Reviewer #1 (Public review):

Summary:

In a heroic effort, Ozanna Burnicka-Turek et al. have made and investigated conduction system-specific Tbx3-Tbx5 deficient mice and investigated their cardiac phenotype. Perhaps according to expectations, given the body of literature on the function of the two T-box transcription factors in the heart/conduction system, the cardiomyocytes of the ventricular conduction system seemed to convert to "ordinary" ventricular working myocytes. As a consequence, loss of VCS-specific conduction system propagation was observed in the compound KO mice, associated with PR and QRS prolongation and elevated susceptibility to ventricular tachycardia.

Strengths:

Great genetic model. Phenotypic consequences at the organ and organismal levels are well investigated. The requirement of both Tbx3 and Tbx5 for maintaining VCS cell state has been demonstrated.

Weaknesses:

The actual cell state of the Tbx3/Tbx5 deficient conducting cells was not investigated in detail, and therefore, these cells could well only partially convert to working cardiomyocytes, and may, in reality, acquire a unique state.

Reviewer #2 (Public review):

Summary:

The goal of this work is to define the functions of T-box transcription factors Tbx3 and Tbx5 in the adult mouse ventricular cardiac conduction system (VCS) using a novel conditional mouse allele in which both genes are targeted in cis. A series of studies over the past 2 decades by this group and others have shown that Tbx3 is a transcriptional repressor that patterns the conduction system by repressing genes associated with working myocardium, while Tbx5 is a potent transcriptional activator of "fast" conduction system genes in the VCS. In a previous work, the authors of the present study further demonstrated that Tbx3 and Tbx5 exhibit an epistatic relationship whereby the relief of Tbx3-mediated repression through VCS conditional haploinsufficiency allows better toleration of Tbx5 VCS haploinsufficiency. Conversely, excess Tbx3-mediated repression through overexpression results in disruption of the fast-conduction gene network despite normal levels of Tbx5. Based on these data the authors proposed a model in which repressive functions of Tbx3 drive the adoption of conduction system fate, followed by segregation into a fast-conducting VCS and slow-conduction AVN through modulation of the Tbx5/Tbx3 ratio in these respective tissue compartments.

The question motivating the present work is: If Tbx5/Tbx3 ratio is important for slow versus fast VCS identity, what happens when both genes are completely deleted from the VCS? Is conduction system identity completely lost without both factors and if so, does the VCS network transform into a working myocardium-like state? To address this question, the authors have generated a novel mouse line in which both Tbx5 and Tbx3 are floxed on the same allele, allowing complete conditional deletion of both factors using the VCS-specific MinK-CreERT2 line, convincingly validated in previous work. The goal is to use these double conditional knockout mice to further explore the model of Tbx3/Tbx5 co-dependent gene networks and VCS patterning. First, the authors demonstrate that the double conditional knockout allele results in the expected loss of Tbx3 and Tbx5 specifically in the VCS when crossed with Mink-CreERT2 and induced with tamoxifen. The double conditional knockout also results in premature mortality. Detailed electrophysiological phenotyping demonstrated prolonged PR and QRS intervals, inducible ventricular tachycardia, and evidence of abnormal impulse propagation along the septal aspect of the right ventricle. In addition, the mutants exhibit downregulation of VCS genes responsible for both fast conduction AND slow conduction phenotypes with upregulation of 2 working myocardial genes including connexin-43. The authors conclude that loss of both Tbx3 and Tbx5 results in "reversion" or "transformation" of the VCS network to a working myocardial phenotype, which they further claim is a prediction of their model and establishes that Tbx3 and Tbx5 "coordinate" transcriptional control of VCS identity.

Overall Appraisal:

As noted above, the present study does not further explore the Tbx5/Tbx3 ratio concept since both genes are completely knocked out in the VCS. Instead, the main claims are that the absence of both factors results in a transcriptional shift of conduction tissue towards a working myocardial phenotype, and that this shift indicates that Tbx5 and Tbx3 "coordinate" to control VCS identity and function. However, only limited data are presented to support the claim of transcriptional reprogramming since the knockout cells are not directly compared to working myocardial cells at the transcriptional level and only a small number of key genes are assessed (versus genome-wide assessment). In addition, the optical mapping dataset is incomplete and has alternative interpretations that are not excluded or thoroughly discussed.

In sum, while this study adds an elegantly constructed genetic model to the field, the data presented fit well within the existing paradigm of established functions of Tbx3 and Tbx5 in the VCS and in that sense do not decisively advance the field. Moreover, the authors' claims about the implications of the data are not always strongly supported by the data presented and do not fully explore alternative possibilities.

Strengths:

(1) Successful generation of a novel Tbx3-Tbx5 double conditional mouse model.

(2) Successful VCS-specific deletion of Tbx3 and Tbx5 using a VCS-specific inducible Cre driver line.

(3) Well-powered and convincing assessments of mortality and physiological phenotypes.

(4) Isolation of genetically modified VCS cells using flow.

Weaknesses:

(1) In general, the data is consistent with a long-standing and well-supported model in which Tbx3 represses working myocardial genes and Tbx5 activates the expression of VCS genes, which seem like distinct roles in VCS patterning. However, the authors move between different descriptions of the functional relationship and epistatic relationship between these factors, including terms like "cooperative", "coordinated", and "distinct" at various points. In a similar vein, sometimes terms like "reversion" are used to describe how VCS cells change after Tbx3/Tbx5 conditional knockout, and other times "transcriptional shift" and at other times "reprogramming". But these are all different concepts. The lack of a clear and consistent terminology for describing the phenomena observed makes the overarching claims of the manuscript more difficult to evaluate.

(2) A more direct quantitative comparison of Tbx5 Adult VCS KO with Tbx5/Tbx3 Adult VCS double KO would be helpful to ascertain whether deletion of Tbx3 on top of Tbx5 deletion changes the underlying phenotype in some discernable way beyond mRNA expression of a few genes. Superficially, the phenotypes look quite similar at the EKG and arrhythmia inducibility level and no optical mapping data from a single Tbx5 KO is presented for comparison to the double KO.

(3) The authors claim that double knockout VCS cells transform to working myocardial fate, but there is no comparison of gene expression levels between actual working myocardial cells and the Tbx3/Tbx5 DKO VCS cells so it's hard to know if the data reflect an actual cell state change or a more non-specific phenomenon with global dysregulation of gene expression or perhaps dedifferentiation. I understand that the upregulation of Gja1 and Smpx is intended to address this, but it's only two genes and it seems relevant to understand their degree of expression relative to actual working myocardium. In addition, the gene panel is somewhat limited and does not include other key transcriptional regulators in the VCS such as Irx3 and Nkx2-5. RNA-seq in these populations would provide a clearer comparison among the groups.

(4) From the optical mapping data, it is difficult to distinguish between the presence of (a) a focal proximal right bundle branch block due to dysregulation of gene expression in the VCS but overall preservation of the right bundle and its distal ramifications; from (b) actual loss of the VCS with reversion of VCS cells to a working myocardial fate. Related to this, the authors claim that this experiment allows for direct visualization of His bundle activation, but can the authors confirm or provide evidence that the tissue penetration of their imaging modality allows for imaging of a deep structure like the AV bundle as opposed to the right bundle branch which is more superficial? Does the timing of the separation of the sharp deflection from the subsequent local activation suggest visualization of more distal components of the VCS rather than the AV bundle itself? Additional clarification would be helpful.

Impact:

The present study contributes a novel and elegantly constructed mouse model to the field. The data presented generally corroborate existing models of transcriptional regulation in the VCS but do not, as presented, constitute a decisive advance.

Reviewer #3 (Public review):

Summary:

In the study presented by Burnicka-Turek et al., the authors generated for the first time a mouse model to cause the combined conditional deletion of Tbx3 and Tbx5 genes. This has been impossible to achieve to date due to the proximity of these genes in chromosome 5, preventing the generation of loss of function strategies to delete simultaneously both genes. It is known that both Tbx3 and Tbx5 are required for the development of the cardiac conduction system by transcription factor-specific but also overlapping roles as seen in the common and diverse cardiac defects found in patients with mutations for these genes. After validating the deletion efficiency and specificity of the line, the authors characterised the cardiac phenotype associated with the cardiac conduction system (CCS)-specific combined deletion of Tbx5 and Tbx3 in the adult by inducing the activation of the CCS-specific tamoxifen-inducible Cre recombination (MinK-creERT) at 6 weeks after birth. Their analysis of 8-9-week-old animals did not identify any major morphological cardiac defects. However, the authors found conduction defects including prolonged PR and QTR intervals and ventricular tachycardia causing the death of the double mutants, which do not survive more than 3 months after tamoxifen induction. Molecular and optical mapping analysis of the ventricular conduction system (VCS) of these mutants concluded that, in the absence of Tbx5 and Tbx3 function, the cells forming the ventricular conduction system (VCS) become working myocardium and lose the specific contractile features characterising VCS cells. Altogether, the study identified the critical combined role of Tbx3 and Tbx5 in the maintenance of the VCS in adulthood.

Strengths:

The study generated a new animal model to study the combined deletion of Tbx5 and Tbx3 in the cardiac conduction system. This unique model has provided the authors with the perfect tool to answer their biological questions. The study includes top-class methodologies to assess the functional defects present in the different mutants analysed, and gathered very robust functional data on the conduction defects present in these mutants. They also applied optical action potential (OAP) methods to demonstrate the loss of conduction action potential and the acquisition of working myocardium action potentials in the affected cells because of Tbx5/Tbx3 loss of function. The study used simpler molecular and morphological analysis to demonstrate that there are no major morphological defects in these mutants and that indeed, the conduction defects found are due to the acquisition of working myocardium features by the VCS cells. Altogether, this study identified the critical role of these transcription factors in the maintenance of the VCS in the adult heart.

Weaknesses:

In the opinion of this reviewer, the weakness in the study lies in the morphological and molecular characterization. The morphological analysis simply described the absence of general cardiac defects in the adult heart, however, whether the CCS tissues are present or not was not investigated. Lineage tracing analysis using the reporter lines included in the crosses described in the study will determine if there are changes in CCS tissue composition in the different mutants studied. Similarly, combining this reporter analysis with the molecular markers found to be dysregulated by qPCR and western blot, will demonstrate that indeed the cells that were specified as VCS in the adult heart, become working myocardium in the absence of Tbx3 and Tbx5 function.

Reviewer #1 (Public review):

Summary:

This paper developed a model of chromosome mosaicism by using a new aneuploidy-inducing drug (AZ3146), and compared this to their previous work where they used reversine, to demonstrate the fate of aneuploid cells during murine preimplantation embryo development. They found that AZ3146 acts similarly to reversine in inducing aneuploidy in embryos, but interestingly showed that the developmental potential of embryos is higher in AZ3146-treated vs. reversine-treated embryos. This difference was associated with changes in HIF1A, p53 gene regulation, DNA damage, and fate of euploid and aneuploid cells when embryos were cultured in a hypoxic environment.

Strengths:

In the current study, the authors investigate the fate of aneuploid cells in the preimplantation murine embryo using a specific aneuploidy-inducing compound to generate embryos that were chimeras of euploid and aneuploid cells. The strength of the work is that they investigate the developmental potential and changes in gene expression profiles under normoxic and hypoxic culture conditions. Further, they also assessed how levels of DNA damage and DNA repair are altered in these culture conditions. They also assessed the allocation of aneuploid cells to the divergent cell lineages of the blastocyst stage embryo.

Weaknesses:

Inconsistent/missing description for sample size, biological/technical replicates, label orientation, the appropriate number of * for each figure panel, and statistical tests used.

Reviewer #2 (Public review):

Summary:

This study by Sanchez-Vasquez is a very innovative approach to inducing aneuploidy and then studying the contribution of treated cells to different lineages, including post-implantation. It connects well to the authors' previous work to induce mosaic aneuploidies. The authors identify sensitivity to HIF1a loss in treated embryos with likely aneuploidy. This work is part of an important line of work with evaluates the consequences of aneuploidy in the mammalian embryo.

Weaknesses:

Given that this is a study on the induction of aneuploidy, it would be meaningful to assess aneuploidy immediately after induction, and then again before implantation. This is also applicable to the competition experiments on page 7/8. What is shown is the competitiveness of treated cells. Because the publication centers around aneuploidy, the inclusion of such data in the main figure at all relevant points would strengthen it. There is some evaluation of karyotypes only in the supplemental - why? It would be good not to rely on a single assay that the authors appear to not give much importance.

Reviewer #1 (Public review):

Summary:

Dopamine neurons contribute to motivated and motor behaviors in many ways, and ample recent evidence has suggested that distinct dopamine neuron subclasses support discrete behavioral and circuit functions. Prior studies have subdivided dopamine neurons by spatial localization, gene expression patterns, and physiological properties. However, many of these studies were bound by previous technical limitations that made comprehensive subclassification efforts difficult or impossible. The main goal of this manuscript was to characterize and further define dopamine neuron heterogeneity in the ventral midbrain. The study uses cutting-edge single nucleus RNA-seq (on the 10X Genomics platform) and spatial transcriptomics (on the MERFISH platform) to define dopamine neuron heterogeneity with unprecedented resolution. The result is a convincing and comprehensive subclassification of dopamine neurons into three main families, each with major branches and subtypes. In addition, the study reports comparisons between wild-type mice and mice that harbor a G2019S mutation in the Lrrk2 gene, which models a common cause of autosomally dominant Parkinson's Disease in humans. These results, while less robust due to the nature of the group comparisons, nevertheless identify vulnerability within specific dopamine neuron subpopulations. This vulnerability may contribute unique risk of dopamine neuron loss in the context of Parkinson's disease. Overall, the study is careful and rigorous and provides a critical resource for the rapidly evolving knowledge of dopamine neuron subtypes.

Strengths:

(1) The creation of a public-facing app where the snRNA-seq data can be investigated by anyone is a major strength.

(2) The manuscript includes careful comparisons to prior datasets that have sought to explore dopamine neuron heterogeneity. The result is a useful synthesis of new findings with previously published work, which is helpful for moving the field forward in this area.

(3) The integration of snRNA-seq with MERFISH results is particularly strong and enables insight not only into subclassification but also into how this relates to spatial localization. The careful neuroanatomy reveals important distinctions between Sox6, Calb1, and Gad2 positive dopamine neuron families, with some degree of spatial overlap.

Weaknesses:

(1) Important details about the nature of DEG comparisons between the wild type and the Lrrk2 G2019S model are missing.

(2) Some aspects of the integration between snRNA-seq and MERFISH data are not clear, and many MERFISH-identified cells do not appear to have a high-confidence cluster transfer into the snRNA-seq data space. Imputation is used to overcome some issues with the MERFISH dataset, but it is not clear that this is appropriate.

Reviewer #2 (Public review):

Gaertner and colleagues present a study examining the transcriptomic diversity and spatial location of dopaminergic neurons from mice and examine the changes in gene expression resulting from knock-in of the Parkinson's LRRK G2019S risk variant. Overall, I found the manuscript presented their study very clearly, well written with very clear figures for the most part. I am not an expert on mouse neuroanatomy but found their classification reasonably well justified and the spatial orientation of dopaminergic neurons within the mouse brain informative and clear. While trends were clear and well presented, the apparent spatial heterogeneity suggests that knowledge of the functional connections and roles of these neurons will be required to better interpret the results presented, but nonetheless their findings exposed significant detail that is required for further understanding.

The study of the transcriptional effects of the LRRK2 KI was also informative and clearly framed in terms of a focused analysis on the effects of the KI only on dopaminergic neurons. However, I think there are issues here in both methodology, narrative, and clarity.

(1) In the GO pathway analyses (both GSEA and DEG GO), I did not see a correction applied to the gene background considered. The study focusses on dopaminergic neurons and thus the gene background should be restricted to genes expressed in dopaminergic neurons, rather than all genes in the mouse genome. The problem arises that if we randomly sample genes from dopaminergic neurons instead of the whole genome, we are predisposed to sampling genes enriched in relevant cell-type-specific roles (and their relevant GO terms) and correspondingly depleted in genes enriched in functions not associated with this cell type. Thus, I am unsure whether the results presented in Figures 8 and 9 may be more likely to be obtained just by randomly sampling genes from a dopaminergic neuron. The background should be limited and these functional analyses rerun.

(2) In the scRDS results, I am unsure what is significant and what isn't. The authors refer to relative measures in the text ("highest") but I do not know whether these differences are significant nor whether any associations are significantly unexpected. Can the x-axis of scRDS results presented in Figure 9 H and I be replaced with a corrected p-value instead of the scRDS score?

(3) The results discussed at the bottom of page 13 state that 48.82% of the proteins encoded by the Calb1 DEGs have pre-synaptic localisations as opposed to 45.83% of the SOX6 DEGs, which does not support the statement that "greater proportions of DEGs are associated with presynaptic locations in cells from vulnerable DA neurons (Sox6 family, [and in particular,Sox6^tafa1]), compared to less vulnerable ones (Calb1 family)".

(4) While an interest in the Sox6^tafa1 subtype is explained through their expression of Anxa1 denoting a previously identified subtype associated with locomotory behaviours, it was unclear to me how to interpret the functional associations made to DEGs in this subtype taken out of context of other subtypes. Given all the other subtypes, it is not possible to ascertain how specific and thus how interesting these results are unless other subtypes are analysed in the same way and this Sox6^tafa1 subtype is demonstrated as unusual given results from other subtypes.

(5) On p12, the authors highlight Mir124a-1hg that encodes miR-124. This is upregulated in Figure 8D but the authors note this has been to be downregulated in PD patients and some PD mouse models. Can the authors comment on the directional difference?

(6) Lastly, can the authors comment on the selection of a LogFC cut-off of 0.15 for their DEG selection? I couldn't see this explained (apologies if I missed it).

Reviewer #1 (Public review):

Mutations in CDHR1, the human gene encoding an atypical cadherin-related protein expressed in photoreceptors, are thought to cause cone-rod dystrophy (CRD). However, the pathogenesis leading to this disease is unknown. Previous work has led to the hypothesis that CDHR1 is part of a cadherin-based junction that facilitates the development of new membranous discs at the base of the photoreceptor outer segments, without which photoreceptors malfunction and ultimately degenerate. CDHR1 is hypothesized to bind to a transmembrane partner to accomplish this function, but the putative partner protein has yet to be identified.

The manuscript by Patel et al. makes an important contribution toward improving our understanding of the cellular and molecular basis of CDHR1-associated CRD. Using gene editing, they generate a loss of function mutation in the zebrafish cdhr1a gene, an ortholog of human CDHR1, and show that this novel mutant model has a retinal dystrophy phenotype, specifically related to defective growth and organization of photoreceptor outer segments (OS) and calyceal processes (CP). This phenotype seems to be progressive with age. Importantly, Patel et al, present intriguing evidence that pcdh15b, also known for causing retinal dystrophy in previous Xenopus and zebrafish loss of function studies, is the putative cdhr1a partner protein mediating the function of the junctional complex that regulates photoreceptor OS growth and stability.

This research is significant in that it:

(1) provides evidence for a progressive, dystrophic photoreceptor phenotype in the cdhr1a mutant and, therefore, effectively models human CRD; and

(2) identifies pcdh15b as the putative, and long sought after, binding partner for cdhr1a, further supporting the theory of a cadherin-based junction complex that facilitates OS disc biogenesis.

Nonetheless, the study has several shortcomings in methodology, analysis, and conceptual insight, which limits its overall impact.

Below I outline several issues that the authors should address to strengthen their findings.

