de l'année de l'examen

La demande formelle et la transmission des pièces se font généralement au moment de l'inscription à l'examen. Le respect des délais est impératif pour permettre à la DEC d'organiser la logistique (ex: la production d'un sujet en braille demande un mois).

Le Processus de Traitement et de Notification

1. Instruction : Les services de la DEC étudient le dossier et vérifient sa conformité réglementaire.

2. Décision : Le recteur prend la décision finale.

3. Notification : La DEC informe officiellement la famille de la décision via l'application Cyclades. Les notifications sont envoyées entre février et mai.

4. Conservation : La notification est à conserver précieusement, à présenter à chaque épreuve avec la convocation, et peut servir de pièce justificative pour de futures demandes.

5. Données Chiffrées et Tendances (Académie de Montpellier)

Les statistiques de l'Académie de Montpellier illustrent une forte augmentation des demandes d'aménagement.

Indicateur

Données 2020

Données 2025 (prévisionnel)

% de candidats avec aménagement

10 %

13,51 %

Nombre total de dossiers

~10 000

14 000

Nombre total de mesures d'aménagement

15 000

76 000

Moyenne de mesures par candidat

~1,5

~5,5

Taux de notifications positives

N/A

99,67 %

Mesures les plus courantes :

• Tiers temps

• Dictée aménagée (DNB)

• Autorisation de la calculatrice

• Aide humaine pour le séquençage ou la reformulation des consignes

6. Points de Vigilance et Conseils Pratiques

• Confiance et Autonomie : Les deux intervenants insistent sur la nécessité de faire confiance aux capacités des enfants, de viser leur autonomie et de s'assurer que les aménagements demandés correspondent réellement à leurs besoins et à leurs habitudes de travail.

• Utilisation de l'ordinateur : Si un ordinateur personnel est autorisé, il doit être entièrement vide de tout dossier et présenté au chef de centre pour vérification avant chaque épreuve.

Il faut bien distinguer la demande de "sujet sur support numérique" de la "composition sur ordinateur".

• Enregistrement régulier : En cas de composition sur ordinateur, il est vital d'enregistrer le travail très régulièrement sur le disque dur ET sur une clé USB pour éviter toute perte en cas de problème technique.

• Contacter le Centre d'Examen : Pour des aménagements lourds ou spécifiques (notamment liés à l'espace, comme un fauteuil roulant), il est conseillé de prendre contact en amont avec le chef du centre d'examen.

• Recours : Si un aménagement accordé n'est pas respecté le jour de l'épreuve, la famille doit adresser un recours écrit au recteur.

La DEC mènera alors une enquête.

Briefing de Préparation à l'Ouverture de Parcoursup 2026

Résumé Exécutif

L'ouverture de la procédure Parcoursup 2026 s'inscrit dans une volonté de simplification et d'accompagnement renforcé pour les élèves et leurs familles. Les points clés à retenir pour cette session sont les suivants :

• Simplification du processus : Un dossier unique, un calendrier commun et une offre centralisée de près de 25 000 formations.

• Dates charnières : Mise à jour de l'offre le 17 décembre 2025, ouverture des inscriptions le 19 janvier 2026, clôture des vœux le 12 mars 2026, et début des réponses le 2 juin 2026.

• Outils d'aide à la décision : Le "simulateur" (basé sur les données des trois dernières années) et les fiches formations détaillées permettent d'évaluer les chances d'admission et de lever l'autocensure.

• Réussite du dispositif : En 2025, deux tiers des lycéens ont reçu une proposition dès le premier jour. En fin de procédure, seuls 38 lycéens sur plus de 160 000 n'avaient pas reçu de proposition.

• Primauté de l'humain : Contrairement aux idées reçues, ce n'est pas un algorithme qui décide de l'admission, mais des commissions d'enseignants qui analysent les dossiers selon des critères affichés en toute transparence.

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I. Nature et Missions de la Plateforme Parcoursup

Parcoursup n'est pas qu'un outil d'affectation ; il se définit comme un support d'amélioration de l'orientation pour favoriser la réussite dans l'enseignement supérieur.

Une procédure simplifiée et transparente

• Unicité : Un seul dossier numérique et un calendrier identique pour les lycéens, les parents et les formations.

• Offre diversifiée : Près de 25 000 formations sont répertoriées, incluant des diplômes nationaux (Licence, BTS, BUT) et des diplômes d'établissement (Écoles d'ingénieurs, Sciences Po, etc.).

• Transparence des critères : Chaque formation doit afficher ses critères d'analyse (résultats scolaires, savoir-être, motivation) et ses frais de scolarité.

L'action pour l'égalité des chances

La plateforme applique des dispositions légales pour soutenir l'accès au supérieur :

• Quotas pour les lycéens boursiers.

• Priorité aux lycéens professionnels pour les BTS et aux lycéens technologiques pour les BUT.

• Prise en compte des situations de handicap, des sportifs de haut niveau et des artistes confirmés.

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II. Calendrier de la Procédure 2026

Le calendrier se décompose en trois phases majeures :

| Phase | Dates Clés | Objectifs | | --- | --- | --- | | Information | À partir du 17 déc. 2025 | Découverte de la carte des formations mise à jour pour 2026. | | Inscription & Vœux | 19 janv. au 1er avril 2026 | Création du dossier et formulation des vœux (Date limite : 12 mars). | | Admission | 2 juin au 11 juillet 2026 | Réception des réponses et choix définitifs des candidats. |

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III. Comprendre l'Offre de Formation

Il est crucial de distinguer les types de formations pour adapter sa stratégie de vœux.

Formations Sélectives (CPGE, BTS, BUT, Écoles)

• La sélection est effective : si une formation dispose de 30 places mais ne retient que 15 candidats, elle n'est pas tenue de remplir ses capacités si les profils ne correspondent pas.

Formations Non Sélectives (Licences)

• Principe de remplissage : L'université doit remplir jusqu'à la hauteur de sa capacité d'accueil.

• Classement : Un classement est effectué uniquement en cas de tension (plus de candidats que de places) pour éviter le tirage au sort. Ce classement repose sur la cohérence entre le dossier de l'élève et les "attendus" de la formation.

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IV. Outils d'Analyse et Aide au Choix

Pour lutter contre l'autocensure et la surconfiance, Parcoursup propose des outils de données historiques.

Le Simulateur d'Admission

Cet outil (utilisé 105 millions de fois l'an dernier) permet de visualiser les chances d'admission selon le profil du candidat (baccalauréat, spécialités, moyenne générale).

• Données : Basées sur les trois dernières années.

• Indicateurs de chance : Rarement (0-5%), Occasionnellement (5-20%), Régulièrement (20-50%), etc.

• Note : La moyenne générale utilisée est une moyenne brute (non pondérée). Les formations regardent toutefois les moyennes par discipline.

La Fiche Formation (Carte d'Identité)

Chaque fiche présente un cadre unique pour faciliter la comparaison :

• Statut de l'établissement (public/privé).

• Taux d'accès (proportion de candidats ayant reçu une offre).

• Taux de réussite et débouchés professionnels (incluant le salaire médian après un an).

• Rapports détaillés de la session précédente (profil des admis).

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V. Modalités de Candidature et Accompagnement

Formulation des vœux

• Nombre : Jusqu'à 10 vœux (plus 10 vœux en apprentissage).

• Sous-vœux : Possibles pour certaines filières (ex: un vœu pour un type de BTS, plusieurs lycées en sous-vœux).

• Absence de hiérarchie : Les vœux ne sont pas classés par l'élève. Les formations ne savent pas quels sont les autres vœux formulés.

• Photo Finish : Ce qui compte est l'état du dossier au 12 mars ; l'ordre chronologique de saisie n'influence pas l'admission.

Dispositifs d'assistance

• Numéro vert : 0 800 400 070 (accessible de France et de l'étranger).

• Réseau AEFE : Les élèves des lycées français à l'étranger sont traités avec une égalité stricte. Ils bénéficient d'une priorité géographique sur tout le territoire français pour les licences (n'ayant pas d'université locale).

• Accompagnement des "sans proposition" : À partir du 2 juin, les élèves n'ayant que des refus sont contactés par téléphone. Une phase complémentaire s'ouvre le 11 juin pour postuler sur les places vacantes.

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VI. Citations et Conseils Clés

"Parcoursup ne fait jamais l’analyse des candidatures. Ce sont bien des enseignants qui reçoivent les dossiers et font l’analyse à partir des critères affichés." — Jérôme Théard

"L’échec sur Parcoursup, ce n'est pas de ne pas avoir de proposition puisqu'on vous accompagne. L’échec, c'est d'avoir exactement ce qu'on veut et de s'apercevoir trois jours après le début que ça ne nous plaît pas." — Jérôme Théard

Conseils aux familles :

• Inscrire les coordonnées parentales : Il est possible d'ajouter l'email et le numéro des parents dans le dossier de l'enfant pour recevoir les mêmes alertes en temps réel.

• Favoriser le dialogue humain : Les journées portes ouvertes (de janvier à mars) sont essentielles pour "donner de la chair" aux informations numériques.

• Diversifier les vœux : Ne pas se mettre en situation de risque en ne formulant qu'un seul vœu ou uniquement des vœux très sélectifs.

Briefing : Bien Choisir ses Spécialités au Lycée

Résumé Exécutif

Le choix des spécialités au lycée constitue un pivot stratégique déterminant pour la réussite au baccalauréat et l'orientation vers l'enseignement supérieur.

Avec un coefficient de 16 pour chaque épreuve terminale, ces matières pèsent lourdement dans l'obtention du diplôme et la qualité du dossier Parcoursup.

La stratégie de sélection doit idéalement équilibrer les compétences réelles de l'élève (stratégie de performance) et les prérequis des formations visées (stratégie de projet).

Un point de vigilance majeur concerne les mathématiques : bien que réintégrées dans le tronc commun, ce niveau est jugé insuffisant pour la quasi-totalité des filières scientifiques et économiques sélectives, rendant le choix de la spécialité mathématique indispensable pour ces parcours.

1. Cadre Général et Enjeux du Choix

Depuis la réforme du baccalauréat, les élèves doivent choisir trois spécialités en classe de Première, pour n'en conserver que deux en Terminale.

• Impact sur le Baccalauréat : Les spécialités représentent un engagement de travail significatif (4 heures par semaine en Première, 6 heures en Terminale) et sont dotées d'un coefficient élevé (16).

• Calendrier de décision :

◦ En Seconde : Choix provisoire en février (2ème conseil de classe) et choix définitif au 3ème conseil de classe.    ◦ En Première : Choix de l'abandon d'une spécialité entre janvier et mars pour une décision finale en fin d'année.

• Flexibilité limitée : Un changement de spécialité en début de Première est possible uniquement avant les vacances de la Toussaint, sous réserve de justification et de capacité à rattraper les cours manqués.

2. Offre et Accessibilité des Enseignements

Il existe actuellement 13 spécialités au total. Cependant, la disponibilité varie selon les établissements.

Les Spécialités "Prioritaires"

Sept spécialités sont jugées prioritaires et sont normalement proposées dans tous les lycées car elles ouvrent les perspectives les plus larges :

1. Mathématiques

2. Physique-Chimie

3. Sciences de la Vie et de la Terre (SVT)

4. Sciences Économiques et Sociales (SES)

5. Histoire-Géographie, Géopolitique et Sciences Politiques (HGGSP)

6. Humanités, Littérature et Philosophie (HLP)

7. Langues, Littératures et Cultures Étrangères (LLCE)

Les Spécialités "Rares" et alternatives

Certaines matières (Arts, Sport - EPPCS, Langues de l'Antiquité) ne sont pas présentes partout. En cas d'absence dans le lycée de secteur, des solutions existent :

• Mutualisation : Partenariats entre lycées (déplacement de l'élève ou cours en visioconférence).

• CNED : Enseignement à distance, nécessitant une grande autonomie de l'élève.

3. Analyse Statistique et Performance

Les effectifs en Première générale (~380 000 élèves) révèlent une hiérarchie marquée dans les choix :

| Spécialité | Part des élèves (approx.) | Observations | | --- | --- | --- | | Mathématiques | 2/3 des élèves | Forte déperdition entre la 1ère et la Terminale. | | Physique-Chimie | 1/2 des élèves | Souvent couplée aux mathématiques. | | SVT | ~45% des élèves | Troisième pilier scientifique classique. | | HGGSP | 1/3 des élèves | Profils diversifiés (sciences po, droit, lettres). |

Le facteur "Effectif / Réussite" : Les données du ministère indiquent que les spécialités à petits effectifs (Italien, Espagnol, Sport) affichent souvent de meilleures moyennes au baccalauréat. Cela s'explique par un accompagnement plus individualisé et un "choix du cœur" qui booste la motivation, contrairement aux choix purement stratégiques parfois subis.

4. Stratégies de Sélection Recommandées

Deux approches principales doivent être croisées pour un choix optimal :

La Stratégie de Performance (Le Dossier)

L'objectif est de maximiser les notes pour le baccalauréat et Parcoursup. Il est conseillé de choisir des matières où l'élève excelle déjà. Un bon dossier dans une spécialité cohérente est plus valorisé qu'un dossier médiocre dans une spécialité jugée "prestigieuse".

La Stratégie de Projet (L'Orientation)

Certaines formations supérieures exigent des parcours spécifiques :

• Filières Scientifiques (Ingénieurs, Prépa MP) : Mathématiques et Physique-Chimie sont quasi-indispensables en Terminale.

• Santé (PASS/L.AS) : Un duo parmi Mathématiques, Physique-Chimie et SVT est requis.

• Économie (Prépa ECG) : La spécialité Mathématiques est essentielle.

• Droit / Sciences Po : Pas de spécialité imposée, mais HGGSP, SES ou HLP sont recommandées pour la cohérence du profil.

5. Le Cas Critique des Mathématiques

L'enseignement des mathématiques est le point de crispation majeur de la réforme.

• Le Tronc Commun : Insuffisant pour la majorité des poursuites d'études scientifiques ou économiques.

• L'Option "Maths Complémentaires" : Destinée aux élèves ayant suivi la spécialité en Première mais souhaitant l'arrêter en Terminale tout en gardant un socle pour des études de santé ou de sciences sociales.

• L'Option "Maths Expertes" : Un ajout de 3 heures pour les profils très scientifiques. Bien que non exigée officiellement, elle facilite grandement l'entrée en prépa mathématiques.

6. Accompagnement et Ressources

Le rôle des parents doit être celui d'un accompagnateur objectif, évitant de projeter ses propres désirs sur l'enfant.

Acteurs à solliciter

1. Le Professeur Principal : Pivot de l'orientation au sein du lycée.

2. Les Psy-EN (Psychologues de l'Éducation Nationale) : Pour des conseils individualisés sur le profil de l'élève (disponibles en lycée ou en CIO).

3. Les Étudiants : Lors des salons spécialisés, ils offrent un retour d'expérience concret sur la charge de travail et la réalité des cours.

Outils disponibles

• Simulateurs de spécialités : Pour visualiser les débouchés en fonction des combinaisons choisies.

• Parcoursup : La "Carte des formations" (mise à jour mi-décembre) permet de consulter les "attendus" de chaque cursus sans avoir besoin de compte.

• Journées Portes Ouvertes (JPO) : Indispensables pour confirmer si une spécialité est réellement nécessaire pour une école spécifique.

"Le choix au lycée ne ferme pas de porte de manière définitive, sauf pour des filières extrêmement spécifiques comme la santé (PASS).

L'enseignement supérieur s'adapte en proposant parfois des remises à niveau, mais la cohérence du parcours reste le meilleur gage de succès."

Briefing sur la Plateforme Avenir : Un Outil d'Accompagnement à l'Orientation de la 5ème à la Terminale

Résumé Exécutif

La plateforme Avenir, développée par l'Office national d'information sur les enseignements et les professions (Onisep), constitue le pivot numérique du « parcours Avenir ».

Conçue pour accompagner les élèves de la classe de 5ème jusqu'à la Terminale, elle vise à rendre la démarche d'orientation plus lisible, interactive et personnalisée.

L'objectif central est de permettre à chaque élève de construire progressivement son projet scolaire et professionnel en fonction de ses compétences et centres d'intérêt, tout en atténuant la pression liée aux choix d'orientation. Intégrée aux établissements scolaires, la plateforme favorise la coéducation en impliquant les équipes éducatives et les familles dans un cadre sécurisé et structuré sur le long terme.

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1. L'Onisep : Missions et Valeurs de l'Opérateur d'État

L'Onisep est un opérateur public placé sous la double tutelle du ministère de l'Éducation nationale et du ministère de l'Enseignement supérieur et de la Recherche. Sa mission repose sur deux piliers : informer et accompagner.

1.1 Un maillage territorial dense

L'Onisep s'appuie sur des services centraux en région parisienne et sur 17 directions territoriales (incluant la Corse et l'Outre-mer). Ce réseau permet :

• L'alimentation continue de bases de données documentaires sur les formations, les métiers et les établissements.

• Un déploiement de la plateforme au plus près des usagers via des présentations en établissement et lors de salons.

1.2 Principes fondamentaux

L'action de l'Onisep et de la plateforme Avenir est guidée par des valeurs d'inclusion et d'équité :

• Égalité d'accès : Un socle commun d'information pour tous.

• Lutte contre les stéréotypes : Déconstruction des préjugés sur les métiers et promotion de l'égalité garçons-filles.

• Inclusion : Ressources spécifiques pour les élèves en situation de handicap.

• Développement durable : Sensibilisation aux enjeux écologiques dans les parcours professionnels.

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2. Structure et Fonctionnalités de la Plateforme Avenir

La plateforme a été conçue de manière intuitive avec le concours d'élèves, d'enseignants, de psychologues de l'Éducation nationale et de parents. Elle s'articule autour de quatre onglets principaux.

2.1 L'Agenda de l'orientation

Il ne s'agit pas d'un cahier de textes scolaire, mais d'un calendrier dédié exclusivement à l'orientation. L'élève y trouve :

• Activités en médiation : Séances programmées par les enseignants.

• Événements suggérés : Forums des métiers, journées portes ouvertes ou salons régionaux.

• Dates institutionnelles : Repères clés (notamment pour Parcoursup en Terminale).

2.2 Les Objectifs annuels

Les objectifs sont adaptés à chaque niveau scolaire pour garantir une progression cohérente.

| Niveau | Exemples d'Objectifs Incontournables | | --- | --- | | Collège (3ème) | Lister ses goûts et points forts ; identifier les voies après la 3ème ; préparer et réaliser un stage de découverte. | | Lycée (Terminale) | Préparer l'accès à l'enseignement supérieur ; finaliser son projet pour Parcoursup. |

2.3 Les Outils d'exploration

La plateforme propose des modules interactifs pour aider l'élève à se découvrir :

• « Je découvre des métiers » : Outil ludique explorant des thématiques modernes (ex: impact de l'intelligence artificielle sur les métiers).

• Fiches métiers et vidéos : Contenus contextualisés permettant de « liker » des professions pour les enregistrer dans son profil.

2.4 L'Espace « Me faire accompagner »

Ce volet rappelle l'importance de l'accompagnement humain. Il facilite la mise en relation avec :

• Le psychologue de l'Éducation nationale (PsyEN) de l'établissement.

• Le service « Mon orientation en ligne », un support gratuit accessible par chat, mail ou téléphone.

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3. Dispositifs Spécifiques pour le Lycée : Le Module « Mon Projet Sup »

Pour les lycéens, la plateforme intègre l'outil Mon Projet Sup, dont la mission principale est de lutter contre l'autocensure.

• Personnalisation : L'outil part des centres d'intérêt, des enseignements de spécialité choisis et des préférences géographiques de l'élève.

• Suggestions intelligentes : Il propose des formations ambitieuses ou des « plans B » réalistes auxquels l'élève n'aurait pas forcément pensé.

• Lien avec Parcoursup : Bien qu'indépendant du système d'affectation, il permet de préparer ses vœux et d'explorer la carte des formations de manière fluide.

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4. Le Portfolio : Une Mémoire du Cheminement

Le portfolio est l'espace où l'élève consigne toutes ses traces d'apprentissage et de réflexion de la 5ème à la Terminale.

• Continuité : Les données suivent l'élève même s'il change d'établissement, d'académie ou de région.

• Conservation : Le contenu est conservé jusqu'à trois ans après la Terminale pour faciliter d'éventuelles réorientations ou reprises d'études.

• Contenus stockés : Projets d'études, CV, lettres de motivation, comptes rendus d'entretiens avec les PsyEN et documents personnels (ex: interviews de professionnels, brochures d'entreprises).

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5. Gouvernance des Données et Rôle des Acteurs

5.1 Accès et Connexion

L'accès à Avenir se fait via les identifiants nationaux sécurisés :

• EduConnect pour la majorité des élèves de l'Éducation nationale.

• EduAgri pour l'enseignement agricole.

• Note : Les parents n'ont pas de compte propre mais sont invités à explorer la plateforme « côte à côte » avec leur enfant.

5.2 Confidentialité et Droits

• RGPD : La plateforme respecte strictement les normes de protection des données personnelles.

• Visibilité restreinte : Les enseignants voient le tableau de bord des objectifs (auto-évaluation de l'élève) et le portfolio pour conseiller l'élève, mais n'ont pas accès à l'espace de stockage privé ni aux comptes rendus confidentiels des psychologues.

• Droit à l'erreur : L'élève est acteur de son profil ; il peut modifier ou supprimer ses centres d'intérêt et métiers favoris à tout moment.

5.3 Déploiement dans les établissements

Le déploiement est progressif.

Le « plan Avenir » prévoit la formation des enseignants, en priorité les professeurs principaux de 3ème.

La mise en œuvre dépend du projet de chaque établissement (utilisation durant les heures dédiées à l'orientation, vie de classe ou demi-journées thématiques).

La plateforme est un outil pédagogique à la main des équipes, respectant leur liberté pédagogique.

Synthèse : Prévention et lutte contre le harcèlement scolaire (Webinaire FCPE-MAE)

Résumé exécutif

Le harcèlement scolaire est une problématique systémique qui touche environ un élève sur dix.

Face à ce constat, le webinaire organisé par la FCPE et la MAE souligne l'impératif d'une action concertée entre parents, professionnels de l'éducation et partenaires institutionnels.

L'approche défendue repose sur trois piliers : la détection précoce des signaux d'alerte, l'utilisation d'outils pédagogiques adaptés à chaque tranche d'âge (de la maternelle au lycée), et une coéducation active.

La MAE, partenaire historique de l'enseignement public, met à disposition des ressources gratuites et agréées par le Ministère de l'Éducation nationale, s'inscrivant notamment dans le cadre du programme national Phare.

L'objectif central est de briser le silence et de passer d'une logique de réaction à une culture de prévention durable.

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1. Analyse du phénomène de harcèlement scolaire

Définitions et mécanismes

Le harcèlement se caractérise par un rapport de force déséquilibré où une ou plusieurs personnes exercent une pression ou un contrôle répété sur une victime.

• Formes constatées : Insultes, moqueries, rumeurs, humiliations et mises à l'écart.

• Évolution : Les situations débutent souvent par des faits perçus comme « pour rire » avant de déraper vers une souffrance physique et psychologique grave.

Le défi du cyber-harcèlement

Le cyber-harcèlement transpose ces violences sur les réseaux sociaux, les messageries, les forums et les jeux vidéo.

• Gravité : La circulation des attaques est extrêmement rapide et peut toucher une audience très large.

• Traces : Les agressions en ligne laissent des marques durables et ne s'arrêtent pas aux portes de l'école.

• Statistique clé : Un collégien sur cinq a déjà été victime d'au moins un acte de cyber-violence répété.

Signaux d'alerte pour les adultes

La vigilance des parents et des enseignants doit se porter sur les changements de comportement :

• État émotionnel : Isolement, colère, tristesse subite.

• Vie scolaire : Baisse des résultats, refus d'aller en cours ou de participer à certaines activités.

• Santé physique : Troubles du sommeil, de l'appétit, maux de tête ou de ventre fréquents.

• Signes matériels : Vêtements abîmés, perte d'effets personnels.

• Rapport au numérique : Enfant qui cache son téléphone ou le consulte avec une angoisse permanente.

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2. Le cadre institutionnel et l'engagement de la MAE

Un acteur historique

Fondée en 1932 par des enseignants, la MAE est une mutuelle issue de l'économie sociale et solidaire. Elle bénéficie de l'agrément national du Ministère de l'Éducation nationale pour intervenir dans les établissements scolaires.

Soutien aux familles et garanties

Au-delà de la prévention, la MAE propose des protections spécifiques dans ses contrats d'assurance :

• Soutien psychologique en cas de harcèlement avéré.

• Assistance juridique en cas d'atteinte à l'image de l'enfant.

• Aide à la suppression de contenus malveillants sur Internet.

Le programme Phare

Les outils présentés s'inscrivent dans le dispositif ministériel Phare, qui repose sur cinq piliers :

1. Éduquer pour prévenir les phénomènes de harcèlement.

2. Former une communauté protectrice autour des élèves.

3. Intervenir efficacement sur les situations de harcèlement.

4. Associer les parents et les partenaires.

5. Mobiliser les instances de démocratie scolaire (CESCE).

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3. Ressources et outils pédagogiques par cycles

Les ressources proposées sont gratuites et conçues en collaboration avec des professionnels de l'éducation (notamment l'AGEEM pour le premier degré).

Pour les 3 - 11 ans (Maternelle et Élémentaire)

| Outil | Description | Objectif | | --- | --- | --- | | Album "Maël le roi des bêtises" | Support de 25 pages avec cahier d'activités. | Apprendre le respect des différences et le vivre-ensemble dès le plus jeune âge. | | BD "Main dans la main" | Format innovant (illustration à gauche, exploitation pédagogique à droite). | Présenter les points de vue de tous les acteurs : victime, harceleur, aidant, suiveur, adulte. | | Jeu de l'oie "Non au harcèlement" | Mallette physique ou version dématérialisée (TBI). | Utiliser le jeu comme prétexte au débat et à l'échange collectif. |

Pour les 11 - 18 ans (Collège et Lycée)

• Jeu de l'oie spécialisé : Orienté vers le harcèlement sexuel, sexiste et homophobe (Cycle 4).

• BD "La Jungle" : Récit d'une rentrée en collège basée sur des témoignages réels, incluant une trousse à outils et des liens utiles.

• "Le Labyrinthe de Nina" (Serious Game) :

◦ Concept : Jeu immersif où le joueur explore le smartphone d'une lycéenne disparue pour comprendre les mécanismes du cyber-harcèlement.  

◦ Partenariat : Développé avec l'association e-Enfance (gestionnaire du 3018).  

◦ Versions : Une version grand public (60 min) et une version "Express" (30 min) pour les ateliers scolaires, facilitant la médiation par l'enseignant.

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4. Supports multimédias et prévention numérique

La MAE développe des formats variés pour s'adapter aux nouveaux usages des familles :

• Podcasts :

◦ Au-delà du miroir : Témoignages de jeunes sur la différence, la discrimination et la résilience.  

◦ Nos enfants, les écrans et Internet : Épisodes dédiés à la pornographie en ligne, aux réseaux sociaux et aux jeux vidéo.   

◦ Parentalité accompagnée : Focus sur la santé mentale et l'égalité filles-garçons.

• Vidéos "3 minutes pour comprendre" : Décryptage par Natacha Waro, psychologue clinicienne, pour identifier les signaux d'alerte et savoir comment agir.

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5. Modalités de déploiement et collaboration territoriale

Accès aux outils

• Numérique : Téléchargement gratuit sur les sites mae.fr ou labyrinthedenina.fr, et sur les stores d'applications mobiles (Android/iOS).

• Physique : Les mallettes et albums sont distribués via les réseaux de délégués départementaux de la MAE. Les parents peuvent solliciter ces délégués via un formulaire sur le site national.

Rôle des parents et coéducation

• Ambassadeurs : Les parents d'élèves sont encouragés à informer les directions d'école de l'existence de ces outils agréés.

• Actions locales : Collaboration possible pour organiser des "Cafés parents", des tables rondes ou des animations lors des assemblées générales de la FCPE.

• Obligations légales : Il est rappelé que depuis 2022, les enseignants ont l'obligation de se former à la lutte contre le harcèlement scolaire.

Vigilance sur les intervenants

Il est crucial de vérifier l'agrément des intervenants extérieurs.

Le Ministère de l'Éducation nationale publie une liste officielle des associations autorisées à intervenir en milieu scolaire afin d'éviter les dérives ou les discours non conformes aux valeurs de la République.

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

Learn more at Review Commons

Reply to the reviewers

Reviewer #1

Evidence, reproducibility and clarity

The manuscript by Wu and Griffin describes a mechanism where CHD4 and BRG1, two chromatin remodelling enzymes, have antagonistic functions to regulate extracellular matrix (ECM) plasmin activity and sterile inflammatory phenotype in the endothelial cells of the developing liver. As a follow up from a previous study, the authors investigate the phenotype of embryonic-lethal endothelial-specific CHD4-knockout, leading to liver phenotype and embryo death, and the rescue of this phenotype when subsequently BRG1 is knocked-out also in the endothelium. First, the authors show that the increase in plasmin activator uPAR (which leads to ECM degradation) in CHD4-KO embryos can be rescued by BRG1-KO, and that both CHD4 and BRG1 interact with the uPAR promoter. However, the authors demonstrate that reducing plasminogen by genetic knockout is unable to rescue the CHD4-KO embryos alone, suggesting an additional mechanism. By RNAseq analysis, the authors identify sterile inflammation as another potential contributor to the lethal phenotype of CHD4-KO embryos through increased expression of ICAM-1 in endothelial cells, also showing binding of both chromatin remodellers to ICAM-1 promoter. Finally, the authors use nonsteroidal anti-inflammatory drug carprofen, alone or in combination with plasminogen genetic knockout, and demonstrate CHD4-KO lethal embryonic phenotype rescue with the combination of plasminogen reduction and inflammation reduction, highlighting the synergistic role of both ECM degradation and sterile inflammation in this genetic KO.

The findings of the manuscript are interesting, experiments well controlled and paper well written. While the work is of potential specialist interest to the field of liver development, there are several issues which authors should address before this paper can be published:

Major issues:

1. The authors still see embryonic lethality of some embryos with endothelial BRG1-KO or combined endothelial CHD4/BRG1-KO - could the authors please show or at least comment in the discussion why those animals are dying?

We observed no dead Brg1-ECko or Brg1/Chd4-ECdko embryos by E14.5. However, at E17.5, there was an 18.8% lethality rate for Brg1-ECko mutants and a 12.5% rate for Brg1/Chd4-ECdko mutants (Fig. 1B). The reasons behind the incomplete rescue of Brg1/Chd4-ECdko embryos and the cause of death in Brg1-ECko mutants remain unknown, as we have mentioned in the revised discussion (see lines 311-316).

1. In the qRT-PCR results Fig.2c, what is each dot?

Each dot represents transcripts acquired from a separate embryo. We have modified the figure legend for clarification.

1. In the same figure, I would expect that in CHD4-KO there is no CHD4 transcript, and in BRG1-KO there is no BRG1 transcript, rather than the reduction shown, which seems quite noisy (though significant) - is it this a result of normalisation? Or is indeed only a certain amount of the transcript reduced?

The VE-Cadherin Cre mouse line utilized in this study is reported to have progressive Cre expression and activity from E8.5 to E13.5 and only to reach full penetrance across all vasculature at E14.51. The liver sinusoidal ECs (LSECs) analyzed in Fig. 2C were isolated at E12.5, before Cre activity reached its full penetrance. This is likely the primary cause of the variability in gene excision seen in this panel.

1. In the same figure, is the statistical testing performed before or after normalisation? This can introduce errors if done after normalisation.

Normalization was performed before statistical analysis to combine relative transcript counts from embryos harvested in multiple litters. This is now clarified in our methods (see lines 486-489).

1. In some cases, the authors show immunofluorescence images but do not specify how many biological replicates this represents (e.g. Fig.1d, 4c-d). This should be added.

We have updated the legends for Figs. 1E, 4C-D, and 6E-F, as suggested.

1. I also encourage the authors to present a supplementary figure with at least one other biological replicate shown for imaging data (optional).

We appreciated this suggestion but opted not to add additional supplemental figures, which might have been confusing to readers.

1. The plasminogen reduction by genetic modulation results in drastic changes to the embryos' appearance - is this a whole embryo KO or endothelial-specific KO? Can authors at least comment on the differences?

The plasminogen-deficient embryos used in this study were global knockouts; this is now clarified on line 177. The Chd4-ECko embryos with varying degrees of plasminogen deficiency that are shown in Fig. 2F were dissected at E17.5, which is ~3 days after the typical time of death for Chd4-ECko embryos. This explains why the dead and partially resorbed mutants in Fig. 2F look so different from their control (Plg-/-) littermate and from the E14.5 Chd4-ECko embryos shown in Fig. 1C.

1. In Fig.2b, do I understand correctly only 1 sample was analysed with different areas plotted on the graph? If so, this experiment should be repeated on another set of embryos to be robust, and data plotted as a mean of each embryo (rather than areas).

Each dot represents the mean value obtained after quantifying 4 fluorescent areas within a liver section from a single embryo. The N number indicates the number of embryos used from each genotype. We have updated the figure legend accordingly.

1. Also in some graphs, authors specify that it was more than n>x embryos, but then - what are the dots on the graph representing? Each embryo? This should be specified (e.g. Fig.2b-c, but please check this in all the figure legends).

Thank you for this question. We have worked to clarify the legends for all our graphs. Overall, for graphs related to embryos, each dot represents data from a single embryo. Since the sample sizes vary across genotypes, we used the smallest sample size taken from the mutant groups when listing our minimum N.

1. "we found Plaur was the only gene that was induced in CHD4-ECko LSECs at E12.5 (Figure S3D)." - I am not sure this is correct, as gene Plau is also increased in 2/3 samples?

Although Plau transcripts were also increased in Chd4-ECko LSECs compared to control samples, our statistical analysis showed a p-value of 0.0564, which was deemed non-significant according to our cutoff criteria of p

1. I find the title and the running title somewhat misleading and too broad; the authors should specify more detail in the title about the content of the paper - the current statement of the title is somewhat true but shown only for one genetic model and not confirmed for all types of "lethal embryonic liver degeneration".

We have updated the title to incorporate this suggestion. The revised title is ‘Plasmin activity and sterile inflammation synergize to promote lethal embryonic liver degeneration in endothelial chromatin remodeler mutants.’ The revised running title is ‘Plasmin and inflammation in endothelial mutant livers.’

Minor issues:

1. If an animal licence was used, its number should be specified in the ethics or methods section

We have added this information to the methods (see line 383).

1. In fig.3g it is very hard to see each of the samples, could authors try to improve this graph for clarity using colours-or split Y axis - or both?

We have revised Fig. 3G to include a split y-axis, as suggested.

1. "This indicates that ECs can play a pro-inflammatory role in embryonic livers and highlights the need for tight regulation to ensure normal liver growth." This sentence for me is misleading, EC are producing inflammatory signals only during the CHD4-KO according to the author's data, and authors do not show such data in normal homeostasis condition. Actually, the pro-inflammatory role here seems detrimental, and ECs should not exhibit it for correct development. The authors should rephrase this to be clearer.