Major comments:

(1) Co-localization of cdhr1a and pcdh15b proteins

The model proposed by the authors is that the interaction of cdhr1a and pcdh15b occurs in trans as a heterodimer. In cochlear hair cells, PCDH15 and CDHR23 are proposed to interact first as dimers in cis and then as heteromeric complexes in trans. This was not shown here for cdhr1a and pcdh15b, but it is a plausible configuration, as are single heteromeric dimers or homodimers. Regardless, this model depends on the differential compartmental expression of the cdhr1a and pcdh15b proteins. Data in Figure 1 show convincing evidence that these two proteins can, at least in some cases, be distributed along the length of photoreceptor membranes that are juxtaposed, as would be the case for OS and CP. If pcdh15b is predominantly expressed in CPs, whereas cdhr1a is predominantly expressed in OS, then this should be confirmed with actin double labeling with cdhr1a and pcdh15b since the apicobasal oriented (vertical) CPs would express actin in this same orientation but not in the OS. This would help to clarify whether cdhr1a and pcdh15b can be trafficked to both OS and CP compartments or whether they are mutually exclusive.

Photoreceptor heterogeneity goes beyond the cone versus rod subtypes discussed here and it is known that in zebrafish, CP morphology is distinct in different cone subtypes as well as cone versus rod. It would be important to know which specific photoreceptor subtypes are shown in zebrafish (Figures 1A-C) and the non-fish species depicted in Figures 1E-L. Also, a larger field of view of the staining patterns for Figures 1E-L would be a helpful comparison (could be added as a supplementary figure).

(2) Cdhr1a function in cell culture

The authors should explain the multiple bands in the anti-FLAG blots. Also, it would be interesting to confirm that the cdhr1a D173 mutant prevents the IP interaction with pcdh15b as well as the additive effects in aggregate assays of Figure 2.

Is it possible that the cultured cells undergo proliferation in the aggregation assays shown in Figure 2? Cells might differentially proliferate as clusters form in rotating cultures. A simple assay for cell proliferation under the different transfection conditions showing no differences would address this issue and lend further support to the proposed specific changes to cell adhesion as a readout of this assay.

Also, the authors report that the number of clusters was normalized to the field of view, but this was not defined. Were the n values different fields of view from one transfection experiment, or were they different fields of view from separate transfection experiments? More details and clarification are needed.

(3) Methodological issues in quantification and statistical analyses

Were all the OS and CP lengths counted in the observation region or just a sample within the region? If the latter, what were the sampling criteria? For CPs, it seems that the length was an average estimate based on all CPs observed surrounding one cone or one-rod cell. Is this correct? Again, if sampled, how was this implemented? In Fig 4M', the cdhr1a-/- ROS mostly looks curvilinear. Did the measurements account for this, or were they straight linear dimension measurements from base to tip of the OS as depicted in Fig 5A-E? A clearer explanation of the OS and CP length quantification methodology is required.

How were cone and rod photoreceptor cell counts performed? The legend in Figure 4 states that they again counted cells in the observation region, but no details were provided. For example, were cones and rods counted as an absolute number of cells in the observation region (e.g., number of cones per defined area) or relative to total (DAPI+) cell nuclei in the region? Changes in cell density in the mutant (smaller eye or thinner ONL) might affect this quantification so it would be important to know how cell quantification was normalized.

In Figure 6I, K, measuring the length of the signal seems problematic. The dimension of staining is not always in the apicobasal (vertical) orientation. It might be more accurate to measure the cdhr1a expression domain relative to the OS (since the length of the OS is already reduced in the mutants). Another possible approach could be to measure the intensity of cdhr1 staining relative to the intensity within a Prph2 expression domain in each group. The authors should provide complementary evidence to support their conclusion.

A better description of the statistical methodology is required. For example, the authors state that "each of the data points has an n of 5+ individuals." This is confusing and could indicate that in Figure 4F alone there were ~5000 individuals assayed (~100 data points per treatment group x n=5 individuals per data point x 10 treatment groups). I don't think that is what the authors intended. It would be clearer if the authors stated how many OS, CP, or cells were counted in their observation region averaged per individual, and then provided the n value of individuals used per treatment group (controls and mutants), on which the statistical analyses should be based.

There are hundreds of data points in the separate treatment groups shown in several of the graphs. It would not be correct to perform the ANOVA on the separate OS or CP length measurements alone as this will bias the estimates since they are not all independent samples. For example, in Figure 6H, 5dpf pcdh15b+/- have shorter CPs compared to WT but pcdh15b-/- have longer compared to WT. This could be an artifact of the analysis. Moreover, the authors should clarify in the Methods section which ANOVA post hoc tests were used to control for multiple pairwise comparisons.

(4) Cdhr1a function in photoreceptors

The cdhr1a IHC staining in 5dpf WT larvae in Figure 3E appears different from the cdhr1a IHC staining in 5dpf WT larvae in Figure 1A or Figure 6I. Perhaps this is just the choice of image. Can the authors comment or provide a more representative image?

The authors show that pcdh15b localization after 5dpf mirrored the disorganization of the CP observed with actin staining. They also show in Figure 5O that at 180dpf, very little pcdh15b signal remains. They suggest based on this data that total degradation of CPs has occurred in the cdhr1a-/- photoreceptors by this time. However, although reduced in length, COS and cone CPs are still present at 180dpf (Figure 5E, E'). Thus, contrary to the authors' general conclusion, it is possible that the localization, trafficking, and/or turnover of pcdh15b is maintained through a cdhr1a-dependent mechanism, irrespective of the degree to which CPs are maintained. The experiments presented here do not clearly distinguish between a requirement for maintenance of localization versus a secondary loss of localization due to defective CPs.

(5) Conceptual insights

The authors claim that cdhr1a and pcdh15b double mutants have synergistic OS and CP phenotypes. I think this interpretation should be revisited.

First, assuming the model of cdhr1a-pcdh15b interaction in trans is correct, the authors have not adequately explained the logic of why disrupting one side of this interaction in a single mutant would not give the same severity of phenotype as disrupting both sides of this interaction in a double mutant.

Second, and perhaps more critically, at 10dpf the OS and CP lengths in cdhr1a-/- mutants (Figure 7J, T) are significantly increased compared to WT. In contrast, there are no significant differences in these measurements in the pcdh15b-/- mutants. Yet in double homozygous mutants, there is a significant reduction of ~50% in these measurements compared to WT. A synergistic phenotype would imply that each mutant causes a change in the same direction and that the magnitude of this change is beyond additive in the double mutants (but still in the same direction). Instead, I would argue that the data presented in Figure 7 suggest that there might be a functionally antagonistic interaction between cdhr1a and pcdh15b with respect to OS and CP growth at 10dpf.

If these proteins physically interacted in vivo, it would appear that the interaction is complex and that this interaction underlies both OS growth-promoting and growth-restraining (stabilizing) mechanisms working in concert. Perhaps separate homodimers or heterodimers subserve distinct CP-OS functional interactions. This might explain the age-dependent differences in mutant CP and OS length phenotypes if these mechanisms are temporally dynamic or exhibit distinct OS growth versus maintenance phases. Regardless of my speculations, the model presented by the authors appears to be too simplistic to explain the data.

Reviewer #2 (Public review):

Summary:

The goal of this study was to develop a model for CDHR1-based Con-rod dystrophy and study the role of this cadherin in cone photoreceptors. Using genetic manipulation, a cell binding assay, and high-resolution microscopy the authors find that like rods, cones localize CDHR1 to the lateral edge of outer segment (OS) discs and closely oppose PCDH15b which is known to localize to calyceal processes (CPs). Ectopic expression of CDHR1 and PCDH15b in K652 cells indicates these cadherins promote cell aggregation as heterophilic interactants, but not through homophilic binding. This data suggests a model where CDHR1 and PCDH15b link OS and CPs and potentially stabilize cone photoreceptor structure. Mutation analysis of each cadherin results in cone structural defects at late larval stages. While pcdh15b homozygous mutants are lethal, cdhr1 mutants are viable and subsequently show photoreceptor degeneration by 3-6 months.

Strengths:

A major strength of this research is the development of an animal model to study the cone-specific phenotypes associated with CDHR1-based CRD. The data supporting CDHR1 (OS) and PCDH15 (CP) binding is also a strength, although this interaction could be better characterized in future studies. The quality of the high-resolution imaging (at the light and EM levels) is outstanding. In general, the results support the conclusions of the authors.

Weaknesses:

While the cellular phenotyping is strong, the functional consequences of CDHR1 disruption are not addressed. While this is not the focus of the investigation, such analysis would raise the impact of the study overall. This is particularly important given some of the small changes observed in OS and CP structure. While statistically significant, are the subtle changes biologically significant? Examples include cone OS length (Figures 4F, 6E) as well as other morphometric data (Figure 7I in particular). Related, for quantitative data and analysis throughout the manuscript, more information regarding the number of fish/eyes analyzed as well as cells per sample would provide confidence in the rigor. The authors should also note whether the analysis was done in an automated and/or masked manner.

Reviewer #3 (Public review):

Summary:

The manuscript by Patel et al investigates the hypothesis that CDHR1a on photoreceptor outer segments is the binding partner for PCDH15 on the calyceal processes, and the absence of either adhesion molecule results in separation between the two structures, eventually leading to degeneration. PCDH15 mutations cause Usher syndrome, a disease of combined hearing and vision loss. In the ear, PCDH15 binds CDH23 to form tip links between stereocilia. The vision loss is less understood. Previous work suggested PCDH15 is localized to the calyceal processes, but the expression of CDH23 is inconsistent between species. Patel et al suggest that CDHR1a (formerly PCDH21) fulfills the role of CDH23 in the retina.

The experiments are mainly performed using the zebrafish model system. Expression of Pcdh15b and Cdhr1a protein is shown in the photoreceptor layer through standard confocal and structured illumination microscopy. The two proteins co-IP and can induce aggregation in vitro. Loss of either Cdhr1a or Pcdh15, or both, results in degeneration of photoreceptor outer segments over time, with cones affected primarily.

The idea of the study is logical given the photoreceptor diseases caused by mutations in either gene, the comparisons to stereocilia tip links, and the protein localization near the outer segments. The work here demonstrates that the two proteins interact in vitro and are both required for ongoing outer segment maintenance. The major novelty of this paper would be the demonstration that Pcdh15 localized to calyceal processes interacts with Cdhr1a on the outer segment, thereby connecting the two structures. Unfortunately, the data presented are inadequate proof of this model.

Strengths:

The in vitro data to support the ability of Pcdh15b and Cdhr1a to bind is well done. The use of pcdh15b and cdhr1a single and double mutants is also a strength of the study, especially being that this would be the first characterization of a zebrafish cdhr1a mutant.

Weaknesses:

(1) The imaging data in Figure 1 is insufficient to show the specific localization of Pcdh15 to calyceal processes or Cdhr1a to the outer segment membrane. The addition of actin co-labelling with Pcdh15/Cdhr1a would be a good start, as would axial sections. The division into rod and cone-specific imaging panels is confusing because the two cell types are in close physical proximity at 5 dpf, but the cone Cdhr1a expression is somehow missing in the rod images. The SIM data appear to be disrupted by chromatic aberration but also have no context. In the zebrafish image, the lines of Pcdh15/Cdhr1a expression would be 40-50 um in length if the scale bar is correct, which is much longer than the outer segments at this stage and therefore hard to explain.

(2) Figure 3E staining of Cdhr1a looks very different from the staining in Figure 1. It is unclear what the authors are proposing as to the localization of Cdhr1a. In the lab's previous paper, they describe Cdhr1a as being associated with the connecting cilium and nascent OS discs, and fail to address how that reconciles with the new model of mediating CP-OS interaction. And whether Cdhr1a localizes to discrete domains on the disc edges, where it interacts with Pcdh15 on individual calyceal processes.

(3) The authors state "In PRCs, Pcdh15 has been unequivocally shown to be localized in the CPs". However, the immunostaining here does not match the pattern seen in the Miles et al 2021 paper, which used a different antibody. Both showed loss of staining in pcdh15b mutants so unclear how to reconcile the two patterns.

(4) The explanation for the CRISPR targets for cdhr1a and the diagram in Figure 3 does not fit with crRNA sequences or the mutation as shown. The mutation spans from the latter part of exon 5 to the initial portion of exon 6, removing intron 5-6. It should nevertheless be a frameshift mutation but requires proper documentation.

(5) There are complications with the quantification of data. First, the number of fish analyzed for each experiment is not provided, nor is the justification for performing statistics on individual cell measurements rather than using averages for individual fish. Second, all cone subtypes are lumped together for analysis despite their variable sizes. Third, t-tests are inappropriately used for post-hoc analysis of ANOVA calculations.

(6) Unclear how calyceal process length is being measured. The cone measurements are shown as starting at the external limiting membrane, which is not equivalent to the origin of calyceal processes, and it is uncertain what defines the apical limit given the multiple subtypes of cones. In Figure 5, the lines demonstrating the measurements seem inconsistently placed.

(7) The number of fish analyzed by TEM and the prevalence of the phenotype across cells are not provided. A lower magnification view would provide context. Also, the authors should explain whether or not overgrowth of basal discs was observed, as seen previously in cdhr1-null frogs (Carr et al., 2021).

(8) The statement describing the separation between calyceal processes and the outer segment in the mutants is not backed up by the data. TEM or co-labelling of the structures in SIM could be done to provide evidence.

(9) "Based on work in the murine model and our own observations of rod CPs, we hypothesize that zebrafish rod CPs only extend along the newly forming OS discs and do not provide structural support to the ROS." Unclear how murine work would support that conclusion given the lack of CPs in mice, or what data in the manuscript supports this conclusion.

(10) The authors state "from the fact that rod CPs are inherently much smaller than cone CPs" without providing a reference. In the manuscript, the measurements do show rod CPs to be shorter, but there are errors in the cone measurements, and it is possible that the RPE pigment is interfering with the rod measurements.

(11) The discussion should include a better comparison of the results with ocular phenotypes in previously generated pcdh15 and cdhr1 mutant animals.

(12) The images in panels B-F of the Supplemental Figure are uncannily similar, possibly even of the same fish at different focal planes.

Reviewer #1 (Public review):

Gray and colleagues describe the identification of Integrator complex subunit 12 (INTS12) as a contributor to HIV latency in two different cell lines and in cells isolated from the blood of people living with HIV. The authors employed a high-throughput CRISPR screening strategy to knock down genes and assess their relevance in maintaining HIV latency. They had used a similar approach in two previous studies, finding genes required for latency reactivation or genes preventing it and whose knockdown could enhance the latency-reactivating effect of the NFκB activator AZD5582. This work builds on the latter approach by testing the ability of gene knockdowns to complement the latency-reactivating effects of AZD5582 in combination with the BET inhibitor I-BET151. This drug combination was selected because it has been previously shown to display synergistic effects on latency reactivation.

The finding that INTS12 may play a role in HIV latency is novel, and the effect of its knockdown in inducing HIV transcription in primary cells, albeit in only a subset of donors, is intriguing. However, there are some data and clarifications that would be important to include to complement the information provided in the current version of the manuscript.

Reviewer #2 (Public review):

Summary:

Identifying an important role for the Integrator complex in repressing HIV transcription and suggesting that by targeting subunits of this complex specifically, INTS12, reversal of latency with and without latency reversal agents can be enhanced.

Strengths:

The strengths of the paper include the general strategy for screening targets that may activate HIV latency and the rigor of exploring the mechanism of INTS12 repression of HIV transcriptional elongation. I found the mechanism of INTS12 interesting and maybe even the most impactful part of the findings.

Weaknesses:

I have two minor comments:

There was an opportunity to examine a larger panel of latency reversal agents that reactivate by different mechanisms to determine whether INTS12 and transcriptional elongation are limiting for a broad spectrum of latency reversal agents.

I felt the authors could have extended their discussion of how exquisitely sensitive HIV transcription is to pausing and transcriptional elongation and the insights this provides about general HIV transcriptional regulation.

Reviewer #3 (Public review):

Summary:

Transcriptionally silent HIV-1 genomes integrated into the host`s genome represent the main obstacle to an HIV-1 cure. Therefore, agents aimed at promoting HIV transcription, the so-called latency reactivating agents (LRAs) might represent useful tools to render these hidden proviruses visible to the immune system. The authors successfully identified, through multiple techniques, INTS12, a component of the Integrator complex involved in 3' processing of small nuclear RNAs U1 and U2, as a factor promoting HIV-1 latency and hindering elongation of the HIV RNA transcripts. This factor synergizes with a previously identified combination of LRAs, one of which, AZD5582, has been validated in the macaque model for HIV persistence during therapy (https://pubmed.ncbi.nlm.nih.gov/37783968/). The other compound, I-BET151, is known to synergize with AZD5582, and is a inhibitor of BET, factors counteracting the elongation of RNA transcripts.

Strengths:

The findings were confirmed through multiple screens and multiple techniques. The authors successfully mapped the identified HIV silencing factor at the HIV promoter.

Weaknesses:

(1) Initial bias:<br /> In the choice of the genes comprised in the library, the authors readdress their previous paper (Hsieh et al.) where it is stated: "To specifically investigate host epigenetic regulators involved in the maintenance of HIV-1 latency, we generated a custom human epigenome specific sgRNA CRISPR library (HuEpi). This library contains sgRNAs targeting epigenome factors such as histones, histone binders (e.g., histone readers and chaperones), histone modifiers (e.g., histone writers and erasers), and general chromatin associated factors (e.g., RNA and DNA modifiers) (Fig 1B and 1C)".

From these figure panels, it clearly appears that the genes chosen are all belonging to the indicated pathways. While I have nothing to object to on the pertinence to HIV latency of the pathways selected, the authors should spend some words on the criteria followed to select these pathways. Other pathways involving epigenetic modifications and containing genes not represented in the indicated pathways may have been left apart.

(2) Dereplication:<br /> From Figure 1 it appears that INTS12 alone reactivates HIV -1 from latency alone without any drug intervention as shown by the MACGeCk score of DMSO-alone controls. If INTS12 knockdown alone shows antilatency effects, why, then were they unable to identify it in their previous article (Hsieh et al., 2023)? The authors should include some words on the comparison of the results using DMSO alone with those of the previous screen that they conducted.

(3) Translational potential:<br /> In order to propose a protein as a drug target, it is necessary to adhere to the "primum non nocere" principle in medicine. It is therefore fundamental to show the effects of INTS12 knockdown on cell viability/proliferation (and, advisably, T-cell activation). These data are not reported in the manuscript in its current form, and the authors are strongly encouraged to provide them.

Finally, as many readers may not be very familiar with the general principles behind CRISPR Cas9 screening techniques, I suggest addressing them in this excellent review: https://pmc.ncbi.nlm.nih.gov/articles/PMC7479249/.

Reviewer #1 (Public review):

Summary:

The authors were attempting to identify the molecular and cellular basis for why modulators of the HR pathway, specifically PARPi, are not effective in CDK12 deleted or mutant prostate cancers and they seek to identify new therapeutic agents to treat this subset of metastatic prostate cancer patients. Overall, this is an outstanding manuscript with a number of strengths and in my opinion represents a significant advance in the field of prostate cancer biology and experimental therapeutics.

Strengths:

The patient data cohort size and clinical annotation from Figure 1 are compelling and comprehensive in scope. The associations between tandem duplications and amplifications of oncogenes that have been well-credentialed to be drivers of cancer development and progression are fascinating and the authors identify that in those that have AR amplification for example, there is evidence for AR pathway activation. The association between CDK12 inactivation and various specific gene/pathway perturbations is fascinating and is consistent with previously published studies - it would be interesting to correlate these changes with cell line-based studies in which CDK12 is specifically deleted or inhibited with small molecules to see how many pathways/gene perturbations are shared between the clinical samples and cell and mouse models with CDK12 perturbation. The short-term inhibitor studies related to changes in HRD genes and protein expression with CDK12/13 inhibition are fascinating and suggest differential pathway effects between short inhibition of CDK12/13 and long-term loss of CDK12. The in vivo studies with the inhibitor of CDK12/13 are intriguing but not definitive

Weaknesses:

Given that there are different mutations identified at different CDK12 sites as illustrated in Figure 1B it would be nice to know which ones have been functionally classified as pathogenic and for which ones that the pathogenicity has not been determined. This would be especially interesting to perform in light of the differences in the LOH scores and WES data presented - specifically, are the pathogenic mutations vs the mutations for which true pathogenicity is unknown more likely to display LOH or TD? For the cell inhibition studies with the CDK12/13 inhibitor, more details characterizing the specificity of this molecule to these targets would be useful. Additionally, could the authors perform short-term depletion studies with a PROTAC to the target or short shRNA or non-selected pool CRISPR deletion studies of CDK12 in these same cell lines to complement their pharmacological studies with genetic depletion studies? Also perhaps performing these same inhibitor studies in CDK12/13 deleted cells to test the specificity of the molecule would be useful. Additionally, expanding these studies to additional prostate cancer cell lines or organdies models would strengthen the conclusions being made. More information should be provided about the dose and schedule chosen and the rationale for choosing those doses and schedules for the in vivo studies proposed should be presented and discussed. Was there evidence for maximal evidence of inhibition of the target CDK12/13 at the dose tested given the very modest tumor growth inhibition noted in these studies?

Reviewer #2 (Public review):

Summary:

The study explores the functional consequence of CDK12 loss in prostate cancer. While CDK12 loss has been shown to confer homologous recombination (HR) deficiency through premature intronic polyadenylation of HR genes, the response of PARPi monotherapy has failed. This study therefore performed an in-depth analysis of genomic sequencing data from mCRPC patient tumors, and showed that tumors with CDK12 loss lack pertinent HR signatures and scars. Furthermore, functional exploration in human prostate cancer cell lines showed that while the acute inhibition of CDK12 resulted in aberrant polyadenylation of HR genes like BRCA1/2, HR-specific effects were overall modest or absent in cell lines or xenografts adapted to chronic CDK12 loss. Instead, vulnerability to genetically targeting CDK13 resulted in a synthetic lethality in tumors with CDK12 loss, as shown in vivo with SR4825, a CDK12/13 inhibitor - thus serving as a potential therapeutic avenue.

The evidence supporting this study is based on in-depth genomic analyses of human patients, acute knockdown studies of CDK12 using a CDK12/13 inhibitors SR4835, adaptive knockout of CDK12 using LuCaP 189.4_CL and inducible re-expression of CDK12, CDK12 single clones in 22Rv1 (KO2 and KO5) and Skov3 (KO1), Tet-inducible knockdown of BRCA2 or CDK12 followed by ionizing radiation and measurement of RAD51 foci, lack of sensitivity generally to PARPi and platinum chemotherapy in cells adapted to CDK12 loss, loss of viability of CDK13 knockout in CDK12 knockout cells, and in vivo testing of SE4825 in LuCaP xenografts with intact and CDK12 loss.

Strengths:

Overall, this study is robust and of interest to the broader homologous recombination and CDK field. First, the topic is clinically relevant given the lack of PARPi response in CDK12 loss tumors. Second, the strength of the genomic analysis in CDK12 lost PCa tumors is robust with clear delineation that BRCA1/2 genes and maintenance of most genes regulating HR are intact. Specifically, the authors find that there is no mutational signature or genomic features suggestive of HR, such as those found in BRCA1/2 tumors. Lastly, novel lines are generated in this study, including de novo LuCaP 189.4_CL with CDK12 loss that can be profound for potential synthetic lethalities.

Weakness:

One caveat that continues to be unclear as presented, is the uncoupling of cell cycle/essentiality of CDK12/13 from HR-directed mechanisms. Is this purely a cell cycle arrest phenotype acutely with associated down-regulation of many genes?

While the RAD51 loading ssRNA experiments are informative, the Tet-inducible knockdown of BRCA2 and CDK12 is confusing as presented in Figure 5, shBRCA2 + and -dox are clearly shown. However, were the CDK12_K02 and K05 also knocked down using inducible shRNA or a stable knockout? The importance of this statement is the difference between acute and chronic deletion of CDK12. Previously, the authors showed that acute knockdown of CDK12 led to an HR phenotype, but here it is unclear whether CDK12-K02/05 are acute knockdowns of CDK12 or have been chronically adapted after single cell cloning from CRISPR-knockout.

Given the multitude of lines, including some single-cell clones with growth inhibitory phenotypes and ex-vivo derived xenografts, the variability of effects with SR4835, ATM, ATR, and WEE1 inhibitors in different models can be confusing to follow. Overall, the authors suggest that the cell lines differ in therapeutic susceptibility as they may have alternate and diverse susceptibilities. It may be possible that the team could present this more succinctly and move extraneous data to the supplement.

The in-vitro data suggests that SR4835 causes growth inhibition acutely in parental lines such as 22RV1. However, in vivo, tumor attenuation appears to be observed in both CDK12 intact and deficient xenografts, LuCAP136 and LuCaP 189.4 (albeit the latter is only nominally significant). Is there an effect of PARPi inhibition specifically in either model? What about the the 22RV1-K02/05? Do these engraft? Given the role of CDK12/13 in RNAP II, these data might suggest that the window of susceptibility in CDK12 tumors may not be that different from CDK12 intact tumors (or intact tissue) when using dual CDK12/13 inhibitors but rather represent more general canonical essential functions of CDK12 and CDK13 in transcription. From a therapeutic development strategy, the authors may want to comment in the discussion on the ability to target CDK13 specifically.

Reviewer #3 (Public review):

Significance:

About 5% of metastatic castration-resistant prostate cancers (mCRPC) display genomic alterations in the transcriptional kinase CDK12. The mechanisms by which CDK12 alterations drive tumorigenesis in this molecularly-defined subset of mCRPC have remained elusive. In particular, some studies have suggested that CDK12 loss confers a homologous recombination deficiency (HRd) phenotype, However, clinical studies have not borne out the benefit to PARP inhibitors in patients with CDK12 alterations, despite the fact that these agents are typically active against tumors with HRd.

In this study, Frank et al. reconcile these findings by showing that: (1) tumors with biallelic CDK12 alterations do not have genomic features of HRd; (2) in vitro, HR gene downregulation occurs with acute depletion of CDK12 but is far less pronounced with chronic CDK12 loss; (3) CDK12-altered cells are uniquely sensitive to genetic or pharmacologic inhibition of CDK13.

Strengths:

Overall, this is an important study that reconciles disparate experimental and clinical observations. The genomic analyses are comprehensive and conducted with a high degree of rigor and represent an important resource to the community regarding the features of this molecular subtype of mCRPC.

Weaknesses:

(1) It is generally assumed that CDK12 alterations are inactivating, but it is noteworthy that homozygous deletions are comparatively uncommon (Figure 1a). Instead many tumors show missense mutations on either one or both alleles, and many of these mutations are outside of the kinase domain (Figure 1b). It remains possible that the CDK12 alterations that occur in some tumors may retain residual CDK12 function, or may confer some other neomorphic function, and therefore may not be accurately modeled by CDK12 knockout or knockdown in vitro. This would also reconcile the observation that knockout of CDK12 is cell-essential while the human genetic data suggest that CDK12 functions as a tumor suppressor gene.

(2) It is not entirely clear whether CDK12 altered tumors may require a co-occurring mutation to prevent loss of fitness, either in vitro or in vivo (e.g. perhaps one or more of the alterations that occur as a result of the TDP may mitigate against the essentiality of CDK12 loss).

Reviewer #1 (Public review):

Summary:<br /> In this study by Fang et al., the authors show how STAMBPL1 promotes TNBC angiogenesis via a feed-forward GRHL3/HIF1a/VEGFA axis. They demonstrate that STAMBPL1 interacts with FOXO1, define the required domains in each protein, and illustrate that this interaction facilitates FOXO1 transcriptional factor activity, which then activating GRHL3/HIF1a/VEGFA signaling. Lastly, they show that the combination of VEGFR and FOXO1 inhibitors can synergistically suppress STAMBPL1-overexpressing TNBC.

Strengths:

The manuscript is clearly written, and the results are well explained. The observation that STAMBPL1 mediates GRHL3 transcription through its interaction with FOXO1 is novel. The findings also have important translational potential.

Weaknesses:<br /> The mechanism by which STAMBPL1 mediates GRHL3 transcription through its interaction with FOXO1 is not sufficiently discussed, especially in relation to how STAMBPL1 regulates FOXO1. Some reported effects are modest.

Reviewer #2 (Public review):

Summary:<br /> In their manuscript, Fang and colleagues make a notable contribution to the field of oncology, particularly in advancing our understanding of triple-negative breast cancer (TNBC). The research delineates the role of STAMBPL1 in promoting angiogenesis in TNBC through its interaction with FOXO1 and the subsequent activation of the GRHL3/HIF1A/VEGFA axis. The evidence presented is robust, with a combination of in vitro experiments, RNA sequencing, and in vivo studies providing a comprehensive view of the molecular mechanisms at play. The strength of the evidence is anchored in the systematic approach and the utilization of multiple methodologies to substantiate the findings.

Strengths:<br /> The manuscript presents a methodologically robust framework, incorporating RNA-sequencing, chromatin immunoprecipitation (ChIP) assays, and a suite of in vitro and in vivo model systems, which collectively substantiate the claims regarding the pro-angiogenic role of STAMBPL1 in TNBC. The employment of multiple cellular models, conditioned media to assess HUVEC functional responses, and xenograft tumor models in murine hosts offers a comprehensive evaluation of STAMBPL1's impact on angiogenic processes.A salient strength of this work is the identification of GRHL3 as a transcriptional target of STAMBPL1 and the demonstration of a physical interaction between STAMBPL1 and FOXO1, which modulates GRHL3-driven HIF1A transcription. The study further suggests a potential therapeutic strategy by revealing the synergistic inhibitory effects of combined VEGFR and FOXO1 inhibitor treatment on TNBC tumor growth.

Weaknesses:<br /> A potential limitation of the study is the reliance on specific cellular and animal models, which may constrain the extrapolation of these findings to the broader spectrum of human TNBC biology. Furthermore, while the study provides evidence for a novel regulatory axis involving STAMBPL1, FOXO1, and GRHL3, the multifaceted nature of angiogenesis may implicate additional regulatory factors not exhaustively addressed in this research.

Appraisal of Achievement and Conclusion Support:<br /> The authors have successfully demonstrated that STAMBPL1 promotes HIF1A transcription and activates the HIF1α/VEGFA axis in a non-enzymatic manner, leading to increased angiogenesis in TNBC. The results are generally supportive of their conclusions, with clear evidence that STAMBPL1 upregulates HIF1α expression and enhances the activity of HUVECs. The study also shows that STAMBPL1 interacts with FOXO1 to promote GRHL3 transcription, which in turn activates HIF1A.

Impact on the Field and Utility:<br /> This research is poised to exert a substantial impact on the oncological research community by uncovering the role of STAMBPL1 in TNBC angiogenesis and by identifying the STAMBPL1/FOXO1/GRHL3/HIF1α/VEGFA axis as a potential therapeutic target. The findings could pave the way for the development of novel therapeutic strategies for TNBC, a subtype characterized by a paucity of effective treatment options. The methodologies utilized in this study are likely to be valuable to the research community, offering a paradigm for investigating the role of deubiquitinating enzymes in oncogenic processes.

Additional Context:<br /> It would be beneficial for readers to understand the broader context of TNBC research and the current challenges in treating this aggressive cancer subtype. The significance of this work is heightened by the lack of effective treatments for TNBC, making the identification of new therapeutic targets particularly important. Furthermore, understanding the specific mechanisms by which STAMBPL1 regulates HIF1α expression could provide insights into hypoxia signaling in other cancer types as well.

Reviewer #3 (Public review):

In this manuscript, Fang et al. describe a new oncogenic function of the STAMBPL1 protein in triple-negative breast cancer (TNBC). STAMBPL1 is a deubiquitinase that has been poorly studied in cancer. Previous reports identify it as a promoter of epithelial to mesenchymal transition or an inhibitor of cisplatin-induced cell death, but its participation to other cancer phenotypes has not been investigated. Fang et al. find that in cell line models of TNBC, STAMBPL1 promotes expression of the transcription factor HIF-1a and its downstream target VEGF, with the consequence of stimulating neo-angiogenesis in vitro and in vivo. Mechanistically, the authors find that this occurs via a non-enzymatic and indirect mechanism, that is by promoting the expression of GRHL3, a transcription factor that in turn binds to the HIF-1a promoter to stimulate its transcription. Interestingly, the way by which STAMPB1 promotes GRHL3 expression is by facilitating the transcriptional activity of FOXO1, a known regulator of GRHL3. Because the authors find that STAMBPL1 and FOXO1 interact, they suggest that STAMBPL1 may promote the formation of an active transcriptional complex containing FOXO1, perhaps by facilitating the recruitment of transcriptional coactivators.<br /> In conclusion, these data position for the first time the STAMBPL1 deubiquitinase in a FOXO-GRHL3 regulatory axis for the control of VEGF expression and tumor angiogenesis.<br /> The main weaknesses of this work are that the relevance of this molecular axis to the pathogenesis of TNBC is not clear, and it is not clearly established whether this is a regulatory pathway that occurs in hypoxic conditions or independently of oxygen levels.<br /> With respect to the first point, both FOXO1 and GRHL3 have been previously described as tumor suppressors, with reports of FOXO1 inhibiting tumor angiogenesis. Therefore, this works describes an apparently contradictory function of these proteins in TNBC. While it is not surprising that the same genes perform divergent functions in different tumor contexts, a stronger evidence in support of the oncogenic function of these two genes should be provided to make the data more convincing. As an example, the data in support of high STAMBPL1, FOXO and GRHL3 gene expression in TNBC TCGA specimens provided in Figure 8 is not very strong and it is not clear what the non-TNBC specimens are (whether other breast cancers or other tumors, perhaps those tumors whether these genes perform tumor suppressive functions). To strengthen the notion that STAMBPL1, FOXO and GRHL3 are overexpressed in TNCB, the authors could provide a comparison with normal tissue, as well as the analysis of other publicly available datasets (like the NCI Clinical Proteomic Tumor Analysis Consortium as an example). Finally, is it not clear what are the basal protein expression levels of STAMBPL1 in the cell lines used in this study, as based on the data presented in Figures 2D and F it appears that the protein is not expressed if not exogenously overexpressed. It would be helpful if the authors addressed this issue and provided further evidence of STAMBPL1 expression in TNBC cell lines.<br /> Linked to these considerations is the second major criticism, namely that it is not made clear if this new regulatory axis is proposed to act in normoxic or hypoxic conditions. The experiments presented in this paper are performed in both conditions but a clear explanation as to why cells are exposed to hypoxia is not given and would be necessary being that HIF-1a transcription and not protein stability is being analyzed. Also, different hypoxic conditions are sometimes used, resulting in different mRNA levels of HIF-1a and its downstream targets and quite significant fluctuations within the same cell line from one experimental setting to the next. The authors should provide an explanation as to why experimental conditions are changed and, more importantly, the experiments presented in Figure 2 should be performed also in normoxia.<br /> Another critical point is that necessary experimental controls are sometimes missing, and this is reducing the strength of some of the conclusions enunciated by the authors. As examples, experiments where overexpression of STAMBPL1 is coupled to silencing of FOXO1 to demonstrate dependency lack FOXO1silencing the absence of STAMBPL1 overexpression. Because diminishing FOXO1 expression affects HIF-1a/VEGF transcription even in the absence of STAMBPL1 (shown in Figure 7C, D), it is not clear if the data presented in Figure 7G are significant. The difference between HIF-1a expression upon FOXO1 silencing should be compared in the presence or absence of STAMBPL1 overexpression to understand if FOXO1 impacts HIF-1a transcription dependently or independently of STAMBPL1.

In addition, some minor comments to improve the quality of this manuscript are provided.<br /> (1) As a general statement, the manuscript is extremely synthetic. While this is not necessarily a negative feature, sometimes results are discussed in the figure legends and not in the main text (as an example, western blots showing HIF-1a expression) and this makes it hard to read thought the data in an easy and enjoyable manner.<br /> (2) The effect of STAMBPL1 overexpression on HIF-1a transcription is minor (Figure 2) The authors should explain why they think this is the case and whether hypoxia may provide a molecular environment that is more permissive to this type of regulation.<br /> (3) HIF-1a does not appear upregulated at the protein level protein by STAMBPL1 or GRLH3 overexpression, even though this is stated in the legends of Figures 2 and 6. The authors should show unsaturated western blots images and provide quantitative data of independent experiments to make this point.<br /> In summary, adding necessary controls and performing additional experiments to substantiate the oncogenic function of these genes in TNCB would strengthen the authors' conclusions.

Reviewer #1 (Public review):

This is a well-done clinical study which provides new information on the effects of metabolic disturbances in the human skeleton. 63 postmenopausal women undergoing hip arthroplasty, consisting of T2D with obesity; obesity alone; and neither T2D nor obesity were studied. Most of the findings relate to T2D. Increased serum TNF-α was found in T2D, as well as increased bone gene expression of TNF- α, which was associated with reduced expression of Wnt pathway genes. mRNA levels of certain of the cytokines correlated with Wnt signaling components. In addition, the increased serum TNF- α in T2D was associated with reduced Young's modulus, a measure of bone strength. A strength of this paper is that it provides information in an area that is not well-understood. However, there are a number of concerns that warrant direct addressing.

(1) Can the authors speculate why the changes in cytokines and Wnt expression do not impact bone microarchitecture?

(2) The authors state that they are showing an association between inflammation and bone strength via the regulation of Wnt signaling. However they have only shown here that serum cytokines correlate with bone strength. It is true that the authors have previously shown that Wnt signaling correlated with bone strength. But here it would be useful to show if bone strength is also correlated with inflammatory genes.

(3) AGEs increase inflammation (by binding to RAGE which triggers an inflammatory cascade). AGEs might also increase SOST. From their previous work, it seems that the authors have bone AGE measures on these patients and they have shown their relationship with SOST. Do the increased AGEs relate to inflammation as measured by serum and bone expression?

(4) Were bone turnover markers done to show how the inflammation and Wnt findings relate to bone resorption and formation?

(5) RNA integrity values should be reported to confirm that the RNA has not degraded.

(6) The discussion of adiponectin could be clearer (studies are cited that show both positive and negative effects). Please clarify that adiponectin effects on bone are complex and what they are.

(7) Were patients excluded for prior as well as current antiresorptive medication use?

(8) Fig 4A. correlation between SOST mRNA and TNF-a mRNA seems to be driven by 1 outlier. Does the relationship persist if it is removed?

Reviewer #2 (Public review):

Summary:

Chronic inflammation of the bone microenvironment conferred by T2DM and obesity may inhibit bone formation and bone strength by decreasing the ratio of Wnt ligands/Wnt inhibitors.

The authors studied 63 postmenopausal women (age >65 years) undergoing hip replacement for osteoarthritis. These were grouped into T2DM and obesity, obesity only, and normal subjects. A set of inflammatory markers was measured in the serum and gene expression of members of the Wnt system in the bone tissue. Bone samples were assessed by micro-CT.

While TNF-α serum levels were higher in T2DM, IL-6 levels were higher in obesity as compared to control. In the bone compartment the most consistent finding was decreased mRNA levels for WNt10b and increased sclerostin mRNA levels, translating into a suppressed Wnt-to-Wnt inhibitor ratio, which was associated with low bone strength.

Strengths:

The study includes clinically well-characterized subjects of three defined subgroups. The analyses were comprehensive.

Weaknesses:

Including data or information on the Wnt inhibitor Dkk1 would be instructive. Analysis were limited to mRNA studies. Validation of protein levels would be supportive (although technically challenging).

Reviewer #3 (Public review):

In this manuscript, the authors examine circulating and bone parameters in patients with T2DM or obesity vs control subjects. Based on their findings they conclude that increased inflammation in bone of subjects with T2DM and obesity is negatively correlated with Wnt pathway signaling and bone strength.