The detrimental inflammation observed when Chd4 was deleted in ECs indicates that endothelial CHD4 normally suppresses inflammation during liver development (Fig. 3F-G, and 4A-B). When endothelial CHD4 functions properly, there is no excessive cytokine activation and inflammation. We have modified the sentence to help clarify this information (see lines 295-297).

Significance

General assessment: The study is well controlled and well written. The findings are interesting. The limitation of the findings is only 1 combination genetic model being studied, and it is unclear if the synergistic effect of sterile inflammation and ECM degradation is broadly applicable to other models, where embryo dies because of liver failure.

Advance: The study makes an incremental advance, following up findings from a previous study. However, it is conceptually interesting.

Audience: The audience for this manuscript would be a liver development specialist. However, broader concepts could also be applicable to liver disease.

Expertise: I research in the field of liver regeneration and disease.

__Reviewer #2 __

Evidence, reproducibility and clarity

In essence, Wu et Al find that Chd4 mutant mice exhibit embryonic liver degeneration due to uPA-mediated plasmin hyperactivity and an ICAM-1-driven hyperinflammation and that additional mutation of BRG1 opposes this liver degeneration, possibly via ICAM-1.

Generally, this is an excellent manuscript with a very logical sequence of experiments, although it has shortcomings such as validating their findings in an independent system, ideally human, and further establishing the translational relevance. Establishing translational relevance through mechanistic experiments that identify specific inflammatory tissue pathways, such as by blocking ICAM-1 and TNF-alpha, could also define developmental aberrations as a model for broader (patho)physiology and thereby enhance the impact on the field.

Major

1. The embryonic and postnatal survival data of Chd4-ECko and Brg1/Chd4-Ecdko mice should be included in Fig. 1

We revised Fig. 1 to add representative photos and lethality rates for control and mutant embryos at E17.5 (see new Fig. 1B). All Chd4-ECko embryos we dissected at E17.5 were dead, which was consistent with our previous report2. Although Brg1/Chd4-ECdko embryos were largely rescued at E17.5, these mutants still die soon after birth due to lung development issues, as we previously reported3.

1. What is the impact of Chd4-ECko and Brg1/Chd4-ECdko on the multicellular microenvironment? At a minimum, IF or spatial transcriptomics for hepatocyte and biliary markers, pericytes, and other mesenchymal cells would be recommended. Can there be a distinction made on what type of endothelial cell is affected? (sinusoidal lineage, vs. venous vs. lymphatic)

To assess whether the multicellular microenvironment of Chd4-ECko livers was altered, we performed immunostaining for various cellular markers from E12.5 to E14.5. These markers included LYVE-1 for liver sinusoids; PROX1 and E-cadherin (ECAD) for hepatocytes; CD41 for platelets and megakaryocytes; CD45 for leukocytes; CD68 and F4/80 for macrophages; MPO for neutrophils; TER119 for erythroid cells; and a-smooth muscle actin (SMA) for pericytes and smooth muscle cells (see Fig. 4D and__ Fig. R1*__). Across all the images we examined, no obvious cell-type-specific differences were observed between control and mutant livers.

Biliary epithelial cells, which begin to differentiate at approximately E15.54, were also assessed using cytokeratin 19 (CK19) immunostaining; however, no CK19-positive cells were detected in control livers at E14.5 (see Fig. R2*). Note that although LYVE-1 is also expressed by lymphatic endothelial cells, lymphatic vessels are not yet established in the liver at E14.52. Therefore, LYVE-1 staining is appropriate for identifying liver sinusoidal ECs at this stage of development. Our data indicate that the affected vasculature in Chd4-ECko livers is predominantly localized to the liver periphery (see Fig. 1D), which LYVE-1 staining shows to be mostly populated by sinusoidal vessels (Fig. R1B and R1F).

*Please see uploaded Response to Reviewers PDF for Figures R1 and R2

1. The experiments showing how endothelial Chd4 loss leads to a hyperinflammatory endothelial-and potentially hepatoblast-state are important. However, the relevance of immune cell infiltration in the hematopoietic-developing liver remains unclear. Which immune cells are presumably recruited to inflame the microenvironment then? Bone-marrow-derived? This aspect would benefit from experimental clarification, for example, using migration and/or direct co-culture versus indirect cell co-culture-ideally with or without ICAM-1 blockade-in vitro assays to determine if direct crosstalk with the CD45+ immune cell compartment explains the hyperinflammatory endothelia phenotype.

In mice, the first hematopoietic cells emerge in the yolk sac at E7.55. Subsequently, embryonic hematopoiesis takes place in the aorta-gonad-mesonephros (AGM) region and the placenta, before immature hematopoietic cells migrate to the fetal liver. After E11.0, the fetal liver becomes the main hematopoietic organ, supporting the expansion and differentiation of hematopoietic stem and progenitor cells into all mature blood cell lineages5-8. Around E16.5, hematopoietic cells migrate to the bone marrow9, so the bone marrow is not a relevant source of infiltrating immune cells in our E12.5-14.5 Chd4-ECko mutants. We therefore examined immune cell populations, including leukocytes, macrophages, and neutrophils, in Chd4-ECko livers. No enrichment of specific immune cell types was observed in Chd4-ECko livers compared with controls at E13.5-14.5 (Fig. R1). Since immune cells develop within fetal livers at this stage, these findings suggest that they are locally activated rather than recruited to Chd4-ECko livers. Moreover, because fetal livers contain a heterogeneous mixture of immature and mature hematopoietic and immune cells, appropriate in vitro cell models to assess immune cell activation in this context are currently lacking. We have added comments to the introduction to address some of these points (see lines 66-68).

1. Related to the previous comment: Can the authors validate their findings in an independent, ideally human, cell-based system?

To explore this, we analyzed PLAUR and ICAM1 transcripts following CHD4 and/or BRG1 knockdown in primary human umbilical vein endothelial cells (HUVECs) for 48 hours. No antagonistic regulation of either gene was detected in HUVECs (Fig. R3*). Moreover, while Icam1 transcription was antagonistically regulated by CHD4 and BRG1 in the mouse MS1 EC line (see Fig. 5A), transcriptional regulation of Plaur by these remodelers was observed only in isolated LSECs and not in cultured MS1 cells. Together, these findings demonstrate that BRG1 and CHD4 play context-specific roles when regulating Icam1 and Plaur transcription in different EC types. Furthermore, in vitro versus in vivo EC environments may additionally influence BRG1 and CHD4 activity.

*Please see uploaded Response to Reviewers PDF for Figure R3

1. Identifying the specific hematopoietic/immune subset could further increase the paper's impact, as it would more definitively clarify the mechanism in the developing endothelial niche.

Please see our response to question # 3.

1. Also, can the authors show experimentally whether, conversely, Chd4 overexpression can limit an endothelial-type of inflammatory liver injury?

We agree that exploring this suggestion would provide useful insights. However, we currently lack a genetic or inducible endothelial-specific Chd4 overexpression model, which makes it challenging to link our embryonic findings to the context of adult liver injury. For now, our study demonstrates that hepatic ECs regulate sterile inflammation to support embryonic liver development. Future development of appropriate genetic tools will allow us to determine if the role of endothelial CHD4 that is demonstrated in the current study is recapitulated in adult inflammatory liver injury models.

Minor

1. A separate figure panel for Chd4fl/fl; Vav-Cre+ appears reasonable, instead of being shown as a table.

Thank you. Please see our new Fig. S1, which includes representative images (and lethality rates) of control and Chd4fl/fl;Vav-Cre+ embryos at E18.5.

Significance:

Generally, this is an excellent manuscript with a strong developmental biology focus, and its translational relevance is not immediately apparent; however, establishing such a link could significantly increase its impact. For example, the significance of these findings in ischemia-reperfusion injury, SOS/VOD, and sepsis could offer therapeutic avenues to stabilize endothelial function.

The advance is the elegant discovery of a multifactorial endothelial-stabilizing mechanism in development, although its applicability to scenarios beyond developmental mutation remains unknown.

The strengths are the clear and transparent experimental interrogation. Rightfully, the authors acknowledge that there would be a benefit in finalizing inflammatory blockade, genetic or antibody-mediated, to pin down the mechanistic circuit.

The reviewer's expertise is: childhood liver diseases, developmental liver organoid generation, stem cells (iPSCs), cell reprogramming

Reviewer #3

Evidence, reproducibility and clarity:

1. Wu et al. report antagonistic roles for chromatin remodelers Chd4 and Brg1, in endothelial cells, during liver development. There is a major flaw in the study which makes it difficult to interpret the conclusions. The genotypes of the mouse models used are flawed. The comparison should be made between two single knockouts (Chd4 single, Brg1 single), double mutants (Chd4/Brg1) and proper controls. For both "single KO", one allele of the other gene is also deleted - Chd4 -Ecko has one allele of Brg1 deleted and vice versa. Also, the proper control should be Chd4 fl/flBrg1fl/fl without the Cre. Since 3 alleles (not just two that belong to the same gene) are deleted in a single knockout, it is impossible to assign the effect to one gene.

We acknowledge the fact that the single Brg1 and Chd4 EC knockouts in this study each carry a heterozygous deletion allele for the other remodeler (exact genotypes are shown in Fig. 1A). The mating strategy that yielded these mutants was chosen for three reasons. First, we have found that genetic background influences the embryonic phenotypes of these chromatin remodeler mutants3. Moreover, embryonic development at the stages analyzed in this study occurs quickly and requires precise timing for comparative analysis between genotypes. Therefore, it is most rigorous to study littermates when comparing single- and double-mutant embryos for BRG1 and CHD4. To achieve this, we used Brg1fl/fl;Chd4fl/fl females rather than Brg1fl/+;Chd4fl/+ females for timed matings. Although the former females cannot produce single knockout embryos without a compound heterozygous allele of the other remodeler, these females allowed us to generate single- and double-knockouts at a rate of 1/8 embryos. If we had used Brg1fl/+;Chd4fl/+ females for timed matings, we would have been able to generate “clean” single mutants with wildtype alleles of the other remodeler, but the single- and double-knockout generation rate would have been 1/32 embryos. This would have been an impractical mutant generation rate for this study. Second, our prior research demonstrates that heterozygous deletion of Chd4 or Brg1 does not produce the liver phenotypes seen with the respective homozygous deletions2,3. Third, the complete lethality of Chd4-ECko (Brg1fl/+;Chd4fl/fl;VE-cadherin-Cre+) mutants in this study demonstrates that deleting one allele of Brg1 cannot rescue Chd4-related lethality.

As for controls in this study, we saw no evidence of phenotypes or of any gene deletion in our Cre- embryos (either in this study or in previous ones analyzing similar phenotypes2,3). Therefore, we used Cre- embryos for controls because they were generated at a 1/2 rate by our timed matings, which boosted our output for analyses.

Specific points

1. Fig 2c Plaur transcript - no statistical comparison between 2nd and 4th column, Chd4 Ecko vs double mutant. If there is not statistical difference, does not explain the rescue in double mutants

Thank you for the suggestion. We have included a comparison between Chd4-ECko and Brg1/Chd4-ECdko in our revised Fig 2C. The Kruskal-Wallis test showed a significant difference between the Chd4-Ecko and Brg1/Chd4-ECdkogroups (p=0.016). This indicates that Plaur induction in Chd4-Ecko LSECs is rescued in Brg1/Chd4-ECdko LSECs.

1. Fig 2e. Comparison should be made between Plg-/- Chd4 fl/fl and Plg-/- Chd4 fl/fl Cre, not other genotypes

This experiment aims to determine whether different levels of plasminogen (Plg) reduction can rescue the lethality caused by Chd4 deletion. To do this, we set up the mating strategy shown in Fig. 2E to produce appropriate littermate controls and to compare lethality among Plg+/+;Chd4-ECko, Plg+/-;Chd4-ECko, and Plg-/-;Chd4-ECko embryos. This comparison would not have been possible with embryos generated only from mice on a Plg-/- background.

1. Fig. 4. How does Chd4 or Brg1 activity in endothelial cells lead to Icam1 activation in epithelial cells?

Since cytokines like IFNg, TNFa, and IL1b can induce ICAM-1 expression in hepatocytes10, we speculate that ICAM-1 expression in hepatoblasts (ECAD+ cells in Fig. 4D) was induced by the elevated TNFa and IL1b produced in Chd4-ECko livers (Fig. 3G).

1. Mice used in Figure 5 are Cdf4 fl/+ and Cdf4 fl/fl, no Brg1 deletion. The authors improperly compare these to Chd4-Ecko which have one allele of Brg1 deleted. The rescue needs to be done in the same genotype Chd4-Ecko.

Please note that data from Fig. 5 were generated from cultured ECs (MS1 cells).

Significance

Wu et al. report antagonistic roles for chromatin remodelers Chd4 and Brg1, in endothelial cells, during liver development. There is a major flaw in the study which makes it difficult to interpret the conclusions. Genotypes that were chosen for the study make the data not interpretable

Please see our response to your Question #1

In summary, we have included the following changes to this revised manuscript:

• New Figure 1B: Representative images and lethality rates for control, Chd4-ECko, Brg1-ECko, and Brg1/Chd4-ECdko embryos at E17.5.
• New Figure 2C: qRT-PCR analysis of Chd4, Brg1, and Plaur gene transcripts in E12.5 control and mutant LSECs.
• Regraphing of Figure 3G: qRT-PCR analysis of Tnf, Il6, and Il1b gene transcripts in E14.5 control and mutant livers.
• New Figure S1: Representative images and lethality rates for control, Chd4fl/+;Vav-Cre+, and Chd4fl/fl;Vav-Cre+embryos at E18.5. References for this revision:

Alva JA, Zovein AC, Monvoisin A, Murphy T, Salazar A, Harvey NL, Carmeliet P, Iruela-Arispe ML. VE-Cadherin-Cre-recombinase Transgenic Mouse: A Tool for Lineage Analysis and Gene Deletion in Endothelial Cells. Dev Dyn. 2006;235:759-767. doi: 10.1002/dvdy.20643 Crosswhite PL, Podsiadlowska JJ, Curtis CD, Gao S, Xia L, Srinivasan RS, Griffin CT. CHD4-regulated plasmin activation impacts lymphovenous hemostasis and hepatic vascular integrity. J Clin Invest. 2016;126:2254-2266. doi: 10.1172/JCI84652 Wu ML, Wheeler K, Silasi R, Lupu F, Griffin CT. Endothelial Chromatin-Remodeling Enzymes Regulate the Production of Critical ECM Components During Murine Lung Development. Arterioscler Thromb Vasc Biol. 2024;44:1784-1798. doi: 10.1161/ATVBAHA.124.320881 Shiojiri N, Inujima S, Ishikawa K, Terada K, Mori M. Cell lineage analysis during liver development using the spfash-heterozygous mouse. Lab Invest. 2001;81:17-25. doi: 10.1038/labinvest.3780208 Soares-da-Silva F, Peixoto M, Cumano A, Pinto-do OP. Crosstalk Between the Hepatic and Hematopoietic Systems During Embryonic Development. Front Cell Dev Biol. 2020;8:612. doi: 10.3389/fcell.2020.00612 Ema H, Nakauchi H. Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. Blood. 2000;95:2284-2288. Kieusseian A, Brunet de la Grange P, Burlen-Defranoux O, Godin I, Cumano A. Immature hematopoietic stem cells undergo maturation in the fetal liver. Development. 2012;139:3521-3530. doi: 10.1242/dev.079210 Freitas-Lopes MA, Mafra K, David BA, Carvalho-Gontijo R, Menezes GB. Differential Location and Distribution of Hepatic Immune Cells. Cells. 2017;6. doi: 10.3390/cells6040048 Christensen JL, Wright DE, Wagers AJ, Weissman IL. Circulation and chemotaxis of fetal hematopoietic stem cells. PLoS Biol. 2004;2:E75. doi: 10.1371/journal.pbio.0020075 Satoh S, Nussler AK, Liu ZZ, Thomson AW. Proinflammatory cytokines and endotoxin stimulate ICAM-1 gene expression and secretion by normal human hepatocytes. Immunology. 1994;82:571-576.

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• Silêncio administrativo = REJEIÇÃO

JCP

Le phénomène du bavardage scolaire : Analyse et perspectives

Synthèse Exécutive

Ce document présente une analyse approfondie du phénomène de bavardage scolaire, un enjeu souvent sous-estimé qui affecte de manière significative le climat de classe et la réussite des élèves.

Basée sur une étude combinant une revue de la littérature scientifique et une enquête de terrain menée auprès d'élèves de 4ème, cette synthèse met en lumière la complexité du bavardage, les perceptions divergentes qu'il suscite et l'efficacité limitée des interventions basées uniquement sur la prise de conscience individuelle.

L'analyse théorique révèle que le bavardage a évolué, passant d'un "chahut traditionnel" structuré à un désordre plus "anomique" et généralisé.

Les travaux de chercheurs comme Florence Ehnuel et Alain Courneloup soulignent un décalage fondamental entre les perceptions des différents acteurs : les élèves le banalisent souvent comme une interaction sociale normale, les parents le perçoivent avec une faible gravité, tandis que les enseignants le vivent comme une source de déprofessionnalisation et d'impuissance.

Les causes identifiées sont multiples, incluant l'ennui, le besoin d'interaction sociale, la pression des pairs et un cadre scolaire parfois perçu comme trop rigide.

L'enquête de terrain, réalisée via un questionnaire suivi d'entretiens individuels, confirme ces constats. Une majorité d'élèves bavards ne se sentent pas personnellement dérangés par le bruit et estiment que leurs propres conversations ne nuisent pas à leurs camarades, se croyant capables de parler et d'écouter simultanément.

L'outil de questionnement a permis une prise de conscience modérée chez environ la moitié des participants, mais n'a entraîné un changement de comportement durable que pour une minorité.

La crainte de sanctions demeure le levier externe le plus efficace, tandis que la motivation interne reste fragile.

En conclusion, la lutte contre le bavardage scolaire ne peut se résumer à des sanctions disciplinaires.

Elle exige une approche globale qui intègre la compréhension des perceptions des élèves, la mise en place de cadres clairs et co-construits, et l'adoption de stratégies pédagogiques actives pour réduire l'ennui.

Si la prise de conscience est une étape nécessaire, elle s'avère insuffisante sans un accompagnement structuré et des règles appliquées avec constance.

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I. Cadre Théorique du Bavardage Scolaire

Définition et Évolution du Phénomène

Le bavardage scolaire est défini comme toute prise de parole non autorisée par l'enseignant durant un temps de cours. Il constitue un phénomène social complexe qui perturbe la transmission des savoirs et le climat d'apprentissage.

L'analyse sociologique de Jacques Testanière (1967) offre une perspective historique sur l'évolution du désordre en classe. Il distingue :

• Le chahut traditionnel : Une "anomalie normale" et collective, souvent ritualisée, qui visait à tester l'autorité de l'enseignant tout en renforçant la cohésion du groupe d'élèves.

• Le chahut anomique : Une forme de désordre plus généralisée, individualiste et sans règles, qui exprime une mauvaise intégration de l'élève au système pédagogique. Le bavardage contemporain s'apparente davantage à cette seconde forme, caractérisée par une multitude de conversations parallèles plutôt qu'une confrontation unifiée.

Perceptions Divergentes des Acteurs

L'une des difficultés majeures dans la gestion du bavardage réside dans le profond décalage de perception entre les différents acteurs de la communauté éducative, comme le démontre l'ouvrage de Florence Ehnuel, « Le bavardage : Parlons-en enfin ! ».

| Acteur | Perception du Bavardage | | --- | --- | | Élèves | Considéré comme une interaction sociale normale et un "non-acte". Beaucoup estiment pouvoir écouter et parler en même temps. Il est souvent justifié par l'ennui, le besoin d'échanger avec les pairs ou le désintérêt pour la matière. | | Enseignants | Vécu comme une nuisance majeure, un manque de respect, et une source de fatigue et de culpabilité. Les réactions varient de la tolérance à la sanction systématique, en passant par un sentiment d'impuissance. | | Parents | Souvent perçu comme un problème mineur, non comparable à l'insolence ou aux mauvais résultats. Certains y voient même un signe de "vitalité" ou d'"aisance relationnelle". | | Didacticiens | Interprété comme une forme de résistance à la norme scolaire, une pratique sociale d'échange, une échappatoire face aux difficultés d'apprentissage, ou un symptôme du décalage entre la culture scolaire et la culture jeune. |

Causes et Motivations du Bavardage

La littérature identifie plusieurs facteurs expliquant la prévalence du bavardage :

• Facteurs Pédagogiques : L'ennui provoqué par un cours jugé trop lent ou inintéressant est une cause majeure. Comme le souligne Alain Courneloup, "un élève qui s'ennuie est un élève qui va trouver à s'occuper".

• Facteurs Sociaux : Le besoin d'interaction avec les pairs est fondamental à l'adolescence. Le groupe agit comme un "médiateur" entre l'individu et les adultes. Répondre à un camarade est souvent perçu comme une obligation sociale pour ne pas le "vexer" ou trahir une amitié.

• Facteurs Sociétaux : La "génération du zapping" est habituée à un environnement bruyant et à la multi-activité. Le silence peut être perçu comme angoissant par certains élèves.

• Facteurs Institutionnels : L'absence de règles claires ou le manque de constance dans l'application des sanctions par les enseignants peut créer un cadre propice au développement du bavardage.

Conséquences et Enjeux

Les méfaits du bavardage sont souvent sous-estimés. Il ne s'agit pas d'un simple désagrément sonore.

• Sur les apprentissages : Le bavardage est une "forme d'absentéisme" intellectuel.

Même si l'élève est physiquement présent, son attention est détournée, ce qui nuit à la concentration, à la compréhension et à la mémorisation.

• Sur le climat de classe : Le bruit constant génère de la fatigue et de la tension pour l'enseignant et pour les élèves qui souhaitent travailler.

Il ralentit le rythme du cours et peut créer un sentiment d'impunité.

• Sur le parcours de l'élève : À long terme, le bavardage persistant, lorsqu'il est le symptôme d'un désintérêt plus profond, peut être un indicateur de risque de décrochage scolaire.

François Dubet, dans « La galère », décrit comment le désengagement scolaire peut mener à des trajectoires de marginalisation.

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II. Enquête de Terrain sur la Prise de Conscience des Élèves

Objectif et Méthodologie de l'Étude

L'enquête visait à déterminer si un outil de questionnement pouvait amener des élèves de 4ème à prendre conscience de l'ampleur et des conséquences de leur propre bavardage, et si cette prise de conscience pouvait induire un changement de comportement. L'expérimentation s'est déroulée en trois phases :

1. Phase 1 : Administration d'un questionnaire en ligne (Google Forms) à 52 élèves pour évaluer leurs pratiques et perceptions.

2. Phase 2 : Une période de plusieurs semaines pour observer d'éventuels changements.

3. Phase 3 : Entretiens individuels avec un échantillon de 8 élèves pour mesurer l'impact de l'intervention.

Principaux Résultats du Questionnaire (N=52, dont 35 "bavards")

L'analyse s'est concentrée sur les 35 élèves s'identifiant comme discutant en cours "de temps en temps", "assez" ou "tout le temps".

• Auto-perception des élèves bavards :

◦ Un paradoxe central : Une grande majorité des élèves bavards (65,7%) déclarent ne pas être dérangés par le bruit en classe.   

◦ La rationalisation du multitâche : Plus de la moitié (54,3%) estiment que leurs propres discussions ne gênent "pas du tout" leurs camarades. La raison principale invoquée (68,4%) est leur conviction de pouvoir "parler à [leur] voisin et écouter le professeur en même temps". 

◦ Les motivations sociales avant tout : La raison principale du bavardage est d'avoir "des choses importantes à dire à leurs amis" (45,7%), devant les difficultés de concentration (40%) et le désintérêt pour la matière (34,3%).

• Conscience de l'Impact :

◦ Un effet modéré : Le questionnaire a permis à 51,4% des élèves de prendre "un peu" conscience des conséquences de leurs conversations.

Seuls 5 élèves (14,3%) ont jugé cette prise de conscience "nécessaire" ou "essentielle". 

◦ Lien avec les résultats scolaires contesté : Les avis sont partagés quant à l'impact du bavardage sur les notes. 37,5% pensent que leurs discussions n'ont "pas d'impact" sur leurs résultats.

• Volonté de Changement :

◦ Une faible envie d'arrêter : Plus de la moitié des élèves bavards n'ont pas l'intention de mettre fin à leurs discussions, considérant que ce n'est "pas si bavard que ça" ou que c'est "plus fort que moi".  

◦ Le poids des sanctions : La "sanction de la part du professeur" est identifiée comme la pression extérieure la plus efficace pour les inciter à diminuer leurs bavardages.   

◦ Des résolutions fragiles : Malgré tout, 16 élèves sur 35 ont décidé de "prendre une résolution" pour se modérer.

Résultats des Entretiens Individuels (N=8)

Les entretiens menés quelques semaines après le questionnaire ont permis de nuancer les résolutions prises.

• Un Impact Limité sur le Comportement Réel : Seuls 3 des 8 élèves interrogés ont déclaré avoir effectivement diminué leur niveau de bavardage. Pour les autres, la situation était "pareille" voire "accentuée".

• La Persistance des Habitudes : Le changement de comportement s'est avéré difficile.

Le placement en classe (proximité avec un ami) reste un facteur déterminant.

Plusieurs élèves reconnaissent que malgré leur bonne volonté, l'habitude reprend le dessus.

• Un Acte Anormal mais Inévitable : La majorité des élèves interrogés conviennent qu'il n'est "pas normal" de discuter en classe.

Cependant, cette reconnaissance intellectuelle ne se traduit que rarement par une auto-discipline efficace, illustrant le fossé entre la conscience d'une règle et sa mise en application.

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III. Synthèse et Recommandations Stratégiques

Synthèse des Constats

1. Le fossé perceptuel comme obstacle majeur : Le principal frein au changement est que les élèves bavards ne perçoivent majoritairement pas leur comportement comme une nuisance, ni pour eux-mêmes ni pour les autres.

La croyance erronée en leur capacité à effectuer plusieurs tâches à la fois est une rationalisation puissante.

2. L'insuffisance de la prise de conscience seule : L'enquête démontre qu'une intervention visant à provoquer une prise de conscience interne, bien qu'utile, est insuffisante pour modifier durablement les comportements.

La volonté de changer est souvent volatile et rapidement supplantée par les habitudes et la dynamique sociale de la classe.

3. L'importance persistante du cadre externe : Les facteurs externes, notamment la clarté des règles et la constance dans l'application des sanctions, restent des leviers d'action déterminants pour la majorité des élèves.

Pistes de Réflexion et Stratégies d'Intervention

En s'appuyant sur les apports de la littérature et les résultats de l'enquête, plusieurs stratégies peuvent être envisagées pour une gestion plus efficace du bavardage.

• Co-construire les règles de vie (Courneloup) : Impliquer les élèves dans l'élaboration des règles de communication en classe.

Cet exercice de citoyenneté permet de rendre les règles plus explicites et de favoriser l'adhésion en montrant qu'elles servent l'intérêt collectif.

• Établir un cadre clair et constant (Ehnuel) : Dès le début de l'année, l'enseignant doit définir clairement ses attentes en matière de silence et de prise de parole.

La constance est cruciale : les élèves identifient rapidement les enseignants dont les avertissements ne sont pas suivis d'effets.

• Adopter une pédagogie active (Courneloup) : Pour contrer l'ennui, il est essentiel de varier les modalités de travail. Alterner les exposés magistraux avec des exercices, des travaux de groupe structurés, et des mises en commun permet de canaliser l'énergie des élèves et de réduire les temps morts propices au bavardage.

• Utiliser la communication non verbale (Courneloup) : Un regard appuyé, un doigt sur la bouche ou un déplacement silencieux vers un groupe d'élèves est souvent plus efficace et moins perturbateur pour le reste de la classe qu'une réprimande verbale à voix haute.

• Privilégier le dialogue individuel (Ehnuel) : En cas de bavardage récurrent d'un élève, une discussion en aparté à la fin du cours peut être bénéfique.

Elle permet de comprendre les raisons du comportement (difficultés, anxiété, etc.) et de responsabiliser l'élève sans l'humilier publiquement.

Synthèse sur la Consommation d'Alcool en France : Enjeux, Conséquences et Stratégies

Résumé Exécutif

Ce document synthétise les enjeux majeurs liés à la consommation d'alcool en France, en se basant sur les analyses d'experts et des témoignages.

L'alcool demeure la deuxième cause de mortalité évitable dans le pays, avec 49 000 décès par an, juste après le tabac.

Bien que la consommation globale soit en baisse, elle reste à un niveau excessivement élevé, posant un problème de santé publique majeur.

Les conséquences de cette consommation sont multiples : sanitaires (cancers, maladies cardiovasculaires), sociales (destruction de familles, stigmatisation des abstinents) et personnelles (perte de contrôle, addiction).

La jeunesse est particulièrement vulnérable, avec une prévalence alarmante du "binge drinking" qui compromet le développement cérébral et augmente le risque de dépendance à l'âge adulte.

Face à ce constat, plusieurs stratégies sont débattues : une prévention jugée insuffisante, notamment en milieu scolaire ; des initiatives comme le "Dry January" portées par la société civile face à un État réticent ; l'émergence d'un marché dynamique de boissons sans alcool comme alternative ; et un cadre réglementaire et fiscal (Loi Évin, taxes) considéré comme moins dissuasif que celui appliqué au tabac, soulevant des questions sur l'influence des lobbys.

Analyse Approfondie des Thématiques Clés

L'Ampleur du Problème de l'Alcool en France

La situation de la consommation d'alcool en France est marquée par une dualité : une tendance à la baisse sur le long terme mais des niveaux qui restent parmi les plus préoccupants en Europe.

• Bilan Humain : L'alcool est directement responsable de 49 000 décès chaque année, ce qui en fait un enjeu de santé publique de premier ordre.

• Le Paradoxe Français : La formule "On boit moins, mais on boit trop" résume la situation. La consommation moyenne a diminué, mais elle excède toujours les seuils de risque recommandés.

• Repères de Consommation : Depuis 2017, Santé Publique France recommande de ne pas dépasser deux verres par jour, et pas tous les jours.

L'Organisation Mondiale de la Santé (OMS) va plus loin en affirmant qu'il n'existe aucune consommation d'alcool sans risque.

Conséquences Sanitaires et Sociales

Les impacts de l'alcool sont profonds et touchent toutes les sphères de la vie de l'individu et de la société. L'âge moyen où les complications graves apparaissent est de 56 ans, mais les dommages peuvent survenir bien plus tôt.

| Type de Conséquence | Description détaillée | | --- | --- | | Sanitaires Aiguës | Liées à l'ivresse : accidents de la voie publique, accidents domestiques, chutes, traumatismes et mises en danger diverses. | | Sanitaires Chroniques | Pathologies graves se développant sur le long terme : cancers (digestifs, foie, sphère ORL), maladies digestives et maladies cardiovasculaires. | | Sociales et Familiales | L'alcool est décrit comme un "ravage dans une famille". L'impact sur les enfants de parents alcooliques est particulièrement dévastateur, créant des perturbations profondes. | | Personnelles | Conséquences professionnelles, financières et judiciaires (retraits de permis, gardes à vue pour bagarre). | | Culturelles | L'alcool est banalisé et associé à la convivialité ("bon vivant"). Inversement, la sobriété est stigmatisée, les non-buveurs étant perçus comme "pas fun", "chiants" ou même "malades". |

Le Mécanisme de l'Addiction et les Facteurs de Vulnérabilité

L'addiction à l'alcool est un processus insidieux qui s'installe progressivement.

1. La "Belle Rencontre" : La consommation débute souvent par la recherche d'effets psychotropes agréables (désinhibition, plaisir).

2. La Perte de Contrôle : Progressivement, l'individu perd la maîtrise de sa consommation (quantité, fréquence, temps consacré). C'est un critère central de l'addiction.

3. La Poursuite Malgré les Conséquences : Le signe définitif de l'addiction est la continuation de la consommation alors même que la personne constate les conséquences négatives sur sa santé, sa famille ou son travail.

Le Dr Delphine Moisan souligne que tout le monde n'est pas égal face à ce risque. Plusieurs facteurs de vulnérabilité individuelle existent :

• Génétiques : Antécédents familiaux d'addiction.

• Biologiques : Différences dans le fonctionnement du "circuit de la récompense" cérébral.

• Psychologiques : Faible estime de soi, sensibilité au stress, introversion.

• Psychiatriques : Comorbidités comme la dépression, la bipolarité ou l'hyperactivité.

• Environnementaux : Traumatismes vécus, contexte social.

L'Alcoolisation des Jeunes : Le Phénomène du "Binge Drinking"

La consommation d'alcool chez les jeunes représente une préoccupation majeure en raison de ses risques spécifiques.

• Définition : Le "Binge Drinking" (ou Alcoolisation Ponctuelle Importante) consiste à consommer une grande quantité d'alcool en un temps très court. Le seuil est fixé à 5 verres ou plus par occasion pour un adolescent.

• Statistiques Inquiétantes :

◦ 36 % des jeunes de 17 ans ont connu un épisode de binge drinking le mois précédant l'enquête.    ◦ 15 % des élèves de 4ème et 3ème rapportent des épisodes similaires.

• Risques Spécifiques :

◦ Développement Cérébral : Le cerveau est en maturation jusqu'à l'âge de 25 ans. L'exposition précoce à l'alcool perturbe ce développement.    ◦ Risque d'Addiction Future : Il est prouvé que plus la consommation d'alcool commence tôt, plus le risque de développer une dépendance à l'âge adulte est élevé.

Stratégies de Réduction et Alternatives

Face à ce tableau, différentes approches sont mises en œuvre ou envisagées, avec des niveaux d'implication variables des acteurs publics et privés.

• Prévention : La sénatrice Cathy Apourceau-Poly dénonce un manque de moyens pour la prévention, notamment dans les établissements scolaires qui voient le nombre d'infirmières et d'assistantes sociales diminuer.

• Initiatives Citoyennes : Le "Dry January", une campagne d'origine britannique invitant à un mois sans alcool, a été adoptée par 1 million de Français. Fait notable, cette initiative n'est pas portée par l'État français, qui s'est rétracté, mais par la société civile.

• Le Marché des Boissons Sans Alcool : Ce secteur est en pleine expansion, avec l'ouverture d'une trentaine de caves spécialisées en France.

◦ Clientèle : Entre 70 % et 80 % des clients de ces caves sont des consommateurs d'alcool qui cherchent à réduire leur consommation, notamment en semaine.   

◦ Adoption : Plus de 25 % des Français déclarent consommer des boissons sans alcool, un chiffre qui monte à 41 % chez les 26-35 ans. 

◦ Usage Thérapeutique : Pour les personnes dépendantes, ces boissons peuvent être une aide mais aussi un risque, le goût pouvant déclencher une envie de consommer de l'alcool.

Le Rôle des Pouvoirs Publics et de la Réglementation

L'action de l'État est jugée ambivalente et souvent insuffisante par les experts interrogés.

• Fiscalité Comportementale : La taxation est un levier efficace mais sous-utilisé pour l'alcool, contrairement au tabac (un paquet de cigarettes comporte 75 % de taxes).

L'exemple de l'Écosse, où une surtaxe a entraîné une baisse de 13 % de la mortalité liée à l'alcool, démontre le potentiel de cette mesure.