Overall, this is a well done clinical study that provides further insights into the pathogenesis of bone loss associated with T2DM. However, there are a number of issues that the authors should address:

(1) The major conceptual problem is that the alterations in circulating and bone factors they observed would predominantly affect bone turnover and thus, bone mass. But bone mass is preserved in T2DM (as their own data show). They postulate that their findings lead to impaired bone quality, but it is not clear how this would occur. For example, the impairment in bone quality could be due to the accumulation of AGEs in bone in T2DM, and the correlations observed be true but unrelated. Along these lines, were serum or bone AGEs measured - and if not, is it possible for the authors to do so? At the least, this issue should be fully addressed in the Discussion if the authors are unable to provide additional data to address this.

(2) The T2DM patients were extremely well controlled. This may have limited some of the differences between groups. Was it not possible to select a group of less well-controlled patients - that is more the norm? This may also explain why the biomechanical indices in Table 3 were only marginally different in the T2DM vs the other groups. This point should also be addressed.

(3) The authors found some interesting differences in bone sclerostin levels. Were circulating sclerostin levels measured? This data would be of interest and should be provided.

(4) Fig 4A - the correlation between TNFa and SOST seems to be driven by one highly influential point. What happens if this point is removed? Is this point a formal statistical outlier? Please check this.

Reviewer #1 (Public review):

This article deals with the chemotactic behavior of E coli bacteria in thin channels (a situation close to 2D). It combines experiments and simulations.

The authors show experimentally that, in 2D, bacteria swim up a chemotactic gradient much more effectively when they are in the presence of lateral walls. Systematic experiments identify an optimum for chemotaxis for a channel width of ~8µm, close to the average radius of the circle trajectories of the unconfined bacteria in 2D. It is known that these circles are chiral and impose that the bacteria swim preferentially along the right-side wall when there is no chemotactic gradient. In the presence of a chemotactic gradient, this larger proportion of bacteria swimming on the right wall yields chemotaxis. This effect is backed by numerical simulations and a geometrical analysis.

If the conclusions drawn from the experiments presented in this article seem clear and interesting, I find that the key elements of the mechanism of this wall-directed chemotaxis are not sufficiently emphasized. Moreover, the paper would be clearer with more details on the hypotheses and the essential ingredients of the analyses.

Reviewer #2 (Public review):

Summary:

In this study, the authors investigated the chemotaxis of E. coli swimming close to the bottom surface in gradients of attractant in channels of increasingly smaller width but fixed height = 30 µm and length ~160 µm. In relatively large channels, they find that on average the cells drift in response to the gradient, despite cells close to the surface away from the walls being known to not be chemotactic because they swim in circles.

They find that this average drift is due to the cell localization close to the side walls, where they slide along the wall. Whereas the bacteria away from the walls have no chemotaxis (as shown before), the ones on the left side wall go down-gradient on average, but the ones on the right side wall go up-gradient faster, hence the average drift. They then study the effect of reducing channel width. They find that chemotaxis is higher in channels with a width of about 8 µm, which approximately corresponds to the radius of the circular swimming R. This higher chemotactic drift is concomitant to an increased density of cells on the RSW. They do simulations and modeling to suggest that the disruption of circular swimming upon collision with the wall increases the density of cells on the RSW, with a maximal effect at w = ~ 2/3 R, which is a good match for their experiments.

Strengths:

The overall result that confinement at the edge stabilises bacterial motion and allows chemotaxis is very interesting although not entirely unexpected. It is also important for understanding bacterial motility and chemotaxis under ecologically relevant conditions, where bacteria frequently swim under confinement (although its relevance for controlling infections could be questioned). The experimental part of the study is nicely supported by the model.

Weaknesses:

Several points of this study, in particular the interpretation of the width effect, need better clarification:

(1) Context:

There are a number of highly relevant previous publications that should have been acknowledged and discussed in relation to the current work:<br /> https://pubs.rsc.org/en/content/articlehtml/2023/sm/d3sm00286a<br /> https://link.springer.com/article/10.1140/epje/s10189-024-00450-7<br /> https://doi.org/10.1016/j.bpj.2022.04.008<br /> https://doi.org/10.1073/pnas.1816315116<br /> https://www.pnas.org/doi/full/10.1073/pnas.0907542106<br /> https://doi.org/10.1038/s41467-020-15711-0<br /> http://doi.org/10.1038/s41467-020-15711-0<br /> http://doi.org/10.1039/c5sm00939a

(2) Experimental setup:

a) The channels are built with asymmetric entrances (Figure 1), which could trigger a ratchet effect (because bacteria swim in circle) that could bias the rate at which cells enter into the channel, and which side they follow preferentially, especially for the narrow channel. Since the channel is short (160 µm), that would reflect on the statistics of cell distribution. Controls with straight entrances or with a reversed symmetry of the channel need to be performed to ensure that the reported results are not affected by this asymmetry.

b) The authors say the motile bacteria accumulate mostly at the bottom surface. This is strange, for a small height of 30 µm, the bacteria should be more-or-less evenly spread between the top and bottom surface. How can this be explained?

c) At the edge, some of the bacteria could escape up in the third dimension (http://doi.org/10.1039/c5sm00939a). What is the magnitude of this phenomenon in the current setup? Does it have an effect?

d) What is the cell density in the device? Should we expect cell-cell interactions to play a role here? If not, I would suggest to de-emphasize the connection to chemotaxis in the swarming paper in the introduction and discussion, which doesn't feel very relevant here, and rather focus on the other papers mentioned in point 1.

e) We are not entirely convinced by the interpretation of the results in narrow channels. What is the causal relationship between the increased density on the RSW and the higher chemotactic drift? The authors seem to attribute higher drift to this increased RSW density, which emerges due to the geometric reasons. But if there is no initial bias, the same geometric argument would induce the same increased density of down-gradient swimmers on the LSW, and so, no imbalance between RSW and LSW density. Could it be the opposite that the increased RSW density results from chemotaxis (and maybe reinforces it), not the other way around? Confinement could then deplete one wall due to the proximity of the other, and/or modify the swimming pattern - 8 µm is very close to the size of the body + flagellum. To clarify this point, we suggest measuring the bacterial distributions in the absence of a gradient for all channel widths as a control.

(3) Simulations:

The simulations treat the wall interaction very crudely. We would suggest treating it as a mechanical object that exerts elastic or "hard sphere" forces and torques on the bacteria for more realistic modeling. Notably, the simulations have a constant (chemotaxis independent) rate of wall escape by tumbling. We would expect that reduced tumbling due to up-gradient motility induces a longer dwell time at the wall.

Reviewer #3 (Public review):

This paper addresses through experiment and simulation the combined effects of bacterial circular swimming near no-slip surfaces and chemotaxis in simple linear gradients. The authors have constructed a microfluidic device in which a gradient of L-aspartate is established to which bacteria respond while swimming while confined in channels of different widths. There is a clear effect that the chemotactic drift velocity reaches a maximum in channel widths of about 8 microns, similar in size to the circular orbits that would prevail in the absence of side walls. Numerical studies of simplified models confirm this connection.

The experimental aspects of this study are well executed. The design of the microfluidic system is clever in that it allows a kind of "multiplexing" in which all the different channel widths are available to a given sample of bacteria.

While the data analysis is reasonably convincing, I think that the authors could make much better use of what must be voluminous data on the trajectories of cells by formulating the mathematical problem in terms of a suitable Fokker-Planck equation for the probability distribution of swimming directions. In particular, I would like to see much more analysis of how incipient circular trajectories are interrupted by collisions with the walls and how this relates to enhanced chemotaxis. In essence, there needs to be a much clearer control analysis of trajectories without sidewalls to understand the mechanism in their presence.

The authors argue that these findings may have relevance to a number of physiological and ecological contexts. Yet, each of these would be characterized by significant heterogeneity in pore sizes and geometries, and thus it is very unclear whether or how the findings in this work would carry over to those situations.

Reviewer #1 (Public review):

Summary:

The authors present a modelling study to test the hypothesis that horizontal gene transfer (HGT) can modulate the outcome of interspecies competition in microbiomes, and in particular promote bistability in systems across scales. The premise is a model developed by the same authors in a previous paper where bistability happens because of a balance between growth rates and competition for a mutual resource pool (common carrying capacity). They show that introducing a transferrable element that gives a "growth rate bonus" expands the region of parameter space where bistability happens. The authors then investigate how often (in terms of parameter space) this bistability occurs across different scales of complexity, and finally under selection for the mobile element (framed as ABR selection).

Strengths:

The authors tackle an important, yet complex, question: how do different evolutionary processes impact the ecology of microbial ecosystems? They do a nice job at increasing the scales of heterogeneity and asking how these impact their main observable: bistability.

Weaknesses:

The author's starting point is their interaction LV model and the manuscript then explores how this model behaves under different scenarios. Because the structure of the model and the underlying assumptions essentially dictate these outcomes, I would expect to see much more focus on how these two aspects relate to the specific scenarios that are discussed. For example:

A key assumption is that the mobile element conveys a multiplicative growth rate benefit (1+lambda). However, the competition between the species is modelled as a factor gamma that modulates the competition for overall resource and thus appears in the saturation term (1+ S1/Nm + gamma2*S2/Nm). This means that gamma changes the perceived abundance of the other species (if gamma > 1, then from the point of view of S1 it looks like there are more S2 than there really are). Most importantly, the relationship between these parameters dictates whether or not there will be bistability (as the authors state).

This decoupling between the transferred benefit and the competition can have different consequences. One of them is that - from the point of view of the mobile element - the mobile element competes at different strengths within the same population compared to between. To what degree introducing such a mobile element modifies the baseline bistability expectation thus strongly depends on how it modifies gamma and lambda.

Thus, this structural aspect needs to be much more carefully presented to help the reader follow how much of the results are just trivial given the model assumptions and which have more of an emergent flavour. From my point of view, this has an important impact on helping the reader understand how the model that the authors present can contribute to the understanding of the question "how microbes competing for a limited number of resources stably coexist". I do appreciate that this changes the focus of the manuscript from a presentation of simulation results to more of a discussion of mathematical modelling.

Reviewer #2 (Public review):

Summary:

In this work, the authors use a theoretical model to study the potential impact of Horizontal Gene Transfer on the number of alternative stable states of microbial communities. For this, they use a modified version of the competitive Lotka Volterra model-which accounts for the effects of pairwise, competitive interactions on species growth-that incorporates terms for the effects of both an added death (dilution) rate acting on all species and the rates of horizontal transfer of mobile genetic elements-which can in turn affect species growth rates. The authors analyze the impact of horizontal gene transfer in different scenarios: bistability between pairs of species, multistability in communities, and a modular structure in the interaction matrix to simulate multiple niches. They also incorporate additional elements to the model, such as spatial structure to simulate metacommunities and modification of pairwise interactions by mobile genetic elements. In almost all these cases, the authors report an increase in either the number of alternative stable states or the parameter region (e.g. growth rate values) in which they occur.

In my opinion, understanding the role of horizontal gene transfer in community multistability is a very important subject. This manuscript is a useful approach to the subject, but I'm afraid that a thorough analysis of the role of different parameters under different scenarios is missing in order to support the general claims of the authors. The authors have extended their analysis to increase their biological relevance, but I believe that the analysis still lacks comprehensiveness.

Understanding the origin of alternative stable states in microbial communities and how often they may occur is an important challenge in microbial ecology and evolution. Shifts between these alternative stable states can drive transitions between e.g. a healthy microbiome and dysbiosis. A better understanding of how horizontal gene transfer can drive multistability could help predict alternative stable states in microbial communities, as well as inspire novel treatments to steer communities towards the most desired (e.g. healthy) stable states.

Strengths:

(1) Generality of the model: the work is based on a phenomenological model that has been extensively used to predict the dynamics of ecological communities in many different scenarios.

(2) The question of how horizontal gene transfer can drive alternative stable states in microbial communities is important and there are very few studies addressing it.

Weaknesses:

(1) There is a need for a more comprehensive analysis of the relative importance of the different model parameters in driving multistability. For example, there is no analysis of the effects of the added death rate in multistability. This parameter has been shown to determine whether a given pair of interacting species exhibits bistability or not (see e.g. Abreu et al 2019 Nature Communications 10:2120). Similarly, each scenario is analyzed for a unique value of species interspecies interaction strength-with the exception of the case for mobile genetic elements affecting interaction strength, which considers three specific values. Considering heterogeneous interaction strengths (e.g. sampling from a random distribution) could also lead to more realistic scenarios - the authors generally considered that all species pairs interact with the same strength. Analyzing a larger range of growth rates effects of mobile genetic elements would also help generalize the results. In order to achieve a more generic assessment of the impact of horizontal gene transfer in driving multistability, its role should be systematically compared to the effects of the rest of the parameters of the model.

(2) The authors previously developed this theoretical model to study the impact of horizontal gene transfer on species coexistence. In this sense, it seems that the authors are exploring a different (stronger interspecies competition) range of parameter values of the same model, which could potentially limit novelty and generality.

(3) The authors analyze several scenarios that, in my opinion, naturally follow from the results and parameter value choices in the first sections, making their analysis not very informative. For example, after showing that horizontal gene transfer can increase multistability both between pairs of species and in a community context, the way they model different niches does not bring significantly new results. Given that the authors showed previously in the manuscript that horizontal gene transfer can impact multistability in a community in which all species interact with each other, one might expect that it will also impact multistability in a larger community made of (sub)communities that are independent of (not interacting with) each-which is the proposed way for modelling niches. A similar argument can be made regarding the analysis of (spatially structured) metacommunities. It is known that, for smaller enough dispersal rates, space can promote regional diversity by enabling each local community to remain in a different stable state. Therefore, in conditions in which the impact of horizontal gene transfer drives multistability, it will also drive regional diversity in a metacommunity.

(4) In some cases, the authors consider that mobile genetic elements can lead to ~50% growth rate differences. In the presence of an added death rate, this can be a relatively strong advantage that makes the fastest grower easily take over their competitors. It would be important to discuss biologically relevant examples in which such growth advantages driven by mobile genetic elements could be expected, and how common such scenarios might be.

Reviewer #3 (Public review):

Hong et al. used a model they previously developed to study the impact of horizontal gene transfer (HGT) on microbial multispecies communities. They investigated the effect of HGT on the existence of alternative stable states in a community. The model most closely resembles HGT through the conjugation of incompatible plasmids, where the transferred genes confer independent growth-related fitness effects. For this type of HGT, the authors find that increasing the rate of HGT leads to an increasing number of stable states. This effect of HGT persists when the model is extended to include multiple competitive niches (under a shared carrying capacity) or spatially distinct patches (that interact in a grid-like fashion). Instead, if the mobile gene is assumed to reduce between-species competition, increasing HGT leads to a smaller region of multistability and fewer stable states. Similarly, if the mobile gene is deleterious an increase in HGT reduces the parameter region that supports multistability.

This is an interesting and important topic, and I welcome the authors' efforts to explore these topics with mathematical modeling. The manuscript is well written and the analyses seem appropriate and well-carried out. However, I believe the model is not as general as the authors imply and more discussion of the assumptions would be helpful (both to readers + to promote future theoretical work on this topic). Also, given the model, it is not clear that the conclusions hold quite so generally as the authors claim and for biologically relevant parameters. To address this, I would recommend adding sensitivity analyses to the manuscript.

Specific points

(1) The model makes strong assumptions about the biology of HGT, that are not adequately spelled out in the main text or methods, and will not generally prove true in all biological systems. These include:<br /> a) The process of HGT can be described by mass action kinetics. This is a common assumption for plasmid conjugation, but for phage transduction and natural transformation, people use other models (e.g. with free phage that adsorp to all populations and transfer in bursts).<br /> b) A subpopulation will not acquire more than one mobile gene, subpopulations can not transfer multiple genes at a time, and populations do not lose their own mobilizable genes. [this may introduce bias, see below].<br /> c) The species internal inhibition is independent of the acquired MGE (i.e. for p1 the self-inhibition is by s1).<br /> These points are in addition to the assumptions explored in the supplementary materials, regarding epistasis, the independence of interspecies competition from the mobile genes, etc. I would appreciate it if the authors could be more explicit in the main text about the range of applicability of their model, and in the methods about the assumptions that are made.

(2) I am not surprised that a mechanism that creates diversity will lead to more alternative stable states. Specifically, the null model for the absence of HGT is to set gamma to zero, resulting in pij=0 for all subpopulations (line 454). This means that a model with N^2 classes is effectively reduced to N classes. It seems intuitive that an LV-model with many more species would also allow for more alternative stable states. For a fair comparison, one would really want to initialize these subpopulations in the model (with the same growth rates - e.g. mu1(1+lambda2)) but without gene mobility.

(3) I am worried that the absence of double gene acquisitions from the model may unintentionally promote bistability. This assumption is equivalent to an implicit assumption of incompatibility between the genes transferred from different species. A highly abundant species with high HGT rates could fill up the "MGE niche" in a species before any other species have reached appreciable size. This would lead to greater importance of initial conditions and could thus lead to increased multistability.

This concern also feels reminiscent of the "coexistence for free" literature (first described here http://dx.doi.org/10.1016/j.epidem.2008.07.001 ) which was recently discussed in the context of plasmid conjugation models in the supplementary material (section 3) of https://doi.org/10.1098/rstb.2020.0478 .

(4) The parameter values tested seem to focus on very large effects, which are unlikely to occur commonly in nature. If I understand the parameters in Figure 1b correctly for instance, lambda2 leads to a 60% increase in growth rate. Such huge effects of mobile genes (here also assumed independent from genetic background) seem unlikely except for rare cases. To make this figure easier to interpret and relate to real-world systems, it could be worthwhile to plot the axes in terms of the assumed cost/benefit of the mobile genes of each species.

Something similar holds for the HGT rate (eta): given that the population of E. coli or Klebsiella in the gut is probably closer to 10^9 than 10^12 (they make up only a fraction of all cells in the gut), the assumed rates for eta are definitely at the high end of measured plasmid transfer rates (e.g. F plasmid transfers at a rate of 10^-9 mL/CFU h-1, but it is derepressed and considered among the fastest - https://doi.org/10.1016/j.plasmid.2020.102489 ). To adequately assess the impact of the HGT rate on microbial community stability it would need to be scanned on a log (rather than a linear) scale. Considering the meta-analysis by Sheppard et al. it would make sense to scan it from 10^-7 to 1 for a community with a carrying capacity around 10^9.

(5) It is not clear how sensitive the results (e.g. Figure 2a on the effect of HGT) are to the assumption of the fitness effect distribution of the mobile genes. This is related to the previous point that these fitness effects seem quite large. I think some sensitivity analysis of the results to the other parameters of the simulation (also the assumed interspecies competition varies from figure to figure) would be helpful to put the results into perspective and relate them to real biological systems.

Reviewer #1 (Public review):

Weiler, Teichert, and Margrie systematically analyzed long-range cortical connectivity, using a retrograde viral tracing strategy to identify layer and region-specific cortical projections onto the primary visual, primary somatosensory, and primary motor cortices. Their analysis revealed several hundred thousand inputs into each region, with inputs originating from almost all cortical regions but dominated in number by connections within cortical sub-networks (e.g. anatomical modules). Generally, the relative areal distribution of contralateral inputs followed the distribution of corresponding ipsilateral inputs. The largest proportion of inputs originated from layer 6a cells, and this layer 6 dominance was more pronounced for contralateral than ipsilateral inputs, which suggests that these connections provide predominantly feedback inputs. The hierarchical organization of input regions was similar between ipsi- and contralateral regions, except for within-module connections, where ipsilateral connections were much more feed-forward than contralateral. These results contrast earlier studies which suggested that contralateral inputs only come from the same region (e.g. V1 to V1) and from L2/3 neurons. Thus, these results provide valuable data supporting a view of interhemispheric connectivity in which layer 6 neurons play an important role in providing modulatory feedback.

The conclusions of this paper are mostly well-supported by the data and analysis, but additional consideration of possible experimental biases is needed.