• Législation : La Loi Évin est jugée "pas satisfaisante" et ne va pas assez loin.

• Publicité : Contrairement au tabac, la publicité pour l'alcool reste autorisée, ce qui est considéré comme un problème majeur.

La régulation est de plus complexifiée par les publicités provenant de l'étranger via les réseaux sociaux.

Témoignages et Études de Cas

Le Parcours de Marine : De l'Addiction à la Sobriété

Le témoignage de Marine, 31 ans, illustre le cheminement vers la dépendance et la possibilité d'en sortir.

• Historique : Consommation initiée à 15 ans, qui s'intensifie avec le "binge drinking" durant ses études supérieures, où l'alcool devient une "béquille" sociale indispensable.

• Le Déclic : Une période de convalescence post-opératoire, marquée par une consommation excessive par ennui, lui fait prendre conscience de son problème.

• La Transition : Elle entame un "Dry January", réduit drastiquement sa consommation, se met au sport, puis décide d'arrêter totalement.

• Les Bénéfices : En 475 jours, elle a économisé près de 3 000 €, évité 171 000 calories et a perdu beaucoup de poids.

• Les Défis Sociaux : Elle se heurte à la pression sociale et à l'image de la personne "rabat-joie". Son témoignage met en lumière la difficulté d'une sobriété choisie mais subie :

"Je préférerais être capable de boire un verre de vin de temps en temps mais j'en suis vraiment pas capable donc je préfère ne rien boire du tout."

Ressources et Informations Utiles

Plusieurs dispositifs d'aide et d'information sont disponibles pour les personnes souhaitant évaluer ou réduire leur consommation.

• Ligne d'écoute : 09 80 980 930

• Site d'information : alcool-info-service.fr

• Outil d'auto-évaluation : alcoometre.fr

Analyse du Microlycée de Sénart : Une Approche Pédagogique Alternative pour les Décrocheurs Scolaires

Synthèse

Le microlycée de Sénart est un établissement public qui incarne une approche pédagogique radicalement différente, conçue pour rescolariser les jeunes de 17 à 26 ans ayant quitté le système traditionnel.

Face au phénomène national de 100 000 décrocheurs annuels, cette structure offre une "seconde chance" à 90 élèves, en s'attaquant aux causes profondes de leur déscolarisation : phobie scolaire, harcèlement, problèmes psychologiques ou mauvaise orientation.

La méthode du microlycée repose sur trois piliers fondamentaux : la flexibilité, la confiance et la co-construction.

Le cadre scolaire est volontairement assoupli : les retards sont tolérés, il n'y a pas de sanctions, et certaines règles des lycées classiques sont levées, comme l'interdiction du téléphone en cours ou de la cigarette (par dérogation).

Les classes à effectifs réduits (neuf élèves) permettent une relation enseignant-élève intime et familière, caractérisée par le tutoiement et un suivi proactif, comme les appels quotidiens aux absents pour les encourager.

L'évaluation est entièrement réinventée pour ne plus être une source de jugement destructeur.

Les notes sur 20 sont remplacées par des pourcentages de réussite que les élèves peuvent discuter, voire négocier, avec leurs professeurs.

Ce système de "co-construction" vise à faire de l'évaluation un outil d'apprentissage, renforçant l'autonomie et la confiance de l'élève.

De même, le conseil de classe est transformé en un format de "speed dating" où chaque élève échange directement avec ses professeurs, devenant ainsi un acteur central de son parcours.

Les parcours d'élèves comme Romain, Lola et Léo témoignent de l'efficacité de cette approche. Ils illustrent la capacité de l'établissement à reconstruire des jeunes brisés par le système traditionnel, en leur redonnant le goût d'apprendre et en leur permettant de se réconcilier avec l'école et avec eux-mêmes.

Le dispositif inclut également un soutien crucial aux familles, via des groupes de parole, qui partagent leur désarroi et leur soulagement.

Malgré un taux d'abandon de 20% en cours d'année, le microlycée parvient à mener 7 élèves sur 10 jusqu'au baccalauréat, prouvant la pertinence de son modèle atypique.

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1. Contexte et Mission du Microlycée de Sénart

Le microlycée de Sénart, ouvert en septembre 2000, est l'une des 61 structures publiques en France dédiées à la rescolarisation des élèves décrocheurs.

Chaque année, 100 000 jeunes quittent le lycée sans diplôme, soit 9% des élèves.

L'établissement accueille 90 de ces jeunes, âgés de 17 à 26 ans, de la seconde à la terminale.

Les raisons du décrochage sont multiples et complexes :

• Phobie scolaire

• Refus du système éducatif traditionnel

• Problèmes psychologiques

• Harcèlement scolaire

• Mauvaise orientation

La mission principale de l'établissement est de "réapprendre à aimer l'école" à ces jeunes en leur offrant un cadre bienveillant et des méthodes alternatives.

2. Une Approche Pédagogique Fondée sur la Confiance et la Flexibilité

Le programme de l'Éducation Nationale est suivi à la lettre, mais les méthodes d'enseignement et le cadre de vie scolaire sont radicalement différents de ceux d'un lycée traditionnel.

2.1. Un Cadre Souple et Non-Punitif

L'objectif est de dédramatiser l'école en supprimant les sources de stress et de conflit.

• Absence de Sanctions : Les retards sont autorisés et il n'y a pas de sanctions disciplinaires. Comme le souligne une enseignante, "on a d'autres moyens aussi de faire en sorte que ces jeunes puissent raccrocher".

• Tolérance et Flexibilité : Les élèves peuvent utiliser leur téléphone portable pour écouter de la musique en cours. La cigarette est autorisée dans des zones dédiées, sur dérogation de l'inspection académique.

• Suivi Proactif : Une professeure, Christine, appelle chaque jour les dizaines d'élèves absents, non pas pour les réprimander, mais pour les encourager à revenir. "Je t'appelle pour t'encourager à revenir à l'école [...] courage".

2.2. La Relation Enseignant-Élève

Le rapport entre les professeurs et les élèves est au cœur du dispositif.

• Effectifs Réduits : Les classes ne comptent que neuf élèves, favorisant une interaction directe et personnalisée.

• Proximité et Familiarité : Le tutoiement est la norme et les élèves sont appelés par leur prénom. Un élève explique : "Ça apporte plutôt je me trouve plus à l'aise en fait avec les profs [...] on commence à prendre confiance".

• Enseignants Volontaires et Formés : Les 14 professeurs sont tous volontaires et suivent des formations spécifiques pour encadrer ces élèves. Une professeure de français, Emmanuel, témoigne de la valeur de son travail : "Ce qu'il m'apporte de plus fondamental, c'est un sens profond à ce que je fais [...] j'ai l'impression de pouvoir accompagner ces jeunes jusqu'à une reprise de confiance en eux".

3. L'Évaluation Réinventée : De la Sanction à la Co-construction

L'un des aspects les plus innovants du microlycée est sa redéfinition complète du système de notation, souvent vécu comme un "jugement de la personne" dans le système classique.

• Pas de Note sur 20 : Les copies ne sont pas notées sur 20 mais reçoivent un pourcentage de réussite.

• La Co-construction : L'évaluation n'est pas un verdict final mais le début d'un dialogue. L'élève peut discuter son résultat avec le professeur.

Emmanuel Catinois, professeure de français, précise : "C'est ce qu'on appelle la co-construction, c'est on construit ensemble l'évaluation. L'idée c'est que l'évaluation, ça ne doit pas être un coup prêt, une note qui sanctionne, mais une note sur laquelle on peut s'appuyer, une note qui aide".

• Le Droit à l'Erreur : Les élèves ont la possibilité de retravailler une partie d'un devoir pour améliorer leur score. Romain, un élève, apprécie cette méthode : "Je peux le refaire, je peux me reprendre et elle va accepter [...] au moins on peut justifier au lieu de perdre des points bêtement".

4. Parcours d'Élèves : Portraits de la Reconstruction

Les profils des élèves sont variés, issus de tous les milieux sociaux, et illustrent les défis auxquels le microlycée répond.

| Élève | Âge | Parcours Avant le Microlycée | Situation au Microlycée | | --- | --- | --- | --- | | Romain | 17 | Ancien "premier de la classe", il tombe en dépression après le divorce de ses parents. Déscolarisé pendant deux ans. | Est devenu le meilleur élève de sa classe. Se réconcilie avec l'école grâce à la nouvelle approche de l'évaluation et des conseils de classe. | | Lola | \- | Déscolarisée pendant deux ans après avoir été harcelée au collège en raison de son homosexualité. A fait une tentative de suicide et a été hospitalisée six mois en psychiatrie. | A réappris à "aimer les lycées, à aimer les cours". Malgré d'importantes lacunes scolaires, l'équipe pédagogique parie sur elle en la faisant passer en classe supérieure. | | Léo | 19 | Décrit comme "je-m'en-foutiste", il a été renvoyé de plusieurs lycées. A décroché pendant deux ans, vivant la nuit et faisant des petits boulots. | Participe activement en cours, a pris le rôle d' "intendant café" et s'est réconcilié avec le français, écrivant désormais des chansons. |

5. Le Rôle Crucial des Familles

Le microlycée reconnaît que le décrochage scolaire est une épreuve pour toute la famille et intègre les parents dans son dispositif.

• Groupe de Parole : Un professeur anime des réunions régulières pour les parents, leur permettant de partager leur "désarroi" et de se soutenir mutuellement.

• Témoignages Émouvants : Les parents expriment un immense soulagement.

◦ Claude, père de Lola : "On est complètement paumé [...] Aujourd'hui [...] regardez c'est le sourire, Lola elle a un soir magnifique".    ◦ Laurence, mère de Romain : "Vous nous avez sauvé [...] le Romain de l'année dernière [...] à se poser des grosses questions est-ce qu'il va pas franchir une autre étape. Et puis aujourd'hui où je retrouve un môme de 17 ans vraiment bien dans ses baskets [...] c'est le jour et la nuit, on respire enfin".

• Forte Demande : L'établissement est perçu comme une "bouée de sauvetage". Chaque année, 40 familles postulent mais 20 candidatures doivent être refusées faute de place.

6. Le Conseil de Classe : Un Modèle de Transparence et d'Implication

Le traditionnel conseil de classe à huis clos est remplacé par un format innovant, conçu pour rendre l'élève acteur de son parcours.

• Format "Speed Dating" : Chaque élève rencontre individuellement chaque professeur pendant trois minutes pour discuter de ses résultats et de ses appréciations.

• Transparence Totale : "Il ne faut pas qu'il y ait des choses qui se disent sans la présence des élèves, rien n'est secret".

• Implication de l'Élève : Les élèves valident ou non les commentaires des professeurs. Romain explique : "On a l'interaction avec le prof et il nous met son commentaire devant nous et on valide ou pas [...] Ça m'apporte que je vois l'avis du prof en face de moi et qu'il fasse pas derrière mon dos".

• Renforcement de la Confiance : Cette pratique est jugée essentielle pour associer des élèves "adultes" (plus de 17 ans) à la construction de leur scolarité, ce qui "permet la confiance et le raccrochage".

7. Résultats, Défis et Perspectives

Le modèle du microlycée de Sénart, bien qu'exigeant, affiche des résultats probants.

• Taux de Réussite : 7 élèves sur 10 qui poursuivent leur scolarité au microlycée décrochent leur baccalauréat.

• Taux d'Abandon : Le parcours reste difficile, et 20% des élèves abandonnent en cours d'année.

• Défis Pédagogiques : L'équipe doit gérer des écarts de niveau considérables, comme celui de Lola qui a un niveau de 5ème en langues. L'établissement fait alors "le pari de la seconde chance" en adaptant ses décisions pour ne pas décourager les élèves malgré leurs lacunes.

• Transformation Personnelle : Au-delà du succès scolaire, l'établissement permet aux élèves de se reconstruire, de reprendre confiance et de développer de nouveaux projets, à l'image de Léo qui compose des chansons : "Apprendre, me cultiver et revenir avec des phrases de prof de français".

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

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Reply to the reviewers

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

SECTION A - Evidence, Reproducibility, and Clarity Summary The study investigates the neurodevelopmental impact of trisomy 21 on human cortical excitatory neurons derived from induced pluripotent stem cells (hiPSCs). Key findings include a modest reduction in spontaneous firing, a marked deficit in synchronized bursting, decreased neuronal connectivity, and altered ion channel expression-particularly a downregulation of voltage‐gated potassium channels and HCN1. These conclusions are supported by a combination of in vitro calcium imaging, electrophysiological recordings, viral monosynaptic tracing, RNA sequencing, and in vivo transplantation with two‐photon imaging.

Major Comments • Convincing Nature of Key Conclusions: The study's conclusions are generally well supported by a diverse set of experimental approaches. However, certain claims regarding the intrinsic properties of the excitatory network would benefit from further qualification. In particular, the assertion that reduced synchronization is solely attributable to altered ion channel expression might be considered somewhat preliminary without additional corroborative experiments.

1.1) We agree with the reviewer and now write in the abstract: 'Together, these findings demonstrate long-lasting impairments in human cortical excitatory neuron network function associated with Trisomy 21 .' And in the Introduction: 'Collectively, the observed changes in ion channel expression, neuronal connectivity, and network activity synchronization may contribute to functional differences relevant to the cognitive and intellectual features associated with Down syndrome.'

• One major limitation of the current experimental design is the reliance on predominantly excitatory neuronal cultures derived from hiPSCs. Although the authors convincingly demonstrate differences in network synchronization and connectivity between trisomic (TS21) and control neurons, the almost exclusive focus on excitatory cells limits the physiological relevance of the in vitro network. In the developing cortex, interneurons and astrocytes play crucial roles in modulating network excitability, synaptogenesis, and plasticity. Therefore, incorporating these cell types-either through co-culture systems or through directed differentiation protocols that yield a more heterogeneous neuronal population-could help to determine whether the observed deficits are intrinsic to excitatory neurons or are compounded by a lack of proper inhibitory regulation and glial support. 1.2) Thank you for this thoughtful comment. We agree that interneurons and astrocytes are crucial for network function. To clarify, astrocytes are generated in this culture system, as we previously reported in our characterisation of the timecourse of network development using this approach (Kirwan et al., Development 2025). However, our primary goal was to first isolate and define the cell-autonomous defects intrinsic to TS21 excitatory neurons, minimizing the complexity introduced by additional neuronal types. This focused approach was chosen also because engineering a stable co-culture system with reproducible excitatory/inhibitory (E/I) proportions is a significant undertaking that extends beyond the scope of this initial investigation, and has proven challenging to date for the field. By establishing this foundational phenotype, our work complements prior studies on interneuron and glial contributions. Future studies building on this work will be essential to dissect the more complex, non-cell-autonomous effects within a heterogeneous network. Importantly, since our initial submission, two highly relevant preprints have emerged-including a notable study from the Geschwind laboratory at UCLA (Vuong et al., bioRxiv, 2025; Risgaard et al., bioRxiv, 2025), as well as our own complementary study Lattke et al, under revision, that highlight widespread transcriptional changes in excitatory cells of the human fetal DS cortex, providing strong validation for our central findings. This convergence of results from multiple groups underscores the timeliness and importance of our work.

• Furthermore, the assessment of neuronal connectivity via pseudotyped rabies virus tracing, while innovative, has inherent limitations. The quantification of connectivity as a ratio of red-to-green fluorescence pixels may be influenced by differential viral infection efficiencies, variations in the expression levels of the TVA receptor, or even by the lower basal activity levels observed in TS21 cultures. Complementary approaches-such as electron microscopy for synaptic density analysis or functional connectivity measurements using multi-electrode arrays (MEAs)-could provide additional structural and functional insights that would validate the rabies tracing data. 1.3) Thank you for this constructive feedback. While we cannot formally exclude that TS21 cells might express the TVA receptor at lower levels due to generalized gene dysregulation, we infected all WT and TS21 cultures in parallel using identical virus preparations and titers to minimize technical variability. Crucially, we also addressed the potential confound of differential basal activity by performing the rabies tracing under TTX incubation (see Suppl. Fig. 7), which blocks network activity and ensures that viral spread reflects structural connectivity alone.

While complementary methods like EM or MEA could provide additional insight, they fall outside the scope of the current study. We are confident that our rigorous controls validate our use of the rabies tracing method to assess structural connectivity.

• Qualification of Claims: Some conclusions, particularly those linking specific ion channel dysregulation (e.g., HCN1 loss) directly to network deficits, might be better presented as preliminary. The authors could temper their language to indicate that while the evidence is suggestive, the mechanistic link remains to be fully established. 1.4) We have revised the text to more clearly indicate that the link between HCN1 dysregulation and network deficits is correlative and remains to be fully established. While our ex vivo recordings suggest altered Ih-like currents consistent with reduced HCN1 expression, we now present these findings as preliminary and hypothesis-generating, pending further functional validation. We write in the discussion: However, further targeted functional validation will be needed to confirm a causal link.

• Need for Additional Experiments: Additional experiments that could further consolidate the current findings include: o Inclusion of Inhibitory Neurons or Co-culture Systems: Incorporating interneurons or astrocytes would help determine whether the observed deficits are solely intrinsic to excitatory neurons. See 1.2 o Alternative Connectivity Assessments: Complementing the rabies virus tracing with electron microscopy or multi-electrode array (MEA) recordings would add structural and functional validation of the connectivity differences. See 1.3 o Extended Temporal Profiling: Monitoring network activity over a longer developmental window would clarify whether the observed deficits represent a delay or a permanent alteration in network maturation. 1.5) In vivo we were able to track the cells for up to five months post-transplantation supporting the interpretation of a permanent alteration.

• Reproducibility and Statistical Rigor: The methods and data presentation are largely clear, with adequate replication and appropriate statistical analyses. Nonetheless, a more detailed description of the experimental replicates, particularly regarding the viral tracing and in vivo transplantation studies, would enhance reproducibility. The availability of raw data and scripts for calcium imaging analysis would also further support independent verification. We thank the reviewer for these suggestions and we now provide a more detailed description of replicates. We also add the raw data.

Minor Comments • Experimental Details: Minor revisions could include clarifying the infection efficiency and expression levels of the viral constructs used in connectivity assays to rule out technical variability.

See 1.3

• Literature Context: The authors reference prior studies appropriately; however, integrating a brief discussion comparing their findings with alternative DS models (e.g., organoids or other hiPSC-derived systems) would improve contextual clarity. We thank the reviewer for this helpful suggestion. We have now added a brief discussion comparing our findings with those reported in alternative Down syndrome models, including brain organoids and other hiPSC-derived systems. This addition helps to contextualize our results within the broader field and highlights the unique strengths and limitations of our in vitro and in vivo xenograft approach. We write: 'Our findings align with and extend previous studies using alternative Down syndrome models, such as brain organoids and other hiPSC-derived systems. Organoid models have provided valuable insights into early neurodevelopmental phenotypes in DS, including altered interneuron proportions (Xu et al Cell Stem Cell 2019) but also suggest that variability across isogenic lines can overshadow subtle trisomy 21 neurodevelopmental phenotypes (Czerminski et al Front in Neurosci 2023). However, these systems often lack the structural complexity, vascularization, and long-term maturation achievable in vivo. By using a xenotransplantation model, we were able to assess the maturation and functional properties of human neurons within a physiologically relevant environment over extended time frames, offering complementary insights into DS-associated circuit dysfunction (Huo et al Stem Cell Reports 2018; Real et al., 2018).

• Presentation and Clarity: Figures are generally clear,.But the manuscript contains a minor labeling error. On page 13, the figure is erroneously labeled as "Fig6A", whereas, based on the context and corresponding data, it should be "Fig5A". I recommend that the authors correct this mistake to ensure consistency and avoid potential confusion for readers. Thank you for pointing this out. This has been corrected in the revised manuscript.

Reviewer #1 (Significance (Required)):

SECTION B - Significance • Nature and Significance of the Advance: The work offers a substantial conceptual advance by providing a mechanistic link between trisomy 21 and impaired neuronal network synchronization. Technically, the study integrates state-of-the-art imaging, electrophysiology, and transcriptomic profiling, thereby offering a multifaceted view of DS-related neural dysfunction. Clinically, the findings have the potential to inform future therapeutic strategies targeting network connectivity and ion channel function in Down syndrome.

We thank the reviewer for this very supportive comment.

• Context in the Existing Literature: The study builds on previous observations of altered network activity in DS patients and DS mouse models (e.g., altered EEG synchronization and reduced synaptic connectivity). It extends these findings to human-derived neuronal models, thus bridging a gap between clinical observations and molecular/cellular mechanisms. Relevant literature includes studies on DS neurodevelopment and the role of ion channels in synaptic maturation. • Target Audience: The reported findings will be of interest to researchers in neurodevelopmental disorders, Down syndrome, and ion channel physiology. Additionally, the study may attract the attention of those working on hiPSC-derived models of neurological diseases, as well as clinicians interested in the pathophysiology of DS. • Keywords and Field Contextualization: Keywords: Down syndrome, trisomy 21, neuronal connectivity, synchronized network activity, hiPSC-derived cortical neurons, ion channel dysregulation.

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

Summary The manuscript by Peter et al., reports on the neuronal activity and connectivity of iPSC-derived human cortical neurons from Down syndrome (DS) that is caused by caused by trisomy of the human chromosome 21 (TS21). Major points: Although the manuscript is potentially interesting, the results appear somehow preliminary and need to be corroborated by control experiments and quantifications of effects to fully sustain the conclusions. (1) The authors have not assessed the percentage of WT and TS21 cells that acquire a neuronal or glia identity in their cultures. Indeed, the origin of alterations in network activity and connectivity observed in TS21 neurons could simply derive from reduced number of neurons arising from TS21 iPSC. Alternatively, the same alteration in network activity and connectivity could derive from a multitude of other factors including deficits in neuronal development, neurite extension, or intrinsic electrophysiological properties. In the current version of the manuscript, none of these has been investigated. 2.1) We thank the reviewer for this thoughtful comment. In response, we included an in vivo characterization of cell-type proportions at the same time points where we observed network activity defects using in vivo calcium imaging (see Supplementary Fig. 6).

Previous work has identified several cellular and molecular phenotypes in human cells, postmortem tissue, and mouse models-including those mentioned by the reviewer. In this study, our focus was on investigating neural network activity, intrinsic electrophysiological properties both in vitro and in vivo, and preliminary bulk RNA sequencing. We have also independently measured cell proportions in the human fetal cortex and conducted a more extensive transcriptomic analysis of Ts21 versus control cells in a separate study (Lattke et al., under revision). We observed a reduction of RORB/FOXP1-expressing Layer 4 neurons in the human fetal cortex at midgestation, as well as increased GFAP+ cells, reduced progenitors and a non significant reduction of Cux2+ cells in late stage DS human cell transplants, along with a gene network dysregulation specifically affecting excitatory neurons (Lattke et al., under revision). Here, we provide complementary findings, demonstrating reduced excitatory neuron network connectivity in vitro and decreased neural network synchronised activity in both in vitro and in vivo models (see also 2.8). We agree with the reviewer that this could be for a number of reasons, both cell autonomous (channel expression and/or function) or non-autonomous (connectivity and/or network composition - as reflected in differences in proportions of SATB2+ neurons generated in TS21 cortical differentiations).

(2) Electrophysiological properties of TS21 and WT neurons at day 53/54 in vitro indicate an extremely immature stage of development (i.e. RMP between -36 and -27 mV with most of the cells firing a single action potential after current injection) in the utilized culture conditions: This is far from ideal for in vitro neuronal-network studies. Finally, reduced activity of HCN1 channels should be confirmed by specific recordings isolating or blocking the related current.

2.2) Thank you for this thoughtful comment. We have also conducted ex vivo electrophysiological recordings and found that the neurons exhibit relatively immature properties, consistent with the known slow developmental trajectory of human neuron cultures. In light of this and the absence of direct confirmatory evidence, we now refer to the observed reduction in HCN1 as preliminary.

Main points highlighting the preliminary character of the study. 1) In Figure 1 immunofluorescence images of the neuronal differentiation markers (Tbr1, Ctip2 and Tuj1) are showed. However, no quantification of the percentage of cells expressing these markers for WT and TS21 neurons is reported. On the other hand, simple inspection of the representative images clearly seams to indicate a difference between the two genotypes, with TS21 cultures showing lower number of cells expressing neuronal markers. This quantification should be corroborated by a similar staining for an astrocyte marker (GFAP, but not S100b since is triplicated in DS). This is an extremely important point since it is obvious that any change in the percentage of neurons (or the neuron/astrocyte ratio) in the cultures will strongly affect the resulting network activity (shown in Figure 2) and the connectivity (showed in Figure 4). Possibly, the quantification should be done at the same time points of the calcium imaging experiments.

2.3) See 2.1. We included an in vivo characterization of cell-type proportions at the same time points where we observed network activity defects using in vivo calcium imaging. (see Supplementary Fig. 6).

2) In Figure 2 the authors show some calcium imaging traces of WT and TS21 cultures at different time points. However, they again do not show any quantification of neuronal activity. A power spectra analysis is shown in Supplementary Figure 2, but only for WT cultures, while in Supplementary Figure 3 a comparison between WT and Ts21 power spectra is done, but only at the 50 day time point, while difference in synchrony are assessed at 60 days. At minimum, the author should include in main Figure 2 the quantification of the mean calcium event rate and mean event amplitude at the different time points and the power spectra analysis for both WT and TS21 cultures at the same timepoints.

2.4) We thank the reviewer for this comment. We now add the power spectra analysis in the main Figure 2 and quantification of the mean calcium burst rate and mean event amplitude in SuppFig. 4.

Of note, the synchronized neuronal activity is present in WT cultures at day 60, but totally lost at subsequent time-points (70 and 80 days). The results of this later time points are different from previous data from the same lab (Kirwan et al., 2015). How might these data be explained? It would be important to rule out any potential issues with the health of the culture that could explain the loss of neuronal activity.It would be beneficial to check cell viability at the different time points to exclude possible confounding factors ? A propidium staining or a MTT assay would strongly improve the soundness of the calcium data.

2.5) We thank the reviewer for this important observation. The difference from the findings reported in Kirwan et al., 2015 is due to the use of a different neuronal differentiation medium in the current study (BrainPhys versus N2B27). BrainPhys medium supports robust early network activity compared to N2B27 (onset before day 60 in BrainPhys, post-day 60 in N2B27), resulting in an earlier decline in synchrony at later stages (day 70-80 in BrainPhys, compared with day 90-100 in N2B27). Importantly, in our in vivo xenograft model, burst activity is sustained up to at least 5 months post-transplantation (mpt), indicating that the neurons retain the capacity for network activity over extended periods in a more physiological environment. We adapted the text accordingly.

3) In Figure 3 there is no quantification of the number and/or density of transplanted neurons for WT and TS21, but only representative images. As above, inspection of the representative images seems to show a decrease in cells labeled by the Tbr1 neuronal marker for TS21 cells. Moreover, the in vivo calcium imaging of transplanted WT and TS21 cells lacks most of the quantification normally done in calcium imaging experiments. Are the event rate and event amplitude different between WT and TS21 neurons ? The measure of neuronal synchrony by mean pixel correlation is not well explained, but it looks somehow simplistic. Neuronal synchrony can be more precisely measured by cross-correlation analysis or spike time tiling coefficients on the traces from single-neuron ROI rather than on all pixels in the field of view, as apparently was done here.

2.6) We thank the reviewer for these valuable points. We now include quantification of the number and density of transplanted neurons for both WT and Ts21 grafts in Extended Data Figure 5 (see 2.1).

Regarding the in vivo calcium imaging, we appreciate the reviewer's suggestion to include additional standard metrics. We have quantified the event rate in Real et al 2018. These analyses reveal that Ts21 neurons show a reduction in event rate.

We agree that our initial description of the synchrony analysis using mean pixel correlation was not sufficiently detailed. We have now clarified this in the Methods and Results, and we acknowledge its limitations. Importantly, we note that the reduced synchronisation is a highly consistent phenotype, observed across at least six independent donor pairs, different differentiation protocols, and both in vitro (and in two independent labs) and in vivo settings. As suggested, future studies using ROI-based approaches-such as cross-correlation or spike-time tiling coefficients-would provide a more refined characterization of synchrony at the single-neuron level (Sintes et al, in preparation). We now include this point in the discussion.

4) The results on reduced neuronal connectivity in Figure 3 look very striking. However, these results should be accompanied by control experiments to verify the number of neuronal cells and neurite extension in WT and Ts21 cultures. These two parameters could indeed strongly influence the results. As the cultures appear to grow in clusters, bright-field images and TuJ1 staining of the cultures will also greatly help to understand the degree of morphological interconnection between the clusters.

We now add Tuj1 staining in Supplementary figure 10.

5) The authors performed RNA-seq experiments on day 50 cultures. Why the authors do not show the complete differential gene expression analysis, but only a small subset of genes? A comprehensive volcano plot and the complete list of identified genes with logFC and FDR values would be helpful. If possible, comparison of the present data (particularly on KCN and HCN expression changes) with published and publicly available expression datasets of other human or human Down syndrome iPSC-derived neurons or human Down syndrome brains will greatly increase the soundness of the present findings. In addition, the gene ontology (GO) results are mentioned in the text, but are not presented. Showing the complete GO analysis for both up and downregulated genes will help the reader to better understand the RNA-seq results. Notably, the results shown in Supplementary Figure on GRIN2A and GRIN2B expression (with values of 300-700 counts versus 2000-4000 counts, respectively) clearly indicate that in both WT and TS21 cultures the NMDA developmental switch has not occurred yet at the 50 days timepoint.

We now show volcano plots in Supplementary Fig. 11.

6) The measure of hyperpolarization-activated currents shown in Figure 5 lack proper control experiments. First, the hyperpolarizing current in TS21 cells do not reach a steady-state as the controls. The two curves are therefore hard to compare. To exclude possible difference in kinetic activation, the authors should have prolonged the current injection period (1-2 seconds). Second, to ultimately prove that such currents are mediated by HCN channels in WT cells the authors should perform some control experiments with a specific HCN blocker. A good example of a suitable protocol, with also current blockers to exclude all other possible current contributions, is the one reported in Matt et al Cell. Mol. Life Sci. 68, 125-137 (2011).

2.7) We thank the reviewer for this detailed and helpful comment. We agree that to definitively identify the recorded currents as Ih, it would be necessary to isolate them pharmacologically using specific HCN channel blockers and appropriate controls, such as those described in Matt et al., Cell. Mol. Life Sci. Unfortunately, due to current constraints, we no longer have access to the animals used in this study and cannot allocate the necessary time or resources, we are unable to perform the additional experiments at this stage.

However, our goal here was to use electrophysiological recordings as an indication of altered HCN channel activity, which we then support with molecular evidence. We now emphasize this point more clearly in the revised manuscript.

7) The manuscript lacks information on the statistical analysis used. Also, the numerosity of samples is not clear. Were the dots shown in some graph technical replicates from a single neuronal induction or were all independent neuronal inductions or a mix of the two ? Please clarify.

We now clarify the numbers in the Figure legend.

8) The method section lacks important information to guarantee reproducibility. Just a few examples: • Only electrophysiology methods for slice are reported, but not for in vitro culture.

We now clarify these details in the methods.

• Details on Laminin coating is lacking. What concentration was used ? Was poly-ornithine or poly-lysine used before Laminin coating ? We now clarify these details in the methods.

• How long cells were switched to BrainPhys medium before calcium imaging ? We now clarify these details in the methods.

Minor point/typos etc.

Introduction • Page 4 line 6: in the line "Trisomy 21 in humans commonly results in a range in developmental and morphological changes in the forebrain ..." "in" could be replaced by "of". We have fixed this. • Page 5 line 2: please remove "an" before the word "another". We have fixed this. • Page 5 line 2: please replace "ecitatory" with "excitatory". We have fixed this typo.

Results • Page 10 line 25: The concept of "pixel-wise" appears for the first time in this section and could be better introduced to facilitate the understanding of the experiment. • In the "results" section, page 11 line 1 and 4, references are made to "Figure 4D" and "4F," but these figures do not appear to be present in the figure section. Upon reviewing the rest of the section, the data seem to refer to "Figure 3D" and "3E." We have fixed this. Discussion • Page 15 line 20: please replace "synchronised" with "synchronized". We have fixed this typo. • Page 16 line 11: please replace "T21" with "TS21". We have fixed this typo. Methods • Page 19 line 12: "Pens/Strep" has to be replaced by Pen/Strep. We have fixed this typo. • Page 20 line 20: "Tocris Biocience" has to be replaced by "Tocris Bioscience". We have fixed this typo. • Page 21 line 2: "Addegene" has to be replaced by "Addgene". We have fixed this typo. Figures • Figure 3: the schematic experimental design (Fig. 3A) could be enlarged to match the width of the images/graphs below. We have fixed this. • Figure 5: the reviewer suggests resizing/repositioning the graphs in Fig. 1A so that they match the width of those below. We have fixed this. • Figure S1D: In all the figures of the paper, the respective controls for the TS21 1 and TS21 2 lines are labelled as "WT1/WT2," while in these graphs, they are called "Ctrl1" and "Ctrl2." To ensure consistency throughout the paper, it is suggested to change the names in these graphs. We have fixed this. • Figure S4L: The graph is not very clear, especially regarding the significance reported at -50 pA, please modify the graphical visualization and/or add a legend in the caption. We have fixed this.

Reviewer #2 (Significance (Required)):

Nature and significance of the advance for the field. The results presented in the manuscript are potentially interesting and useful, but not completely novel (currents deregulation has already been highlighted in mouse models of Down Syndrome).

2.8) We thank the reviewer for this comment. While we agree that current deregulation has been observed in mouse models of Down syndrome, the novelty and significance of our study lie in demonstrating these alterations directly in human neurons using both in vitro and in vivo xenograft models.

This is a critical advance because the human cortex has distinct developmental and functional properties not fully recapitulated in mice. In fact, three recent studies have already highlighted significant defects mainly in excitatory neurons within the fetal human DS cortex (Vuong et al., bioRxiv, 2025; Risgaard et al., bioRxiv, 2025; Lattke et al, under revision). Our work builds directly on these observations by providing, for the first time, an electrophysiological and network-level characterization of these human-specific deficits.

Our findings thus provide translationally relevant insight that is not merely confirmatory but extends previous work by grounding it in a human cellular context.

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Referee #2

Evidence, reproducibility and clarity

Summary

The manuscript by Peter et al., reports on the neuronal activity and connectivity of iPSC-derived human cortical neurons from Down syndrome (DS) that is caused by caused by trisomy of the human chromosome 21 (TS21).

Major points:

Although the manuscript is potentially interesting, the results appear somehow preliminary and need to be corroborated by control experiments and quantifications of effects to fully sustain the conclusions.

(1) The authors have not assessed the percentage of WT and TS21 cells that acquire a neuronal or glia identity in their cultures. Indeed, the origin of alterations in network activity and connectivity observed in TS21 neurons could simply derive from reduced number of neurons arising from TS21 iPSC. Alternatively, the same alteration in network activity and connectivity could derive from a multitude of other factors including deficits in neuronal development, neurite extension, or intrinsic electrophysiological properties. In the current version of the manuscript, none of these has been investigated.

(2) Electrophysiological properties of TS21 and WT neurons at day 53/54 in vitro indicate an extremely immature stage of development (i.e. RMP between -36 and -27 mV with most of the cells firing a single action potential after current injection) in the utilized culture conditions: This is far from ideal for in vitro neuronal-network studies. Finally, reduced activity of HCN1 channels should be confirmed by specific recordings isolating or blocking the related current.

Main points highlighting the preliminary character of the study.

1) In Figure 1 immunofluorescence images of the neuronal differentiation markers (Tbr1, Ctip2 and Tuj1) are showed. However, no quantification of the percentage of cells expressing these markers for WT and TS21 neurons is reported. On the other hand, simple inspection of the representative images clearly seams to indicate a difference between the two genotypes, with TS21 cultures showing lower number of cells expressing neuronal markers. This quantification should be corroborated by a similar staining for an astrocyte marker (GFAP, but not S100b since is triplicated in DS). This is an extremely important point since it is obvious that any change in the percentage of neurons (or the neuron/astrocyte ratio) in the cultures will strongly affect the resulting network activity (shown in Figure 2) and the connectivity (showed in Figure 4). Possibly, the quantification should be done at the same time points of the calcium imaging experiments.

2) In Figure 2 the authors show some calcium imaging traces of WT and TS21 cultures at different time points. However, they again do not show any quantification of neuronal activity. A power spectra analysis is shown in Supplementary Figure 2, but only for WT cultures, while in Supplementary Figure 3 a comparison between WT and Ts21 power spectra is done, but only at the 50 day time point, while difference in synchrony are assessed at 60 days. At minimum, the author should include in main Figure 2 the quantification of the mean calcium event rate and mean event amplitude at the different time points and the power spectra analysis for both WT and TS21 cultures at the same timepoints.

Of note, the synchronized neuronal activity is present in WT cultures at day 60, but totally lost at subsequent time-points (70 and 80 days). The results of this later time points are different from previous data from the same lab (Kirwan et al., 2015). How might these data be explained? It would be important to rule out any potential issues with the health of the culture that could explain the loss of neuronal activity.It would be beneficial to check cell viability at the different time points to exclude possible confounding factors ? A propidium staining or a MTT assay would strongly improve the soundness of the calcium data.

3) In Figure 3 there is no quantification of the number and/or density of transplanted neurons for WT and TS21, but only representative images. As above, inspection of the representative images seems to show a decrease in cells labeled by the Tbr1 neuronal marker for TS21 cells. Moreover, the in vivo calcium imaging of transplanted WT and TS21 cells lacks most of the quantification normally done in calcium imaging experiments. Are the event rate and event amplitude different between WT and TS21 neurons ? The measure of neuronal synchrony by mean pixel correlation is not well explained, but it looks somehow simplistic. Neuronal synchrony can be more precisely measured by cross-correlation analysis or spike time tiling coefficients on the traces from single-neuron ROI rather than on all pixels in the field of view, as apparently was done here.

4) The results on reduced neuronal connectivity in Figure 3 look very striking. However, these results should be accompanied by control experiments to verify the number of neuronal cells and neurite extension in WT and Ts21 cultures. These two parameters could indeed strongly influence the results. As the cultures appear to grow in clusters, bright-field images and TuJ1 staining of the cultures will also greatly help to understand the degree of morphological interconnection between the clusters.

5) The authors performed RNA-seq experiments on day 50 cultures. Why the authors do not show the complete differential gene expression analysis, but only a small subset of genes? A comprehensive volcano plot and the complete list of identified genes with logFC and FDR values would be helpful. If possible, comparison of the present data (particularly on KCN and HCN expression changes) with published and publicly available expression datasets of other human or human Down syndrome iPSC-derived neurons or human Down syndrome brains will greatly increase the soundness of the present findings. In addition, the gene ontology (GO) results are mentioned in the text, but are not presented. Showing the complete GO analysis for both up and downregulated genes will help the reader to better understand the RNA-seq results. Notably, the results shown in Supplementary Figure on GRIN2A and GRIN2B expression (with values of 300-700 counts versus 2000-4000 counts, respectively) clearly indicate that in both WT and TS21 cultures the NMDA developmental switch has not occurred yet at the 50 days timepoint.

6) The measure of hyperpolarization-activated currents shown in Figure 5 lack proper control experiments. First, the hyperpolarizing current in TS21 cells do not reach a steady-state as the controls. The two curves are therefore hard to compare. To exclude possible difference in kinetic activation, the authors should have prolonged the current injection period (1-2 seconds). Second, to ultimately prove that such currents are mediated by HCN channels in WT cells the authors should perform some control experiments with a specific HCN blocker. A good example of a suitable protocol, with also current blockers to exclude all other possible current contributions, is the one reported in Matt et al Cell. Mol. Life Sci. 68, 125-137 (2011).

7) The manuscript lacks information on the statistical analysis used. Also, the numerosity of samples is not clear. Were the dots shown in some graph technical replicates from a single neuronal induction or were all independent neuronal inductions or a mix of the two ? Please clarify.

8) The method section lacks important information to guarantee reproducibility. Just a few examples: - Only electrophysiology methods for slice are reported, but not for in vitro culture. - Details on Laminin coating is lacking. What concentration was used ? Was poly-ornithine or poly-lysine used before Laminin coating ? - How long cells were switched to BrainPhys medium before calcium imaging ?

Minor point/typos etc.

Introduction

• Page 4 line 6: in the line "Trisomy 21 in humans commonly results in a range in developmental and morphological changes in the forebrain ..." "in" could be replaced by "of".
• Page 5 line 2: please remove "an" before the word "another".
• Page 5 line 2: please replace "ecitatory" with "excitatory"

Results

• Page 10 line 25: The concept of "pixel-wise" appears for the first time in this section and could be better introduced to facilitate the understanding of the experiment.
• In the "results" section, page 11 line 1 and 4, references are made to "Figure 4D" and "4F," but these figures do not appear to be present in the figure section. Upon reviewing the rest of the section, the data seem to refer to "Figure 3D" and "3E."

Discussion

• Page 15 line 20: please replace "synchronised" with "synchronized".
• Page 16 line 11: please replace "T21" with "TS21".

Methods

• Page 19 line 12: "Pens/Strep" has to be replaced by Pen/Strep.
• Page 20 line 20: "Tocris Biocience" has to be replaced by "Tocris Bioscience".
• Page 21 line 2: "Addegene" has to be replaced by "Addgene".

Figures

• Figure 3: the schematic experimental design (Fig. 3A) could be enlarged to match the width of the images/graphs below.
• Figure 5: the reviewer suggests resizing/repositioning the graphs in Fig. 1A so that they match the width of those below.
• Figure S1D: In all the figures of the paper, the respective controls for the TS21 1 and TS21 2 lines are labelled as "WT1/WT2," while in these graphs, they are called "Ctrl1" and "Ctrl2." To ensure consistency throughout the paper, it is suggested to change the names in these graphs.
• Figure S4L: The graph is not very clear, especially regarding the significance reported at -50 pA, please modify the graphical visualization and/or add a legend in the caption.

Significance

Nature and significance of the advance for the field. The results presented in the manuscript are potentially interesting and useful, but not completely novel (currents deregulation has already been highlighted in mouse models of Down Syndrome).

Work in the context of the existing literature. This work follows the line of evidence that characterizes Down Syndrome in human neurons (Huo, H.-Q. et al. Stem Cell Rep. 10, 1251-1266 (2018); Briggs, J. A. et al. Etiology. Stem Cells 31, 467-478 (2013)), both in vitro and in xenotransplanted mice, by corrborating some important findings already found in animal models (Stern, S., Segal, M. & Moses, E. EBioMedicine 2, 1048-1062 (2015); Cramer, N. P., Xu, X., F. Haydar, T. & Galdzicki, Z. Physiol. Rep. 3, e12655 (2015); Stern, S., Keren, R., Kim, Y. & Moses, E. http://biorxiv.org/lookup/doi/10.1101/467522 (2018) doi:10.1101/467522.

Audience. Scientists in the field of pre-clinical biomedical research, especially those working on neurodevelopmental disorders and iPSC-based non-animal models.

Field of expertise. In vitro electrophysiology, Neurodevelopmental disorders, Down Syndrome, ips cells.

How typewriters are making a comeback<br /> by [[CNN]] on YouTube<br /> accessed on 2026-01-14T09:01:22

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary: 

This study is an evaluation of patient variants in the kidney isoform of AE1 linked to distal renal tubular acidosis. Drawing on observations in the mouse kidney, this study extends findings to autophagy pathways in a kidney epithelial cell line. 

Strengths: 

Experimental data are convincing and nicely done.

Thank you

Weaknesses: 

Some data are lacking or not explained clearly. Mutations are not consistently evaluated throughout the study, which makes it difficult to draw meaningful conclusions.

We have revised our manuscript to clarify some earlier explanations and provided rationale for focusing on specific variants throughout the study.

Reviewer #2 (Public review):

Context and significance: 

Distal renal tubular acidosis (dRTA) can be caused by mutations in a Cl-/HCO3- exchanger (kAE1) encoded by the SLC4A1 gene. The precise mechanisms underlying the pathogenesis of the disease due to these mutations are unclear, but it is thought that loss of the renal intercalated cells (ICs) that express kAE1 and/or aberrant autophagy pathway function in the remaining ICs may contribute to the disease. Understanding how mutations in SLC4A1 affect cell physiology and cells within the kidney, a major goal of this study, is an important first step to unraveling the pathophysiology of this complex heritable kidney disease. 

Summary: 

The authors identify a number of new mutations in the SLC4A1 gene in patients with diagnosed dRTA that they use for heterologous experiments in vitro. They also use a dRTA mouse model with a different SLC4A1 mutation for experiments in mouse kidneys. Contrary to previous work that speculated dRTA was caused mainly by trafficking defects of kAE1, the authors observe that their new mutants (with the exception of Y413H, which they only use in Figure 1) traffic and localize at least partly to the basolateral membrane of polarized heterologous mIMCD3 cells, an immortalized murine collecting duct cell line. They go on to show that the remaining mutants induce abnormalities in the expression of autophagy markers and increased numbers of autophagosomes, along with an alkalinized intracellular pH. They also reported that cells expressing the mutated kAE1 had increased mitochondrial content coupled with lower rates of ATP synthesis. The authors also observed a partial rescue of the effects of kAE1 variants through artificially acidifying the intracellular pH. Taken together, this suggests a mechanism for dRTA independent of impaired kAE1 trafficking and dependent on intracellular pH changes that future studies should explore. 

Strengths: 

The authors corroborate their findings in cell culture with a well-characterized dRTA KI mouse and provide convincing quantification of their images from the in vitro and mouse experiments

Thank you  

Weaknesses: 

The data largely support the claims as stated, with some minor suggestions for improving the clarity of the work. Some of the mutants induce different strengths of effects on autophagy and the various assays than others, and it is not clear why this is from the present manuscript, given that they propose pHi and the unifying mechanism

We have modified our manuscript to discuss the various strengths of the mutants and emphasize that alteration of cytosolic pH by kAE1 variants may not be the only mechanism leading to dRTA.  

Reviewer #3 (Public review):

Summary: 

The authors have identified novel dRTA causing SLC4A1 mutations and studied the resulting kAE1 proteins to determine how they cause dRTA. Based on a previous study on mice expressing the dRTA kAE1 R607H variant, the authors hypothesize that kAE1 variants cause an increase in intracellular pH, which disrupts autophagic and degradative flux pathways. The authors clone these new kAE1 variants and study their transport function and subcellular localization in mIMCD cells. The authors show increased abundance of LC3B II in mIMCD cells expressing some of the kAE1 variants, as well as reduced autophagic flux using eGFP-RFP-LC3. These data, as well as the abundance of autophagosomes, serve as the key evidence that these kAE1 mutants disrupt autophagy. Furthermore, the authors demonstrate that decreasing the intracellular pH abrogates the expression of LC3B II in mIMCD cells expressing mutant SLC4A1. Lastly, the authors argue that mitochondrial function, and specifically ATP synthesis, is suppressed in mIMCD cells expressing dRTA variants and that mitochondria are less abundant in AICs from the kidney of R607H kAE1 mice. While the manuscript does reveal some interesting new results about novel dRTA causing kAE1 mutations, the quality of the data to support the hypothesis that these mutations cause a reduction in autophagic flux can be improved. In particular, the precise method of how the western blots and the immunofluorescence data were quantified, with included controls, would enhance the quality of the data and offer more supportive evidence of the authors' conclusions. 

Strengths: 

The authors cloned novel dRTA causing kAE1 mutants into expression vectors to study the subcellular localization and transport properties of the variants. The immunofluorescence images are generally of high quality, and the authors do well to include multiple samples for all of their western blots.

Thank you

Weaknesses: 

Inconsistent results are reported for some of the variants. For example, R295H causes intracellular alkalinization but also has no effect on intracellular pH when measured by BCECF. The authors also appear to have performed these in vitro studies on mIMCD cells that were not polarized, and therefore, the localization of kAE1 to the basolateral membrane seems unlikely, based upon images included in the manuscript. Additionally, there is no in vivo work to demonstrate that these kAE1 variants alter intracellular pH, including the R607H mouse, which is available to the authors. The western blots are of varying quality, and it is often unclear which of the bands are being quantified. For example, LAMP1 is reported at 100kDa, the authors show three bands, and it is unclear which one(s) are used to quantify protein abundance. Strikingly, the authors report a nonsensical value for their quantification of LCRB II in Figure 2, where the ratio of LCRB II to total LCRB (I + II) is greater than one. The control experiments with starvation and bafilomyocin are not supportive and significantly reduce enthusiasm for the authors' findings regarding autophagy. There are labeling errors between the manuscript and the figures, which suggest a lack of vigilance in the drafting process.

The R295H variant was identified in a dRTA patient and as such, it was important to report it. However, this is the first mutation located in the amino-terminus of the protein, which may be involved in protein-protein interactions, so other mechanisms may cause dRTA for this variant. We have therefore modified our manuscript to state that alteration of cytosolic pH may not be the only mechanism leading to dRTA. At this time, we are not able to measure cytosolic pH in vivo and hope to be able to do it in the future.

In our revised manuscript, we also show cell surface biotinylation results supporting that plasma membrane abundance of the kAE1 S525F and R589H variants is not significantly different than WT in non-polarized mIMCD3 cells (Figure 3 A&B), in line with the predominant basolateral localization of the variants in polarized cells (Figure 1C). Therefore, these two mutant proteins are not mis-trafficked in non-polarized cells.  Finally, we have clarified which bands have been used for quantification and corrected quantifications (including ratio measurements).

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

(1) R295H is recessively inherited, whereas Y413H is dominantly inherited: this is interesting and may be linked to their cellular expression and function. Is this information known for the other mutations examined in this study? 

The S25F and R589H dRTA variants have both been reported to exhibit autosomal dominant inheritance. This information is now updated in lines 146 and 158-159.

(2) R589H expression levels are evaluated in the Western blot of Figure 1, but localization and activity are not examined in Figure 2. However, R589H is included in autophagy experiments shown in later figures. Similarly, mutant R607H is the subject of several experiments further into the manuscript, but no initial analysis is provided for this variant. 

Protein abundance and localization of the R589H mutant in mIMCD3 cells have been shown in our previous publication in Supplementary Fig 5D and Supplementary Fig 2J [1]. This now indicated on lines 158-159. Our previous paper also presented a detailed study of the R607H dRTA mutant, the mouse model corresponding to the human R589H mutation. This is now indicated on lines 70, 118-119 and 180. The present study builds upon those published findings.

(3) This inconsistency is confusing, detracts from the usefulness of the study, and makes the comparative analysis of mutations incomplete. It is difficult to extrapolate from published studies in MDCK1 cells, which show different results on trafficking. 

The mIMCD3 cell line, which more closely resembles the physiology of the mouse collecting duct than MDCK cells, was selected for this study and our previous one [1]. Accordingly, the results obtained are better aligned with in vivo evidence. In contrast, differences in mutant protein expression and localization observed in other cell lines, like the MDCK cells, are likely attributable to differences in their cellular origin. 

(4) In Figure 2, could the authors explain why total LC3B is graphed for the data shown in mouse lysates, whereas the ratio of bands is analysed for cell lysates? Both sets of data show the two LC3B bands.

Total LC3B levels were significantly increased in the mutant compared to WT; however, no significant difference was observed in the lipidation ratio. For this reason, that graph is not shown in the main paper but has been included in the Supplementary Figure 1D. 

(5) In Figure 3, representative fluorescence images should be shown for all cell lines.

We have now included representative immunofluorescence images for all cell lines in Figure 3C.

(6) pH effects: Suggest that steady state pHi (Figure 3E) and rate of alkalization (Figure 1F) would be more effective together in Figure 1. The authors should show data for the effect of nigericin on cytoplasmic pH in Figure 3. If the rate of alkalinization in the mutant cells is reduced, shouldn't the intracellular steady state pH be more acidic? A cartoon depicting the transporter activity in the cell and the expected changes in pHi would be helpful. Is there a way to activate/inhibit NHE1 and rescue the effect of the mutant kAE1? It is unclear if the link between the mutant kAE1 and mitochondrial ATP production is a consequence of the intracellular pH or an indirect effect.

We opted to keep the effect of nigericin on pHi in Supplementary Fig1A given that Figure 3 already contains 11 panels. Also, in intercalated cells, the kAE1 protein physiologically exports 1 molecule of bicarbonate in exchange of 1 chloride ion import hence a reduced transport activity would result in a more alkaline intracellular pH. To clarify this point, we have included a diagram in Figure 1E as suggested. However, to calculate the rate of intracellular alkalinisation, the transporter is functioning in the opposite direction, i.e. extruding chloride and importing bicarbonate (see methods protocol for transport assay). Therefore, in this assay (Figure 1G), a defective chloride/bicarbonate activity results in a reduced rate of intracellular alkalinisation rate. This is now explained on lines 169-172.

Disruption of NHE1 function would impair sodium homeostasis and as such, potentially affect the activity of other proteins associated with acid-base balance and autophagy in collecting duct cells. Therefore, any resulting effects may not be confidently attributed specifically to the mutant kAE1. With nigericin, we aimed to alter pHi while affecting the least possible other ion concentration. Due to space considerations, Figure 1 has been reorganised to include the rate of alkalinisation and pHi (panels F and G). 

Reviewer #2 (Recommendations for the authors):

(1) The authors could improve the readability of this manuscript for a general audience by clarifying and summarizing the respective phenotype(s)/effect(s) of the different mutants in some kind of table in the main figures. It is hard to keep track of the different disease mutants alongside the KI mouse mutations, as the text frequently discusses multiple mutants at a time. 

As requested, we added two tables (Supplementary Tables 1 & 2) in Supplementary files summarizing the data obtained in this study. We hope this will help the readership to keep track of each variant’s phenotype.

(2) The subtitle of the results section of Figure 2 should be reworded to reflect that  whole kidney lysates are used for the KI mice and not the other mutants.

As requested, the title in the Results section has been modified (lines 178-179).

(3) More discussion of why the different mutants cause different strengths of phenotypes should be included.

Different variants induce different degree of functional defects as seen in Figure 1F & G. The kAE1 R295H, the only amino acid substitution in the amino-terminal cytosol causing dRTA, does not affect the transporter’s function or cells’ pHi. Therefore, this variant may cause dRTA via a different pathway than transport-defective S525F or partially inactive R589H variants that both affect pHi. Our study does not exclude that dRTA may be caused by other defects than pHi alterations, including defective proteinprotein interactions. This discussion is now included in the manuscript on lines 386-391.

Reviewer #3 (Recommendations for the authors):

In general, I found the subject matter of this manuscript interesting and of value to the scientific community. The interpretation of the data and how much it supports the conclusion that "kAE1 variants increases pHi which alters mitochondrial function and leads to reduced cellular energy levels that eventually attenuate energy-dependent autophagic pathways" is largely incomplete. There are significant concerns about the quantification of Western blot data. Additionally, including the R607H variant in the in vitro experiments would improve the interpretation and extrapolation of in vitro data to the kidney.

We apologize for the confusion with R589H and R607H variants. The R607H mutant is the murine ortholog to the human R589H dRTA variation. To clarify this, we have added this information on line 180, in addition to lines 118-119 and line 70.

Suggestions:

(1) Can an anion replacement experiment be performed in the mIMCD cells (no Cl or no HCO3) to determine that bicarbonate transport through AE1 is responsible for the reduced ATP rates in Figure 5? Inclusion of WT +dox control would be helpful to convince the reader of the effects.

Because Seahorse real-time cell metabolism ATP rates measurements require specific and patented buffers with un-specified compositions, it was not possible to modify the Cl⁻ or HCO₃⁻ content during the ATP measurement assay. All cell lines, including empty vector cells (EV) were treated with doxycycline; thus, WT + dox was already included. The empty vector cell line treated with doxycycline allowed the exclusion of specific effects of doxycycline on mitochondrial activity as a control. This is now clarified in Figure 5 legend, lines 655-656.

(2) Can the authors measure pHi in fresh kidney sections from the R607H mouse?

Unfortunately, we are not currently able to measure pHi in fresh kidney sections and although we recognize it would benefit greatly to our study, establishing a new collaboration to perform this measurement would significantly delay the publication of this work; therefore, these results will not be available for the present manuscript. 

(3) Does pH 7.0 media have any effect on autophagy, as shown in Figure 3? Why was pH 6.6 selected?

The idea was to artificially acidify pHi in mutant cell lines (that have a steady state alkaline pHi) and assess whether this acidification corrects autophagy defects. We first determined that incubation in cell culture medium at pH 6.6 with 0.033 µM nigericin (final potassium concentration: 168 mM) for 2 hours provided optimal conditions, i.e. ensuring cell viability over the 2-hour period while effectively lowering intracellular pH to 6.9, as demonstrated in Supplementary Figure 1A-C.

(4) In vitro experiments should be performed on polarized cells with kAE1 properly inserted in the basolateral membrane. Experiments on subconfluent, non-polarized cells do not support the hypothesis that transport functions of AE1 initiate the cascade of events attributed to these SLC4A1 mutations.

To address this point, we have performed cell surface biotinylations on 70-80 % confluent mIMCD3 cells expressing kAE1 WT, S525F or R589H mutants and show that cell surface abundance of the mutants is not significantly different from the WT protein. This is now shown in Figure 3 A&B. As cell surface biotinylation provides a more quantitative assessment of protein cell surface abundance, we have removed the immunofluorescence images from non-polarised cells and replaced them with representative immunoblots from a cell surface biotinylation assay.

Concerns:

(1) No information about the B1 ATPase antibody used.

Now provided in Supplementary Material, ATP6V1B1 Antibody from Bicell cat#20901.

(2) No actin band in Figure 1E (as prepared).

Actin bands are provided for each blot in Figure 1D.

(3) Figures 1E and 1F are labelled wrong in the figure versus the results section. 

Thank you for letting us know, this is now corrected.

(4) The cortical sections shown in Figure 4 for the KI/KI do not appear to have the morphology of a CCD. The authors may want to consider including glomeruli to convince the reader of the localization of the tubules. Same concern with Figure 5G and I. The WT image in 5G does not have the morphology of a CCD. Principal cells should be predominant, and ICs should be dispersed.

Both figures 4 and 5 have been updated with images showing glomeruli (light blue “G” on figure) with neighbour and dispersed IC staining.

(5) The quantification of LAMP1 in Figure 4 is unclear. How did the authors determine the boundary of AICs, and how did they calculate the volume of lysosomes? If a zstack was used, how are the authors sure that their 10um section includes the entire AIC?

The quantification of LAMP1 is detailed under “Image analysis”, then “Volocity” sections in Supplementary Material. The boundary of A-IC was manually detected in Volocity based on the presence of the H⁺-ATPase before Volocity analysis for lysosomal volume as described in the Methods.

The 10 micron sections are expected to include full AIC as well as partial AIC, but the frequency of these events should be the same between WT and variants’ sections, therefore they were all included in the analysis if cells displayed H⁺-ATPase signal. 

(6) Figure 5: There is no description of how ATP rates are calculated from the provided traces.

We used Agilent Seahorse XF ATP rate assay kit for this experiment. In this assay, the total ATP rate is the sum of ATP production rate from both glycolysis and oxidative phosphorylation. Glycolysis releases protons in a 1:1 ratio with ATP hence the glycolytic ATP rate is calculated from the glycolytic proton efflux rate (glycoPER). GlycoPER is determined by subtracting respiration linked proton efflux from total proton efflux by inhibiting complex I and III. This information is now added to Supplementary Material, in the “Metabolic Flux analysis” section.

(7) Figure labels in Figure 5 are wrong. It seems 5H (as presented) should actually be labeled 5G. In 5H (G?), why did some cells not have any TOM20 pixel intensity for S525F and R589H variants?

Confocal image acquisition in this experiment was kept under the same settings to allow comparison between samples. Therefore, some cells show dimer fluorescence than others. From the figure 5 panels, all cells showed TOM 20 pixel intensity. Figure 5H panel has been relabelled Figure 5G.

(8) In Figure 2, the summary graphs show analysis of more samples than are visible on the included western blots. What is the rationale for this? Why does S525F have 9 samples in BafA1 while R295H only has 3 (2H)? Yet, R295H has 6 samples in 2I. In 2D, S525F has at least 9 samples. Explain.

Figure 2A-C shows representative immunoblots, among several ones independently conducted. Therefore, the final number of samples is higher than showed on Figure 2. This is now indicated in Figure 2 legend, line 603. It became clear quite early in our study that the recessive kAE1 R295H variant does not behave similarly to the other variants studied, maybe because it affects the cytosolic domain, so we did not perform as many replicates for this variant as we did for the others. However, we felt it was valuable to the research community to report the characterization of this variant and decided to keep it in our study. 

(9) In general, the actin loading does not appear to be equal between samples. And some figures show the same actin blot twice (2A, C) while some show independent actin bands for LC3B and p62. Equal loading seems a fairly significant control, considering the importance of quantification in the figures.

In addition to performing protein assays, we systematically conduct immunoblot with anti-b-actin antibody to control for loading variability. When possible, two or three proteins, including actin, are detected on the same blot, when molecular weight differ enough. This sometimes results in b-actin being used as a loading control for two different proteins, as seen on Figure 2A and 2C. This is now indicated on lines 605606.

(10) In the Supplemental Figure 2, which band is being quantified for mature CTSD at 33kDa? Same for intermediate CTSD. The quantification of V-ATPase seems questionable based on the actin variance shown in the blot. Surely the ratio of the fourth sample is greater than 1.

Supplementary Figure 2 has been updated to include arrows indicating which band was selected for the quantification. After verifying the measurements of band intensities from “Image Lab” quantification software, we confirm the results, including that fourth KI/KI sample has a ratio of 0.78 (Adj Total Band Vol (Int), lanes 10). Screen shots of quantifications are attached below.

Author response image 1.

Author response image 2.

(11) Why are the experiments performed on non-confluent IMCD cells? Figure 1D shows good basolateral localization of AE1, yet the other experiments in the manuscript appear to use IMCD cells in low confluent states, without proper localization of AE1. Figure 3A shows AE1 dispersed throughout the cytoplasm. Why have the authors decided to study the effects of an anion exchanger without it being properly localized to the basolateral membrane? Shouldn't all experiments be performed in polarized IMCDs? If AE1 isnt properly in the membrane, and the cells do not have defined apico-basolateral polarity, then what role can AE1-mediated intracellular pH change have on the results of the experiments? Were the pHi experiments in 3E performed on polarized cells? Or even 1F?

To address this point, we have performed cell surface biotinylations on 70-80 % confluent mIMCD3 cells expressing kAE1 WT, S525F or R589H mutants and show that cell surface abundance of the mutants is not significantly different from the WT protein. This is now shown in Figure 3A & B. As it provides a more quantitative assessment of protein cell surface abundance, we have removed the immunofluorescence images from non-polarised cells and replaced them with a representative immunoblot from a cell surface biotinylation assay.

(12) As mentioned in the public comments, how is the ratio A/(A+B) greater than 1? With A and B > 0. In Figure 3, the data is reasonable, but in Figure 2, the data is simply impossible. What is the explanation for this phenomenon? Why was this presentation of data approved? Is it supposedly a fold of WT, like 2K and 2L? Is the reader also to believe that total LC3B is 2-fold greater in KI/KI mice, as shown in 2K? My eyes, though not densitometry equipment, cannot confirm this. The actin bands are not equal. Yet again, there are 4 lanes of KI/KI mice, but the quantification shows 5 samples.

The ratios in figure 2D, 2F, 2H and 2L have been re-calculated and corrected. As indicated above, immunoblots are representative and quantification of additional blots has been included in the graphs.

(12) Spelling error Figure 4B: cels.

Corrected

References 

(1) Mumtaz, R. et al. Intercalated Cell Depletion and Vacuolar H+-ATPase Mistargeting in an Ae1 R607H Knockin Model. Journal of the American Society of Nephrology 28, 1507–1520 (2017).

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Lahtinen et al. evaluated the association between polygenic scores and mortality. This question has been intensely studied (Sakaue 2020 Nature Medicine, Jukarainen 2022 Nature Medicine, Argentieri 2025 Nature Medicine), where most studies use PRS as an instrument to attribute death to different causes. The presented study focuses on polygenic scores of non-fatal outcomes and separates the cause of death into "external" and "internal". The majority of the results are descriptive, and the data doesn't have the power to distinguish effect sizes of the interesting comparisons: (1) differences between external vs. internal (2) differences between PGI effect and measured phenotype. I have two main comments:

(1) The authors should clarify whether the p-value reported in the text will remain significant after multiple testing adjustment. Some of the large effects might be significant; for example, Figure 2C

We have now added Benjamini-Hochberg multiple-testing adjusted p-values in the text each time we present nominal p-values. Additionally, supplementary tables S5 and S6 provide multiple-adjusted p-values for all analysed PGIs.

Although this was not always the case, many comparisons remained significant after multiple testing adjustments, especially in Figure 2C that the reviewer commented on. In the revised version, we have placed more emphasis on describing these HRs that have low p-values after multiple-test adjustment. The revised text for Figure 2C in the Results section now reads:

Panel C analyses mortality in three age-specific follow-up periods. The PGIs were more predictive of death in younger age groups, although the difference between the 25–64 and 65–79 age groups was small, except for the PGI of ADHD (HR=1.14, 95% CI 1.08; 1.21 for 25–64-year-olds; HR=1.04, 95% CI 1.00; 1.08 for 65–79-year-olds; p=0.008 for difference, p=0.27 after multiple-testing adjustment). PGIs predicted death only negligibly among those aged 80+, and the largest differences between the age groups 25–64 and 80+ were for PGIs of self-rated health (HR 0.87, 95% CI 0.82; 0.93 for 25–64-year-olds, HR 1.00, 95% CI 0.94; 1.04 for 80+ year-olds, p=2*10^-4 for difference, p=0.006 after multiple-testing adjustment), ADHD (HR 1.14, 95% CI 1.08; 1.21 for 25–64-year-olds, HR 0.99, 95% CI 0.95; 1.03 for 80+ year-olds, p=7*10^-4 for difference, p=0.012 after multiple-testing adjustment) and depressive symptoms (HR 1.12, 95% CI 1.06; 1.18 for 25–64-year-olds, HR 1.00, 95% CI 0.96; 1.04 for 80+ year-olds, p=0.002 for difference, p=0.032 after multiple-testing adjustment). Additionally, the difference in HRs between these age groups achieved significance after multiple testing adjustment at the conventional 5% level for PGIs of cigarettes per day, educational attainment, and ever smoking.

We have also included the recent study by Argentieri et al. (2025) in the literature review, which was missing from our previous version. We appreciate the reference. Other references mentioned were already included in the previous version of the manuscript.

(note that the small prediction accuracy of PGI in older age groups has been extensively studied, see Jiang, Holmes, and McVean, 2021, PLoS Genetics).

We would like to thank the reviewer for suggesting the relevant reference by Jiang et al. We have now expanded on the discussion of age-specific differences in the discussion section and included this reference.

(2) The authors might check if PGI+Phenotype has improved performance over Phenotype only. This is similar to Model 2 in Table 1, but slightly different.

The reviewer raises an interesting angle to approach the analysis. We have now added an analysis assessing the information criteria and the significance of improvement between nested models in Supplementary table S8. All the tested PGI+phenotype models show improvement over the phenotype-only model that is statistically significant at all conventional levels when tested by likelihood-ratio tests between nested models . Additionally,  improvement was found when using Akaike and Bayesian (Schwarz) information criteria (albeit sometimes modest in size). We have added a passage in the results section briefly summarising this analysis:

Supplementary table S8 presents information criteria and significance tests on corresponding models. Models with PGI+phenotype (Models 2a–f) showed improvement over models with the phenotype only (Models 1a, 1c, 1e, 1g, 1i, 1k, with a p=0.0006 or lower) in terms of both Akaike information criterion (AIC) as well as Bayesian (Schwarz) information criterion (BIC) with a p=0.0006 or lower in all comparisons. The full Model 4 again showed improvement over the model with all PGIs jointly (Model 3b, with a p=0.0002 or p=0.00002, depending on continuous/categorical phenotype measurement), which had a lower AIC but not BIC.

Reviewer #2 (Public review): 

Summary:

This study provides a comprehensive evaluation of the association between polygenic indices (PGIs) for 35 lifestyle and behavioral traits and all-cause mortality, using data from Finnish population- and family-based cohorts. The analysis was stratified by sex, cause of death (natural vs. external), age at death, and participants' educational attainment. Additional analyses focused on the six most predictive PGIs, examining their independent associations after mutual adjustment and adjustment for corresponding directly measured baseline risk factors.

Strengths:

Large sample size with long-term follow-up.

Use of both population- and family-based analytical approaches to evaluate associations.

Weaknesses:

It is unclear whether the PGIs used for each trait represent the most current or optimal versions based on the latest GWAS data.

To our reading, this comment is closely related to the “recommendations for the author” number 3 by reviewer 2, and we thus address them together. 

If the Finnish data used in this study also contributed to the development of some of the PGIs, there is a risk of overestimating their associations with mortality due to overfitting or "double-dipping." Similar inflation of effect sizes has been observed in studies using the UK Biobank, which is widely used for PGI construction.

To our reading, this comment is closely related to the “recommendations for the author” 4 by reviewer 2, and we thus address them together.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Specific comments:

(1) Cited reference 1 also investigated the PRS association with life span; cited reference 8 explains PRS association with healthy lifespan. Can authors be clearer about what is new in the context of these references? Specifically, what are the PGIs studied here that were not analyzed in the cited analyses?

Although some previous studies on the topic do exist, our analysis arguably has novelty in touching upon several unstudied or scarcely studied themes. These include:

A set of PGIs focusing on social, psychological, and behavioural phenotypes or PGIs for typically non-fatal health conditions.

An assessment of direct genetic effects/ confounding with a within-sibship design.

An assessment of potential heterogeneous effects by several socio-demographic characteristics.

An analysis of external causes of deaths (which can be hypothesised to be particularly relevant here, given the choice of our PGIs not focusing directly on typical causes of death).

A detailed assessment of the interplay of the most predictive PGIs with their corresponding phenotypes.

We have substantially revised the Introduction section focusing on making these novel contributions more explicit.