Further discussion or analysis is needed about possible biases in uptake efficiency for different cell types. Is it possible that the nuclear retro-AAV has a tropism for layer 6 axons? Quantitative comparisons with results obtained with alternative methods such as rabies virus (Yao et al., 2023) or anterograde tracing (Harris et al., 2019) may be helpful for this.

Quantitative analysis of the injection sites should be included to account for possible biases. For example, L6 neurons are known to be the main target of contralateral inputs into the visual cortex (Yao et al., 2023). Thus, if the injections are biased towards or against layer 6 neurons, this may change the layer distribution of retrogradely labeled input cells. Comparison across biological replicates may help reveal sensitivity to particular characteristics of the injections.

The possibility of labeling axons of passage within the white matter should be addressed. This could potentially lead to false positive connections, contributing to the broad connectivity from most cortical regions that were observed.

Reviewer #2 (Public review):

Summary:

Weiler et al use retrograde tracers, two-photon tomography, and automatic cell detection to provide a detailed quantitative description of the laminar and area sources of ipsi- and contralateral cortico-cortical inputs to two primary sensory areas and a primary motor area. They found considerable bilateral symmetry in the areas providing cortico-cortical inputs. However, although the same regions in both hemispheres tended to supply inputs, a larger proportion of inputs from contralateral areas originated from deeper layers (L5 and L6).

Strengths:

The study applies state-of-the-art anatomical methods, and the data is very effectively presented and carefully analyzed. The results provide many novel insights into the similarities and differences of inputs from the two hemispheres. While over the past decade there have been many studies quantitively and comprehensively describing cortico-cortical connections, by directly comparing inputs from the ipsi and contralateral hemispheres, this study fills in an important gap in the field. It should be of great utility and an important reference for future studies on inter-hemispheric interactions.

Weaknesses:

Overall, I do not find any major weakness in the analyses or their interpretation. However, one must keep in mind that the study only analyses inputs projecting to three areas. This is not an inherent flaw of the study; however, it warrants caution when extrapolating the results to callosal projections terminating in other areas. As inputs to two primary sensory areas and one is the primary motor cortex are studied, some of the conclusions could potentially be different for inputs terminating in high-order sensory and motor areas. Given that primary areas were injected, there are few instances of feedforward connections sampled in the ipsilateral hemisphere. The study finds that while ipsi-projections from the visual cortex to the barrel cortex are feedforward given its fILN values, those from the contralateral visual cortex are feedback instead. One is left to wonder whether this is due to the cross-modal nature of these particular inputs and whether the same rule (that contralateral inputs consistently exhibit feedback characteristics regardless of the hierarchical relationship of their ipsilateral counterparts with the target area,) would also apply to feedforward inputs within the same sensory cortices.

Another issue that is left unexplored is that, in the current analyses the barrel and primary visual cortex are analyzed as a uniform structure. It is well established that both the laminar sources of callosal inputs and their terminations differ in the monocular and binocular areas of the visual cortex (border with V2L). Similarly, callosal projections differ when terminating the border of S1 (a row of whiskers), and then in other parts of S1. Thus, some of the conclusions regarding the laminar sources of callosal inputs might depend on whether one is analyzing inputs terminating or originating in these border regions.

Finally, while the paper emphasizes that projections from L6 "dominate" intra and contralateral cortico-cortical inputs, the data shows a more nuanced scenario. While it is true that the areas for which L6 neurons are the most common source of cortico-cortical projections are the most abundant, the picture becomes less clear when considering the number of neurons sending these connections. In fact, inputs from L2/3 and L5 combined are more abundant than those from L6 (Figure 3B), challenging the view that projections from L6 dominate ipsi- and contralateral projecting cortico-cortical inputs.

Reviewer #1 (Public review):

D'Oliviera et al. have demonstrated cleavage of human TRMT1 by the SARS-CoV-2 main protease in vitro. Following, they solved the structure of Mpro (Nsp5)-C145A bound to TRMT1 substrate peptide, revealing binding conformation distinct from most viral substrates. Overall, this work enhances our understanding of substrate specificity for a key drug target of CoV2. The paper is well-written and the data is clearly presented. It complements the companion article by demonstrating interaction between Mpro and TRMT1, as well as TRMT1 cleavage under isolated conditions in vitro. They show that cleaved TRMT1 has reduced tRNA binding affinity, linking a functional consequence to TRMT1 cleavage by MPro. Importantly, the revelation for flexible substrate binding of Nsp5 is fundamental for understanding Nsp5 as a drug target. Trmt1 cleavage assays by Mpro revealed similar kinetics for TRMT1 cleavage as compared to nsp8/9 viral polyprotein cleavage site. They purify TRMT1-Q350K, in which there is a mutation in the predicted cleavage consensus sequence, and confirm that it is resistant to cleavage by recombinant Mpro. I am unable to comment critically on the structural analyses as it is outside of my expertise. Overall, I think that these findings are important for confirming TRMT1 as a substrate of Mpro, defining substrate binding and cleavage parameters for an important drug target of SARS-CoV-2, and may be of interest to researchers studying RNA modifications.

Reviewer #2 (Public review):

Summary:

The manuscript 'Recognition and Cleavage of Human tRNA Methyltransferase TRMT1 by the SARS-CoV-2 Main Protease' from Angel D'Oliviera et al., uncovers that TRMT1 can be cleaved by SARS-CoV-2 main protease (Mpro) and defines the structural basis of TRMT1 recognition by Mpro. They use both recombinant TRMT1 and Mpro as well as endogenous TRMT1 from HEK293T cell lysates to convincingly show cleavage of TRMT1 by the SARS-CoV-2 protease. Using in vitro assays, the authors demonstrate that TRMT1 cleavage by Mpro blocks its enzymatic activity leading to hypomodification of RNA. To understand how Mpro recognizes TRMT1, they solved a co-crystal structure of Mpro bound to a peptide derived from the predicted cleavage site of TRMT1. This structure revealed important protein-protein interfaces and highlights the importance of the conserved Q530 for cleavage by Mpro. They then compare their structure with previous X-ray crystal structures of Mpro bound to substrate peptides derived from the viral polyprotein and propose the concept of two distinct binding conformations to Mpro: P3´-out and P3´-in conformations (here P3´ stands for the third residue downstream of the cleavage site). It remains unknown what is the physiological role of these two binding conformations on Mpro function, but the authors established that Mpro has dramatically different cleavage efficiencies for three distinct substrates. In an effort to rationalize this observation, a series of mutations in Mpro's active site and the substrate peptide were tested but unexpectedly had no significant impact on cleavage efficiency. While molecular dynamic simulations further confirmed the propensity of certain substrates to adopt the P3´-out or P3´-in conformation, it did not provide additional insights into the dramatic differences in cleavage efficiencies between substrates. This led the authors to propose that the discrimination of Mpro for preferred substrates might occur at a later stage of catalysis after binding of the peptide. Overall, this work will be of interest to biologists studying proteases and substrate recognition by enzymes and RNA modifications as well as help efforts to target Mpro with peptide-like drugs.

Strengths:

• The authors' statements are well supported by their data, and they used relevant controls when needed. Indeed, they used the Mpro C145A inactive variant to unambiguously show that the TRMT1 cleavage detected in vitro is solely due to Mpro's activity. Moreover, they used two distinct polyclonal antibodies to probe TRMT1 cleavage.<br /> • They demonstrate the impact of TRMT1 cleavage on RNA modification by quantifying both its activity and binding to RNA.<br /> • Their 1.9 Å crystal structure is of high quality and increases the confidence in the reported protein-protein contacts seen between TRMT1-derived peptide and Mpro.<br /> • Their extensive in vitro kinetic assay was performed in ideal conditions although it is sometimes unclear how many replicates were performed.<br /> • They convincingly show how Mpro cleavage is conserved among most but not all mammalian TRMT1 bringing an interesting evolutionary perspective on virus-host interactions.<br /> • The authors test multiple hypotheses to rationalize the preference of Mpro for certain substrates.<br /> • While this reviewer is not able to comment on the rigor of the MD simulations, the interpretations made by the authors seem reasonable and convincing.<br /> • The concept of two binding conformations (P3´-out or P3´-in) for the substrate in the active site of Mpro is significant and can guide drug design.

Weaknesses:

• The two polyclonal antibodies used by the authors seem to have strong non-specific binding to proteins other than TRMT1 but did not impact the author's conclusions or statements. This is a limitation of the commercially available antibodies for TRMT1.<br /> • Despite the reasonable efforts of the authors, it remains unknown why Mpro shows higher cleavage efficiency for the nsp4/5 sequence compared to TRMT1 or nsp8/9 sequences. This is a challenging problem that will take substantially more effort by several labs to decipher mechanistically.<br /> • The peptide cleavage kinetic assay used by the authors relies on a peptide labelled with a fluorophore (MCA) on the N-terminus and a quencher (Dpn) on the C-terminus. This design allows high-throughput measurements compatible with plate readers and is a robust and convenient tool. Nevertheless, the authors did not control for the impact of the labels (MCA and Dpn) on the activity of Mpro. While in most cases the introduced fluorophore/quencher do not impact activity, sometimes it can.<br /> • An unanswered question not addressed by the authors is if the peptides undergo conformational changes upon Mpro binding or if they are pre-organized to adopt the P3´-out and P3´-in conformations. This might require substantially more work outside the scope of this immediate article.

Reviewer #3 (Public review):

Summary:

In this manuscript, the authors have used a combination of enzymatic, crystallographic, and in silico approaches to provide compelling evidence for substrate selectivity of SARS-CoV-2 Mpro for human TRMT1.

Strengths:

In my opinion, the authors came close to achieving their intended aim of demonstrating the structural and biochemical basis of Mpro catalysis and cleavage of human TRMT1 protein. The revised version of the manuscript has addressed most of the questions I had posed in my earlier review.

Weaknesses:

Although several new hypotheses are generated from the Mpro structural data, the manuscript falls a bit short of testing them in functional assays, which would have solidified the conclusions the authors have drawn.

Reviewer #2 (Public review):

A high fraction of cells in early embryos carry aneuploid karyotypes, yet even chromosomally mosaic human blastocysts can implant and lead to healthy newborns with diploid karyotypes. Previous studies in other models have shown that genotoxic and proteotoxic stresses arising from aneuploidy lead to the activation of the p53 pathway and autophagy, which helps eliminate cells with aberrant karyotypes. These observations have been here evaluated and confirmed in human blastocysts. The study also demonstrates that the second lineage and formation of primitive endoderm are particularly impaired by aneuploidy.

Comments on revisions:

The authors have addressed the critical issues sufficiently. In particular, they improved the data analysis and added additional data from embryonal samples.

Reviewer #3 (Public review):

This study provides valuable insights into the cellular responses to complex aneuploidy in human preimplantation embryos. The authors have significantly expanded their sample size and conducted additional analysis and experiments to address previous concerns. The revised manuscript presents stronger evidence for gene dosage-dependent effects of aneuploidy on stress responses and lineage segregation. Overall, the findings contribute important knowledge to our understanding of how human embryos respond to chromosomal abnormalities.

Overall, the revision has substantially improved the manuscript and addressed the major concerns raised in the initial review.

Reviewer #1 (Public review):

Summary:

In this manuscript, Yan and colleagues introduce a modification to the previously published PETRI-seq bacterial single cell protocol to include a ribosomal depletion step based on a DNA probe set that selectively hybridizes with ribosome-derived (rRNA) cDNA fragments. They show that their modification of the PETRI-seq protocol increases the fraction of informative non-rRNA reads from ~4-10% to 54-92%. The authors apply their protocol to investigating heterogeneity in a biofilm model of E. coli, and convincingly show how their technology can detect minority subpopulations within a complex community.

Strengths:

The method the authors propose is a straightforward and inexpensive modification of an established split-pool single cell RNA-seq protocol that greatly increases its utility, and should be of interest to a wide community working in the field of bacterial single cell RNA-seq.

Comments on revised version:

The reviewers have responded thoughtfully and comprehensively to all of my comments. I believe the details of the protocol are now much easier to understand, and the text and methods have been significantly clarified. I have no further comments.

Reviewer #2 (Public review):

Summary:

This work introduces a new method of depleting the ribosomal reads from the single-cell RNA sequencing library prepared with one of the prokaryotic scRNA-seq techniques, PETRI-seq. The advance is very useful since it allows broader access to the technology by lowering the cost of sequencing. It also allows more transcript recovery with fewer sequencing reads. The authors demonstrate the utility and performance of the method for three different model species and find a subpopulation of cells in the E.coli biofilm that express a protein, PdeI, which causes elevated c-di-GMP levels. These cells were shown to be in a state that promotes persister formation in response to ampicillin treatment.

Strengths:

The introduced rRNA depletion method is highly efficient, with the depletion for E.coli resulting in over 90% of reads containing mRNA. The method is ready to use with existing PETRI-seq libraries which is a large advantage, given that no other rRNA depletion methods were published for split-pool bacterial scRNA-seq methods. Therefore, the value of the method for the field is high. There is also evidence that a small number of cells at the bottom of a static biofilm express PdeI which is causing the elevated c-di-GMP levels that are associated with persister formation. This finding highlights the potentially complex role of PdeI in regulation of c-di-GMP levels and persister formation in microbial biofilms.

Comments on revised version:

The authors edited the manuscript thoroughly in response to the comments, including both performing new experiments and showing more data and information. Most of the major points raised between both reviewers were addressed. The authors explained the seeming contradiction between c-di-GMP levels and PdeI expression.

Reviewer #1 (Public review):

Summary:

This work investigated the role of CXXC-finger protein 1 (CXXC1) in regulatory T cells. CXXC1-bound genomic regions largely overlap with Foxp3-bound regions and regions with H3K4me3 histone modifications in Treg cells. CXXC1 and Foxp3 interact with each other, as shown by co-immunoprecipitation. Mice with Treg-specific CXXC1 knockout (KO) succumb to lymphoproliferative diseases between 3 to 4 weeks of age, similar to Foxp3 KO mice. Although the immune suppression function of CXXC1 KO Treg is comparable to WT Treg in an in vitro assay, these KO Tregs failed to suppress autoimmune diseases such as EAE and colitis in Treg transfer models in vivo. This is partly due to the diminished survival of the KO Tregs after transfer. CXXC1 KO Tregs do not have an altered DNA methylation pattern; instead, they display weakened H3K4me3 modifications within the broad H3K4me3 domains, which contain a set of Treg signature genes. These results suggest that CXXC1 and Foxp3 collaborate to regulate Treg homeostasis and function by promoting Treg signature gene expression through maintaining H3K4me3 modification.

Strengths:

Epigenetic regulation of Treg cells has been a constantly evolving area of research. The current study revealed CXXC1 as a previously unidentified epigenetic regulator of Tregs. The strong phenotype of the knockout mouse supports the critical role CXXC1 plays in Treg cells. Mechanistically, the link between CXXC1 and the maintenance of broad H3K4me3 domains is also a novel finding.

Weaknesses:

(1) It is not clear why the authors chose to compare H3K4me3 and H3K27me3 enriched genomic regions. There are other histone modifications associated with transcription activation or repression. Please provide justification.

(2) It is not clear what separates Clusters 1 and 3 in Figure 1C. It seems they share the same features.

(3) The claim, "These observations support the hypothesis that FOXP3 primarily functions as an activator by promoting H3K4me3 deposition in Treg cells." (line 344), seems to be a bit of an overstatement. Foxp3 certainly can promote transcription in ways other than promoting H3K3me3 deposition, and it also can repress gene transcription without affecting H3K27me3 deposition. Therefore, it is not justified to claim that promoting H3K4me3 deposition is Foxp3's primary function.

(4) For the in vitro suppression assay in Figure S4C, and the Treg transfer EAE and colitis experiments in Figure 4, the Tregs should be isolated from Cxxc1 fl/fl x Foxp3 cre/wt female heterozygous mice instead of Cxxc1 fl/fl x Foxp3 cre/cre (or cre/Y) mice. Tregs from the homozygous KO mice are already activated by the lymphoproliferative environment and could have vastly different gene expression patterns and homeostatic features compared to resting Tregs. Therefore, it's not a fair comparison between these activated KO Tregs and resting WT Tregs.

(5) The manuscript didn't provide a potential mechanism for how CXXC1 strengthens broad H3K4me3-modified genomic regions. The authors should perform Foxp3 ChIP-seq or Cut-n-Taq with WT and Cxxc1 cKO Tregs to determine whether CXXC1 deletion changes Foxp3's binding pattern in Treg cells.

Reviewer #2 (Public review):

FOXP3 has been known to form diverse complexes with different transcription factors and enzymes responsible for epigenetic modifications, but how extracellular signals timely regulate FOXP3 complex dynamics remains to be fully understood. Histone H3K4 tri-methylation (H3K4me3) and CXXC finger protein 1 (CXXC1), which is required to regulate H3K4me3, also remain to be fully investigated in Treg cells. Here, Meng et al. performed a comprehensive analysis of H3K4me3 CUT&Tag assay on Treg cells and a comparison of the dataset with the FOXP3 ChIP-seq dataset revealed that FOXP3 could facilitate the regulation of target genes by promoting H3K4me3 deposition.

Moreover, CXXC1-FOXP3 interaction is required for this regulation. They found that specific knockdown of Cxxc1 in Treg leads to spontaneous severe multi-organ inflammation in mice and that Cxxc1-deficient Treg exhibits enhanced activation and impaired suppression activity. In addition, they have also found that CXXC1 shares several binding sites with FOXP3 especially on Treg signature gene loci, which are necessary for maintaining homeostasis and identity of Treg cells.

The findings of the current study are pretty intriguing, and it would be great if the authors could fully address the following comments to support these interesting findings.

Major points:

(1) There is insufficient evidence in the first part of the Results to support the conclusion that "FOXP3 functions as an activator by promoting H3K4Me3 deposition in Treg cells". The authors should compare the results for H3K4Me3 in FOXP3-negative conventional T cells to demonstrate that at these promoter loci, FOXP3 promotes H3K4Me3 deposition.

(2) In Figure 3 F&G, the activation status and IFNγ production should be analyzed in Treg cells and Tconv cells separately rather than in total CD4+ T cells. Moreover, are there changes in autoantibodies and IgG and IgE levels in the serum of cKO mice?

(3) Why did Cxxc1-deficient Treg cells not show impaired suppression than WT Treg during in vitro suppression assay, despite the reduced expression of Treg cell suppression assay -associated markers at the transcriptional level demonstrated in both scRNA-seq and bulk RNA-seq?

(4) Is there a disease in which Cxxc1 is expressed at low levels or absent in Treg cells? Is the same immunodeficiency phenotype present in patients as in mice?

Reviewer #3 (Public review):

In the report entitled "CXXC-finger protein 1 associates with FOXP3 to stabilize homeostasis and suppressive functions of regulatory T cells", the authors demonstrated that Cxxc1-deletion in Treg cells leads to the development of severe inflammatory disease with impaired suppressive function. Mechanistically, CXXC1 interacts with Foxp3 and regulates the expression of key Treg signature genes by modulating H3K4me3 deposition. Their findings are interesting and significant. However, there are several concerns regarding their analysis and conclusions.

Major concerns:

(1) Despite cKO mice showing an increase in Treg cells in the lymph nodes and Cxxc1-deficient Treg cells having normal suppressive function, the majority of cKO mice died within a month. What causes cKO mice to die from severe inflammation?

Considering the results of Figures 4 and 5, a decrease in Treg cell population due to their reduced proliferative capacity may be one of the causes. It would be informative to analyze the population of tissue Treg cells.

(2) In Figure 5B, scRNA-seq analysis indicated that Mki67+ Treg subset are comparable between WT and Cxxc1-deficient Treg cells. On the other hand, FACS analysis demonstrated that Cxxc1-deficient Treg shows less Ki-67 expression compared to WT in Figure 5I. The authors should explain this discrepancy.

In addition, the authors concluded on line 441 that CXXC1 plays a crucial role in maintaining Treg cell stability. However, there appears to be no data on Treg stability. Which data represent the Treg stability?