(2) In the Methods section, it is not very clear why the authors specifically study the "within-sibship" samples. Is this for avoiding nurturing effects from parental genotypes or for controlling assortative mating? The authors should clarify the rationale behind the design.

The substance-related rationale behind this approach was briefly discussed in the Introduction section while in the Methods section, we focused more on the technical description of our analyses. However, it is certainly worthwhile to clarify to the reader why within-sibship methods have been used. The revised passage in the methods section now states:

“In addition to this population sample, we used a within-sibship analysis sample to assess the extent of direct and indirect genetic associations captured by the PGIs, as discussed in the introduction.”

(3) Residual correlations of PGIs were no more than 0.050..." As a minor comment, since PGIs is a noisy variable, the correlation would be low; however, I don't think there are better ways to evaluate Cox assumptions, and in many cases, this assumption is not correct for strong predictors.

Yes, these points are true. Overall, it is often implausible that empirical distributions exactly match distributional assumptions in statistical models. For example, it may not be realistic to expect that the mortality hazards across categories of independent variables stay exactly proportional during long mortality-follow-ups; some deviations from constant proportions are almost inevitable. However, there are reasonable grounds to argue that in case of moderate violations of the proportional hazards assumption, the estimates still remain interpretable for practical uses. They can be read as approximating average relative hazards over the study period (for discussion, see pages 42–47 in Allison P. 2014. Event history and survival analysis: Regression for longitudinal event data (second edition). Thousand Oaks: SAGE).

(4) "PGI of ADHD (HR=1.08 95%CI 1.04;1.11 among men; HR=1.01 95%CI 0.97;1.05 among women; p=0.012 for difference)." Is this difference significant after multiple testing correction?

We have presented multiple-testing adjusted p-values together with nominal ones in this and in all other instances where they are mentioned in the text. Additionally, Supplementary tables S5–S6 present multiple-adjusted p-values for each PGIs studied.

(5) "Panel D displays that most PGIs had stronger associations with external (accidents, violent, suicide, and alcohol related deaths) than natural causes of death." Similar to the comment above, are there any results that are significantly different between internal and external?

We have added the p-values of those variables that had larger differences in the revised text. Quoting from the revised article: “The HR differences between external and natural causes of death were nominally significant at the conventional 5% level for cannabis use (p=0.016), drinks per week (p=0.028), left out of social activity (p=0.029), ADHD (p=0.031), BMI (p=0.035) and height (p=0.049), but none of these differences remained significant after adjusting for 35 multiple tests. “

(6) Table 1: The effect of the phenotype is stronger than the PGI; this is expected as PGI is a weak predictor and can be considered as "noised" measurement of true genetic value (Becker 2021 Nature Human behavior). Is there a way to adjust for the impact of noise in PGI at tagging genetic value and compare if the PGI effect is different from the phenotype effect?

PGIs are certainly imperfect measures that contain a lot of noise. However, extracting new information from what is unknown is an extremely demanding exercise, and still further complicated for example, by that we do not know the exact benchmark of total genetic effect we should be aiming at. Different methods of heritability estimation, for instance, often give dramatically differing results – for reasons that are still up to scrutiny.

We are thus not familiar with a method that could achieve satisfactory answer for this challenging task.

Reviewer #2 (Recommendations for the authors):

(3) Justification and Selection of PGIs:

For several traits, such as BMI, multiple polygenic indices (PGIs) are currently available. The criteria used to select specific PGIs for this study are not clearly described. A more systematic and reproducible approach-for example, leveraging metadata from the PGS Catalog-could strengthen the justification for PGI selection and enhance the study's generalizability.

There are numerous PGIs developed in the extensive GWAS literature, but a finite set of PGIs always needs to be chosen for any analysis. The rationale behind our decision to include every PGI from the repository of Becker et al. 2021 (full reference in the manuscript, see also https://www.thessgac.org/pgi-repository) that was available for the Finnish data (including the possibility to exclude overlapping samples, see our response to the next comment for more discussion) was to provide rigorous analysis by limiting the researchers degrees of freedom in arbitrarily choosing PGIs. Although it would have been tempting to not use some PGIs that were not expected to substantially correlate with mortality, we believe that our conservative strategy increases the credibility of the reported p-values, particularly the multiple adjustment should now work as intended. 

We also mention now this rationale when discussing the chosen PGIs in the methods section: “As the independent variables of main interest, we used 35 different PGIs in the Polygenic Index repository by Becker et al., which were mainly based on GWASes using UK Biobank and 23andMe, Inc. data samples, but also other data collections. They were tailored for the Finnish data, i.e., excluding overlapping individuals between the original GWAS and our analysis and performing linkage-disequilibrium adjustment. We used every single-trait PGI defined in the repository (except for subjective well-being, for which we were unable to obtain a meta-analysis version that excluded the overlapping samples). By limiting the researchers’ freedom in selecting the measures, this conservative strategy should increase the validity of our estimates, particularly with regards to multiple-testing adjusted p-values.”

(4) Overlap Between PGI Training Data and Study Sample:

The authors should describe any overlap between the data used to develop the PGIs and the current study sample. If such overlap exists, it may lead to overestimation of effect sizes due to "double-dipping." A discussion of this issue and its potential implications is warranted, as similar concerns have been raised in studies using UK Biobank data.

This is, fortunately, not a concern of our analysis. Overlapping samples were excluded in creating the PGIs that we used. We have now described this more clearly in the revised methods section.

(1) Clarify the Methodology for Family-Based Cox Analysis:

It is unclear what specific method was used to perform Cox regression in the family-based analysis. Please provide additional methodological details. ”

We have described the method further and added an additional reference in the revision. The text now stands:

“We compared these models to the corresponding within-sibship models, using the sibship identifier as the strata variable. This method employs a sibship-specific (instead of a whole-sample-wide baseline hazard in the population models) baseline hazard, and corresponds to a fixed-effects model in some other regression frameworks (e.g., linear model with sibship-specific intercepts)”

(2) Clarify Timing of Measured Risk Factors Relative to Follow-Up:

The main text should provide more detailed information regarding the timing of data collection for directly measured risk factors. Specifically, it should be clarified whether the measurements used correspond to the first available data for each individual after the start of follow-up, or if a different criterion was applied.

BMI, self-rated health, alcohol consumption and smoking status were measured at the baseline survey of each dataset. Education was registered as the highest completed degree up to the end of 2019. Depression was a composite of survey self-report (at the time of the baseline survey), as well as depression-related medicine purchases and hospitalizations over a two-year period before the start of the individual’s follow-up.

We have added more comprehensive information on the measurement of the phenotypes of interest in Supplementary table 2, including the timing of the measurement.

Est-ce que ce paragraphe n'aurait pas plus sa place après celui sur les étapes du travail ? Si on est pris, je pourrai participer à la rédaction de cette partie (surtout que les bibli ont pas toujours les infrastructures, selon les institutions, c'est parfois les presses, parfois les bibli, parfois la DSI... et ça renforce justement le côté "qui est le responsable" ?)

ça me semble très clair ! :)

Pour ma part, le 1er débrancher me semble très pertinent, et je proposerais "a disparu" ici

pour éviter la répétition et jouer sur la métaphore : "jeter au pilon" ?

EZt tegyük át légyszi . A vizsgálatok blokk alá a DTT tábla fölé

Na szóval helyesen a mondat : Alletétkezelőtől, elszámolóháztól bejövő (MT567) szátuszüzenetek egy un. forgatótábla szerint kerülnek kiküldésre vagy megállításra ...

Kerüljön törlésre a fázis a CIB_nál

Ide nem kell a, b. csak sime bullet pontok legyenek

Author response:

Point-by-point description of the revisions:

Reviewer #1 (Evidence, reproducibility and clarity):

Summary

In this article, the authors used the synthetic TALE DNA binding proteins, tagged with YFP, which were designed to target five specific repeat elements in Trypanosoma brucei genome, including centromere and telomeres-associated repeats and those of a transposon element. This is in order to detect and identified, using YFP-pulldown, specific proteins that bind to these repetitive sequences in T. brucei chromatin. Validation of the approach was done using a TALE protein designed to target the telomere repeat (TelR-TALE) that detected many of the proteins that were previously implicated with telomeric functions. A TALE protein designed to target the 70 bp repeats that reside adjacent to the VSG genes (70R-TALE) detected proteins that function in DNA repair and the protein designed to target the 177 bp repeat arrays (177R-TALE) identified kinetochore proteins associated T. brucei mega base chromosomes, as well as in intermediate and mini-chromosomes, which imply that kinetochore assembly and segregation mechanisms are similar in all T. brucei chromosome.

Major comments:

Are the key conclusions convincing?

The authors reported that they have successfully used TALE-based affinity selection of proteinassociated with repetitive sequences in the T. brucei genome. They claimed that this study has provided new information regarding the relevance of the repetitive region in the genome to chromosome integrity, telomere biology, chromosomal segregation and immune evasion strategies. These conclusions are based on high-quality research, and it is, basically, merits publication, provided that some major concerns, raised below, will be addressed before acceptance for publication.

(1) The authors used TALE-YFP approach to examine the proteome associated with five different repetitive regions of the T. brucei genome and confirmed the binding of TALE-YFP with Chip-seq analyses. Ultimately, they got the list of proteins that bound to synthetic proteins, by affinity purification and LS-MS analysis and concluded that these proteins bind to different repetitive regions of the genome. There are two control proteins, one is TRF-YFP and the other KKT2-YFP, used to confirm the interactions. However, there are no experiment that confirms that the analysis gives some insight into the role of any putative or new protein in telomere biology, VSG gene regulation or chromosomal segregation. The proteins, which have already been reported by other studies, are mentioned. Although the author discovered many proteins in these repetitive regions, their role is yet unknown. It is recommended to take one or more of the new putative proteins from the repetitive elements and show whether or not they (1) bind directly to the specific repetitive sequence (e.g., by EMSA); (2) it is recommended that the authors will knockdown of one or a small sample of the new discovered proteins, which may shed light on their function at the repetitive region, as a proof of concept.

The main request from Referee 1 is for individual evaluation of protein-DNA interaction for a few candidates identified in our TALE-YFP affinity purifications, particularly using EMSA to identify binding to the DNA repeats used for the TALE selection. In our opinion, such an approach would not actually provide the validation anticipated by the reviewer. The power of TALE-YFP affinity selection is that it enriches for protein complexes that associate with the chromatin that coats the target DNA repetitive elements rather than only identifying individual proteins or components of a complex that directly bind to DNA assembled in chromatin.

The referee suggests we express recombinant proteins and perform EMSA for selected candidates, but many of the identified proteins are unlikely to directly bind to DNA – they are more likely to associate with a combination of features present in DNA and/or chromatin (e.g. specific histone variants or histone post-translational modifications). Of course, a positive result would provide some validation but only IF the tested protein can bind DNA in isolation – thus, a negative result would be uninformative.

In fact, our finding that KKT proteins are enriched using the 177R-TALE (minichromosome repeat sequence) identifies components of the trypanosome kinetochore known (KKT2) or predicted (KKT3) to directly bind DNA (Marciano et al., 2021; PMID: 34081090), and likewise the TelR-TALE identifies the TRF component that is known to directly associate with telomeric (TTAGGG)n repeats (Reis et al 2018; PMID: 29385523). This provides reassurance on the specificity of the selection, as does the lack of cross selectivity between different TALEs used (see later point 3 below). The enrichment of the respective DNA repeats quantitated in Figure 2B (originally Figure S1) also provides strong evidence for TALE selectivity.

It is very likely that most of the components enriched on the repetitive elements targeted by our TALE-YFP proteins do not bind repetitive DNA directly. The TRF telomere binding protein is an exception – but it is the only obvious DNA binding protein amongst the many proteins identified as being enriched in our TelR-TALE-YFP and TRF-YFP affinity selections.

The referee also suggests that follow up experiments using knockdown of the identified proteins found to be enriched on repetitive DNA elements would be informative. In our opinion, this manuscript presents the development of a new methodology previously not applied to trypanosomes, and referee 2 highlights the value of this methodological development which will be relevant for a large community of kinetoplastid researchers. In-depth follow-up analyses would be beyond the scope of this current study but of course will be pursued in future. To be meaningful such knockdown analyses would need to be comprehensive in terms of their phenotypic characterisation (e.g. quantitative effects on chromosome biology and cell cycle progression, rates and mechanism of recombination underlying antigenic variation, etc) – simple RNAi knockdowns would provide information on fitness but little more. This information is already publicly available from genome-wide RNAi screens (www.tritrypDB.org), with further information on protein location available from the genome-wide protein localisation resource (Tryptag.org). Hence basic information is available on all targets selected by the TALEs after RNAi knock down but in-depth follow-up functional analysis of several proteins would require specific targeted assays beyond the scope of this study.

(2) NonR-TALE-YFP does not have a binding site in the genome, but YFP protein should still be expressed by T. brucei clones with NLS. The authors have to explain why there is no signal detected in the nucleus, while a prominent signal was detected near kDNA (see Fig.2). Why is the expression of YFP in NonR-TALE almost not shown compared to other TALE clones?

The NonR-TALE-YFP immunolocalisation signal indeed is apparently located close to the kDNA and away from the nucleus. We are not sure why this is so, but the construct is sequence validated and correct. However, we note that artefactual localisation of proteins fused to a globular eGFP tag, compared to a short linear epitope V5 tag, near to the kinetoplast has been previously reported (Pyrih et al, 2023; PMID: 37669165).

The expression of NonR-TALE-YFP is shown in Supplementary Fig. S2 in comparison to other TALE proteins. Although it is evident that NonR-TALE-YFP is expressed at lower levels than other TALEs (the different TALEs have different expression levels), it is likely that in each case the TALE proteins would be in relative excess.

It is possible that the absence of a target sequence for the NonR-TALE-YFP in the nucleus affects its stability and cellular location. Understanding these differences is tangential to the aim of this study.

However, importantly, NonR-TALE-YFP is not the only control for used for specificity in our affinity purifications. Instead, the lack of cross-selection of the same proteins by different TALEs (e.g. TelR-TALE-YFP, 177R-TALE-YFP) and the lack of enrichment of any proteins of interest by the well expressed ingiR-TALE-YFP or 147R-TALE-YFP proteins each provide strong evidence for the specificity of the selection using TALEs, as does the enrichment of similar protein sets following affinity purification of the TelR-TALE-YFP and TRF-YFP proteins which both bind telomeric (TTAGGG)n repeats. Moreover, control affinity purifications to assess background were performed using cells that completely lack an expressed YFP protein which further support specificity (Figure 6).

We have added text to highlight these important points in the revised manuscript:

Page 8:

“However, the expression level of NonR-TALE-YFP was lower than other TALE-YFP proteins; this may relate to the lack of DNA binding sites for NonR-TALE-YFP in the nucleus.”

Page 8:

“NonR-TALE-YFP displayed a diffuse nuclear and cytoplasmic signal; unexpectedly the cytoplasmic signal appeared to be in the vicinity the kDNA of the kinetoplast (mitochrondria). We note that artefactual localisation of some proteins fused to an eGFP tag has previously been observed in T. brucei (Pyrih et al, 2023).”

Page 10:

Moreover, a similar set of enriched proteins was identified in TelR-TALE-YFP affinity purifications whether compared with cells expressing no YFP fusion protein (No-YFP), the NonR-TALE-YFP or the ingiR-TALE-YFP as controls (Fig. S7B, S8A; Tables S3, S4). Thus, the most enriched proteins are specific to TelR-TALE-YFP-associated chromatin rather than to the TALE-YFP synthetic protein module or other chromatin.

(3) As a proof of concept, the author showed that the TALE method determined the same interacting partners enrichment in TelR-TALE as compared to TRF-YFP. And they show the same interacting partners for other TALE proteins, whether compared with WT cells or with the NonR-TALE parasites. It may be because NonR-TALE parasites have almost no (or very little) YFP expression (see Fig. S3) as compared to other TALE clones and the TRF-YFP clone. To address this concern, there should be a control included, with proper YFP expression.

See response to point 2, but we reiterate that the ingi-TALE -YFP and 147R-TALE-YFP proteins are well expressed (western original Fig. S3 now Fig. S2) but few proteins are detected as being enriched or correspond to those enriched in TelR-TALE-YFP or TRF-YFP affinity purifications (see Fig. S9). Therefore, the ingi-TALE -YFP and 147R-TALE-YFP proteins provide good additional negative controls for specificity as requested. To further reassure the referee we have also included additional volcano plots which compare TelR-TALE-YFP, 70R-TALE-YFP or 177R-TALE-YFP to the ingiR-TALE-YFP affinity selection (new Figure S8). As with No-YFP or NonR-TALE-YFP controls, the use of ingiR-TALE-YFP as a negative control demonstrates that known telomere associated proteins are enriched in TelR-TALE-YFP affinity purification, RPA subunits enriched with 70R-TALE-YFP and Kinetochore KKT poroteins enriched with 177RTALE-YFP. These analyses demonstrate specificity in the proteins enriched following affinity purification of our different TALE-YFPs and provide support to strengthen our original findings.

We now refer to use of No-YFP, NonR-TALE-YFP, and ingiR-TALE -YFP as controls for comparison to TelR-TALE-YFP, 70R-TALE-YFP or 177R-TALE-YFP in several places:

Page10:

“Moreover, a similar set of enriched proteins was identified in TelR-TALE-YFP affinity purifications whether compared with cells expressing no YFP fusion protein (No-YFP), the NonR-TALE-YFP or the ingiR-TALE-YFP as controls (Fig. S7B, S8A; Tables S3, S4).”

Page 11:

“Thus, the nuclear ingiR-TALE-YFP provides an additional chromatin-associated negative control for affinity purifications with the TelR-TALE-YFP, 70R-TALE-YFP and 177R-TALE-YFP proteins (Fig. S8).”

“Proteins identified as being enriched with 70R-TALE-YFP (Figure 6D) were similar in comparisons with either the No-YFP, NonR-TALE-YFP or ingiR-TALE-YFP as negative controls.”

Top Page 12:

“The same kinetochore proteins were enriched regardless of whether the 177R-TALE proteomics data was compared with No-YFP, NonR-TALE or ingiR-TALE-YFP controls.”

Discussion Page 13:

“Regardless, the 147R-TALE and ingiR-TALE proteins were well expressed in T. brucei cells, but their affinity selection did not significantly enrich for any relevant proteins. Thus, 147R-TALE and ingiR-TALE provide reassurance for the overall specificity for proteins enriched TelR-TALE, 70R-TALE and 177R-TALE affinity purifications.”

(4) After the artificial expression of repetitive sequence binding five-TALE proteins, the question is if there is any competition for the TALE proteins with the corresponding endogenous proteins? Is there any effect on parasite survival or health, compared to the control after the expression of these five TALEs YFP protein? It is recommended to add parasite growth curves, for all the TALE proteins expressing cultures.

Growth curves for cells expressing TelR-TALE-YFP, 177R-TALE-YFP and ingiR-TALE-YFP are now included (New Fig S3A). No deficit in growth was evident while passaging 70R-TALE-YFP, 147R-TALE-YFP, NonR-TALE-YFP cell lines (indeed they grew slightly better than controls).

The following text has been added page 8:

“Cell lines expressing representative TALE-YFP proteins displayed no fitness deficit (Fig. S3A).”

(5) Since the experiments were performed using whole-cell extracts without prior nuclear fractionation, the authors should consider the possibility that some identified proteins may have originated from compartments other than the nucleus. Specifically, the detection of certain binding proteins might reflect sequence homology (or partial homology) between mitochondrial DNA (maxicircles and minicircles) and repetitive regions in the nuclear genome. Additionally, the lack of subcellular separation raises the concern that cytoplasmic proteins could have been co-purified due to whole cell lysis, making it challenging to discern whether the observed proteome truly represents the nuclear interactome.

In our experimental design, we confirmed bioinformatically that the repeat sequences targeted were not represented elsewhere in the nuclear or mitochondrial genome (kDNA). The absence of subcellular fractionation could result in some cytoplasmic protein selection, but this is unlikely since each TALE targets a specific DNA sequence but is otherwise identical such that cross-selection of the same contaminating protein set would be anticipated if there was significant non-specific binding. We have previously successfully affinity selected 15 chromatin modifiers and identified associated proteins without major issues concerning cytoplasmic protein contamination (Staneva et al 2021 and 2022; PMID: 34407985 and 36169304). Of course, the possibility that some proteins are contaminants will need to be borne in mind in any future follow-up analysis of proteins of interest that we identified as being enriched on specific types of repetitive element in T. brucei. Proteins that are also detected in negative control, or negative affinity selections such as No-YFP, NoR-YFP, IngiR-TALE or 147R-TALE must be disregarded.

(6) Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

As mentioned earlier, the author claimed that this study has provided new information concerning telomere biology, chromosomal segregation mechanisms, and immune evasion strategies. But there are no experiments that provides a role for any unknown or known protein in these processes. Thus, it is suggested to select one or two proteins of choice from the list and validate their direct binding to repetitive region(s), and their role in that region of interaction.

As highlighted in response to point 1 the suggested validation and follow up experiments may well not be informative and are beyond the scope of the methodological development presented in this manuscript. Referee 2 describes the study in its current form as “a significant conceptual and technical advancement” and “This approach enhances our understanding of chromatin organization in these regions and provides a foundation for investigating the functional roles of associated proteins in parasite biology.”

The Referee’s phrase ‘validate their direct binding to repetitive region(s)’ here may also mean to test if any of the additional proteins that we identified as being enriched with a specific TALE protein actually display enrichment over the repeat regions when examined by an orthogonal method. A key unexpected finding was that kinetochore proteins including KKT2 are enriched in our affinity purifications of the 177R-TALE-YFP that targets 177bp repeats (Figure 6F). By conducting ChIP-seq for the kinetochore specific protein KKT2 using YFP-KKT2 we confirmed that KKT2 is indeed enriched on 177bp repeat DNA but not flanking DNA (Figure 7). Moreover, several known telomere-associated proteins are detected in our affinity selections of TelRTALE-YFP (Figure 6B, FigS6; see also Reis et al, 2018 Nuc. Acids Res. PMID: 29385523; Weisert et al, 2024 Sci. Reports PMID: 39681615).

Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

The answer for this question depends on what the authors want to present as the achievements of the present study. If the achievement of the paper was is the creation of a new tool for discovering new proteins, associated with the repeat regions, I recommend that they add a proof for direct interactions between a sample the newly discovered proteins and the relevant repeats, as a proof of concept discussed above, However, if the authors like to claim that the study achieved new functional insights for these interactions they will have to expand the study, as mentioned above, to support the proof of concept.

See our response to point 1 and the point we labelled ‘6’ above.

Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.

I think that they are realistic. If the authors decided to check the capacity of a small sample of proteins (which was unknown before as a repetitive region binding proteins) to interacts directly with the repeated sequence, it will substantially add of the study (e.g., by EMSA; estimated time: 1 months). If the authors will decide to check the also the function of one of at least one such a newly detected proteins (e.g., by KD), I estimate the will take 3-6 months.

As highlighted previously the proposed EMSA experiment may well be uninformative for protein complex components identified in our study or for isolated proteins that directly bind DNA in the context of a complex and chromatin. RNAi knockdown data and cell location data (as well as developmental expression and orthology data) is already available through tritrypDB.org and trtyptag.org

Are the data and the methods presented in such a way that they can be reproduced? Yes

Are the experiments adequately replicated, and statistical analysis adequate?

The authors did not mention replicates. There is no statistical analysis mentioned.

The figure legends indicate that all volcano plots of TALE affinity selections were derived from three biological replicates. Cutoffs used for significance: P < 0.05 (Student's t-test).

For ChiP-seq two biological replicates were analysed for each cell line expressing the specific YFP tagged protein of interest (TALE or KKT2). This is now stated in the relevant figure legends – apologies for this oversight. The resulting data are available for scrutiny at GEO: GSE295698.

Minor comments:

Specific experimental issues that are easily addressable.

The following suggestions can be incorporated:

(1) Page 18, in the material method section author mentioned four drugs: Blasticidine, Phleomycin and G418, and hygromycin. It is recommended to mention the purpose of using these selective drugs for the parasite. If clonal selection has been done, then it should also be mentioned.

We erroneously added information on several drugs used for selection in our labaoratory. In fact all TALE-YFP construct carry the Bleomycin resistance genes which we select for using Phleomycin. Also, clones were derived by limiting dilution immediately after transfection. We have amended the text accordingly:

Page 17/18:

“Cell cultures were maintained below 3 x 106 cells/ml. Pleomycin 2.5 µg/ml was used to select transformants containing the TALE construct BleoR gene.”

“Electroporated bloodstream cells were added to 30 ml HMI-9 medium and two 10-fold serial dilutions were performed in order to isolate clonal Pleomycin resistant populations from the transfection. 1 ml of transfected cells were plated per well on 24-well plates (1 plate per serial dilution) and incubated at 37°C and 5% CO2 for a minimum of 6 h before adding 1 ml media containing 2X concentration Pleomycin (5 µg/ml) per well.”

(2) In the method section the authors mentioned that there is only one site for binding of NonR-TALE in the parasite genome. But in Fig. 1C, the authors showed zero binding site. So, there is one binding site for NonR-TALE-YFP in the genome or zero?

We thank the reviewer for pointing out this discrepancy. We have checked the latest Tb427v12 genome assembly for predicted NonR-TALE binding sites and there are no exact matches. We have corrected the text accordingly.

Page 7:

“A control NonR-TALE protein was also designed which was predicted to have no target sequence in the T. brucei genome.”

Page 17:

“A control NonR-TALE predicted to have no recognised target in the T. brucei geneome was designed as follows: BLAST searches were used to identify exact matches in the TREU927 reference genome. Candidate sequences with one or more match were discarded.”

(3) The authors used two different anti-GFP antibodies, one from Roche and the other from Thermo Fisher. Why were two different antibodies used for the same protein?

We have found that only some anti-GFP antibodies are effective for affinity selection of associated proteins, whereas others are better suited for immunolocalisation. The respective suppliers’ antibodies were optimised for each application.

(4) Page 6: in the introduction, the authors give the number of total VSG genes as 2,634. Is it known how many of them are pseudogenes?

This value corresponds to the number reported by Consentino et al. 2021 (PMID: 34541528) for subtelomeric VSGs, which is similar to the value reported by Muller et al 2018 (PMID: 30333624) (2486), both in the same strain of trypanosomes as used by us. Based on the earlier analysis by Cross et al (PMID: 24992042), 80% of the identified VSGs in their study (2584) are pseudogenes. This approximates to the estimation by Consentino of 346/2634 (13%) being fully functional VSG genes at subtelomeres, or 17% when considering VSGs at all genomic locations (433/2872).

(5) I found several typos throughout the manuscript.

Thank you for raising this, we have read through the manuscipt several times and hopefully corrected all outstanding typos.

(6) Fig. 1C: Table: below TOTAL 2nd line: the number should be 1838 (rather than 1828)

Corrected- thank you.

- Are prior studies referenced appropriately? Yes

- Are the text and figures clear and accurate? Yes

- Do you have suggestions that would help the authors improve the presentation of their data and conclusions? Suggested above

Reviewer #1 (Significance):

Describe the nature and significance of the advance (e.g., conceptual, technical, clinical) for the field:

This study represents a significant conceptual and technical advancement by employing a synthetic TALE DNA-binding protein tagged with YFP to selectively identify proteins associated with five distinct repetitive regions of T. brucei chromatin. To the best of my knowledge, it is the first report to utilize TALE-YFP for affinity-based isolation of protein complexes bound to repetitive genomic sequences in T. brucei. This approach enhances our understanding of chromatin organization in these regions and provides a foundation for investigating the functional roles of associated proteins in parasite biology. Importantly, any essential or unique interacting partners identified could serve as potential targets for therapeutic intervention.

- Place the work in the context of the existing literature (provide references, where appropriate). I agree with the information that has already described in the submitted manuscript, regarding its potential addition of the data resulted and the technology established to the study of VSGs expression, kinetochore mechanism and telomere biology.

- State what audience might be interested in and influenced by the reported findings. These findings will be of particular interest to researchers studying the molecular biology of kinetoplastid parasites and other unicellular organisms, as well as scientists investigating chromatin structure and the functional roles of repetitive genomic elements in higher eukaryotes.

- (1) Define your field of expertise with a few keywords to help the authors contextualize your point of view. Protein-DNA interactions/ chromatin/ DNA replication/ Trypanosomes

- (2) Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. None

Reviewer #2 (Evidence, reproducibility and clarity):

Summary

Carloni et al. comprehensively analyze which proteins bind repetitive genomic elements in Trypanosoma brucei. For this, they perform mass spectrometry on custom-designed, tagged programmable DNA-binding proteins. After extensively verifying their programmable DNA-binding proteins (using bioinformatic analysis to infer target sites, microscopy to measure localization, ChIP-seq to identify binding sites), they present, among others, two major findings: 1) 14 of the 25 known T. brucei kinetochore proteins are enriched at 177bp repeats. As T. brucei's 177bp repeatcontaining intermediate-sized and mini-chromosomes lack centromere repeats but are stable over mitosis, Carloni et al. use their data to hypothesize that a 'rudimentary' kinetochore assembles at the 177bp repeats of these chromosomes to segregate them. 2) 70bp repeats are enriched with the Replication Protein A complex, which, notably, is required for homologous recombination. Homologous recombination is the pathway used for recombination-based antigenic variation of the 70bp-repeat-adjacent variant surface glycoproteins.

Major Comments

None. The experiments are well-controlled, claims well-supported, and methods clearly described. Conclusions are convincing.

Thank you for these positive comments.

Minor Comments

(1) Fig. 2 - I couldn't find an uncropped version showing multiple cells. If it exists, it should be linked in the legend or main text; Otherwise, this should be added to the supplement.

The images presented represent reproducible analyses, and independently verified by two of the authors. Although wider field of view images do not provide the resolution to be informative on cell location, as requested we have provided uncropped images in new Fig. S4 for all the cell lines shown in Figure 2A.

In addition, we have included as supplementary images (Fig. S3B) additional images of TelRTALE-YFP, 177R-TALE-YFP and ingiR-TALE YFP localisation to provide additional support their observed locations presented in Figure 1. The set of cells and images presented in Figure 2A and in Fig S3B were prepared and obtained by a different authors, independently and reproducibly validating the location of the tagged protein.

(2) I think Suppl. Fig. 1 is very valuable, as it is a quantification and summary of the ChIP-seq data. I think the authors could consider making this a panel of a main figure. For the main figure, I think the plot could be trimmed down to only show the background and the relevant repeat for each TALE protein, leaving out the non-target repeats. (This relates to minor comment 6.) Also, I believe, it was not explained how background enrichment was calculated.

We are grateful for the reviewer’s positive view of original Fig. S1 and appreciate the suggestion. We have now moved these analysis to part B of main Figure 2 in the revised manuscript – now Figure 2B. We have also provided additional details in the Methods section on the approaches used to assess background enrichment.

Page 19:

“Background enrichment calculation

The genome was divided into 50 bp sliding windows, and each window was annotated based on overlapping genomic features, including CIR147, 177 bp repeats, 70 bp repeats, and telomeric (TTAGGG)n repeats. Windows that did not overlap with any of these annotated repeat elements were defined as "background" regions and used to establish the baseline ChIP-seq signal. Enrichment for each window was calculated using bamCompare, as log₂(IP/Input). To adjust for background signal amongst all samples, enrichment values for each sample were further normalized against the corresponding No-YFP ChIP-seq dataset.”

Note: While revising the manuscript we also noticed that the script had a nomalization error. We have therefore included a corrected version of these analyses as Figure 2B (old Fig. S1)

(3) Generally, I would plot enrichment on a log2 axis. This concerns several figures with ChIP-seq data.

Our ChIP-seq enrichment is calculated by bamCompare. The resulting enrichment values are indeed log2 (IP/Input). We have made this clear in the updated figures/legends.

(4) Fig. 4C - The violin plots are very hard to interpret, as the plots are very narrow compared to the line thickness, making it hard to judge the actual volume. For example, in Centromere 5, YFP-KKT2 is less enriched than 147R-TALE over most of the centromere with some peaks of much higher enrichment (as visible in panel B), however, in panel C, it is very hard to see this same information. I'm sure there is some way to present this better, either using a different type of plot or by improving the spacing of the existing plot.

We thank the reviewer for this suggestion; we have elected to provide a Split-Violin plot instead. This improves the presentation of the data for each centromere. The original violin plot in Figure 4C has been replaced with this Split-Violin plot (still Figure 4C).

(5) Fig. 6 - The panels are missing an x-axis label (although it is obvious from the plot what is displayed).

Maybe the "WT NO-YFP vs" part that is repeated in all the plot titles could be removed from the title and only be part of the x-axis label?

In fact, to save space the X axis was labelled inside each volcano plot but we neglected to indicate that values are a log2 scale indicating enrichment. This has been rectified – see Figure 6, and Fig. S7, S8 and S9.

(6) Fig. 7 - I would like to have a quantification for the examples shown here. In fact, such a quantification already exists in Suppl. Figure 1. I think the relevant plots of that quantification (YFPKKT2 over 177bp-repeats and centromere-repeats) with some control could be included in Fig. 7 as panel C. This opportunity could be used to show enrichment separated out for intermediate-sized, mini-, and megabase-chromosomes. (relates to minor comment 2 & 8)

The CIR147 sequence is found exclusively on megabase-sized chromosomes, while the 177 bp repeats are located on intermediate- and mini-sized chromosomes. Due to limitations in the current genome assembly, it is not possible to reliably classify all chromosomes into intermediate- or mini- sized categories based on their length. Therefore, original Supplementary Fig. S1 presented the YFP-KKT2 enrichment over CIR147 and 177 bp repeats as a representative comparison between megabase chromosomes and the remaining chromosomes (corrected version now presented as main Figure 2B). Additionally, to allow direct comparison of YFP-KKT2 enrichment on CIR147 and 177 bp repeats we have included a new plot in Figure 7C which shows the relative enrichment of YFP-KKT2 on these two repeat types.

We have added the following text , page 12:

“Taking into account the relative to the number of CIR147 and 177 bp repeats in the current T.brucei genome (Cosentino et al., 2021; Rabuffo et al., 2024), comparative analyses demonstrated that YFP-KKT2 is enriched on both CIR147 and 177 bp repeats (Figure 7C).”

(7) Suppl. Fig. 8 A - I believe there is a mistake here: KKT5 occurs twice in the plot, the one in the overlap region should be KKT1-4 instead, correct?

Thanks for spotting this. It has been corrected

(8) The way that the authors mapped ChIP-seq data is potentially problematic when analyzing the same repeat type in different regions of the genome. The authors assigned reads that had multiple equally good mapping positions to one of these mapping positions, randomly.

This is perfectly fine when analysing repeats by their type, independent of their position on the genome, which is what the authors did for the main conclusions of the work.

However, several figures show the same type of repeat at different positions in the genome. Here, the authors risk that enrichment in one region of the genome 'spills' over to all other regions with the same sequence. Particularly, where they show YFP-KKT2 enrichment over intermediate- and mini-chromosomes (Fig. 7) due to the spillover, one cannot be sure to have found KKT2 in both regions.

Instead, the authors could analyze only uniquely mapping reads / read-pairs where at least one mate is uniquely mapping. I realize that with this strict filtering, data will be much more sparse. Hence, I would suggest keeping the original plots and adding one more quantification where the enrichment over the whole region (e.g., all 177bp repeats on intermediate-/mini-chromosomes) is plotted using the unique reads (this could even be supplementary). This also applies to Fig. 4 B & C.

We thank the reviewer for their thoughtful comments. Repetitive sequences are indeed challenging to analyze accurately, particularly in the context of short read ChIP-seq data. In our study, we aimed to address YFP-KKT2 enrichment not only over CIR147 repeats but also on 177 bp repeats, using both ChIP-seq and proteomics using synthetic TALE proteins targeted to the different repeat types. We appreciate the referees suggestion to consider uniquely mapped reads, however, in the updated genome assembly, the 177 bp repeats are frequently immediately followed by long stretches of 70 bp repeats which can span several kilobases. The size and repetitive nature of these regions exceeds the resolution limits of ChIP-seq. It is therefore difficult to precisely quantify enrichment across all chromosomes.

Additionally, the repeat sequences are highly similar, and relying solely on uniquely mapped reads would result in the exclusion of most reads originating from these regions, significantly underestimating the relative signals. To address this, we used Bowtie2 with settings that allow multi-mapping, assigning reads randomly among equivalent mapping positions, but ensuring each read is counted only once. This approach is designed to evenly distribute signal across all repetitive regions and preserve a meaningful average.

Single molecule methods such as DiMeLo (Altemose et al. 2022; PMID: 35396487) will need to be developed for T. brucei to allow more accurate and chromosome specific mapping of kinetochore or telomere protein occupancy at repeat-unique sequence boundaries on individual chromosomes.

Reviewer #2 (Significance):

This work is of high significance for chromosome/centromere biology, parasitology, and the study of antigenic variation. For chromosome/centromere biology, the conceptual advancement of different types of kinetochores for different chromosomes is a novelty, as far as I know. It would certainly be interesting to apply this study as a technical blueprint for other organisms with minichromosomes or chromosomes without known centromeric repeats. I can imagine a broad range of labs studying other organisms with comparable chromosomes to take note of and build on this study. For parasitology and the study of antigenic variation, it is crucial to know how intermediate- and mini-chromosomes are stable through cell division, as these chromosomes harbor a large portion of the antigenic repertoire. Moreover, this study also found a novel link between the homologous repair pathway and variant surface glycoproteins, via the 70bp repeats. How and at which stages during the process, 70bp repeats are involved in antigenic variation is an unresolved, and very actively studied, question in the field. Of course, apart from the basic biological research audience, insights into antigenic variation always have the potential for clinical implications, as T. brucei causes sleeping sickness in humans and nagana in cattle. Due to antigenic variation, T. brucei infections can be chronic.

Thank you for supporting the novelty and broad interest of our manuscript

My field of expertise / Point of view:

I'm a computer scientist by training and am now a postdoctoral bioinformatician in a molecular parasitology laboratory. The laboratory is working on antigenic variation in T. brucei. The focus of my work is on analyzing sequencing data (such as ChIP-seq data) and algorithmically improving bioinformatic tools.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors assess the role of map3k1 in adult Planaria through whole body RNAi for various periods of time. The authors' prior work has shown that neoblasts (stem cells that can regenerate the entire body) for various tissues are intermingled in the body. Neoblasts divide to produce progenitors that migrate within a "target zone" to the "differentiated target tissues" where they differentiate into a specific cell type. Here the authors show that map3k1-i animals have ectopic eyes that form along the "normal" migration path of eye progenitors (Fig. 1), ectopic neurons and glands along the AP axis (Fig. 2) and pharynx in ectopic anterior positions (Fig. 3). The rest of the study show that positional information is largely unaffected by loss of map3k1 (Fig. 4,5). However, loss of map3k1 leads to premature differentiated of progenitors along their normal migratory route (Fig. 6). They also show that an ill-defined "long-term" whole body depletion of map3k1 results in mis-specified organs and teratomas.

Strengths:

(1) The study has appropriate controls, sample sizes and statistics.

(2) The work appears to be high-quality.

(3) The conclusions are supported by the data.

(4) Planaria is a good system to analyze the function of map3k1, which exists in mammals but not in other invertebrates.

Weaknesses:

(1) The paper is largely descriptive with no mechanistic insights. 

The mechanistic insights we aim to address are primarily at the cellular systems level – how adult progenitor cells produce pattern. Specifically, we uncovered strong evidence that regulation of differentiation is an active process occurring in migratory progenitors and that this regulation is a major component of pattern formation during the adult processes of tissue turnover and regeneration. The map3k1 phenotype provided a tool used to reveal the existence of this regulation, and to understand the patterning abnormalities prevented by this regulatory mechanism. We updated the text in several places to make clearer some of this emphasis. For example, in the Discussion: "We suggest that differentiation is restricted during migratory targeting as an essential component of pattern formation, with the map3k1 RNAi phenotype indicating the existence and purpose of this element of patterning." 

Naturally, identifying a particular molecule involved in this process is of interest for understanding molecular mechanism; this would allow for comparison to other cellular systems in other organisms and would focus future molecular inquiry. Future molecular studies into the mechanism of Map3k1 regulation and its downstream signaling will be fascinating as next steps towards understanding the process at the molecular level more deeply. We also added some discussion considering the types of upstream activation cues that could potentially be associated with Map3k1 regulation to suppress differentiation. 

(2) Given the severe phenotypes of long-term depletion of map3k1, it is important that this exact timepoint is provided in the methods, figures, figure legends and results. 

We removed the use of the term “long-term” and instead added timepoints used to all figure legends. We also added a summary of timepoints used in the methods section and included RNAi timepoint labels in figures where a phenotype progression over time is relevant to interpretation. For timecourses, we also added suitable time information to text in the results. 

(3) Figure 1C, the ectopic eyes are difficult to see, please add arrows. 

To improve visualization, we replaced the example animal in the original Figure 1C with one that has a stronger phenotype, including arrows pointing to every ectopic event. Additionally, we included magnified images of optic cup cells and photoreceptor neurons in the trunk and tail region. This is now Figure 1B.

(4) line 217 - why does the n=2/12 animals not match the values in Figure 3B, which is 11/12 and 12/12. The numbers don't add up. Please correct/explain. 

In Figure 3B in the submitted version (3/18 had cells in the tail) had more animals scored (6 animals from a replicate experiment where 1/6 showed the cells in the tail) than the total scored (2/12 had cells in the tail) in the text, which did not have the animals from the replicate added during writing. The results are the same, just different sample sizes were noted in those locations and we fixed this issue. In the updated Figure 3, the order of presentation has shifted (e.g., prior 3B is now in 3C and Figure 3_figure supplement 1). We made sure to include numbers to all figure panels. 

(5) Figure panels do not match what is written in the results section. There is no Figure 6E. Please correct.

Thank you for catching this. We have gone through figures and text after editing to make sure that text callouts are appropriately matched to the figures. 

Reviewer #2 (Public review):

Summary:

The flatworm planarian Schmidtea mediterranea is an excellent model for understanding cell fate specification during tissue regeneration and adult tissue maintenance. Planarian stem cells, known as neoblasts, are continuously deployed to support cellular turnover and repair tissues damaged or lost due to injury. This reparative process requires great precision to recognize the location, timing, and cellular fate of a defined number of neoblast progeny. Understanding the molecular mechanisms driving this process could have important implications for regenerative medicine and enhance our understanding of how form and function are maintained in long-lived organisms such as humans. Unfortunately, the molecular basis guiding cell fate and differentiation remains poorly understood.

In this manuscript, Canales et al. identified the role of the map3k1 gene in mediating the differentiation of progenitor cells at the proper target tissue. The map3k1 function in planarians appears evolutionarily conserved as it has been implicated in regulating cell proliferation, differentiation, and cell death in mammals. The results show that the downregulation of map3k1 with RNAi leads to spatial patterning defects in different tissue types, including the eye, pharynx, and the nervous system. Intriguingly, long-term map3k1-RNAi resulted in ectopic outgrowths consistent with teratomas in planarians. The findings suggest that map3k1 mediates signaling, regulating the timing and location of cellular progenitors to maintain correct patterning during adult tissue maintenance.

Strengths:

The authors provide an entry point to understanding molecular mechanisms regulating progenitor cell differentiation and patterning during adult tissue maintenance.

The diverse set of approaches and methods applied to characterize map3k1 function strengthens the case for conserved evolutionary mechanisms in a selected number of tissue types. The creativity using transplantation experiments is commendable, and the findings with the teratoma phenotype are intriguing and worth characterizing.

Thank you to the reviewer for the positive feedback

Weaknesses:

The article presents a provocative idea related to the importance of positional control for organs and cells, which is at least in part regulated by map3k1. Nonetheless, the role of map3k1 or its potential interaction with regulators of the anterior-posterior, mediolateral axes, and PCGs is somewhat superficial. The authors could elaborate or even speculate more in the discussion section and the different scenarios incorporating these axial modulators into the map3k1 model presented in Figure 8 

First, to strengthen the support for our finding that positional information is largely unaffected in map3k1 RNAi animals, we added data regarding the expression of additional relevant position control genes (PCGs) –ndl-4, ptk7, sp5, and wnt11-1 – to the PCG panel in Figure 5. The expression domain of ndl-4, an FGF receptor-like protein family member that contributes to head patterning and anterior pole maintenance, was normal in map3k1 RNAi. wnt11-1, a PCG with expression concentrated in the posterior end of the animal and with expression dependent on general Wnt activity, was also normal in map3k1 RNAi animals. ptk7, RNAi of which can result in supernumerary pharynges, also showed normal expression in map3k1 RNAi animals. Finally, sp5, a Wnt-activated gene with expression in the tail, also showed normal expression in map3k1 RNAi animals. 

Second, to further support the conclusion that cells are not suitably responding to positional information after map3k1 RNAi, which we argue normally dictates where differentiation should occur, we added examples of differentiated cell types that are ectopically positioned within an atypical PCG expression domain for that cell type (Figure 5C). This underscores that following map3k1 RNAi the PCG expression domains do not change, but the pattern of differentiated cell types relative to these domains does shift. We also added data showing that regenerating tails had a proper wntP-2 gradient, but an anterior regenerating pharynx appeared outside of this wntP-2⁺ zone and inside of an ndl-5⁺ zone (Figure 5- figure supplement 1E). We added some discussion of these new data in the Figure 5 results section. We also noted, regarding independent recent map3k1 work (Lo, 2025), some evidence exists that a minor posterior shift in ndl-5 expression can occur after map3k1 RNAi.

Next, we added a new element to the model figure (Figure 8B) depicting that PCG expression domains remain normal after map3k1 RNAi, with ectopic differentiation occurring in an incorrect positional information environment. We refer to this new panel in the discussion: "We suggest that map3k1 is not required for the spatial distribution of progenitor-extrinsic differentiation-promoting cues themselves, but for progenitors to be restricted from differentiating until these cues are received (Figure 8B)."; we then follow this statement with a summary in the Discussion of six pieces of evidence that support this model.

Finally, we added some additional text to the discussion section about candidate mechanisms by which extrinsic cues could potentially regulate Map3k1, pointing to potential future inquiry directions. We suggest that map3k1 is not involved in regulating PCG activity domains themselves, but instead acts as a brake on differentiation within migratory progenitors through active signaling. This brake is then lifted when the progenitors hit their correct PCG-based migratory target, or when they hit their target tissue. How that occurs mechanistically is unknown. One scenario is that each progenitor is tuned to respond to a particular PCG-regulated environment (such as a particular ECM or signaling environment) to generate a molecular change that inactivates Map3K1 signaling, such as by inactivating or disengaging an RTK signal. Alternatively, the migratory process in progenitors could engage the Map3K1 signal, enabling signal cessation with arrival at a target location. When Map3K1 is active it could result in a transcriptional state that prevents full expression of differentiated factors required for maturation, tissue incorporation, and cessation of migration. These considerations are now added to the discussion.

The article can be improved by addressing inconsistencies and adding details to the results, including the main figures and supplements. This represents one of the most significant weaknesses of this otherwise intriguing manuscript. Below are some examples of a few figures, but the authors are expected to pay close attention to the remaining figures in the paper.

Details associated with the number of animals per experiment, statistical methods used, and detailed descriptions of figures appear inconsistent or lacking in almost all figures. In some instances, the percentage of animals affected by the phenotype is shown without detailing the number of animals in the experiment or the number of repeats. Figures and their legends throughout the paper lack details on what is represented and sometimes are mislabeled or unrelated. 

We endeavored to ensure that these noted details are present throughout the legends and figures for all figure panels.

Specifically, the arrows in Figure 1A are different colors. Still, no reasoning is given for this, and in the exact figure, the top side (1A) shows the percentages and the number of animals below. 

The only reason for the different colored arrows was for visibility purposes. To avoid confusion, we now use white arrows for all FISH images in figure 1, and where ever else possible. We also replaced the percentages with n numbers in the bottom left corner of the live images in Figure 1A. 

Conversely, in Figures 1B, C, and D, no details on the number of animals or percentages are shown, nor an explanation of why opsin was used in Figure 1A but not 1B. 

The original Figure 1B represented a few different examples of ectopic eye/eye cell patterns in the map3k1 RNAi animals to demonstrate the variable and disorganized nature of the phenotype. To address this, we added further explanation in the legend. We also merged 1A and 1B for simplicity of interpretation. opsin was used in Figure 1A to label cell bodies of photoreceptors. anti-Arrestin was used in the example FISH images to see if these cells were interconnected via projections, which we now clarify in the legend and in the text. 

Is Figure 1B missing an image for the respective control? Figure 1C needs details regarding what the two smaller boxes underneath are. 

The control for Figure 1B was in Figure 1A; the merger of Figures 1A/B should address this. Boxes in Figure 1C were labelled with numbers corresponding to the image above them.

Figure 1C could use an AP labeling map in 10 days (e.g., AP6 has one optic cup present). Figure 1C and F counts do not match. 

We added a cartoon to 1C to show the region imaged. Note that the 36d trunk image (now Fig. 1B) has now been replaced with a full animal image and magnified boxes that show locations of example ectopic cells. That cell in 1C was categorized as in AP5. Note that additional animals were analyzed and data added to the distribution (now Fig. 1D). 

In Figure 1C, we do not know the number of animals tested, controls used, the scale bar sizes in the first two images, nor the degree of magnification used despite the pharynx region appearing magnified in the second image.  Figure 1C is also shown out of chronological order; 36 days post RNAi is shown before 10 days post RNAi. Moreover, the legends for Figures 1C and 1D are swapped.

We have endeavored to ensure sample numbers, control images, and appropriate scale bar notation in legends are present for all images. Figure 1C has now been split into two panels: Figure 1B and Figure 1C. It does not follow a chronological order in presentation for the following logic flow: we uncover and describe the phenotype; then, with knowledge of the defect, we walk back to see how early the phenotype starts after RNAi and what the pattern of ectopic cell distribution is when the phenotype starts to emerge (using the knowledge of which cells are affected from the overt phenotype described in 1A/B). 

Additionally, Figure 1F and many other figures throughout the paper lack overall statistical considerations. Furthermore, Figure 1F has three components, but only one is labeled. Labeling each of them individually and describing them in the corresponding figure legend may be more appropriate.

The main point of the graphs in 1F (now 1D) was the overt overall pattern difference with the wild-type, which never has ectopic eye cells in the midbody or tail, and that the ectopic eye cells progress throughout the entire AP axis. However, we concur that a statistical test is a reasonable thing to show here and that is now included in the legend. The 3 components (in Figure 1F, now Figure 1D) where kept together with one figure label (D) for simplicity, but were rearranged (top and bottom) with a cartoon to the side and with modified labeling for extra clarity. 

Figure 2C shows images of gene expression for two genes, but the counts are shown for only one in Figure 2D. It is challenging to follow the author's conclusions without apparent reasoning and by only displaying quantitative considerations for one case but not the other. These inconsistencies are also observed in different figures. 

In Figure 2C, FISH images of cintillo+ and dd_17258+ neurons are shown to display the specificity of this effect to some neurons and not others. Because cintillo+ cells did not expand at all (n=24/24 animals), the counts for them would all be zero values. We only counted data for dd_17258 cells because it was the neuron that expanded compared to the control animals. We have now added a note in the legend explaining this.

In Figure 2D, 24/24 animals were reported to show the phenotype, but only eight were counted (is there a reason for this?).

8 animals were used to quantitatively characterize the spread of cells along the AP axis, as it was deemed an adequate sample size to capture the change in distribution of 17258+ cells from being head restricted to being present throughout the body. Through multiple cohorts of animals in replicates, a total of 24/24 examined animals showed this expansion phenotype. Double FISH experiments were additionally carried out using dd_17258 and various PCGs; these data are now included in Figure 5C, and these animals were added to the total counts regarding quantitative analysis of the phenotype in Figure 2D. 

In Figure 2E, the expression for three genes is shown, with some displaying anterior and posterior regions while others only show the anterior picture. Is there a particular reason for this? 

The original first panel in Figure 2E showed an example of a non-expanding gland cell type, dd_9223, which is very restricted to the head in both control and map3k1 RNAi animals. Because we did not observe a phenotype for this cell type (no cells in all control and map3k1 RNAi animal tails), we only included tail images of cell types that showed an abnormal phenotype with clear expanded to the posterior (dd_8476 and dd_7131). However, we have now included tail images of dd_9223 cells and added data for dd_9223 to the graph in Figure 2E. 

Also, in Figure 2F, the counts are shown for only the posterior region of two genes out of the three displayed in Figure 2E. It is unclear why the authors do not show counts for the anterior areas considered in Figure 2E. Furthermore, the legend for Figure 2D is missing, and the legend for 2F is mislabeled as a description for Figure 2D.

We now include tail images for dd_9223 in Figure 2E to show that there are no ectopic cells in tails. We did not originally include counts of dd_9223 because there was no phenotype observed. dd_7131 and dd_8476 cell types appeared in the posterior of even control animals at a low frequency, unlike dd_9223 cells. However, we did now add counts for dd_9223 tail regions in the graph. We did not count the anterior regions of the animal because our goal was to show data for the visible phenotype (ectopic cells in the tail) not only with an example image, but also by showing the number of cells in the tail with a graph and statistical test. Legends have been updated with correct details.

Supplement Figure 1 B reports data up to 6 weeks, but no text in the manuscript or supplement mentions any experiment going up to 6 weeks. There are no statistics for data in Supplement Figure 1E. Any significance between groups is unclear.

More details about the RNAi feeding schedules have been added in the methods section. All RNAi timepoints are now specified specifically in the legends. The Figure 1F and Figure 1- figure supplement 1E (additional data: ovo⁺; smedwi-1^- cell counts) and legends now mention the statistical tests performed and annotations (not significant *ns) or p values have been added to the graphs. For simplicity, we decided to include all smedwi-1+ counts together rather than splitting them into low and high smedwi-1+ cells, because we weren't really making any claims about low and high cells. 

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

It would be good to acknowledge in the discussion the recent paper from the Petersen lab on map3k1, published in PLoS Genet 2025, especially if the results differ between the two labs.

We added reference/discussion regarding the recent PLoS Genetics Lo, 2025 map3k1 paper at several suitable points in the manuscript.

Reviewer #2 (Recommendations for the authors):

Please pay close attention to the description of experimental details and the consistency throughout the paper. It seems like the reader has to assume or come across information that is not readily available from the text or the legends in the paper. This is an interesting paper with intriguing findings. However, the version presented here appears rushed or put together on the flight.

Thank you for your thorough feedback. We have endeavored to ensure all appropriate details are present in figures and/or figure legends.

make this not bold

Reviewer #1 (Public review):

Summary:

This manuscript by Joshi and colleagues demonstrates that the precise theta-phase timing of spikes is causal for CA1 hippocampal theta sequences during locomotion on a linear track and is necessary for learning the cognitively demanding outbound component of a hippocampus-dependent alternation task (W-maze), independently of replay during immobility. To reach these conclusions, the authors developed a theta-phase-specific, closed-loop manipulation that used optogenetic activation of medial septal parvalbumin (PV) interneurons at the ascending phase of theta during locomotion. This protocol preserved immobility periods, allowing a clean and elegant dissociation from SWR-associated replay.

The manuscript is well written and was a pleasure to read. The work described is of high quality and introduces several notable advances to the field:

(a) It extends prior studies that manipulated theta oscillations by examining precise temporal structure (specifically theta sequences) rather than only LFP features.

(b) The closed-loop manipulation enabled dissociation between deficits in theta sequences during a behavioural task and SWR-associated replay activity.

(c) As controls, the authors included rats with suboptimal viral transduction or optic-fibre placement, and, within subjects, both stimulation-on (stim-on) and stimulation-off (stim-off) trials. Notably, sequence disruption persisted into stim-off periods within the same session.

Overall, this is a strong manuscript that will provide valuable insights to the field. I have only minor comments:

(1) As the authors note, it is striking that both behavioural performance and spike patterns are altered during stim-off trials. They propose that "disruption of theta sequences during the initial experience in an environment is sufficient to have lasting effects," implying that rapid, experience-dependent plasticity is driven by sequential firing. Does this imply that if rats were previously trained on the task, subsequent stim-on and stim-off trials would yield different outcomes, with stim-off trials showing improved performance and intact theta sequences? For example, if the sequence of one-third stim-on, one-third stim-off, one-third stim-on were inverted to off-on-off, would theta sequences be expected to emerge, disappear, and potentially re-emerge? While I am not asking for additional experiments, I think the discussion could be extended in this aspect.

Alternatively, could the number of stim-off trials (one third of the total) be insufficient to support learning/induce plasticity? In the controls, ~50-100 trials appear necessary to achieve high performance.

(2) In line with the point above, the authors characterise the behavioural changes induced by MS optogenetic stimulation specifically as a "learning deficit," as rats failed to improve across 300 trials in an initially novel environment (W-maze). While they present this as complementary to prior demonstrations of impaired performance on previously learned tasks (Zutshi et al., 2018; Quirk et al., 2021; Etter et al., 2023; Petersen et al., 2020), an alternative interpretation is a working-memory deficit. This would produce the same behavioural pattern, with reference memory (the less cognitively demanding trials) remaining intact despite stimulation and concomitant changes in theta sequences. This interpretation would also be consistent with work in certain disease models, where reduced synaptic plasticity and working-memory deficits co-occur with preserved place coding despite impaired theta sequences (e.g., Viana da Silva et al., 2024; Donahue et al., 2025).

(3) It was not immediately clear whether SWR-associated activity was derived from the interleaved ~15-min rest sessions in a rest box, or from periods of immobility or reward consumption in the maze (aSWR, as in Jadhav et al 2012). Regardless, it would be informative to compare aSWR events within the maze to rest-box SWRs that may occur during more prolonged slow-wave episodes (even if not full sleep). This contrasts with Liu et al. (2024), who analysed replay during ~1.5-h sleep sessions.

Synthèse : Enfants Violents à l'École - Entre Aide et Répression

Résumé Exécutif

Ce document de synthèse analyse les tensions et les débats entourant la gestion de la violence chez les jeunes enfants au sein du système scolaire et de la société française.

Il ressort que l'école se trouve démunie face à des comportements extrêmes, conduisant à la création de structures expérimentales comme "r'école" pour éviter la déscolarisation.

Parallèlement, une tendance croissante à la médicalisation des troubles du comportement, incarnée par le diagnostic d'hyperactivité et la prescription de Ritaline, suscite une vive controverse.

Des experts dénoncent l'influence du lobbying pharmaceutique et une simplification qui ignore les causes profondes de la souffrance de l'enfant.

Cette approche s'inscrit dans un contexte de "psychose médiatique" qui exagère le phénomène de la violence infantile, contredit par la réalité judiciaire qui atteste de la rareté des cas criminels chez les très jeunes.

L'analyse des cas individuels révèle que la violence est souvent le symptôme d'une souffrance psychique profonde, liée à des contextes familiaux difficiles (ruptures, violence parentale) et socio-économiques précaires.

Face à des réponses répressives ou médicamenteuses, des initiatives de prévention de proximité, comme l'association "Mission Possible", démontrent qu'un accompagnement axé sur l'écoute et le soutien aux familles est non seulement plus humain, mais aussi considérablement moins coûteux et plus efficace à long terme pour la société.

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1. Le Défi de l'École Face aux Comportements Extrêmes

Le système éducatif est confronté à une difficulté croissante pour gérer les comportements violents et ingérables de certains très jeunes élèves.

Les enseignants et les directions d'école expriment un sentiment d'impuissance et de manque de formation, menant à des situations d'échec et à l'exclusion des enfants concernés comme ultime recours.

• Le Cas d'Ethan Paul et Hamadi (6 ans) : Ces deux élèves de CP ont été exclus de leur école pour "comportement violent et ingérable".

Ethan Paul aurait tenté d'étrangler un camarade, conduisant des parents à porter plainte. Sa maîtresse reconnaît son échec :

"Je n'ai pas réussi à ce qu'ils puissent être intégrés en classe et faire les apprentissages de la place de façon satisfaisante." Elle décrit un enfant en "très très très très grande souffrance".

• La Structure "r'école" : Mise en place en janvier 2009 par le rectorat de Paris, cette structure unique en France accueille pour trois mois des enfants exclus.

◦ Objectif : Éviter la déscolarisation en offrant une "surveillance éducative renforcée" et en réapprenant aux enfants les règles de la vie en groupe.  

◦ Fonctionnement : Les enfants sont pris en charge par un personnel mixte (enseignante, auxiliaire de vie scolaire), mais seule l'enseignante est spécifiquement formée. Les crises de violence y sont fréquentes et difficiles à gérer pour le personnel.   

◦ Limites : Bien que présentée comme le "seul espoir", cette solution est coûteuse et soulève le risque de créer des filières alternatives pour enfants "difficiles", comme le souligne Jean-Louis Barateau, initiateur du projet : "Ça pourrait être dangereux si on multipliait des r'écoles au point d'avoir finalement des écoles alternatives."

2. La Médicalisation de la Violence Infantile : Une Solution Controversée

Face à l'inquiétude grandissante, une approche tendant à considérer les troubles du comportement comme des pathologies médicales à traiter a émergé, non sans susciter de vives critiques.

2.1. Le Rapport de l'INSERM et la Récupération Politique

• Le Rapport (2006) : Consacré aux "troubles des conduites", ce rapport d'experts visait à dépister les "facteurs de risque" et de "vulnérabilité" chez l'enfant.

• La Controverse : Le rapport a servi de caution scientifique à un projet de loi sur la délinquance des mineurs, porté par Nicolas Sarkozy, alors ministre de l'Intérieur.

Celui-ci affirmait : "Plutôt on n'intervient plus mieux on a de chances d'éviter le drame d'un enfant qui évolue vers la délinquance."

• La Réponse de la Société Civile : Des experts ayant participé au rapport ont précisé n'avoir "jamais écrit un rapport sur la prévention de la délinquance".

En réaction, la pétition "Pas de zéro de conduite pour les enfants de 3 ans" a recueilli près de 200 000 signatures, dénonçant le risque de transformer des comportements normaux (morsures, colères) en symptômes d'un trouble mental à rééduquer.

2.2. L'Hyperactivité et la Ritaline : Remède ou Simplification ?

Le diagnostic de "l'hyperactivité avec déficit de l'attention" (TDAH) et son traitement par la Ritaline, une amphétamine, sont au cœur du débat.

• La Défense du Traitement : La pédopsychiatre Marie-France Le Heuzey justifie son usage pour "améliorer la qualité de vie et d'améliorer le quotidien de ces enfants", soulignant la souffrance liée au rejet social et familial.

Pour elle, si le médicament permet à l'enfant de ne plus être puni et aux parents de moins se disputer, "on a aussi fait du bien largement à l'enfant".

• La Critique de la Sur-médicalisation :

◦ Rareté de la pathologie : Selon le Pr Bernard Golse (Hôpital Necker), l'hyperactivité "vraie" est très rare (1 à 2 cas pour 1000), loin des 5 à 10% avancés par certains. Il dénonce "l'effet direct du lobbying pharmaceutique qui veut élargir coûte que coûte la prescription de médicaments".   

◦ Création de la demande : Philippe Pignarre, ancien cadre de l'industrie pharmaceutique, explique la stratégie marketing : "L'industrie pharmaceutique travaille à créer à la fois l'offre et la demande... On va la créer la demande en disant aux gens... ce que vous saviez pas, c'est qu'il a un trouble mental et qu'on peut soigner ce trouble mental."   

◦ Traitement des symptômes, pas des causes : La Ritaline, surnommée "pilule de l'obéissance", agit sur les symptômes mais ne traite pas les causes sous-jacentes de la souffrance.

2.3. L'Expérience Vécue : Le Cas d'Aymeric

Aymeric, 16 ans, a été traité à la Ritaline pendant des années.

Son témoignage illustre l'ambivalence du traitement : "C'était bien mais c'est pas bien. Pourquoi c'était bien ? Parce que ça me calmait d'un côté. Mais... c'était bien pour eux, mais pour moi c'était pas bien parce que là je mangeais plus... j'étais tout le temps fatigué."

3. Psychose Médiatique et Réalité Judiciaire

La perception publique de la violence infantile est fortement influencée par un traitement médiatique qui tend à se focaliser sur des faits divers extrêmes, créant une forme de psychose collective.

• L'Affaire d'Uckange (2009) : Un Emballement Révélateur :

◦ Un garçon de 5 ans est accusé d'avoir poignardé sa sœur de 8 ans, prétendument sous l'influence de jeux vidéo.

L'affaire est largement médiatisée, et la thèse de l'enfant coupable est acceptée par les médias, la police et la justice.   

◦ Quelques jours plus tard, la mère avoue être l'auteure du coup de couteau. L'affaire démontre la rapidité avec laquelle "les médias ont véhiculé un peu trop vite le scénario de l'enfant criminel".

• La Perspective des Magistrats : La juge pour enfants Marie-Pierre Hourcade affirme que les affaires de violence au pénal impliquant de très jeunes enfants sont très rares.

Le critère de la responsabilité pénale est le discernement, qui apparaît vers 7-8 ans. "En aucune façon le parquet ne nous saisit pour des situations de violences commises par des très jeunes enfants."

4. Derrière la Violence : Souffrance Psychique et Contexte Familial

L'analyse approfondie des cas révèle que les comportements violents sont presque toujours l'expression d'une souffrance profonde, souvent enracinée dans des histoires familiales et sociales complexes.

• L'Expression de la Souffrance : Le Cas de Sami (13 ans) :

◦ Retiré à 8 ans d'un contexte familial violent, Sami a été ballotté de foyer en foyer. Sa violence est une manifestation de sa tristesse face aux ruptures affectives répétées.  

◦ Le Dr Roger Teboul, psychiatre, explique : "Bien souvent, quand vous parlez de la violence, vous parlez de la tristesse... Le seul truc qui permet de tenir, c'est d'être en colère."

L'objectif de son service est de permettre à ces jeunes d'exprimer leur tristesse pour ne plus avoir à l'agir par la violence.

• L'Impact du Contexte Socio-économique : Le Cas de Florian (7 ans) :

◦ Florian vit dans un quartier précaire d'Amiens. Sa mère élève seule 5 enfants avec le RMI. L'État s'est largement désengagé du quartier.  

◦ Cet environnement de précarité rend l'éducation extrêmement difficile. La mère de Florian exprime sa peur que son fils devienne délinquant si elle n'est pas soutenue.

5. Stratégies d'Intervention : La Prévention comme Alternative

Face aux approches répressives ou médicales, les initiatives de prévention axées sur l'accompagnement et le soutien des familles démontrent leur pertinence humaine et économique.

• "Mission Possible" : Un Modèle de Prévention de Proximité :

◦ Créée par le juge des enfants Claude Baud, cette association à Amiens accueille librement des familles sans obligation judiciaire.  

◦ Elle offre un soutien aux parents, souvent démunis et en rupture avec les services sociaux, sans les juger ni les culpabiliser.   

◦ Elle apprend aux enfants les règles de vie en société par le dialogue et un cadre clair, en cherchant à comprendre le sens de leurs comportements plutôt qu'à les étiqueter.

• L'Analyse d'un Juge : Coût et Efficacité : Claude Baud souligne les avantages de la prévention :

◦ Moins Stigmatisant : "Un parcours judiciaire pour un enfant est dix fois plus stigmatisant qu'un parcours de prévention."   

◦ Moins Coûteux : Il établit une comparaison financière éloquente :      

▪ Prévention (Mission Possible) : 8 € par jour et par enfant.     

▪ Placement Éducatif : 200 à 400 € par jour.    

▪ Détention en section mineurs : 700 à 1000 € par jour.

La conclusion est claire : investir dans des moyens humains, de l'écoute et du personnel bien formé pour aider les familles et les enfants en souffrance est infiniment moins coûteux que de devoir gérer, quelques années plus tard, les conséquences de cette souffrance non traitée.

Cher Tony Gheeraert,

Merci pour cet article, que j'ai vraiment apprécié. Parce que c'est une chronique, elle pourrait être publiée telle quelle. j'ai fait un certain nombre de commentaires, que vous être libre d'utiliser ou non, qui ne s'insèrent pas toujours dans la logique de l'article (reprendre la théorie marxiste de la création de valeurs et essayer de l'appliquer à la situation présente de l'IA) et que vous êtes bien évidemment libre de reprendre ou non.

Reprenons les différents critères d'évaluation :

Pertinence de la réflexion

Cet article propose une relecture marxiste de l'économie de l'IA générative, de la bulle spéculative qu'elle forme, etc. Même pour un non-marxiste, cette relecture est intéressante: dans mon expérience de chercheur non-marxiste, les écrits marxistes (orthodoxes ou hétérodoxes) m'ont toujours forcé à regarder des situations avec un autre regard et, donc, à varier mes analyses.

Deux exemple: le livre de Gavin Mueller qui essaye de réconcilier luddisme et marxisme (Mueller Gavin, Breaking things at work: the Luddites are right about why you hate your job, London New York, Verso, 2021.) J'avoue que je me contrefiche un peu de la compatibilité du luddisme avec le marxisme (sauf par intérêt historique). Mais cela a poussé Mueller à analyser la nature du mouvement du logiciel libre comme forme de luddisme technophile, idée que je repends depuis à mon tompte.

Second exemple, très loin de ce qui nous préoccupe ici: ce sont notamment des analyses marxistes hétérodoxes qui ont poussé les historiens et historiennes à regarder de plus près les politiques financière, budgétaires des dictatures fascistes et nazies, dès la fin des années 1940.

Le seul problème de cette démarche de relecture marxiste est qu'elle peut être souvent frustrante pour le lecteur. Est-ce qu'il y a des moyens d'éviter cela, je n'en suis pas sûr.

Je reste cela dit peu convaincu par l'entrée en matière de l'article. Le texte de gemini est vraiment mauvais et caricatural (et en outre, j'aime bien avoir les prompts quand quelqu'un cite une IA).

Subjectivité et démarche

Je renvoie à la section précédente sur ce point, car la démarche, une relecture marxiste, y est bien commentée.