(3) The authors found that Cxxc1-deficient Treg cells exhibit weaker H3K4me3 signals compared to WT in Figure 7. This result suggests that Cxxc1 regulates H3K4me3 modification via H3K4 methyltransferases in Treg cells. The authors should clarify which H3K4 methyltransferases contribute to the modulation of H3K4me3 deposition by Cxxc1 in Treg cells.

Furthermore, it would be important to investigate whether Cxxc1-deletion alters Foxp3 binding to target genes.

(4) In Figure 7, the authors concluded that CXXC1 promotes Treg cell homeostasis and function by preserving the H3K4me3 modification since Cxxc1-deficient Treg cells show lower H3K4me3 densities at the key Treg signature genes. Are these Cxxc1-deficient Treg cells derived from mosaic mice? If Cxxc1-deficient Treg cells are derived from cKO mice, the gene expression and H3K4me3 modification status are inconsistent because scRNA-seq analysis indicated that expression of these Treg signature genes was increased in Cxxc1-deficient Treg cells compared to WT (Figure 5F and G).

Reviewer #1 (Public review):

Summary:

The manuscript by Rowell et al aims to identify differences in TCR recombination and selection between foetal and adult thymus in mice. Authors sequenced the unpaired bulk TCR repertoire in foetal and adult mice thymi and studied both TCRB and TCRa characteristics in the double negative (DN, CD4-CD8-) and single positive (SP4 CD4+CD8- and SP8 CD4-CD8+) populations. They identified age-related differences in TCRa and TCRB segment usage, including a preferential bias toward 3'TRAV and 5' TRAJ rearrangements in foetal cells compared to adults who had a larger perveance for 5'TRAV segments. By depleting the thymocyte population in adult thymi using hydrocortisone, the authors demonstrated that the repertoire became more foetal like, they, therefore, argue that the preferential 5'TRAV rearrangements in adults may be resulting from prolonged/progressive TCRa rearrangements in the adult thymocytes. In line with previous studies, Authors demonstrate that the foetal TCR repertoire was less diverse, less evenly distributed and had fewer non-template insertions while containing more clonal expansions. In addition, the authors claim that changes in V-J usage and CDR1 and CDR2 in the DN vs SP repertoires indicated that positive selection of foetal thymocytes are less dependent on interactions with the MHC.

Strengths:

Overall, the manuscript provides an extensive analysis of the foetal and adult TCR repertoire in the thymus, resulting in new insights in T cell development in foetal and adult thymi.

Weaknesses:

Three major concerns arise:<br /> (1) the authors have analysed TCR repertoires of only 4 foetal and 4 adult mice, considering the high spread the study may have been underpowered.

- The sample size was increased in the revised version

(2) Gating strategies are missing and

- These have now been provided in the revised version

(3) The manuscript is very technical and clearly aimed for a highly specialised audience with expertise in both thymocyte development and TCR analysis. Considering eLife is a scientific journal with a broader readership, Authors are recommended to provide schematics of the TCR rearrangements/their findings and include a summary of conclusions/implications of their findings at the end of each results section rather than waiting till the discussion. This will help the reader to interpret their findings while reading the results.

- These have now been included in the revised version

Reviewer #2 (Public review):

Summary:

The authors comprehensively assess differences in the TCRB and TCRA repertoires in the fetal and adult mouse thymus by deep sequencing of sorted cell populations. For TCRB and TCRA they observed biased gene segment usage, less diversity, and greater repertoire sharing among individuals in fetal thymocytes. The TCRB repertoire was less evenly distributed and displayed more evidence of clonal expansions in fetal thymocytes. Both fetal and adult thymocytes demonstrated repertoire skewing in CD4 and CD8 as compared to DP thymocytes, which was attributed to MHC-I- vs MHC-II-restriction during positive selection. Effects of MHC-restriction were notably weaker in fetal thymocytes. The authors conclude that in multiple respects fetal repertoires are distinct from adult repertoires.

Strengths:

The analyses of the F18.5 and adult thymic repertoires are comprehensive with respect to the cell populations analyzed and the diversity of statistical approaches used to characterize the repertoires. Because repertoires were analyzed in pre- and post-selection thymocyte subsets, the data allowed assessment of repertoire selection at different developmental stages. Intriguing differences between fetus and adult are identified.

Weaknesses:

Some of the repertoire characteristics reported are already fairly well documented in the literature. Moreover, an unaddressed limitation of the study is that fetal thymocytes were analyzed at single time-point in their development. As a result, at least some of the conclusions about the fetal repertoire may be viewed not as general conclusions, but rather, due to the synchronous development of fetal thymocytes, as pertaining to the one day of fetal/early neonatal development assayed. Statements suggesting that (1) "progressive TCRa rearrangements occur less frequently in foetal DP cells" (Abstract), (2) "One possible explanation for this bias is that in the foetus progressive rounds of TCRa rearrangement are less common than in young adult" (Discussion), and (3) "Overall, the differences between the foetal and adult thymus TCR repertoires are consistent with the foetal thymus producing abT-cells ... with preference for particular gene segment usage" (Discussion), are oversimplified and potentially misleading.

Reviewer #1 (Public Review):

The authors report compound heterozygous deleterious variants in the kinase domains of the non-receptor tyrosine kinases (NRTK) TNK2/ACK1 in familial SLE. They suggest that ACK1 and BRK deficiencies are associated with human SLE and impair efferocytosis.

The experiments in this revision showing that a weekly injection of ACK1 or BRK inhibitors induced various kinds of lupus-related autoantibodies in BALB/c supported the pivotal role of ACK1/BRK in systemic autoimmunity, although treated mice failed to demonstrate the full picture of lupus.

Reviewer #2 (Public Review):

In this manuscript, the authors revealed that genetic deficiencies of ACK1 and BRK are associated with human SLE. First, the authors found that compound heterozygous deleterious variants in the kinase domains of the non-receptor tyrosine kinases (NRTK) TNK2/ACK1 in one multiplex family and PTK6/BRK in another family. Then, by an experimental blockade of ACK1 or BRK in a mouse SLE model, they found an increase in glomerular IgG deposits and circulating autoantibodies. Furthermore, they reported that ACK and BRK variants from the SLE patients impaired the MERTK-mediated anti-inflammatory response to apoptotic cells in human induced pluripotent stem cells (hiPSC)-derived macrophages. This work identified new SLE-associated ACK and BRK variants and a role for the NRTK TNK2/ACK1 and PTK6/BRK in efferocytosis, providing a new molecular and cellular mechanism of SLE pathogenesis.

Reviewer #1 (Public Review):

This article is interesting because the phenotype of the virus with mutations that alter the affinity of HS has been associated with how the viral particle interacts with HS and, thus, with binding and entry. However, the data in this manuscript is compelling and strongly suggests that the mutation that increases the affinity of HS alters capsid stability. To my knowledge, this is the first evidence that such mutation causes capsid destabilization. Furthermore, the idea that this mutation increases infectivity in cell lines by also using a pH-independent route and that, in vivo, this mutation attenuates the virus is very novel. Last year Wa-Chu's lab proposed that encephalopathic Alphaviruses produce capsids with different sizes and that this helps to attenuate highly pathogenic viruses (which might not be the case for non encepahlopatic Alphavirsues). However, they did not demonstrate whether these alterations attenuate the virus and if the altered morphology affects capsid stability. Therefore, this manuscript is fundamental as it contributes to understanding how the assembly/disassembly mechanism can be used to attenuate a virus. Furthermore, it is possible this mechanism could not be restricted to viruses that belong to the Picornaviridae family and opens a new door to understanding viral attenuation in other icosahedral viruses.

Reviewer #3 (Public Review):

Heparan Sulphate is a general association factor in the extracellular matrix which assists in host cell entry for a multitude of viral and bacterial pathogens by concentrating them in the vicinity of cellular membranes. The neurotropic picornavirus, EV-71 utilizes a protein receptor SCARB-2, in conjunction with Heparan Sulfate, in order to enter cells through the endo-lysosomal pathway. The uncoating and release of viral genome requires both receptor binding and late endosomal pH conditions. The authors have attempted to address a seeming contradiction in the in vitro and in vivo infectivity of strain MP4 variants of EV-71. One of the cell culture adapted strains MP4-L97R/E167G has stronger association with HS, which translates to higher infectivity in cell culture models; however, viral virulence is significantly lower in animal models.

Using an elegant and methodical set of experiments, the authors have probed the steps in the cellular entry pathway of MP4 and its L97R/E167G variant. Their experiments strongly suggest a difference in capsid uncoating mechanisms in the variant, with the L97R/E167G variant being significantly less robust and prone to destabilize earlier in the pathway. While this confers an advantage in terms of cell culture based infectivity, it is posited that the particles will not survive the gastric pH intact, which compromises virulence in the animal model. While the cell culture based uncoating experiments somewhat support this hypothesis, the main weakness of this work is a lack of explanation for the mechanism(s) of capsid destabilization conferred by overall increased positive charge. The structural bioinformatics study in the supplementary section does not explain how receptor binding, pocket factor expulsion, subunit interactions and low pH based capsid dynamics may be influenced by the mutations. Capsid destabilization could be an outcome in alteration of any or all of these processes. It is also unclear whether it is suggested that all mutations enhancing the net positive charge of VP1, or any other structural protein, will cause capsid destabilization by similar pathways. A clearer analysis of the influence of overall charge alterations, or individual mutations, on subunit interaction or particle conformation is needed. The enhancement in cell culture infectivity of the L97R/E167G variant under elevated endosomal pH is also unclear and requires further experimentation.

It has been suggested earlier that increased HS binding in vivo results in virus "trapping" and decreased infectivity. This may still be a major reason for reduced infectivity in vivo, in addition to the capsid destabilization as proposed in this work.

Reviewer #4 (Public Review):

In this work, Tee et al. study the implications of Heparan Sulfate (HS) binding mutations observed on the Enterovirus A71 (EV-A71) capsid. HS-binding mutations are observed for several virus infections and are often presumed to be a cell culture adaptation. However, in the case of EV-A71, the presence of HS-binding mutations in clinical samples and the contradictory findings in animal studies have made the clinical relevance of HS-binding a subject of debate. Therefore, to better understand the role of HS-binding in EV-A71, the authors use a mouse-adapted EV-A71 variant (MP4) and compare it to a cell-adapted strong HS-binder (MP4-97R/167G). Using these two variants, the authors show that the strong HS-binder does not require acidification for uncoating and genome release. Furthermore, it is demonstrated that the capsid stability of the HS-binding variant is compromised, resulting in pH-independent uncoating. Overall, this study provides new insights demonstrating that seemingly beneficial mutations increasing viral replication may be counterbalanced by other unintended consequences.

Strengths:

The thoroughness of the experiments performed to demonstrate that the HS-binding phenotype results in pH-independent entry and capsid destabilisation is worth highlighting. In this regard, the authors have explored viral entry using a range of approaches involving lysosomotropic drugs, viral binding assays, and neutral red-labelled viruses coupled with diverse techniques such as FISH, RNAscope, and transient expression of constitutively active molecules to inhibit parts of the viral cycle. In my opinion, this is necessary to rule out the other downstream effects of the lysomotropic drugs and to confirm the role of the HS-binding mutation in the entry phase. The use of in silico analysis coupled with negative staining electron microscopy and environmental challenge assays is notable. Finally, the demonstration of some of the work using a human-relevant strain is commendable.

Weaknesses:

A major weakness in this study is the focus on using a mouse-adapted EV-A71 strain (MP4). In the introduction, it is argued that HS-binding mutations are controversial due to their occurrence in cell culture. However, due to host limitations, mice are not the natural hosts for EV-A71 and thus, the same argument can be made for a mouse-adapted strain. It is not clear how different this strain is from circulating EV-A71 strains and the relevance of these findings to the human situation is questionable. This is particularly made evident in the discussion where it is highlighted that HS-binding variants (VP1-145G/Q mutants) have been associated with severe neurological cases while the same variants show attenuated phenotypes in mice and monkeys. This contrast between clinical data and animal studies should be highlighted in the introduction, rather than later in the discussion, as currently the in vivo animal studies are presented as the optimal situation and may lead to misconstrued conclusions from the results.

An important consideration is that the results are based primarily on image analysis. The inclusion of RT-qPCR and/or plaque assays as supplementary data will help strengthen the findings. Moreover, there are suggestions of an intermediate binder having a different phenotype. As this intermediate binder is the clinical phenotype, data on the entry of this intermediate binder will be valuable.

Another weakness in the study is the lack of contextualization of the results to current EV-A71 literature. For instance, SCARB2 is referred to as the internalization receptor but a recent study has shown that SCARB2 is not required for internalization (https://doi.org/10.1128%2Fjvi.02042-21). The findings from this study are consistent with the localization of SCARB2 in the lysosomal membranes. Furthermore, the same study has highlighted host sulfation as a key factor in EV-A71 entry. Post-translational sulfation introduces negatively charged residues on host proteins including HS and SCARB2. This increases the binding of HS-binding strains to these proteins. In this regard, the reduced infectivity upon soluble SCARB2 treatment may simply be due to enhanced binding rather than capsid opening as suggested in the results. Therefore, additional experiments (e.g. nSEM following soluble SCARB2 treatment) must be performed to support the conclusion of capsid opening, due to inherent instability, upon SCARB2 binding.

In addition to the above, other existing literature on EV-A71 pathogenesis using organoids contradicts some of the explanations of differential phenotype in clinical observations versus mice models. In the introduction, it is suggested that reduced neurovirulence of HS-binding strains is due to binding to the vascular endothelia. However, the correlation of clinical severity to viremia (https://doi.org/10.1186/1471-2334-14-417) and the association of HS-binding mutants to clinical disease counteract this suggestion. Similarly, viral infection in human organoids with EV-A71 results in as low as 0.4% of the cells being infected (https://doi.org/10.1038/s41564-023-01339-5). In this case, if viral binding to (ubiquitously expressed) HS results in viral trapping then the HS-binding mutants should show lowered infectivity in organoid models rather than the observed higher infectivity (https://doi.org/10.3389/fmicb.2023.1045587, https://doi.org/10.1038/s41426-018-0077-2). Finally, EV-A71 release has also been shown to occur in exosomes (https://doi.org/10.1093%2Finfdis%2Fjiaa174) which effectively provides a protective lipid membrane. These recent findings must be incorporated into the article and will help better contextualize their findings.

Overall, the authors present new findings with convincing methodology. The manuscript can be improved in the contextualization of the findings and highlighting the weakness in translating these findings to resolve the debate surrounding the relevance of HS-binding phenotype. The inclusion of additional experiments and data recommended to the authors will also help strengthen the manuscript.

Reviewer #1 (Public Review):

This manuscript was previously reviewed and this earlier evaluation resulted in two conflicting assessments. I fully endorse the favourable opinion of former Reviewer 1 and find most negative comments of former Reviewer 2 inappropriate.

This work is absolutely necessary. Even though the authors find it difficult to be fully assertive in the end, their ground work in trying to demonstrate the existence of bias in PPI data is undeniably valuable. Other authors have tried before to show the limitation of unequivocally assigning the degree distribution to a power law but these doubts have had a weak impact. This new study is a great opportunity to discuss further a concern for a simplistic view of PPI network topology. The recent contribution of Broido & Clauset was definitely one to bounce on. The approach of this new manuscript is compelling. Dividing the study in several parts, each reflecting an attempt to bring out commonly used shortcuts in PPI network analyses, makes sense.

Surprisingly, the authors do not refer to the endless controversy of labeling hubs as party or date, which is another manifestation of the interpretative bias of PPI data.

The only worthy point prompted by former Reviewer 2 is the effect of spoke expansion. In their response, the authors suggest that it would probably extend questioning and even if it is considered as future work, it could be mentioned in the main manuscript.

In the end, this submission is an invitation to constructively rethink the analysis of PPI networks and it feeds the discussion on modelling degree distributions that should not be considered as a solved issue.

Reviewer #2 (Public Review):

Many naturally occurring networks are assumed to have a power-law (PL) degree distribution. This assumption has certainly been widely held in the field of protein interactomes (PPIs), although important studies around 2010 have conclusively shown that many of these PL distributions are either the result of data mis-handling or of sloppy statistical procedures (see e.g. Porter and Stumpf in Science around 2014, which I would advise the authors to cite). The value of the present study is to introduce a new mechanism, experiment bias, to explain the appearance of such distributions in the PPI case, and in particular to show how correcting empirically for this mechanism can lead to a reappraisal of which proteins are genuine hubs in these networks. The claims are well supported by empirical evidence and some theoretical analysis. Overall, this is a worthwhile contribution although its significance is somewhat dented by the fact that the PL enthusiasm of many had already been tempered by the studies mentioned above.

Reviewer #3 (Public Review):

I would like to congratulate the authors to an impressive piece of work highlighting important real and potential biases, which may lead to power-law distributed node degrees in protein-protein interaction networks.<br /> This manuscript is easy to follow and very well written manuscript.<br /> I truly enjoyed the concise and convincing scientific presentation.<br /> Even if some of the concerns have already been discussed or raised in the past, the manuscript assesses potential biases in PPIs in a rigorous manner.

I deem the following observations highly relevant to be communicated to the community again:<br /> (1) PL-like distributions emerge by aggregation of data sets alone.<br /> (2) Research interest in itself is PL-distributed and drives PL-like properties in PPI networks<br /> (3) Bait usage is a major driver of PL-like behaviour.<br /> (4) Accounting for biases changes the biological interpretation of the networks<br /> (5) Simulation studies further corroborate these findings.

Reviewer #1 (Public review):

This is a very relevant study, with the potential of having high impact on future research on the evolution of chemical defense mechanisms in animals. The authors present a substantial number of new and surprising experimental results, i.e., the presence in low quantities of alkaloids in amphibians previously deemed to lack these toxins. These data are then combined with literature data to weave the importance of passive accumulation mechanisms into a 4-phases scenario of the evolution of chemical defense in alkaloid-containing poison frogs.

In general, the new data presented in the manuscript are of high quality and high scientific interest, the suggested scenario compelling, and the discussion thorough. Also, the revised version of the manuscript has been carefully prepared with a high quality of illustrations. UI did not detect typos in the text

Understanding that the majority of dendrobatid frogs, including species considered undefended, can contain low quantities of alkaloids in their skin provides an entirely new perspective to our understanding of how the amazing specializations of poison frogs evolved. Although only few non-dendrobatids were included in the alkaloid screening, some of these also included minor quantities of alkaloids, and the capacity of passive alkaloid accumulation may therefore characterize numerous other frog clades, or even amphibians in general.

The overall quality of the work is exceptional. The authors also have done a fantastic job restructuring the manuscript in response to my initial comments, and it is now very clear which new hypotheses are presented and which testable predictions for future studies derive from these hypotheses. This study will be highly influential in informing and guiding future research on toxicity, alkaloid sequestration and resistance, and evolution of aposematism.

Reviewer #2 (Public review):

Summary:

This was a well-executed and well-written paper. The authors have provided important new datasets that expand on previous investigations substantially. The discovery that changes in diet are not so closely correlated with the presence of alkaloids (based on the expanded sampling of non-defended species) is important, in my opinion.

Strengths:

Provision of several new expanded datasets using cutting edge technology and sampling a wide range of species that had not been sampled previously. A conceptually important paper that provides evidence for the importance of intermediate stages in the evolution of chemical defense and aposematism.

Weaknesses:

There were some aspects of the paper that I thought could be revised. One thing I was struck by is lack of discussion of the potentially negative effects of toxin accumulation, and how this might play out in terms of different levels of toxicity in different species. Further, are there aspects of ecology or evolutionary history that might make some species less vulnerable to the accumulation of toxins than others? This could be another factor that strongly influences the ultimate trajectory of a species in terms of being well-defended. I think the authors did a good job in terms of describing mechanistic factors that could affect toxicity (e.g. potential molecular mechanisms), but did not make much of an attempt to describe potential ecological factors that could impact trajectories of the evolution of toxicity. This may have been done on purpose (to avoid being too speculative), but I think it would be worth some consideration.