Contribution au champ disciplinaire

Il est indéniable que cet artcile participe à quelque chose d'important, démystifier l'IA générative. Ce que je regrette un peu, c'est le fait que l'auteur ne fait pas complètement le tri (de manière explicite, s'entend) entre certains mythes de l'IA et la réalité économique de l'IA. Je reconnais que c'est assez difficile, dans la mesure où certains éléments relèvent des deux, la question de la bulle spéculative en premier lieu. Mais on a parfois l'impression que lauteur succombe lui-même un peu au mythe de l'IA (ce que je ne juge pas, moi-même y succombant parfois aussi). Mais je ne peux qu'être d'accord avec la logique d'ensemble de l'article et notamment la section "Par-delà « le libre »".

Avis général sur la publication

Comme c'est une chronique, je dirais que l'article peut être publié sans modification. À l'auteur, ou à l'éditeurice, de voir s'il souhaite prendre en compte certaines de mes remarques.

Bien à vous.

oui! C'est aussi le raisonnement de Bender et Hannah dans The AI Con.

À titre personnel, je ne peux qu'approuver ces trois points. La question reste de savoir s'ils sont applicables, car: - quelles conditions économiques pour qu'ils soient applicables? Les grandes plateformes sont quand même assez douées pour étouffer ce type de modèles alternatifs, - quelles conditions sociales, disons, et institutionnelles?

Sur ce second point, ce que je veux dire, c'est qu'il faudrait notamment -- pour juste considérer le milieu universitaire -- que ce milieu s'empare de l'IA générative, pour être prête à proposer des modèles alternatifs de développement de l'IA générative (et au-delà). Or celles et ceux qui veulent s'en emparer et en faire quelque chose d'autre vont de plus en plus vers un rejet complet de l'IA générative (voir les deux prises de position sur la liste DH: https://groupes.renater.fr/sympa/arc/dh/2025-12/msg00047.html , https://www.lobrassard.net/carnet/2025-12-19-ia-agnotologie.html et https://atecopol.hypotheses.org/13082)

Le résultat est que beaucoup d'usage de l'IA dans le monde universitaire se fait à bas bruit (du côté des chercheurs et chercheuses en SHS, s'entend, pas du côté des institutions qui elles font grand bruit de beaucoup de bullshit). Bref, les conditions ne sont pas réunies pour un tel programme.

Il y a des travaux des Digital Humanities sur la frugalité informatique: https://dhq-static.digitalhumanities.org/pdf/000646.pdf

Et ce texte de Lauren Klein, qui reprend des éléments de son Data feminism (avec d'Ignazio) et essaye de l'appliquer à l'IA (et doute): https://zenodo.org/records/6567985

Sans vouloir faire de la pub, il y a les travaux de pleias, startup, qui a mis en place un corpus d'entraînement constitué de données du domaine public: https://huggingface.co/blog/Pclanglais/common-corpus

(le fait que deux des co-fondateurs, si mes souvenirs sont bons, de pleias viennent des SHS est probablement lié à leur recherche d'utiliser les communs pour l'IA).

Ce qui n'est pas totalement nouveau, en effet -- Graeber l'a montré, mais beaucoup d'économistes (je n'ai pas ici de références précises, pardon, sauf Robert Solow et le paradoxe de la productivité) ont mis en doute les gains de productivité proclamés de l'informatique dès les années 1980.

quid des métiers intellectuels? quid des universitaires? Quid des pamphlétaires? quid des intellectuels médiatiques semi-éduqués? L'IA générative telle que nous la connaissant vient aussi troubler le jeux de ces professions.

Oui!

OUI! Suivant Gavin Mueller, je suis un luddite technophile.

Ou l'époque du bon roi Ludd! Les machines que détruisaient les luddites en 1811-1812 étaient moins performantes que les ouvriers, mais les patrons voulaient s'affranchir des ouvriers.

OUI!

Une autre références intéressante pourrait être celle-là: Mueller Gavin, Breaking things at work: the Luddites are right about why you hate your job, London New York, Verso, 2021.

Je regarderais un peu plus qui émet les mises en garde -- ces derniers temps, elles viennent du secteur de l'IA lui-même, et peuvent faire partie de la même tactique de surinvestissement: investissez chez moi car je survivrai à une crise, mais pas le voisin.

Aussi pour partie le schéma de la crise des dotcoms. Et pour partie, des acteurs communs (google, amazon), mais aussi de nouveaux acteurs (certes openai et anthropic -- mais leurs investisseurs sont souvent aussi les historiques, dont microsoft, mais aussi les entreprises chinoises comme deepseek: il serait intéressant de se demander si une telle crise ne changerait pas cette fois la configuration geopolitique du secteur des nouvelles technologies).

Modèle de la crise des dotcoms en 2000-2001, d'aileurs. Mais cette crise a débouché sur le fameux web2 d'O'Reilly en 2005, plus participatif, etc mais surtout fondé sur un nouveau modèle économique prédateur, le capitalisme de surveillance décrit par Zuboff.

Je ne suis pas fan des prédictions (la damnation des historiens), même si je pense qu'il y aura éclatement de la bulle: toutefois, il n'y a pas de certitude qu'elle éclate effectivement. Il y a d'autres trajectoires de sortie des bulles spéculatives.

Là aussi, je suis un peu gêné, car ces propos ne sont valables que pour les plus grands modèles de données, aujourd'hui pour la plupart (exception de deepseek et mistral3) accessibles que par les chatbots et/ou des APIs spécifiques (pour anthropic et openai notamment, google également). C'est beaucou pmoins vrai pour les petits modèles, qui sont pourtant de plus en plus performants.

J'ai un doute ici. Il faudrait peut-être éclaircir les choses: il y a tout un champs de recherche sur les données synthétiques et leur efficacité / inefficacité pour entraîner un modèle de langue. PleIAs par exemple a sorti un jeu de données synthétiques pour l'entraînement de petits modèles, jeux de donées extrapolé à partir de plus de 50 000 articles de wikipedia (si j'ai bien compris). Dans votre exemple, il me semble que vous pensez plutôt à la possibilité que les grandes composantes des données d'entraînement (wikipedia pour les articles qui sont semble-t-il de plus en plus générés par des bots IA, commoncrawl qui intègre une grande partie du web, donc de nombreuses pages désormais générées par IA, etc) contiennent de plus en plus de données engendrées automatiquement, et surtout du domaine du 'AI Slop'. Je pense que ce sont deux cas bien différent, et l'usage du terme 'synthétique' est trop ambigu ici.

oui! Je ne vois pas de référence à A. Casilli, mai cela pourrait être pertinent de la rajouter.

Je pense particulièrement au rapport du diplab qu'il a co-écrit sur le digital labor dans deepseek: https://ip-paris.hal.science/hal-04952735/file/THE%20HUMAN%20COST%20OF%20DEEPSEEK-DiPLab%20Policy%20Memo%201%281%29.pdf

Je me demande ce que serait l'usure, ici d'un modèle de langue ou d'un chatbot? Ce serait intéressant d'avoir une petite réflexion là-dessus.

Je pense qu'il y a des arguments qui contredisent cette phrase. Alors, n'étant pas un spécialiste de Marx, je suis susceptble de n'avoir compris ce que vous impliquez ici. Mais je vois aussi dans cette affirmation une référence implicite à l'article 'stochastic parrots (bender et al.). Or il a été contesté depuis.

Surtout, pour être le plus rigoureux possible, il faudrait séparer la référence au modèle de langue et la référene au chatbot: il y a beaucoup plus qu'un modèle de langue derrière un chatbot, même si c'en est le coeur, avec de très nombreuses opérations avant et après la requête au chatbot elle-même. Quid de ces opérations en termes de 'travail mort'?

La question de la différence entre chatbot et modèle au sens strict, me semble importante, car l'on pourrait argumenter que la création de valeur se situe non pas dans les réponses des chatbots, mais bien dans l'interaction entre l'utilisateur et le chatbot.

En dehors du fantastique "que sauver de Marx?", le texte de gemini laisse un peu le lecteur sur sa faim, car d'une très grande platitude. Il serait intéressant de donner au lecteur pour le paragraphe 18, des citations peut-être plus concrètes du discours public des plateformes (il y a beaucoup de textes de ce type chez Sam Altman, par exemple). Il y a certes la référence au blog de microsoft, mais pas tellement plus.

Faire un lien avec des exemples passés? Je pense aux dotcoms de la find es années 1990, qui se sont effondrées en 2000-2001, sauf celles qui ont pu adopter le capitalisme de la surveillance, bien sûr.

Cher auteur -- les remarques ci-dessous sont faites au fur et à mesure de ma lecture de votre article. Il peut donc y avoir des interrogations auxquelles vous répondez plus tard dans l'article.

Synthèse sur la Maltraitance Infantile : Thèmes, Intervenants et Cas d'Étude

Synthèse Exécutive

Ce document de synthèse analyse les thèmes centraux, les dynamiques et les conséquences de la maltraitance infantile, en se basant sur une série d'études de cas et d'interventions d'experts.

L'analyse révèle que la maltraitance est un phénomène polymorphe, incluant la violence physique extrême, le syndrome du bébé secoué, les abus sexuels et les négligences graves.

Une conclusion alarmante émerge : dans la majorité des cas (neuf sur dix), les sévices sont infligés au sein même de la cellule familiale, transformant le lieu de sécurité supposé en principal foyer de danger.

Le silence des victimes, la complicité passive ou active de certains membres de la famille et l'aveuglement de l'entourage constituent des obstacles majeurs à la protection des enfants.

La chaîne d'intervention, bien que complexe, est clairement définie : elle commence par une alerte (via le 119 ou un signalement médical), se poursuit par une enquête policière (Brigade des Mineurs), aboutit à une réponse judiciaire (Procureur, Juge des enfants) et se conclut par une prise en charge spécialisée (placement, suivi psychologique).

Les séquelles de la maltraitance sont profondes et durables, affectant les victimes sur les plans physique, psychologique et comportemental.

Néanmoins, les témoignages de résilience, illustrés par des parcours de reconstruction personnelle et la recréation de liens affectifs, soulignent que la guérison, bien que longue et ardue, reste possible grâce à un soutien adéquat et continu.

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1. Les Multiples Visages de la Maltraitance

La maltraitance infantile se manifeste sous diverses formes, souvent cumulatives, allant des sévices physiques aux abus psychologiques et sexuels.

Violences Physiques et Sévices Graves

La violence physique est la forme la plus visible de la maltraitance. Les statistiques présentées sont alarmantes : chaque semaine en France, trois enfants meurent des suites de mauvais traitements.

• Le cas de Gaël : Adolescent de 15 ans, il a été victime de violences extrêmes de la part de son père pendant 21 mois, à l'âge de 8 ans. Ses témoignages décrivent un calvaire :

◦ Brûlures de cigarettes.  

◦ Coups portés avec divers objets : manche à balai, fourchette à poulet, bouteille de verre, assiette, pare-chocs de voiture.  

◦ Tentative de noyade dans la baignoire.  

◦ Humiliations extrêmes, comme être forcé à manger des excréments de chien.

Son père a été condamné à 14 ans de prison ferme.

Gaël utilise aujourd'hui la boxe à haut niveau pour "dégager la haine" et se reconstruire.

• Le cas de Dylan : Enfant de 4 ans décédé en 2003, son corps présentait d'innombrables traces de coups, de morsures et de brûlures de cigarettes, infligées par son beau-père.

Il était devenu son "souffre-douleur" depuis plusieurs mois.

Le Syndrome du Bébé Secoué

Une forme de violence spécifique aux nourrissons est mise en évidence : le syndrome du bébé secoué.

• Mécanisme : Le Dr Philippe Meyer explique qu'il ne s'agit pas d'un simple jeu, mais de "mouvements très répétitifs" et "extrêmement violents".

La tête du bébé, très lourde et mal soutenue par les muscles du cou, subit des accélérations et décélérations qui provoquent des hémorragies cérébrales (hématome sous-dural).

• Prévalence : L'hôpital Necker reçoit plus de 50 bébés par an présentant ces symptômes.

• Conséquences : Les séquelles peuvent être irréversibles, et un bébé secoué sur dix en meurt.

• Cas étudiés :

◦ Louis (6 jours) : Arrivé pour un hématome sous-dural, son cas est d'autant plus suspect que son frère est décédé cinq ans plus tôt dans des circonstances similaires, conduisant les médecins à faire un signalement au procureur.  

◦ Willy (3,5 mois) : Admis pour le même symptôme, son père avoue lui avoir porté un coup lors d'une dispute.

Il reconnaît son geste : "J'ai craqué [...] j'ai fait ces gestes là j'ai regretté".

Abus Sexuels

Les abus sexuels, souvent perpétrés par des proches, sont une autre facette de la maltraitance.

• Le cas d'Elena (7 ans) : La fillette se plaint d'avoir été touchée par Yvon, l'ami de sa grand-mère.

L'enquête de la Brigade des Mineurs révèle que l'agresseur présumé a déjà des antécédents pour "agression sexuelle sur mineur" en 1998. Confronté, il avoue les faits.

• Le cas d'Estelle : Violée de 2 à 12 ans par son grand-père maternel, elle n'a osé en parler que dix ans plus tard.

Son parcours illustre la difficulté de la révélation et le poids de la culpabilité et de la honte, qui se sont traduits par des conduites à risque (tentatives de suicide, drogue) à l'adolescence.

Négligences Graves et Violences Psychologiques

La maltraitance ne se limite pas aux actes de commission.

• Le placement d'un enfant de 8 ans : La Brigade des Mineurs intervient pour retirer un enfant de sa famille suite à des "graves négligences". L'enfant n'est pas scolarisé et les services sociaux n'ont plus accès à la famille.

• Le cas de Marie : Placée à 15 ans, elle a fui une famille où, au-delà des violences physiques, régnait une "violence psychologique permanente". Elle témoigne : "chaque fois je faisais quelque chose ma mère me disait que ça allait pas tout le temps tout le temps". Cette emprise psychologique l'a conduite à des pensées suicidaires.

2. L'Environnement Familial : Principal Foyer de Danger

Le documentaire souligne de manière récurrente que le danger provient le plus souvent de l'entourage immédiat de l'enfant.

La Responsabilité des Auteurs et le Silence Complice

Les auteurs des violences sont les parents, beaux-parents ou des proches. Le silence d'un des parents peut être assimilé à une forme de complicité.

• Le cas d'Adeline, mère de Dylan : Elle est jugée pour ne pas avoir dénoncé les violences infligées par son compagnon à son fils.

Elle a retiré Dylan de l'école pour cacher ses blessures. Son procès en appel aboutit à une peine alourdie à 20 ans de réclusion criminelle.

Pour l'avocat du père de Dylan, son comportement n'était pas un simple silence mais une "dissimulation" active des faits, court-circuitant toute aide possible.

L'Aveuglement et la Culpabilité de l'Entourage

L'entourage élargi peine souvent à percevoir ou à admettre la réalité de la maltraitance, ce qui engendre une profonde culpabilité a posteriori.

• L'entourage de Gaël : La mère de Gaël, Carole, a lutté seule pendant deux ans pour récupérer son fils, séquestré par son ex-mari.

Les grands-parents expriment leur regret : "Carole disait toujours mon enfant est en danger et nous autour d'elle, on le croyait pas [...] on ne peut pas imaginer qu'on s'est rendu compte de rien." Ils avouent même avoir pensé qu'elle "amplifiait la chose".

• L'indifférence du voisinage : Gaël raconte avoir dormi en slip sur le toit du garage, visible par des centaines de personnes, y compris les parents et enfants de l'école voisine. "Personne a jugé bon de signaler qu'il y avait un souci, c'est inadmissible."

3. La Chaîne d'Intervention : Du Signalement à la Protection

Le processus de prise en charge d'un enfant en danger implique une succession d'acteurs institutionnels.

| Étape | Acteurs Clés | Actions et Observations | | --- | --- | --- | | L'Alerte | Ligne 119, entourage, écoles, médecins | Le service du 119 reçoit plus de 4000 appels par jour. L'alerte est le point de départ crucial qui déclenche l'intervention. | | Le Diagnostic Médical | Médecins hospitaliers (pédiatres, réanimateurs) | Ils sont en première ligne pour détecter les signes physiques (hématomes, fractures). Leur rôle est de soigner mais aussi de signaler les suspicions aux autorités judiciaires, comme dans le cas de Louis. | | L'Enquête Policière | Brigade de Protection des Mineurs | Les policiers mènent des auditions et des interrogatoires pour établir les faits. Leur travail consiste à démêler le vrai du faux face aux dénégations initiales des parents (cas du bébé secoué) ou à obtenir les aveux (cas d'Elena). | | La Réponse Judiciaire | Procureur de la République, Juge des enfants | Le procureur décide des suites à donner (mise en examen, contrôle judiciaire, procès). Le juge des enfants prend les mesures de protection nécessaires (enquête sociale, placement) et évalue la sécurité de l'enfant dans son milieu familial (cas d'Elena et de Marie). | | Le Placement et le Soin | Foyers, pouponnières, éducateurs spécialisés, pédopsychiatres | Lorsque le danger est avéré, les enfants sont retirés de leur famille et placés dans des structures spécialisées. Le placement est souvent un traumatisme, comme le montre l'intervention forcée pour l'enfant de 8 ans. Le soin vise à "réparer" les traumatismes (cas de Roxane et Charlotte à la pouponnière). |

4. Les Séquelles et le Chemin de la Reconstruction

Les conséquences de la maltraitance sont profondes et nécessitent un travail de reconstruction de longue haleine.

Traumatismes Physiques et Psychologiques

• Séquelles physiques : Gaël conserve de multiples cicatrices de ses blessures.

• Séquelles psychologiques : Me Brun Meyrin, l'avocate de Gaël, souligne : "Il a surtout des séquelles morales dont on se demande bien comment elles pourraient ne pas avoir de conséquences dans son futur."

La psychologue Martine Nisse explique que la communication paradoxale dans les familles maltraitantes ("c'est pour ton bien que je te frappe") rend les enfants "difficiles à comprendre".

• Comportements post-traumatiques : Les enfants placés en pouponnière manifestent des troubles du comportement :

Roxane, exposée à la violence, développe de l'agressivité et des difficultés relationnelles ; Charlotte, bébé secoué, a appris à "éviter la relation" en se protégeant du contact physique.

La Thérapie comme Voie de Guérison

Le suivi psychologique est essentiel pour surmonter le traumatisme.

• Le cas d'Estelle : Après quatre ans de thérapie, elle a pu mettre des mots sur l'inceste subi et déconstruire le sentiment de culpabilité. "Là j'ai compris vraiment que j'y étais pour rien [...] la honte elle reste mais elle s'estompe."

• L'importance de la parole : L'éducateur de Marie souligne que "le fait qu'il y ait une intervention du commissariat [...] n'a pas réglé les problèmes". Il a fallu deux ans et demi pour qu'elle arrive progressivement à "prendre en main sa vie".

La Résilience et la Reconstruction des Liens

Malgré la gravité des faits, des parcours de résilience sont possibles.

• Gaël : La boxe lui sert d'exutoire et il retisse un lien fort avec sa mère, Carole. Il parvient à formuler : "Grâce à ma mère, je suis là."

• Marie : Bien qu'inquiète de sa majorité, elle demande à la juge de continuer à la protéger, montrant sa volonté de se construire un avenir stable.

• Cindy, mère de Roxane : En désintoxication et séparée de son conjoint violent, elle s'engage dans un processus pour recréer un lien avec ses enfants et espère pouvoir un jour les récupérer.

5. Citations Clés

• Gaël, victime de violences paternelles : "C'est pour pouvoir me défendre, c'est pour pouvoir dégager la haine que j'ai sur lui."

• Père de Willy, auteur de secouement : "J'ai craqué [...] je pardonne pas parce qu'on fait pas ça à un bébé mais je sais que ça peut arriver à n'importe qui."

• Grand-père de Gaël, sur sa culpabilité : "Carole disait toujours mon enfant est en danger et nous autour d'elle, on le croyait pas [...] je m'imaginais jamais ce qui se passe."

• Maître Bejo, avocat du père de Dylan : "On n'est pas dans le silence, on est dans un comportement actif de dissimulation des faits et c'est ce comportement actif qui a court-circuité toutes les velléités d'intervention."

• Dr Renier, pionnier sur le syndrome du bébé secoué : "Ce qui fait la différence entre un bien-traitant pour un bébé et un non bien-traitant [...] c'est la maîtrise et la maîtrise elle est indispensable en toutes circonstances."

• Françoise Achard, médecin scolaire, aux enseignants : "On sait que tout le monde peut être maltraitant, c'est-à-dire que ces parents qui avaient l'air bien sympathiques, et ben ça veut pas dire pour autant qu'ils soient pas maltraitants dans l'intimité de leur maison."

• Martine Nisse, psychologue : "Je crois que les principaux sévices c'est la famille, c'est le principal danger pour l'enfant."

• Carole, mère de Gaël : "On essaie de récupérer mais on récupérera jamais ces années, c'est des années qui vont nous manquer toujours."

Note d'information : La Stratégie d'Expansion du Groupe Emeis (ex-Orpea) dans le Secteur de la Psychiatrie en France

Synthèse Exécutive

Cette note d'information analyse la stratégie d'expansion du groupe privé lucratif Emeis (anciennement Orpea) dans le secteur de la psychiatrie en France.

Elle s'appuie sur une enquête journalistique qui met en lumière comment le groupe, marqué par le scandale de ses EHPAD, capitalise sur la crise profonde de la psychiatrie publique pour s'implanter sur ce marché jugé très rentable.

L'analyse révèle une situation de crise systémique dans le secteur public : un sous-financement chronique, un manque criant de personnel (seulement 600 pédopsychiatres en France), des infrastructures vétustes et une explosion de la demande de soins, notamment chez les jeunes depuis la crise du Covid (+77 % d'épisodes dépressifs chez les 18-24 ans).

Dans ce contexte, Emeis déploie une stratégie agressive pour s'imposer, illustrée par un projet de clinique de 80 lits près de Strasbourg.

Cette implantation, menée via sa filiale Clinea, s'est initialement appuyée sur une alliance "étonnante" avec un concurrent, Clinipsy.

L'enquête suggère que cette alliance aurait pu servir de "cheval de Troie" pour Emeis, lui permettant d'obtenir des autorisations administratives que le groupe, sous le nom d'Orpea, s'était vu refuser à plusieurs reprises depuis 2007.

Les principales préoccupations soulevées sont le risque d'affaiblissement de l'hôpital public par le débauchage de son personnel, une complémentarité illusoire où le privé se concentrerait sur les cas les plus rentables en laissant les plus complexes au public, et un modèle économique basé sur la rentabilité qui pourrait se faire au détriment de la qualité des soins par la réduction des effectifs.

Enfin, le document souligne l'opacité de l'Agence Régionale de Santé (ARS) Grand Est, qui a refusé de communiquer des documents essentiels sur ce projet malgré les importants financements publics engagés.

1. Le Contexte : Une Psychiatrie Publique en Crise Profonde

La psychiatrie en France est décrite comme étant "malade" et "abandonnée par les pouvoirs publics".

Ce secteur est devenu le "parent pauvre de la santé", confronté à un manque critique de moyens alors que les besoins de soins explosent.

• Explosion de la demande : La crise du Covid et les confinements ont provoqué une forte augmentation des pathologies mentales.

◦ +77 % d'épisodes dépressifs chez les 18-24 ans.    ◦ +133 % d'hospitalisations pour tentative de suicide ou automutilation.

• Manque de moyens structurel : Le secteur public souffre d'un sous-investissement chronique.

◦ Une politique ambulatoire non financée : Depuis les années 1980, une politique de fermeture de lits a été menée au profit de soins ambulatoires (hors de l'hôpital).

Cependant, les moyens financiers n'ont pas suivi pour développer ces structures alternatives comme les Centres Médico-Psychologiques (CMP).  

◦ Pénurie de personnel : La France compte environ 600 pédopsychiatres, laissant des départements entiers sans spécialiste.  

◦ Diminution des capacités : L'hôpital public a perdu près de 7 000 places de prise en charge psychiatrique à temps complet en 15 ans.

• Vétusté des infrastructures : L'état des bâtiments publics est alarmant.

À Strasbourg, le secteur de la pédopsychiatrie des Hôpitaux Universitaires est logé dans des "bâtiments complètement vétustes" et des "préfabriqués".

L'Inspection générale des affaires sociales (Igas) a signalé des risques d'incendie et demandé un déménagement en urgence, qui n'a été annoncé que 15 ans plus tard.

"On a alerté qu'on allait droit dans le mur et le mur aujourd'hui on se le prend en pleine face." - Un soignant, cité dans le documentaire de Laurence Deur.

2. L'Émergence d'un Nouvel Eldorado : Le Secteur Privé Lucratif

La défaillance du système public crée une opportunité majeure pour les groupes privés à but lucratif, qui considèrent la psychiatrie comme un "marché très rentable".

• Un secteur profitable : Selon un rapport récent du Sénat, la psychiatrie est l'un des secteurs de la santé les plus rentables, avec des marges estimées entre 5 % et 8 %.

L'investissement principal étant l'humain, la réduction du personnel est le principal levier pour augmenter les profits.

• Une croissance rapide : La part du secteur privé lucratif dans l'offre de soins psychiatriques a considérablement augmenté :

◦ 1975 : 11 % des lits.    ◦ Aujourd'hui : Plus de 30 % des lits.

• Un parallèle avec les EHPAD : La situation actuelle en psychiatrie est comparée à la privatisation du secteur des EHPAD dans les années 1980.

Face à des établissements publics vieillissants et coûteux à rénover, l'État avait ouvert la porte au privé qui promettait de "faire moins cher, plus vite".

• Le rôle des ARS : Les Agences Régionales de Santé, autrefois réticentes à ouvrir la psychiatrie au privé, sont aujourd'hui plus enclines à le faire.

Face à l'incapacité du public à répondre à la demande immense, elles autorisent l'ouverture de cliniques et d'hôpitaux de jour privés.

3. Étude de Cas : La Stratégie d'Implantation d'Emeis à Strasbourg

L'enquête se concentre sur un projet de clinique psychiatrique privée de 80 lits à Schiltigheim, près de Strasbourg, porté par le groupe Emeis (ex-Orpea), rebaptisé pour faire oublier le scandale révélé par le livre Les Fossoyeurs. Ce projet est jugé "démesuré" et "anachronique" par les acteurs locaux.

Une Alliance Stratégique Inédite

Le projet est né d'une alliance "étonnante" entre deux concurrents :

1. Clinea : La filiale sanitaire d'Emeis/Orpea.

2. Clinipsy : Un acteur plus petit, déjà connu pour une enquête du Parquet National Financier (PNF) concernant des autorisations obtenues en région Rhône-Alpes par d'anciens fonctionnaires de l'ARS locale, ensuite embauchés par des filiales du groupe.

Cette collaboration entre concurrents directs est jugée inhabituelle, comparable à "si Intermarché et Leclerc montaient un supermarché ensemble".

L'Hypothèse du "Cheval de Troie"

L'enquête soulève l'hypothèse que cette alliance aurait servi de stratégie à Emeis pour contourner des obstacles réglementaires.

• Dissimulation : Emeis se serait "dissimulé un petit peu" derrière le nom de Clinipsy, un groupe plus petit avec une "moins mauvaise image" auprès des ARS, pour obtenir plus facilement les autorisations.

• Historique des refus : Des documents montrent qu'Orpea tentait d'ouvrir une clinique psychiatrique dans la région depuis au moins 2007 et avait essuyé au moins deux refus de la part de l'agence régionale (alors ARH).

Depuis, Clinipsy s'est désengagé du projet de clinique de 80 lits pour se concentrer sur des hôpitaux de jour, des structures moins coûteuses et "extrêmement rentables", laissant le champ libre à Emeis pour le projet principal.

"La question [...] se pose de savoir si Clinipsy a été un petit peu le cheval de Troie d'Orpea dans cette affaire." - Laurence Deur, journaliste.

4. Les Risques Systémiques de la Privatisation

L'arrivée massive d'acteurs privés lucratifs comme Emeis dans la psychiatrie fait peser plusieurs risques majeurs sur l'équilibre global du système de santé mentale.

Le "Pillage" des Ressources Humaines du Public

La principale inquiétude est que les nouvelles cliniques privées, en offrant de meilleures conditions de travail ou de rémunération, ne débauchent le personnel médical et soignant déjà en sous-effectif dans le secteur public.

• Un exemple concret : Un courrier de 2022 révèle qu'une clinique privée près de Nancy a débauché cinq médecins de l'hôpital public local, fragilisant ce dernier.

• L'inquiétude de la Mairie de Strasbourg : La maire, Jeanne Barségan, craint que le projet de 80 lits n'aggrave la pénurie de psychiatres et ne "vide" l'hôpital public de ses forces.

Une Complémentarité Illusoire : Le "Triage" des Patients

L'offre privée est souvent présentée comme "complémentaire" du public. Cependant, l'analyse montre qu'elle ne remplit pas les mêmes missions.

• Évitement des cas complexes : Le privé évite généralement les missions les plus lourdes et les moins rentables, comme l'hospitalisation sous contrainte, qui nécessite plus de personnel et de temps.

• Gestion des urgences "à la carte" : Dans le projet d'Emeis, la prise en charge des urgences se ferait "de gré à gré", sans obligation contraignante. Le médecin du privé peut accepter ou refuser un patient envoyé par le public.

• La conclusion : "Tout ce qui est complexe reste dans l'hôpital public", tandis que le privé se positionne sur des missions "plus faciles à assurer".

Le Modèle Économique : Profit vs. Qualité des Soins

Emeis est une entreprise cotée en bourse qui doit générer du profit pour ses actionnaires.

• Le levier du personnel : En psychiatrie, où "l'investissement, c'est l'humain", la principale méthode pour augmenter la rentabilité est de réduire les effectifs.

• Conflits sociaux : Plusieurs conflits sociaux ont éclaté dans des cliniques psychiatriques d'Emeis (Thionville, Nord, Isère) où le personnel dénonçait un manque d'effectifs et une réorganisation du travail impactant la qualité des soins.

Une grève de trois semaines a eu lieu à Seyssins, un événement "extrêmement rare dans le privé".

"Une entreprise est là pour faire du profit alors que l'hôpital public on lui demande pas d'être profitable, on lui demande d'être à l'équilibre." - Laurence Deur, journaliste.

5. Le Rôle et l'Opacité des Autorités de Régulation

L'enquête met en cause le manque de transparence de l'Agence Régionale de Santé (ARS) Grand Est.

• Rétention d'information : La journaliste a été "baladée pendant un mois et demi" sans obtenir de réponse ni les documents demandés concernant le projet de clinique Emeis.

L'ARS a fini par envoyer un document public générique qui ne correspondait pas à la demande.

• Recours à la CADA : Il a fallu saisir la Commission d'Accès aux Documents Administratifs (CADA) pour obtenir une partie des informations.

• Enjeux financiers publics : Cette opacité est jugée problématique car le projet engage d'importants fonds publics.

Les autorisations délivrées "valent des millions d'euros" et le groupe peut prétendre à une "dotation d'amorçage" de l'État pour financer son démarrage.

Cette situation soulève des questions sur le contrôle et la régulation de l'expansion du secteur privé lucratif, financée en partie par de l'argent public, dans un domaine aussi sensible que la santé mentale.

Synthèse du rapport : Protection de l’enfance et maltraitances — État des lieux 2025

Résumé Exécutif

Ce document de synthèse présente les principales conclusions du rapport "Protection de l’enfance et maltraitances — État des lieux 2025", publié par l’Observatoire national de la protection de l’enfance (ONPE).

L'analyse des données, arrêtées au 31 décembre 2023, révèle plusieurs tendances structurelles profondes qui redéfinissent le paysage de la protection de l'enfance en France.

Au 31 décembre 2023, 364 200 prestations et mesures étaient en cours pour les mineurs et 33 400 pour les jeunes majeurs, des chiffres en augmentation significative sur la dernière décennie.

Les dynamiques clés sont les suivantes :

1. Une croissance globale et continue : Le nombre total d'interventions pour les mineurs a augmenté de 22 % entre 2013 et 2023.

Le taux de prise en charge pour 1 000 mineurs a quant à lui progressé de 27 % sur la même période, passant de 20,3 ‰ à 25,8 ‰, une hausse accentuée par la baisse démographique de cette tranche d'âge.

2. Un basculement structurel vers l'accueil : Pour la première fois depuis le début du suivi, l'accueil (placement hors du domicile familial) est devenu majoritaire, représentant 52,2 % des interventions pour les mineurs.

Cette inversion de tendance, amorcée en 2018, marque un changement profond par rapport au suivi en milieu ouvert (à domicile).

3. La prédominance de l'hébergement en établissement : Une seconde inversion de tendance est observée dans les modalités d'accueil.

L'hébergement en établissement (41 %) dépasse désormais l'accueil familial traditionnel chez les assistants familiaux (36 %), qui voit sa part diminuer de manière continue.

4. Une judiciarisation accrue des mesures : La part des interventions décidées par un juge ne cesse de croître, atteignant 82,4 % de l'ensemble des mesures pour mineurs en 2023, contre 78,6 % en 2013.

Cette tendance est particulièrement marquée pour les mesures d'accueil (92,1 %).

5. L'impact majeur des mineurs non accompagnés (MNA) : La forte augmentation du nombre de MNA pris en charge (46 200 mineurs et jeunes majeurs fin 2023) influence profondément les statistiques globales, notamment la hausse des accueils, la prédominance masculine chez les adolescents et l'augmentation des saisines judiciaires.

6. Des disparités territoriales persistantes et croissantes : Des écarts considérables subsistent entre les départements, que ce soit pour les taux de prise en charge globaux, les taux de judiciarisation ou les modalités d'intervention. Ces disparités tendent à se creuser au fil du temps.

7. Une attention renforcée aux jeunes majeurs : Bien qu'en légère baisse depuis un pic en 2021, le soutien aux 18-20 ans a fortement augmenté sur dix ans (+53 %).

Le taux de poursuite de l'accompagnement après la majorité a atteint 52 % en 2023, retrouvant son niveau d'avant 2013, signe d'une politique active contre les "sorties sèches".

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1. Vue d'ensemble de la prise en charge en protection de l'enfance

1.1. Augmentation continue des interventions

Le nombre d'interventions en protection de l'enfance pour les mineurs (0-17 ans) a connu une croissance soutenue sur la dernière décennie.

• Nombre total d'interventions : Au 31 décembre 2023, 364 200 prestations administratives et mesures judiciaires étaient en cours, soit une augmentation de 22 % par rapport à 2013 (297 500).

• Taux de prise en charge : Le taux d'intervention pour 1 000 mineurs est passé de 20,3 ‰ en 2013 à 25,8 ‰ en 2023, une augmentation de 27 %.

Cette hausse est plus rapide que celle des effectifs en raison d'une diminution de 4 % de la population des moins de 18 ans sur la même période.

• Estimation du nombre de mineurs : En croisant diverses sources (DREES, Olinpe), le nombre de mineurs uniques suivis est estimé à environ 351 500 au 31 décembre 2023.

| Année | Nombre de prestations et mesures | Taux pour 1 000 mineurs | | --- | --- | --- | | 2013 | 297 500 | 20 ‰ | | 2017 | 342 900 | 23 ‰ | | 2020 | 338 600 | 24 ‰ | | 2022 | 347 100 | 24 ‰ | | 2023 | 364 200 | 26 ‰ |

Source : DREES, DPJJ, Insee, calculs ONPE

La croissance a été particulièrement marquée entre 2022 et 2023, avec une hausse de 5 %.