In the discussion, the authors make the claim that poison frogs don't (seem to) suffer from eating alkaloids. I don't think this claim has been properly tested (the cited references don't adequately address it). To do so would require an experimental approach, ideally obtained data on both lifespan and lifetime reproductive success.

Update: Revised version: The authors carefully addressed the comments and suggestions on the first draft of the manuscript. In my opinion, these revisions were sufficient and the authors have adequately addressed the previously noted weaknesses in the manuscript.

Reviewer #1 (Public review):

Summary:

Utilizing transgenic lineage tracing techniques and tissue clearing-based advanced imaging and three-dimensional slices reconstruction, the authors comprehensively mapped the distribution atlas of NFATc1+ and PDGFR-α+ cells in dental and periodontal mesenchyme and tracked their in vivo trajectories. This important work expands our understanding of both single and double positive NFATc1 and PDGFR-α cells in maintaining dental and periodontal mesenchyme homeostasis, and will provide impact on clinical application and investigation. The strength of this work is convincing, as it employed CRISPR/Cas9-mediated gene editing to generate two dual recombination systems, and mapped gNFATc1+ and PDGFR-α+ cells residing in dental and periodontal mesenchyme, their capacity for progeny cell generation, and their inclusive, exclusive and hierarchical relations in homeostasis, generating a spatiotemporal atlas of these skeletal stem cell population.

This work has theoretical or practical implications in the periodontal field. The methods, data and analyses support the claims.

Comments on revised version:

The authors have addressed my main concerns.

Reviewer #1 (Public review):

In this work, Veseli et al. present a computational framework to infer the functional diversity of microbiomes in relation to microbial diversity directly from metagenomic data. The framework reconstructs metabolic modules form metagenomes and calculates the per-population copy number of each module, resulting in the proportion of microbes in the sample carrying certain genes. They applied this framework to a dataset of gut microbiomes from 109 inflammatory bowel disease (IBD) patients, 78 patients with other gastrointestinal conditions, and 229 healthy controls. The found that the microbiomes of IBD patients were enriched in a high fraction of metabolic pathways, including biosynthesis pathways such as those for amino acids, vitamins, nucleotides, and lipids. Hence, they had higher metabolic independence compared with healthy controls. To an extent, the authors also found a pathway enrichment suggesting higher metabolic independence in patients with gastrointestinal conditions other than IBD indicating this could be a signal for a general loss in host health. Finally, a machine learning classifier using high metabolic independence in microbiomes could predict IBD with good accuracy. Overall, this is an interesting and well-written article and presents a novel workflow that enables a comprehensive characterization of microbiome cohorts.

Comments on revisions:

I believe that after the second round of revisions, the Reviewers sufficiently addressed the comments and improved the manuscript. Open questions have been answered. I have no further comments.

Reviewer #2 (Public review):

This study builds upon the team's recent discovery that antibiotic treatment and other disturbances favours the persistence of bacteria with genomes that encode complete modules for the synthesis of essential metabolites (Watson et al. 2023). Veseli and collaborators now provide an in-depth analysis of metabolic pathway completeness within microbiomes, finding strong evidence for an enrichment of bacteria with high metabolic independence in the microbiomes associated with IBD and other gastrointestinal disorders. Importantly, this study provides a new open-source software to facilitate the reconstruction of metabolic pathways, estimate their completeness and normalize their results according to species diversity. Finally, this study also shows that metabolic independence of microbial communities can be used as a marker of dysbiosis. The function-based health index proposed here is more robust to individual's lifestyles and geographic origin than previously proposed methods based on bacterial taxonomy.

The implications of this study have the potential to spur a paradigm shift in the field. It shows that certain bacterial taxa that have been consistently associated with disease might not be harmful to their host as previously thought. These bacteria seem to be the only species that are able to survive in a stressed gut environment. They might even be important to rebuild a healthy microbiome (although the authors are careful in not making this speculation).

This paper provides an in-depth discussion of the results, and limitations are clearly addressed throughout the manuscript (see also the supplementary files for an in-depth assessment of the robustness of the methods). Some of the potential limitations relate to the use of large publicly available datasets, where sample processing and the definition of healthy status varies between studies. The authors have recognised these issues and their results were robust to analyses performed at a per-cohort basis. The potential limitations therefore are unlikely to have affected the conclusions of this study.

Overall, this is manuscript is a magnificent contribution to the field, likely to inspire many other studies to come.

Comments on revisions:

The authors have performed a detailed assessment of the accuracy and robustness of their new methods, and included an informative session comparing their new approach with existing ones. The new analyses have strengthened the manuscript, and the results support the biological interpretations of the study.<br /> I commend the authors for the effort and the excellent research.

Reviewer #3 (Public review):

The major strength of this manuscript is the "anvi-estimate-metabolism' tool, which is already accessible online, extensively documented, and potentially broadly useful to microbial ecologists. Inclusion of extensive benchmarking and validation on simulated metagenomes has further increased confidence in this approach. Further, the conceptual insights raise interesting hypotheses that could be pursued in follow-on experimental work.

Comments on revisions:

Thank you for the very thorough response and congratulations!

Joint Public Review:

The subject area will have general appeal to those interested in the study of Pavlovian conditioning. The paper is important, showcasing a rigorous experimental design, several different approaches to data analysis, careful consideration of prior literature, and a thorough introduction. The results indicate that the rate of Pavlovian learning is determined by the ratio of reward rate during cue to the overall reward rate, and that the asymptotic response rate is determined by the reward rate during cue. These findings provide context to many conflicting recent results on this topic and are supported by strong/convincing evidence.

It is additionally claimed that the parameter that governs the acquisition and asymptote of responding in rats is exactly the same as that which governs the acquisition and asymptote of responding in the Gibbon and Balsam (1981) study that used pigeons as experimental subjects; and that the rates of responding during the inter-trial interval and the cue are proportional to the corresponding reward rates with the same proportionality constant. In both of these respects, there are several points that stand in need of clarification - at present, the strength of the evidence in support of these claims is solid. More generally, there are some points that could clarify aspects of rate estimation theory and, thereby, increase the rating of the paper from important to fundamental. These points range from analytical to conceptual and are presented below.

ANALYTICAL

(1) A key claim made here is that the same relationship (including the same parameter) describes data from pigeons by Gibbon and Balsam (1981; Figure 1) and the rats in this study (Figure 3). The evidence for this claim, as presented here, is not as strong as it could be. This is because the measure used for identifying trials to criterion in Figure 1 appears to differ from any of the criteria used in Figure 3, and the exact measure used for identifying trials to criterion influences the interpretation of Figure 3***. To make the claim that the quantitative relationship is one and the same in the Gibbon-Balsam and present datasets, one would need to use the same measure of learning on both datasets and show that the resultant plots are statistically indistinguishable, rather than simply plotting the dots from both data sets and spotlighting their visual similarity. In terms of their visual characteristics, it is worth noting that the plots are in log-log axis and, as such, slight visual changes can mean a big difference in actual numbers. For instance, between Figure 3B and 3C, the highest information group moves up only "slightly" on the y-axis but the difference is a factor of 5 in the real numbers. Thus, in order to support the strong claim that the quantitative relationships obtained in the Gibbon-Balsam and present datasets are identical, a more rigorous approach is needed for the comparisons.

***The measure of acquisition in Figure 3A is based on a previously established metric, whereas the measure in Figure 3B employs the relatively novel nDKL measure that is argued to be a better and theoretically based metric. Surprisingly, when r and r2 values are converted to the same metric across analyses, it appears that this new metric (Figure 3B) does well but not as well as the approach in Figure 3A. This raises questions about why a theoretically derived measure might not be performing as well on this analysis, and whether the more effective measure is either more reliable or tapping into some aspect of the processes that underlie acquisition that is not accounted for by the nDKL metric.

(2) Another interesting claim here is that the rates of responding during ITI and the cue are proportional to the corresponding reward rates with the same proportionality constant. This too requires more quantification and conceptual explanation. For quantification, it would be more convincing to calculate the regression slope for the ITI data and the cue data separately and then show that the corresponding slopes are not statistically distinguishable from each other. Conceptually, it is not clear why the data used to test the ITI proportionality came from the last 5 conditioning sessions. What were the decision criteria used to decide on averaging the final 5 sessions as terminal responses for the analyses in Figure 5? Was this based on consistency with previous work, or based on the greatest number of sessions where stable data for all animals could be extracted?

If the model is that animals produce response rates during the ITI (a period with no possible rewards) based on the overall rate of rewards in the context, wouldn't it be better to test this before the cue learning has occurred? Before cue learning, the animals would presumably only have attributed rewards in the context to the context and thus, produce overall response rates in proportion to the contextual reward rate. After cue learning, the animals could technically know that the rate of rewards during ITI is zero. Why wouldn't it be better to test the plotted relationship for ITI before cue learning has occurred? Further, based on Figure 1, it seems that the overall ITI response rate reduces considerably with cue learning. What is the expected ITI response rate prior to learning based on the authors' conceptual model? Why does this rate differ from pre and post-cue learning? Finally, if the authors' conceptual framework predicts that ITI response rate after cue learning should be proportional to contextual reward rate, why should the cue response rate be proportional to the cue reward rate instead of the cue reward rate plus the contextual reward rate?

(3) There is a disconnect between the gradual nature of learning shown in Figures 7 and 8 and the information-theoretic model proposed by the authors. To the extent that we understand the model, the animals should simply learn the association once the evidence crosses a threshold (nDKL > threshold) and then produce behavior in proportion to the expected reward rate. If so, why should there be a gradual component of learning as shown in these figures? In terms of the proportional response rule to the rate of rewards, why is it changing as animals go from 10% to 90% of peak response? The manuscript would be greatly strengthened if these results were explained within the authors' conceptual framework. If these results are not anticipated by the authors' conceptual framework, this should be explicitly stated in the manuscript.

(4) Page 27, Procedure, final sentence: The magazine responding during the ITI is defined as the 20 s period immediately before CS onset. The range of ITI values (Table 1) always starts as low as 15 s in all 14 groups. Even in the case of an ITI on a trial that was exactly 20 s, this would also mean that the start of this period overlaps with the termination of the CS from the previous trial and delivery (and presumably consumption) of a pellet. It should be indicated whether the definition of the ITI period was modified on trials where the preceding ITI was < 20 s, and if any other criteria were used to define the ITI. Were the rats exposed to the reinforcers/pellets in their home cage prior to acquisition?

(5) For all the analyses, the exact models that were fit and the software used should be provided. For example, it is not necessarily clear to the reader (particularly in the absence of degrees of freedom) that the model discussed in Figure 3 fits on the individual subject data points or the group medians. Similarly, in Figure 6 there is no indication of whether a single regression model was fit to all the plotted data or whether tests of different slopes for each of the conditions were compared. With regards to the statistics in Figure 6, depending on how this was run, it is also a potential problem that the analyses do not correct for the potentially highly correlated multiple measurements from the same subjects, i.e. each rat provides 4 data points which are very unlikely to be independent observations.

CONCEPTUAL

(1) We take the point that where traditional theories (e.g., Rescorla-Wagner) and rate estimation theory (RET) both explain some phenomenon, the explanation in terms of RET may be preferred as it will be grounded in aspects of an animal's experience rather than a hypothetical construct. However, like traditional theories, RET does not explain a range of phenomena - notably, those that require some sort of expectancy/representation as part of their explanation. This being said, traditional theories have been incorporated within models that have the representational power to explain a broader array of phenomena, which makes me wonder: Can rate estimation be incorporated in models that have representational power; and, if so, what might this look like? Alternatively, do the authors intend to claim that expectancy and/or representation - which follow from probabilistic theories in the RW mould - are unnecessary for explanations of animal behaviour?***

***If the authors choose to reply to these points, they should consider taking advantage of an "Ideas and Speculation" subsection within the Discussion that is supported by eLife [ https://elifesciences.org/inside-elife/e3e52a93/elife-latest-including-ideas-and-speculation-in-elife-papers ].

(2) The discussion of Rescorla's (1967) and Kamin's (1968) findings needs some elaboration. These findings are already taken to mean that the target CS in each design is not informative about the occurrence of the US - hence, learning about this CS fails. In the case of blocking, we also know that changes in the rate of reinforcement across the shift from stage 1 to stage 2 of the protocol can produce unblocking. Perhaps more interesting from a rate estimation perspective, unblocking can also be achieved in a protocol that maintains the rate of reinforcement while varying the sensory properties of the US (Wagner). How does rate estimation theory account for these findings and/or the demonstrations of trans-reinforcer blocking (Pearce-Ganesan)? Are there other ways that the rate estimation account can be distinguished from traditional explanations of blocking and contingency effects? If so, these would be worth citing in the discussion. More generally, if one is going to highlight seminal findings (such as those by Rescorla and Kamin) that can be explained by rate estimation, it would be appropriate to acknowledge findings that challenge the theory - even if only to note that the theory, in its present form, is not all-encompassing. For example, it appears to me that the theory should not predict one-trial overshadowing or the overtraining reversal effect - both of which are amenable to discussion in terms of rates. I assume that the signature characteristics of latent inhibition and extinction would also pose a challenge to rate estimation theory, just as they pose a challenge to Rescorla-Wagner and other probability-based theories. Is this correct?

Reviewer #1 (Public Review):

Summary:

A key challenge at the second chemical step of splicing is the identification of the 3' splice site of an intron. This requires recruitment of factors dedicated to the second chemical step of splicing and exclusion of factors dedicated to the first chemical step of splicing. Through the highest resolution cyroEM structure of the spliceosome to-date, the authors show the binding site for Fyv6, a factor dedicated to the second chemical step of splicing, is mutually exclusive with the binding site for a distinct factor dedicated to the first chemical step of splicing, highlighting that splicing factors bind to the spliceosome at a specific stage not only by recognizing features specific to that stage but also by competing with factors that bind at other stages. The authors further reveal that Fyv6 functions at the second chemical step to promote selection of 3' splice sites distal to a branch point and thereby discriminate against proximal, suboptimal 3' splice site. Lastly, the authors show by cyroEM that Fyv6 physically interacts with the RNA helicase Prp22 and by genetics Fyv6 functionally interacts with this factor, implicating Fyv6 in 3'SS proofreading and mRNA release from the spliceosome. The evidence for this study is robust, with the inclusion of genomics, reporter assays, genetics, and cyroEM. Further, the data overall justify the conclusions, which will be of broad interest.

Strengths:

(1) The resolution of the cryoEM structure of Fyv6-bound spliceosomes at the second chemical step of splicing is exceptional (2.3 Angstroms at the catalytic core; 3.0-3.7 Angstroms at the periphery), providing the best view of this spliceosomal intermediate in particular and the core of the spliceosome in general.<br /> (2) The authors observe by cryoEM three distinct states of this spliceosome, each distinguished from the next by progressive loss of protein factors and/or RNA residues. The authors appropriately refrain from overinterpreting these states as reflecting distinct states in the splicing cycle, as too many cyroEM studies are prone to do, and instead interpret these observations to suggest interdependencies of binding. For example, when Fyv6, Slu7, and Prp18 are not observed, neither are the first and second residues of the intron, which otherwise interact, suggesting an interdependence between 3' splice site docking on the 5' splice site and binding of these second step factors to the spliceosome.<br /> (3) Conclusions are supported from multiple angles.<br /> (4) The interaction between Fyv6 and Syf1, revealed by the cyroEM structure, was shown to account for the temperature-sensitive phenotypes of a fyv6 deletion, through a truncation analysis.<br /> (5) Splicing changes were observed in vivo both by indirect copper reporter assays and directly by RT-PCR.<br /> (6) Changes observed by RNA-seq are validated by RT-PCR.<br /> (7) The authors go beyond simply observing a general shift to proximal 3'SS usage in the fyv6 deletion by RNA-seq by experimentally varying branch point to 3' splice site distance experimentally in a reporter and demonstrating in a controlled system that Fyv6 promotes distal 3' splice sites.<br /> (8) The importance of the Fyv6-Syf1 interaction for 3'SS recognition is demonstrated by truncations of both Fyv6 and of Syf1.<br /> (9) In general, the study was executed thoroughly and presented clearly.

Comments on revisions:

The authors have satisfactorily addressed the comments.

Reviewer #2 (Public Review):

In this manuscript, Senn, Lipinski, and colleagues report on the structure and function of the conserved spliceosomal protein Fyv6. Pre-mRNA splicing is a critical gene expression step that occurs in two steps, branching and exon ligation. Fyv6 had been recently identified by the Hoskins' lab as a factor that aids exon ligation (Lipinski et al., 2023), yet the mechanistic basis for Fyv6 function was less clear. Here, the authors combine yeast genetics, transcriptomics, biochemical assays, and structural biology to reveal the function of Fyv6. Specifically, they describe that Fyv6 promotes the usage of distal 3'SSs by stabilizing a network of interactions that include the RNA helicase PRP22 and the spliceosome subunit SYF1. They discuss a generalizible mechanism for splice site proofreading by spliceosomsal RNA helicases that could be modulated by other, regulatory splicing factors.

This is a very high quality study, which expertly combines various approaches to provide new insights into the regulation of 3'SS choice, docking, and undocking. The cryo-EM data is also of excellent quality, which substantially extends on previous yeast P complex structures. This is also supported by the authors use of the latest data analysis tools (Relion-5, AlphaFold2 multimer predictions, Modelangelo). The authors re-evaluate published EM densities of yeast spliceosome complexes (B*, C,C*,P) for the presence or absence of Fyv6, substantiate Fyv6 as a 2nd step specific factor, confirm it as the homolog of the human protein FAM192A, and provide a model for how Fyv6 may fit into the splicing pathway. The biochemical experiments on probing the splicing effects of BP to 3'SS distances after Fyv6 KO, genetic experiments to probe Fyv6 and Syf1 domains, and the suppressor screening add substantially to the study and are well executed. The manuscript is clearly written and we particularly appreciated the nuanced discussions, for example for an alternative model by which Prp22 influences 3'SS undocking. The research findings will be of great interest to the pre-mRNA splicing community.

Comments on revisions:

I'm satisfied with the changes.

Reviewer #3 (Public Review):

In this manuscript the authors expand their initial identification of Fyv6 as a protein involved in the second step of pre-mRNA splicing to investigate the transcriptome-wide impact of Fyv6 on splicing and gain a deeper understanding of the mechanism of Fyv6 action.

They first use deep sequencing of transcripts in cells depleted of Fyv6 together with Upf1 (to limit loss of mis-spliced transcripts) to identify broad changes in the transcriptome due to loss of Fyv6. This includes both changes in overall gene expression, that are not deeply discussed, as well as alterations in choice of 3' splice sites - which is the focus of the rest of the manuscript

They next provide the highest resolution structure of the post-catalytic spliceosome to date; providing unparalleled insight into details of the active site and peripheral components that haven't been well characterized previously.

Using this structure they identify functionally critical interactions of Fyv6 with Syf1 but not Prp22, Prp8 and Slu7. Finally, a suppressor screen additionally provides extensive new information regarding functional interactions between these second step factors.

Overall this manuscript reports new and essential information regarding molecular interactions within the spliceosome that determine the use of the 3' splice site. It would be helpful, especially to the non-expert, to summarize these in a table, figure or schematic in the discussion.

Comments on revisions:

I'm satisfied with the changes made in the revision.

Reviewer #1 (Public review):

Summary:

This study reports single-cell RNA sequencing results of lung adenocarcinoma, comparing 4 treatment-naive and 5 post-neoadjuvant chemotherapy tumor samples.<br /> The authors claim that there are metabolic reprogramming in tumor cells as well as stromal and immune cells after chemotherapy.<br /> The most significant findings are in the macrophages that there are more pro-tumorigenic cells after chemotherapy, i.e. CD45+CD11b+ARG+ cells. In the treatment-naive samples, more anti-tumorigenic CD45+CD11b+CD86+ macrophages are found. They sorted each population and performed functional analyses.