Cette dynamique fait écho à l'augmentation des saisines des juges des enfants observée après la crise sanitaire.

1.2. Disparités territoriales croissantes

Les écarts de prise en charge entre les départements non seulement persistent mais se sont accentués entre 2013 et 2023.

• Écart des taux : Au 31 décembre 2023, le taux de prise en charge des mineurs variait de 13,5 ‰ (Yvelines) à 48,1 ‰ (Nièvre). En 2013, l'écart était moins prononcé, allant de 10,9 ‰ à 37 ‰.

• Tendances géographiques :

◦ Les taux les plus faibles se concentrent majoritairement en Île-de-France et en Auvergne-Rhône-Alpes.  

◦ Les taux les plus élevés sont observés dans des territoires souvent moins densément peuplés.

• Évolutions hétérogènes : Entre 2013 et 2023, le taux de prise en charge a augmenté dans 98 départements, mais avec des variations extrêmes, allant de -7 % (Hauts-de-Seine, Loiret) à +101 % (Lozère).

2. La dynamique des types d'intervention

2.1. Une judiciarisation accrue

La prise en charge des mineurs est majoritairement décidée par l'autorité judiciaire, et cette tendance se renforce.

• Part des mesures judiciaires : Au 31 décembre 2023, 82,4 % des 364 200 interventions résultaient d'une décision judiciaire, contre 78,6 % en 2013.

• Répartition par type d'intervention :

◦ Accueil : 92,1 % des mesures sont judiciaires.    ◦ Milieu ouvert : 71,7 % des mesures sont judiciaires.

• Disparités départementales : Le "taux de judiciarisation" varie fortement, de 66,9 % (Morbihan) à 94,9 % (Seine-Saint-Denis).

Les départements des Hauts-de-France et du Grand-Est affichent des taux particulièrement élevés.

2.2. Le basculement vers l'accueil au détriment du milieu ouvert

Un changement structurel majeur s'est opéré : le nombre de placements d'enfants (accueil) dépasse désormais le nombre d'interventions à domicile (milieu ouvert).

• Inversion de la tendance : En 2023, les mesures d'accueil s'élèvent à près de 190 300 (52,2 % du total), tandis que les mesures en milieu ouvert sont de 174 000 (47,8 %). Le point de bascule s'est produit en 2018.

• Croissance différentielle (2013-2023) :

◦ Le taux de mineurs accueillis a augmenté de 40 % (passant de 9,7 ‰ à 13,5 ‰).    ◦ Le taux de mineurs suivis en milieu ouvert a augmenté de 16 % (passant de 10,7 ‰ à 12,3 ‰).

• Facteurs explicatifs : Cette évolution est notamment liée à la forte augmentation des accueils de mineurs non accompagnés (MNA) et au développement de nouvelles mesures comme le "placement à domicile", comptabilisées comme de l'accueil.

3. L'accueil des mineurs : modalités et profils

3.1. Évolution des modes d'hébergement : l'établissement devance la famille d'accueil

Pour la deuxième année consécutive, l'accueil en établissement est la modalité la plus fréquente, devant l'accueil familial traditionnel.

| Mode d'hébergement | Part en 2013 | Part en 2023 | Évolution en effectifs (2013-2023) | | --- | --- | --- | --- | | Établissement | 38 % | 41 % | +50 % | | Famille d'accueil | 52 % | 36 % | \-4 % (depuis 2019) | | Hébergement autonome | 4 % | 6 % | +143 % | | Autres modes d'hébergement | 6 % | 17 % | +321 % |

Source : DREES, calculs ONPE. Champ : Mineurs et jeunes majeurs confiés à l'ASE.

• La catégorie "Autres modes d'hébergement" inclut les placements chez un tiers digne de confiance, en internat, l'accueil durable et bénévole, etc. Son explosion est un facteur clé de la restructuration du secteur.

• Cette tendance coïncide avec une baisse de 11 % du nombre d'assistants familiaux employés par les départements entre 2016 et 2023.

3.2. Le placement direct : le recours croissant au "tiers digne de confiance"

Le placement direct, décidé par un juge sans passer par une mesure de confiement à l'ASE, évolue également.

• Au 31 décembre 2023, 17 100 enfants bénéficiaient d'un placement direct.

• La part des placements chez un "tiers digne de confiance" a fortement augmenté, passant de 69 % en 2013 à 86 % en 2023.

• Cette évolution est directement liée à la loi du 7 février 2022, qui systématise la recherche d'un membre de la famille ou d'un proche pour accueillir l'enfant.

3.3. Profils démographiques des enfants accueillis

• Prédominance masculine : Les garçons représentent 59 % des mineurs accueillis (hors placement direct).

Ce déséquilibre s'accentue avec l'âge, atteignant 69 % chez les 16-17 ans, principalement en raison de la population de MNA (à plus de 90 % masculine).

• Répartition par âge : Entre 2015 et 2023, la croissance des accueils a été la plus forte aux âges extrêmes : +38 % pour les moins de 6 ans et +50 % pour les 16-17 ans.

• Profil en placement direct : La population en placement direct est très différente, avec un équilibre quasi parfait entre les sexes (50,2 % de filles) et une part plus importante de 11-15 ans (39 % contre 34 %).

4. La situation spécifique des jeunes majeurs (18-20 ans)

4.1. Tendances générales et disparités

L'accompagnement des jeunes majeurs a connu une croissance massive, bien qu'en léger recul depuis 2021.

• Effectifs : 33 400 jeunes majeurs étaient pris en charge fin 2023, soit une augmentation de 53 % depuis 2013. Le pic a été atteint en 2021 avec 35 100 jeunes.

• Nature de l'intervention : La prise en charge est quasi exclusivement administrative (99,8 %) et consiste très majoritairement en un accueil (92,2 %).

• Taux de prise en charge : Le taux national est de 13,6 ‰, mais les disparités départementales sont extrêmes, allant de 1,6 ‰ (Hautes-Alpes) à 28,5 ‰ (Allier).

4.2. La poursuite de l'accompagnement après 18 ans

Un nouvel indicateur, le "taux de poursuite en Accueil Provisoire Jeunes Majeurs (APJM)", mesure la probabilité pour un jeune confié à 17 ans de continuer à être hébergé après sa majorité.

• Après une chute à un niveau plancher de 37 % en 2018, ce taux a connu une remontée spectaculaire pour atteindre 52 % en 2023.

• Cette hausse s'explique par les mesures liées à la crise sanitaire puis par la Stratégie nationale de prévention et de protection de l’enfance, qui a fait de la lutte contre les "sorties sèches" un objectif prioritaire.

5. Facteurs d'influence et dynamiques transversales

5.1. L'impact des mineurs non accompagnés (MNA)

Les MNA constituent une part croissante et influente de la population protégée.

• Effectifs : Au 31 décembre 2023, 46 200 mineurs et jeunes majeurs MNA étaient pris en charge, une hausse de 17 % en un an.

• Répartition : 65 % sont mineurs et 35 % sont de jeunes majeurs. Cette proportion de jeunes majeurs a diminué depuis son pic à 50 % en 2021.

• Influence statistique : Les MNA contribuent significativement à la hausse du nombre d'accueils, à la surreprésentation des garçons de 16-17 ans et à l'augmentation des saisines judiciaires.

5.2. L'augmentation des saisines des juges des enfants

L'activité judiciaire en assistance éducative est en forte croissance.

• En 2023, les juges des enfants ont été saisis pour 124 117 nouveaux mineurs, un chiffre en hausse de 10 % par rapport à 2022 et de 50 % depuis 2013.

• Le rapport note une corrélation entre la courbe des saisines judiciaires et celle des évaluations de minorité pour les MNA, suggérant un lien de cause à effet partiel.

Analyse de l'Avis du CESE sur les Temps de Vie de l'Enfant

Résumé Exécutif

Cet avis du Conseil économique, social et environnemental (CESE), intitulé « Satisfaire les besoins fondamentaux des enfants et garantir leurs droits », dresse un constat critique de la situation des enfants en France, dont les temps de vie sont davantage structurés par les contraintes des adultes que par leurs propres besoins fondamentaux.

Fruit d'une saisine gouvernementale faisant suite à une Convention citoyenne, le rapport souligne un décalage majeur entre les droits constitutionnels et internationaux de l'enfant et leur application effective, particulièrement pour les plus vulnérables.

Les principales conclusions révèlent des inégalités sociales, territoriales et économiques profondes qui entravent le développement, la santé et le bien-être des enfants.

L'avis pointe du doigt des rythmes scolaires inadaptés, une sédentarité croissante, un manque de sommeil chronique, une surexposition aux écrans, et une déconnexion préoccupante de la nature.

La pression sur les familles, notamment monoparentales, et le manque de coordination entre les acteurs éducatifs aggravent ces constats.

Pour y remédier, le CESE formule 19 préconisations interdépendantes visant une transformation systémique. Celles-ci incluent des mesures politiques fortes comme l'instauration d'une « clause impact enfance » dans chaque projet de loi, une réforme ambitieuse des rythmes scolaires sur la base des besoins physiologiques, et la création d'un Service Public de la Continuité Éducative (SPCE) pour assurer une meilleure coordination des acteurs.

L'avis appelle également à renforcer le soutien à la parentalité, à garantir l'accès de tous les enfants aux loisirs, à la culture et aux activités de plein air, et à allouer des financements publics pérennes pour faire de l'enfance un véritable investissement d'avenir.

Introduction et Contexte

En réponse à une saisine du Premier ministre de mai 2025, le CESE a élaboré cet avis suite aux travaux d'une Convention citoyenne dédiée aux temps de vie des enfants. Cent trente-trois citoyens et un panel de vingt enfants et adolescents ont été invités à répondre à la question :

« Comment mieux structurer les différents temps de la vie quotidienne des enfants afin qu’ils soient plus favorables à leurs apprentissages, à leur développement et à leur santé ? ».

Le constat principal de la Convention citoyenne, repris par le CESE, est que les enfants subissent les rythmes effrénés d'une société qui construit leurs temps autour des contraintes des adultes plutôt qu'en réponse à leurs besoins biologiques et de développement.

Le rapport du CESE, s'appuyant sur les 20 propositions citoyennes, formule 19 préconisations qui constituent une position commune de la société civile organisée.

Cet avis s'inscrit dans la continuité de travaux antérieurs du CESE sur l'éducation, la protection de l'enfance et la santé mentale, et vise à proposer des réponses globales et articulées.

Partie 1 : Droits et Besoins Fondamentaux de l'Enfant : Un Constat Alarmant

A. L'Écart entre Droits Reconnus et Réalité Vécue

La France a consacré les droits de l'enfant dans sa Constitution et a ratifié la Convention Internationale des Droits de l'Enfant (CIDE) en 1990, s'engageant sur quatre principes fondamentaux : le droit à la vie, l'intérêt supérieur de l'enfant, la non-discrimination et le respect de son opinion.

Cependant, l'avis du CESE met en lumière une ineffectivité préoccupante de ces droits pour une part significative des enfants.

• Pauvreté et Précarité : En 2023, 21,9 % des enfants de moins de 18 ans vivent sous le seuil de pauvreté monétaire.

À la rentrée 2025, au moins 2 159 enfants se sont retrouvés sans solution d'hébergement.

Ces réalités percutent violemment la capacité de la société à répondre à leurs besoins fondamentaux.

• Critiques Internationales : Le Comité des droits de l'enfant de l'ONU a enjoint la France en 2023 à prendre des mesures urgentes concernant la violence, la protection de l'enfance, la détention d'enfants étrangers, la pauvreté et l'inclusion des enfants en situation de handicap.

• L'« Infantisme » : Le rapport dénonce la persistance de l'« infantisme », un concept désignant les préjugés et la discrimination fondée sur l'âge, qui considère les enfants comme des êtres inférieurs et moins dignes de respect.

Cette culture conduit à ignorer leur parole et leur capacité à être des acteurs sociaux. Pour le combattre, le CESE réaffirme la nécessité d'un débat de société et la création d'un Code de l'enfance.

• Clause « Impact Enfance » : S'inspirant de la « clause impact jeunesse », le CESE préconise (Préconisation #1) d'intégrer un volet enfance dans chaque étude d'impact des projets de loi afin de s'assurer que toute politique publique soit fondée sur le respect des droits de l'enfant.

B. Le Rôle de la Famille et les Obstacles Socio-économiques

La famille est le premier lieu de développement de l'enfant, mais elle fait face à de nombreux obstacles.

• Soutien à la Parentalité : Face à la diversité des modèles familiaux (nucléaire, monoparentale, recomposée...), un soutien renforcé à la parentalité est jugé nécessaire pour aider les parents à répondre aux besoins de leurs enfants (Préconisation #7).

• Inégalités de Genre : Les femmes continuent d'assumer l'essentiel des responsabilités familiales et de la charge mentale, ce qui impacte leur santé et leur carrière.

Le rapport souligne la nécessité d'une répartition équitable des tâches.

• Conciliation Vie Professionnelle/Familiale : Les contraintes professionnelles empiètent sur le temps familial.

Le CESE préconise (Préconisation #2) la transposition complète de la directive européenne sur l'équilibre vie professionnelle-vie personnelle, en créant un droit à des « formules souples de travail » (aménagement du temps, télétravail) négocié dans les branches et la fonction publique.

• Enfants Séparés de leur Famille :

◦ Parents séparés : Il est crucial de soutenir les dispositifs comme les Espaces de rencontre pour préserver la relation parent-enfant tout en prenant en compte le point de vue de l'enfant (Préconisation #3).   

◦ Aide Sociale à l'Enfance (ASE) : L'avis dénonce une crise systémique de la protection de l'enfance, où les droits des enfants confiés, notamment l'accès aux loisirs et à la culture, sont négligés.

Il est préconisé (Préconisation #4) que le Projet Pour l'Enfant (PPE) soit co-construit avec les parents et l'enfant, et qu'il intègre l'ensemble de ses besoins.

Partie 2 : Les Enjeux des Temps et des Espaces de Vie

L'avis analyse en profondeur la manière dont les temps et les espaces de l'enfant sont organisés, révélant de multiples fractures et inadéquations.

A. Les Temps de Vie : Entre Contraintes et Qualité

La vie de l'enfant est rythmée par trois grands temps : familial, scolaire, et les "tiers temps" (périscolaire, extrascolaire).

• Qualité des Temps : Le rapport insiste sur la nécessité d'un équilibre entre temps contraints et temps libre, temps individuel et collectif, activité et repos.

La qualité des interactions avec les adultes et un environnement sécurisant sont déterminants.

Le CESE préconise (Préconisation #6) d'intégrer des temps libres de qualité dans toutes les activités d'apprentissage.

• Le Temps Scolaire : La France se distingue par des journées scolaires longues et un temps d'instruction élevé, sans que cela se traduise par de meilleurs résultats.

Le rythme de la semaine de quatre jours est jugé contraire aux besoins des enfants. Le CESE estime que le statu quo n'est plus tenable et appelle (Préconisation #8) à une évolution des rythmes scolaires :

◦ Premier degré : Réorganiser la journée et la semaine scolaire après concertation.   

◦ Second degré : Adapter les amplitudes horaires aux besoins physiologiques des jeunes (ex: commencer plus tard).   

◦ Calendrier scolaire : Organiser le calendrier hexagonal autour de deux zones de vacances, avec une alternance de 7 semaines de cours et 2 semaines de vacances.

• Les Tiers Temps et le Droit aux Loisirs : Les activités périscolaires et extrascolaires, portées par les associations et les collectivités, sont essentielles mais menacées par le désengagement de l'État et la marchandisation.

L'accès à ces activités, ainsi qu'aux vacances, est fortement marqué par les inégalités sociales.

Un enfant sur deux ne part pas en vacances. Le CESE réaffirme (Préconisation #9) que chaque enfant a droit aux vacances et aux loisirs, et appelle à renforcer le financement des accueils collectifs de mineurs et l'information sur les aides existantes.

B. Les Espaces de Vie : De l'« Enfant d'Intérieur » à la Reconnexion au Dehors

L'environnement physique joue un rôle crucial dans le développement de l'enfant.

• L'« Enfant d'Intérieur » : Le rapport alerte sur le phénomène des « enfants d'intérieur », qui passent de moins en moins de temps à l'extérieur et en contact avec la nature, en raison de la peur du risque, de l'urbanisation centrée sur la voiture et de l'attrait des écrans.

• Repenser l'Aménagement : Il est impératif de repenser l'aménagement des territoires « à hauteur d'enfant », en créant des espaces publics (rues, places) sécurisés, propices au jeu, à la socialisation et aux mobilités douces.

Le CESE préconise (Préconisation #11) d'associer les enfants à l'élaboration des projets d'urbanisme.

• Le Bâti et le Cadre de Vie : Les bâtiments accueillant des enfants (écoles, centres de loisirs) sont souvent inadaptés, notamment face aux enjeux climatiques (vagues de chaleur).

Leur rénovation écologique et leur accessibilité sont des priorités. Toute rénovation doit faire l'objet d'une concertation incluant les enfants et les jeunes (Préconisation #12).

Partie 3 : Leviers d'Action pour la Santé et le Bien-être

L'avis identifie quatre domaines d'action prioritaires pour améliorer la santé physique et mentale des enfants.

• Reconnecter à la Nature : Le contact avec la nature est fondamental pour la santé.

Le CESE appelle à valoriser et accompagner l'éducation au dehors (Préconisation #10) et à garantir que chaque enfant bénéficie d'un accès à des espaces naturels, de sorties régulières et d'au moins un séjour en classe de découverte par cycle scolaire (Préconisation #13).

• Lutter contre le Manque de Sommeil : Le déficit de sommeil touche plus de 30 % des enfants et 70 % des adolescents, avec des conséquences graves sur l'apprentissage et la santé.

Le CESE demande une campagne nationale de sensibilisation (Préconisation #14) et la garantie de temps de repos et de sieste dans toutes les structures, notamment en maternelle (Préconisation #15).

• Favoriser l'Activité Physique : Face à une sédentarité alarmante, il est crucial de faciliter l'accès au sport pour tous. Le CESE préconise (Préconisation #16) une tarification sociale et l'élargissement du dispositif Pass'Sport, récemment restreint.

• Mieux Réguler les Écrans : L'omniprésence des écrans a des effets néfastes documentés (sommeil, sédentarité, exposition à des contenus inappropriés). L'avis souligne la nécessité d'une meilleure régulation et d'un accompagnement à la parentalité numérique.

Partie 4 : Gouvernance, Coordination et Financement

Pour que ces changements soient effectifs, une transformation de la gouvernance des politiques de l'enfance est indispensable.

• Coordination des Acteurs : L'action publique est jugée trop fragmentée. Le CESE préconise (Préconisation #17) de réhabiliter le Projet Éducatif Territorial (PEDT) et d'en faire le volet éducation des Conventions Territoriales Globales (CTG) pour assurer une coordination efficace au niveau local.

• Un Service Public de la Continuité Éducative (SPCE) : Pour garantir une offre éducative cohérente sur tous les temps de l'enfant, l'avis propose la création d'un SPCE (Préconisation #18).

Ce service, confié aux collectivités locales, serait chargé de diagnostiquer les besoins et de planifier les actions en associant tous les acteurs.

• Formation et Financement : La revalorisation des métiers éducatifs et le développement d'une culture commune des droits de l'enfant sont essentiels.

Enfin, le CESE alerte sur l'insuffisance des budgets alloués aux politiques de l'enfance et appelle (Préconisation #19) à un effort budgétaire conséquent et pérenne de l'État et de la Sécurité sociale, considérant ces dépenses comme un investissement fondamental pour l'avenir.

Synthèse des 19 Préconisations du CESE

| Numéro | Thème Principal | Résumé de la Préconisation | | --- | --- | --- | | #1 | Droits de l'enfant | Mettre en œuvre une « clause impact enfance » dans chaque étude d'impact de projet de loi ou de texte réglementaire pour garantir que les politiques publiques respectent les droits de l'enfant. | | #2 | Parentalité & Travail | Créer un droit aux « formules souples de travail » (aménagement du temps, télétravail) pour les parents, par la négociation dans les branches et la fonction publique. | | #3 | Séparation parentale | Développer et soutenir financièrement les Espaces de rencontre pour aider les parents séparés à assumer leurs responsabilités parentales en prenant en compte le point de vue de l'enfant. | | #4 | Protection de l'enfance (ASE) | Rendre le Projet pour l'enfant (PPE) systématiquement co-construit avec les parents et l'enfant, et y intégrer tous les besoins, y compris les loisirs et la culture. Simplifier la gestion des actes usuels. | | #5 | Accès à la culture | Soutenir financièrement et développer tous les dispositifs culturels et artistiques pour les enfants (scolaires, ACM), via des contrats multipartites (État, collectivités, réseau culturel). | | #6 | Qualité des temps | Intégrer des temps libres de qualité dans les activités d'apprentissage, ce qui implique de former les adultes et personnels encadrants. | | #7 | Soutien à la parentalité | Mieux faire connaître, rendre accessibles et valoriser financièrement les lieux et actions d'aide aux parents (maisons des familles, groupes de parole, LAEP, PMI...). | | #8 | Rythmes scolaires | Faire évoluer les rythmes scolaires : réorganiser la journée et la semaine au primaire ; adapter les horaires aux besoins physiologiques au secondaire ; organiser un calendrier national à 2 zones (7 semaines de cours / 2 de vacances). | | #9 | Droit aux vacances et loisirs | Mobiliser les pouvoirs publics pour rendre effectif le droit aux vacances. Renforcer l'information sur les aides et financer davantage les accueils collectifs de mineurs (ACM). | | #10 | Éducation à la nature | Valoriser et accompagner l'éducation au dehors et en lien avec la nature (formation des acteurs, verdissement des espaces, aires éducatives, terrains d'aventure...). | | #11 | Aménagement du territoire | Aménager les territoires « à hauteur d'enfant » dans une démarche participative, en repensant les espaces publics comme lieux de sociabilité, de mixité et de jeu. | | #12 | Bâti et cadre de vie | Rendre obligatoire la concertation avec les enfants et les jeunes pour tout projet d'aménagement ou de rénovation de bâtiments (écoles, centres de loisirs, gymnases...). | | #13 | Lien à la nature | Garantir que chaque enfant bénéficie d'un accès à des espaces naturels, de sorties régulières, et d'au moins un séjour en classe de découverte par cycle de scolarité. | | #14 | Sommeil | Organiser une campagne nationale d'information et de sensibilisation sur le rôle fondamental du sommeil et les facteurs qui lui nuisent. | | #15 | Temps de repos | Prévoir des temps de repos, de calme et de sieste (préservée en maternelle) dans toutes les structures accueillant des enfants, et repenser les locaux pour créer une atmosphère paisible. | | #16 | Activité physique et sportive | Soutenir une tarification sociale pour l'accès au sport. Étendre et revaloriser le Pass'Sport, en y incluant les associations sportives scolaires. | | #17 | Coordination locale | Réhabiliter le Projet Éducatif Territorial (PEDT) et en faire le volet "éducation" des Conventions Territoriales Globales (CTG) pour une coordination globale des acteurs. | | #18 | Gouvernance | Créer un Service Public de la Continuité Éducative (SPCE), confié aux collectivités, pour diagnostiquer les besoins et planifier les actions éducatives sur le territoire. | | #19 | Financement | Assurer un effort budgétaire conséquent et pérenne de l'État et de la Sécurité sociale pour financer les politiques publiques en faveur de l'enfance. |

Synthèse de l'Étude sur la Protection des Mineurs en Ligne

Synthèse Exécutive

Cette étude, menée par l'Arcom en septembre 2025, révèle que les plateformes numériques sont devenues un pilier central et inévitable de la vie des adolescents de 11 à 17 ans, avec des implications majeures en matière d'exposition aux risques et d'efficacité des mesures de protection.

L'accès à ces services est quasi universel, de plus en plus précoce, et se fait souvent en contournant les restrictions d'âge conçues pour protéger les plus jeunes.

Les principaux points à retenir sont les suivants :

• Usage quasi universel et intensif : 99 % des 11-17 ans utilisent au moins une plateforme en ligne, et 83 % fréquentent quotidiennement une très grande plateforme (VLOP).

En moyenne, les adolescents utilisent 3,6 plateformes différentes chaque jour, motivés principalement par le besoin de lien social, de divertissement et d'accès à l'information.

• Contournement systématique des restrictions d'âge : L'âge moyen de la première utilisation des réseaux sociaux est de 12,3 ans, bien en deçà du seuil légal de 13 ans.

Une part significative (62 %) des adolescents reconnaît avoir menti sur son âge lors de l'inscription, principalement pour accéder à des services pour lesquels ils n'avaient pas l'âge requis (65 %).

Cette tendance à une inscription précoce s'accentue chez les plus jeunes générations.

• Faiblesse des mécanismes de vérification : Les systèmes de vérification d'âge des plateformes s'avèrent largement inefficaces.

Seulement 18 % des mineurs déclarent avoir déjà dû prouver leur âge ou avoir vu leur compte bloqué.

Les observations techniques montrent que le contournement des blocages à l'inscription est souvent simple, notamment sur des plateformes majeures comme Instagram, Snapchat et Facebook.

• Encadrement parental ambivalent et contourné : Bien que 94 % des foyers instaurent des règles sur l'usage du numérique, près de la moitié des adolescents (45 %) admettent les contourner régulièrement.

Il existe une perception partagée des risques entre parents et enfants, mais les parents se montrent nettement plus inquiets et moins convaincus des bénéfices des plateformes.

• Perception dichotomique : Les adolescents et leurs parents entretiennent un rapport ambivalent aux plateformes, les considérant à la fois comme des outils d'intégration sociale et de divertissement indispensables, mais aussi comme des sources d'inquiétude et d'exposition à des risques graves.

1. Contexte et Méthodologie de l'Étude

Objectifs de l'Étude

L'étude menée pour l'Arcom vise à dresser un état des lieux complet de la protection des mineurs dans l'univers numérique. Elle s'articule autour de trois axes principaux d'investigation :

1. L'Exposition : Mesurer le degré de conscience des mineurs face aux risques en ligne et leur exposition réelle.

2. La Protection : Analyser les moyens de prévention mis en place par les mineurs et leur entourage, ainsi que leurs réactions post-exposition.

3. Les Attentes : Recueillir les attentes des mineurs, des parents et des professionnels pour une meilleure protection.

L'objectif est de comprendre les compétences que les adolescents mobilisent pour naviguer en ligne, dans un contexte oscillant entre la conscience des dangers et la prise de risques.

Approche Méthodologique

Pour garantir une vision exhaustive, l'étude a été réalisée en quatre volets complémentaires entre novembre 2024 et avril 2025, en partenariat avec Ipsos BVA et OpinionWay.

| Volet | Type d'étude | Période | Participants et Méthodes | | --- | --- | --- | --- | | 1 | Entretiens préparatoires | Nov - Déc 2024 | Entretiens avec des experts, des représentants de plateformes. | | 2 | Étude qualitative | Fév - Mars 2025 | Entretiens avec des experts (associations, psychologue, pédiatre), 16 entretiens individuels et 4 triades avec des mineurs (11-17 ans). | | 3 | Étude sémiologique et observations | Avril 2025 | Analyse des outils et CGU des plateformes ; simulation de parcours utilisateurs avec 8 profils fictifs ; focus sur les thèmes de la maigreur et du masculinisme. | | 4 | Étude quantitative | Avril 2025 | Questionnaire en ligne auprès de 2 000 mineurs (11-17 ans) et de leurs parents. |

Le périmètre de l'étude couvre les réseaux sociaux (Snapchat, TikTok, Facebook, Instagram, etc.), les plateformes de partage de vidéos (YouTube, Twitch, etc.) et les messageries instantanées (WhatsApp, Discord, etc.).

2. L'Usage Incontournable des Plateformes par les Mineurs

Omniprésence et Intensité d'Usage

Les plateformes en ligne sont omniprésentes dans la vie des 11-17 ans. L'étude révèle des chiffres qui témoignent d'une adoption quasi totale et d'un usage quotidien intensif.

• 99 % des 11-17 ans utilisent au moins une plateforme en ligne.

• 83 % utilisent au moins une Très Grande Plateforme en Ligne (VLOP) chaque jour.

• En moyenne, les adolescents utilisent 3,6 plateformes différentes quotidiennement.

La ventilation par catégorie de services montre une forte pénétration de tous les types de plateformes.

| Catégorie de Service | Taux d'Utilisation (11-17 ans) | | --- | --- | | Plateformes de vidéos en ligne | 98 % | | Messageries instantanées | 91 % | | Réseaux sociaux | 88 % | | Jeux en ligne | 87 % | | Sites de rencontres | 15 % |

YouTube, Snapchat, TikTok et WhatsApp sont les plateformes les plus utilisées au quotidien par plus de la moitié des 11-17 ans. L'usage quotidien des VLOP augmente de manière significative avec l'âge, passant de 62 % chez les 11 ans à 96 % chez les 17 ans.

Motivations Principales des Adolescents

Trois motivations majeures expliquent pourquoi les plateformes sont devenues incontournables pour les adolescents.

1. Le besoin d'appartenance et de lien social : Les plateformes sont perçues comme un vecteur essentiel d'intégration sociale et de communication avec les pairs.

2. La recherche de divertissement et d'évasion : Les contenus ludiques et humoristiques sont massivement plébiscités pour se détendre et s'évader du quotidien.

3. L'accès à l'information : Les plateformes servent également de canal d'information pour se tenir au courant de l'actualité et des sujets d'intérêt.

3. Le Contournement Systématique des Restrictions d'Âge

Malgré les dispositifs de restriction, l'accès des mineurs aux plateformes est de plus en plus précoce, grâce à des stratégies de contournement généralisées et à une faible application des règles par les services en ligne.

Précocité de l'Accès

L'âge de la première utilisation des plateformes se situe bien en dessous des seuils réglementaires.

• Âge moyen déclaré de la 1ère utilisation :

◦ 11,2 ans pour les plateformes vidéos.    ◦ 12,3 ans pour les réseaux sociaux.

L'étude met en évidence une tendance à un accès toujours plus précoce : 22 % des jeunes de 11 ans actuels déclarent avoir utilisé les réseaux sociaux pour la première fois à 10 ans ou moins, contre seulement 4 % des jeunes de 17 ans.

Déclaration d'Âge et Manquements à la Vérification

Le contournement de l'âge minimum requis est une pratique massive et assumée par les adolescents.

• 62 % des adolescents reconnaissent ne pas avoir mis leur vraie date de naissance sur au moins une de leurs inscriptions.

• 17 % l'ont fait sur toutes leurs inscriptions.

La principale raison invoquée est l'impossibilité de s'inscrire autrement :

• 65 % n'avaient pas l'âge minimum requis.

• 31 % ne voulaient pas donner leurs données personnelles.

• 12 % voulaient passer pour plus âgés.

"Tout le monde peut y aller, parce que quand tu t'inscris, tu as juste à mettre une fausse date de naissance, ils ne la vérifient pas." - Garçon, 15 ans.

Face à cette pratique, les mesures de contrôle des plateformes apparaissent très limitées :

• Seulement 18 % des 11-17 ans ont déjà dû prouver leur âge ou ont vu leur compte bloqué.

• Facebook est la plateforme où les contrôles sont les plus fréquents (12 % des utilisateurs concernés), suivie par TikTok (10 %) et Instagram (7 %).

Failles Techniques et Contournement à l'Inscription

Les observations de parcours utilisateurs confirment la facilité avec laquelle les restrictions peuvent être contournées.

• L'interdiction d'inscription pour les moins de 13 ans n'est pas clairement explicitée lors du processus.

• Sur Instagram, Snapchat et Facebook, il est possible de contourner un premier refus en modifiant simplement sa date de naissance lors d'une nouvelle tentative.

• Le contournement est plus complexe sur d'autres plateformes comme TikTok, YouTube ou X, nécessitant des manipulations comme la réinitialisation de l'application ou la création d'une nouvelle adresse mail.

4. Perceptions Ambivalentes et Encadrement Familial

Une Perception Dichotomique des Risques et Bénéfices

Les adolescents et leurs parents partagent une vision ambivalente des plateformes, oscillant entre l'attrait des bénéfices et l'inquiétude face aux risques. Cependant, les parents se montrent systématiquement plus préoccupés et moins convaincus des avantages.

| Perception des plateformes (% d'accord) | Mineurs | Parents | | --- | --- | --- | | Permettent d’avoir une vie sociale riche | 80 % | 37 % | | Permettent d’accéder à des contenus éducatifs | 76 % | 56 % | | Exposent les mineurs à des risques graves | 77 % | 89 % | | Inquiètent quant à leur impact sur moi / votre enfant | 83 % | 86 % |

L'Encadrement Parental : Règles et Contournement

L'encadrement familial est une réalité dans la quasi-totalité des foyers, mais son efficacité est relative.

• 94 % des familles ont instauré au moins une règle concernant l'usage du numérique, avec une moyenne de 3,5 règles par foyer.

• Les règles les plus fréquentes sont l'interdiction du téléphone pendant les repas (63 %) et au coucher (55 %).

Malgré ce cadre, 45 % des mineurs admettent contourner ces règles régulièrement (8 % "souvent" et 37 % "de temps en temps"). Les adolescents reconnaissent la finalité protectrice de ces règles mais développent des stratégies pour s'y soustraire.

Utilisation des Comptes Supervisés

Une majorité de jeunes déclarent utiliser des dispositifs de protection intégrés aux plateformes, mais une part non négligeable ignore leur statut.

• 71 % des 11-17 ans déclarent utiliser au moins un compte paramétré pour un adolescent ou supervisé par un adulte.

• Le taux d'utilisation de ces comptes varie selon les plateformes : 63 % sur Snapchat, 60 % sur TikTok, 58 % sur Instagram et 49 % sur YouTube.

• Cependant, une part importante des jeunes (par exemple, 26 % sur Instagram) ne savent pas si leur compte est un compte "adulte" ou un compte "ado/supervisé", ce qui questionne la clarté et l'efficacité de ces dispositifs.

Rapport sur l’Éducation aux Médias, à l’Information et à la Citoyenneté Numérique 2024-2025

Résumé Exécutif

Ce rapport de l'Arcom pour l'année 2024-2025 analyse les initiatives en matière d’éducation aux médias, à l’information et à la citoyenneté numérique (EMI&CN) menées par les acteurs de l'audiovisuel et du numérique.

L'engagement global est en nette progression, avec une augmentation de 35 % des actions déclarées par les chaînes de télévision et de radio.

La croissance la plus spectaculaire concerne les actions de terrain, qui ont bondi de 75 %, témoignant d'une volonté d'aller à la rencontre des publics.

Cette dynamique s'accompagne d'une diversification des publics cibles, touchant non seulement le public scolaire mais aussi de plus en plus les étudiants, le grand public, les seniors et même le public carcéral.

Cependant, si les thématiques de la lutte contre la désinformation et la découverte du journalisme dominent, un effort reste à fournir pour diversifier les sujets abordés.

De plus, la proportion de programmes spécifiquement dédiés au décryptage des médias reste faible sur les antennes (12 %) et les plateformes numériques (27 %).

Les plateformes en ligne concentrent leurs efforts sur des campagnes de sensibilisation à la désinformation et à la détection des contenus générés par l'IA, en s'appuyant sur des partenariats stratégiques.