Strengths:

Comparison of the treatment-naive and post-chemotherapy samples of lung adenocarcinoma.

Weaknesses:

After the revision, issues remain with lengthy descriptive clustering type analysis, insufficient statistical support, and inefficient figure presentation.

Reviewer #1 (Public review):

Summary:

This study examined the associations of healthy lifestyles with comprehensive and organ-specific biological ages. It emphasized the importance of lifestyle factors in determining biological ages, which were using common blood biomarkers and body measures.

Strengths:

The data were from a large cohort study and defined comprehensive and six-specified BA.

Weaknesses highlighted previously:

(1) Since only 8.5% of participants from the CMEC were included in the study, has any section bias happened?

(2) The author should specify the efficiency of FFQ. How FFQ can genuinely reflect the actual intake? Moreover, how was the aMED calculated in your study?

(3) HLI (range) and HLI (category) should be clearly defined.

(4) The rationale of comprehensive and specific BA construction should be clearly defined and discussed. For example, can cardiopulmonary BA be reflected only by using cardiopulmonary status? I do not think so.

(5) The lifestyle index is defined based on an equal-weight approach, but this does not reflect reality and can not fully answer the research questions it raises.

Comments on the revised version:

The author answered most of the questions raised. However, since wine is the most important component of aMED, removing wine or alcohol may result in biased estimates. In addition, The authors acknowledge the limitations of this approach, namely that some biomarkers may not fully capture the complete aging process of the system; this weakness is particularly remarkable in organ-specific BA. The authors emphasize that it is cost-effective and easy to implement. However, the results associated with organ-specific BA may not be credible because they do not fully reflect the state of a particular organ. It is recommended that these shortcomings and the applicability of the results should be discussed in the text.

Reviewer #1 (Public review):

The authors sought to determine the impact of early antiretroviral treatment on the size, composition, and decay of the HIV latent reservoir. This reservoir represents the source of viral rebound upon treatment interruption and therefore constitutes the greatest challenge to achieving an HIV cure. A particular strength of this study is that it reports on reservoir characteristics in African women, a significantly understudied population, of whom some have initiated treatment within days of acute HIV diagnosis. With the use of highly sensitive and current technologies, including digital droplet PCR and near full-length genome next-generation sequencing, the authors generated a valuable dataset for investigation of proviral dynamics in women initiating early treatment compared to those initiating treatment in chronic infection. The authors confirm previous reports that early antiretroviral treatment restricts reservoir size, but further show that this restriction extends to defective viral genomes, where late treatment initiation was associated with a greater frequency of defective genomes. Furthermore, an additional strength of this study is the longitudinal comparison of viral dynamics post-treatment, wherein early treatment was shown to be associated with a more rapid rate of decay in proviral genomes, regardless of intactness, over a period of one year post-treatment. While it is indicated that intact genomes were not detected after one year following early treatment initiation, sampling depth is noted as a limitation of the study by the authors, and caution should thus be taken with interpretation where sequence numbers are low. Defective genomes are more abundant than intact genomes and are therefore more likely to be sampled. Early treatment was also associated with reduced proviral diversity and fewer instances of polymorphisms associated with cytotoxic T-lymphocyte immune selection. This is expected given that rapid evolution and extensive immune selection are synonymous with HIV infection in the absence of treatment, yet points to an additional benefit of early treatment in the context of immune therapies to restrict the reservoir.

This is one of the first studies to report the mapping of longitudinal intactness of proviral genomes in the globally dominant subtype C. The data and findings from this study therefore represent a much-needed resource in furthering our understanding of HIV persistence and informing broadly impactful cure strategies. The analysis on clonal expansion of proviral genomes may be limited by higher sequence homogeneity in hyperacute infection i.e., cells with different proviral integration sites may have a higher likelihood of containing identical genomes compared to chronic infection.

Overall, these data demonstrate the distinct benefits of early treatment initiation at reducing the barrier to a functional cure for HIV, not only by restricting viral abundance and diversity but also potentially through the preservation of immune function and limiting immune escape. It therefore provides clues to curative strategies even in settings where early diagnosis and treatment may be unlikely.

Reviewer #2 (Public review):

HIV infection is characterized by viral integration into permissive host cells - an event that occurs very early in viral-host encounter. This constitutes the HIV proviral reservoir and is a feature of HIV infection that provides the greatest challenge for eradicating HIV-1 infection once an individual is infected.

This study looks at how starting HIV treatment very early after infection, which substantially reduces the peak viral load detectable (compared to untreated infection), affects the amount and characteristics of the viral reservoir. The authors studied 35 women in South Africa who were at high risk of getting HIV. Some of these women started HIV treatment very soon after getting infected, while others started later. This study is well-designed and has as its focus a very well characterized cohort. Comparison groups are appropriately selected to address proviral DNA characterization and dynamics in the context of acute and chronic treated HIV-1. The amount of HIV and various characteristics of the genetic makeup of the virus (intact/defective proviral genome) was evaluated over one year of treatment. Methods employed for proviral DNA characterization are state-of-the-art and provide in-depth insights into the reservoir in peripheral blood.

While starting treatment early didn't reduce the amount of HIV DNA at the outset, it did lead to a gradual decrease in total HIV DNA quantity over time. In contrast, those who started treatment later didn't see much change in this parameter. Starting treatment early led to a faster decrease in intact provirus (a measure of replication-competence), compared to starting treatment later. Additionally, early treatment reduced genetic diversity of the viral DNA and resulted in fewer immune escape variants within intact genomes. This suggests that collectively having a smaller intact replication-competent reservoir, less viral variability, and less opportunity for virus to evade the immune system - are all features that are likely to facilitate more effective clearance of viral reservoir, especially when combined with other intervention strategies.

Major strengths of the study include the cohort of very early treated persons with HIV and the depth of study. These are important findings, particularly as the study was conducted in HIV-1 subtype C infected women (more cure studies have focussed on men and with subtype B infection)- and in populations most affected by HIV and in need of HIV cure interventions. This is highly relevant because it cannot be assumed that any interventions employed for reducing/clearing the HIV reservoir would perform similarly in men and women or across different populations. Other factors also deserve consideration and include age, and environment (e.g. other comorbidities and coinfections).

Reviewer #1 (Public review):

Previous studies have highlighted some of these paracrine activities of Toxoplasma - and Rasogi et al (mBio, 2020) used a single cell sequencing approach of cells infected in vitro with the WT or MYR KO parasites - and one of their conclusions was that MYR-1 dependent paracrine activities counteract ROP-dependent processes. Similarly, Chen et al (JEM 2020) highlighted that a particular rhoptry protein (ROP16) could be injected into uninfected macrophages and move them to an anti-inflammatory state that might benefit the parasite.

Caveats around immunity and as yet no insight into how this works. In Fig 2 there is a marked defect in the ability of the parasites to expand at day 2 and day 5. Together, these data sets suggest that this paracrine effect mediated by MYR-1 works early - well before the development of adaptive responses.

Comments on revisions:

The authors have provided their perspective on the original review. There were some previous comments that revolved around whether some of the early changes were masked by pooling data sets where they have reiterated that it is not statistically different. Would have been nice to have seen out addressed by having experiments that were appropriately powered. But it's their call.

Reviewer #2 (Public review):

Summary:

In this manuscript by Torelli et al., the authors propose that the major function of MYR1 and MYR1-dependent secreted proteins is to contribute to parasite survival in a paracrine manner rather than to protect parasites from cell-autonomous immune response. The authors conclude that these paracrine effects rescue ∆MYR1 or knockouts of MYR1-dependent effectors within pooled in vivo CRISPR screens.

Strengths:

The authors raised a more general concern that pooled CRISPR screens (not only in Toxoplasma but also other microbes or cancers) would miss important genes by "paracrine masking effect". Although there is no doubt that pooled CRISPR screens (especially in vivo CRISPR screens) are powerful techniques, I think this topic could be of interest to those fields and researchers.

Weaknesses:

In this version, the reviewer is not entirely convinced of the 'paracrine masking effect' because the in vivo experiments should include appropriate controls (see major point 2) in the first submission.

After the revision, although no experiments were added, this reviewer considered that the points have been sufficiently discussed and commented on.

Reviewer #1 (Public review):

In their manuscript, authors Isotani et al used in vivo and ex vivo models to show that nicotine could promote stemness and tumorigenicity in murine model. The authors further provided data supporting that the effects of nicotine on stem cell proliferation and tumor initiation were mediated by the Hippo-YAP/TAZ and Notch signal pathway.

The major strength of this study is the using a set of tools, including Lgr5 reporter mice (Lgr5-EGFP-IRES-CreERT2 mice), stem cell-specific Apc knockout mice (Lgr5CreER Apcfl/fl mice), organoids derived from these mice and chemical compounds (agonists and antagonists) to demonstrate nicotine affects stem cells rather than Paneth cells, leading to increased intestinal stemness and tumorigenicity. Whereas, all models are restricted to mice, lacking analysis of human samples or human intestinal organoids to prove the human relevance of these findings.

Overall, the presented results support their conclusions. A previous study reported that nicotine acts through the α2β4 nAChR to enhance Wnt production by Paneth cells, which subsequently affects ISCs. In contrast, this manuscript demonstrated that nicotine directly promotes ISCs through α7-nAChR, independent of Paneth cells. Therefore, this manuscript offers novel insights into the mechanism of nicotine's effects on the mouse intestine.

Reviewer #2 (Public review):

Summary:

The manuscript by Isotani et al characterizes the hyperproliferation of intestinal stem cells (ISCs) induced by nicotine treatment in vivo. Employing a range of small molecule inhibitors, the authors systematically investigated potential receptors and downstream pathways associated with nicotine-induced phenotypes through in vitro organoid experiments. Notably, the study specifically highlights a signaling cascade involving α7-nAChR/PKC/YAP/TAZ/Notch as a key driver of nicotine-induced stem cell hyperproliferation. Utilizing a Lgr5CreER Apcfl/fl mouse model, the authors extend their findings to propose a potential role of nicotine in stem cell tumorgenesis. The study posits that Notch signaling is essential during this process.

Strengths and Weaknesses:

One noteworthy research highlight in this study is the indication, as shown in Figure 2 and S2, that the trophic effect of nicotine on ISC expansion is independent of Paneth cells. In the Discussion section, the authors propose that this independence may be attributed to distinct expression patterns of nAChRs in different cell types. To further substantiate these findings, the authors provided qPCR analysis of nAchRs in ISCs and Paneth cells from isolated whole small intestine, indicating that α7-nAChR uniquely responds to nicotine treatment among various nAChRs. The authors further strengthen the clinical relevance of the study by exploring human scRNA-seq dataset, in which α7-nAChR is indeed also expressed in human ISCs and Paneth cells.

As shown in the same result section, the effect of nicotine on ISC organoid formation appears to be independent of CHIR99021, a Wnt activator. In the Lgr5CreER Apcfl/fl mouse model, it is known that APC loss results in a constitutive stabilization of β-catenin, thus the hyperproliferation of ISCs by nicotine treatment in this mouse model is likely beyond Wnt activation. The authors have included such discussion.

In Figure 4, the authors investigate ISC organoid formation with a pan-PKC inhibitor, revealing that PKC inhibition blocks nicotine-induced ISC expansion. It's noteworthy that PKC inhibitors have historically been used successfully to isolate and maintain stem cells by promoting self-renewal. Therefore, it is surprising to observe no or reversal effect on ISCs in this context. The authors have now included an additional PKC inhibitor Sotrastaurin to confirm the role of PKC in nicotine-induced ISC expansion.

Overall, the manuscript has provided sufficient experimental evidence to address my concerns and also significantly enhanced its quality.

Reviewer #2 (Public review):

In this manuscript, Tiedje and colleagues longitudinally track changes in parasite numbers across four time points as a way of assessing the effect of malaria control interventions in Ghana. Some of the study results have been reported previously, and in this publication, the authors focus on age-stratification of the results. Malaria prevalence was lower in all age groups after IRS. Follow-up with SMC, however, maintained lower parasite prevalence in the targeted age group but not the population as a whole. Additionally, they observe that diversity measures rebound more slowly than prevalence measures. This adds to a growing literature that demonstrates the relevance of asymptomatic reservoirs.

Strengths:

Overall, I found these results clear, convincing, and well-presented. There is growing interest in developing an expanded toolkit for genomic epidemiology in malaria, and detecting changes in transmission intensity is one major application. As the authors summarize, there is no one-size-fits-all approach, and the Bayesian MOIvar estimate developed here has the potential to complement currently used methods, particularly in regions with high diversity/transmission. I find its extension to a calculation of absolute parasite numbers appealing as this could serve as both a conceptually straightforward and biologically meaningful metric.

Weaknesses:

While I understand the conceptual importance of distinguishing among parasite prevalence, mean MOI, and absolute parasite number, I am not fully convinced by this manuscript's implementation of "census population size". The authors reference the population genetic literature, but within the context of that field, "census population size" refers to the total population size (which, if not formally counted, can be extrapolated) as opposed to "effective population" size, which accounts for a multitude of demographic factors. There is often interesting biology to be gleaned from the magnitude of difference between N and Ne. In this manuscript, however, "census population size" is used to describe the number of distinct parasites detected within a sample, not a population. As a result, the counts do not have an immediate population genetic interpretation and cannot be directly compared to Ne. This doesn't negate their usefulness but does complicate the use of a standard population genetic term. In contrast, I think that sample parasite count will be most useful in an epidemiological context, where the total number of sampled parasites can be contrasted with other metrics to help us better understand how parasites are divided across hosts, space and time. However, for this use, I find it problematic that the metric does not appear to correct for variations in participant number. For instance, in this study, participant numbers especially varied across time for 1-5 year-olds (N=356, 216, 405, and 354 in 2012, 2014, 2015, and 2017 respectively). This sample size variability is accounted for with other metrics like mean MOI. In sum, while the manuscript opens up an interesting discussion, I'm left with an incomplete understanding of the robustness and interpretability of the new proposed metric.

Reviewer #3 (Public review):

Summary:

The manuscript coins a term "the census population size" which they define from the diversity of malaria parasites observed in the human community. They use it to explore changes in parasite diversity in more than 2000 people in Ghana following different control interventions.

Strengths:

This is a good demonstration of how genetic information can be used to augment routinely recorded epidemiological and entomological data to understand the dynamics of malaria and how it is controlled. The genetic information does add to our understanding, though by how much is currently unclear (in this setting it says the same thing as age stratified parasite prevalence), and its relevance moving forward will depend on the practicalities and cost of the data collection and analysis. Nevertheless, this is a great dataset with good analysis and a good attempt to understand more about what is going on in the parasite population.

Weaknesses:

None

Reviewer #1 (Public review):

Previous experimental studies demonstrated that membrane association drives avidity for several potent broadly HIV-neutralizing antibodies and its loss dramatically reduces neutralization. In this study, the authors present a tour de force analysis of molecular dynamics (MD) simulations that demonstrate how several HIV-neutralizing membrane-proximal external region (MPER)-targeting antibodies associate with a model lipid bilayer.

First, the authors compared how three MPER antibodies, 4E10, PGZL1, and 10E8, associated with model membranes, constructed with two lipid compositions similar to native viral membranes. They found that the related antibodies 4E10 and PGZL1 strongly associate with a phospholipid near heavy chain loop 1, consistent with prior crystallographic studies. They also discovered that a previously unappreciated framework region between loops 2-3 in the 4E10/PGZL1 heavy chain contributes to membrane association. Simulations of 10E8, an antibody from a different lineage, revealed several differences from published X-ray structures. Namely, a phosphatidylcholine binding site was offset and includes significant interaction with a nearby framework region. The revised manuscript demonstrates that these lipid interactions are robust to alterations in membrane composition and rigidity. However, it does not address the reverse-that phospholipids known experimentally not to associate with these antibodies (if any such lipids exist) also fail to interact in MD simulations.

Next, the authors simulate another MPER-targeting antibody, LN01, with a model HIV membrane either containing or missing an MPER antigen fragment within. Of note, LN01 inserts more deeply into the membrane when the MPER antigen is present, supporting an energy balance between the lowest energy conformations of LN01, MPER, and the complex. These simulations recapitulate lipid binding interactions solved in published crystallographic studies but also lead to the discovery of a novel lipid binding site the authors term the "Loading Site", which could guide future experiments with this antibody.

The authors next established course-grained (CG) MD simulations of the various antibodies with model membranes to study membrane embedding. These simulations facilitated greater sampling of different initial antibody geometries relative to membrane. These CG simulations , which cannot resolve atomistic interactions, are nonetheless compelling because negative controls (ab 13h11, BSA) that should not associate with membrane indeed sample significantly less membrane.

Distinct geometries derived from CG simulations were then used to initialize all-atom MD simulations to study insertion in finer detail (e.g., phospholipid association), which largely recapitulate their earlier results, albeit with more unbiased sampling. The multiscale model of an initial CG study with broad geometric sampling, followed by all-atom MD, provides a generalized framework for such simulations.

Finally, the authors construct velocity pulling simulations to estimate the energetics of antibody membrane embedding. Using the multiscale modelling workflow to achieve greater geometric sampling, they demonstrate that their model reliably predicts lower association energetics for known mutations in 4E10 that disrupt lipid binding. However, the model does have limitations: namely, its ability to predict more subtle changes along a lineage-intermediate mutations that reduce lipid binding are indistinguishable from mutations that completely ablate lipid association. Thus, while large/binary differences in lipid affinity might be predictable, the use of this method as a generative model are likely more limited.

The MD simulations conducted throughout are rigorous and the analysis are extensive, creative, and biologically inspired. Overall, these analyses provide an important mechanistic characterization of how broadly neutralizing antibodies associate with lipids proximal to membrane-associated epitopes to drive neutralization.

Reviewer #2 (Public review):

In this study, Maillie et al. have carried out a set of multiscale molecular dynamics simulations to investigate the interactions between the viral membrane and four broadly neutralizing antibodies that target the membrane proximal exposed region (MPER) of the HIV-1 envelope trimer. The simulation recapitulated in several cases the binding sites of lipid head groups that were observed experimentally by X-ray crystallography, as well as some new binding sites. These binding sites were further validated using a structural bioinformatics approach. Finally, steered molecular dynamics was used to measure the binding strength between the membrane and variants of the 4E10 and PGZL1 antibodies.

The use of multiscale MD simulations allows for a detailed exploration of the system at different time and length scales. The combination of MD simulations and structural bioinformatics provides a comprehensive approach to validate the identified binding sites. Finally, the steered MD simulations offer quantitative insights into the binding strength between the membrane and bnAbs.

While the simulations and analyses provide qualitative insights into the binding interactions, they do not offer a quantitative assessment of energetics. The coarse-grained simulations exhibit artifacts and thus require careful analysis.

This study contributes to a deeper understanding of the molecular mechanisms underlying bnAb recognition of the HIV-1 envelope. The insights gained from this work could inform the design of more potent and broadly neutralizing antibodies.

Reviewer #1 (Public review):

Summary:

In their manuscript, Gomez-Frittelli and colleagues characterize the expression of cadherin6 (and -8) in colonic IPANs of mice. Moreover, they found that these cdh6-expressing IPANs are capable of initiating colonic motor complexes in the distal colon, but not proximal and midcolon. They support their claim by morphological, electrophysiological, optogenetic, and pharmacological experiments.

Strengths:

The work is very impressive and involves several genetic models and state-of-the-art physiological setups including respective controls. It is a very well-written manuscript that truly contributes to our understanding of GI-motility and its anatomical and physiological basis. The authors were able to convincingly answer their research questions with a wide range of methods without overselling their results.

Weaknesses:

The authors put quite some emphasis on stating that cdh6 is a synaptic protein (in the title and throughout the text), which interacts in a homophilic fashion. They deduct that cdh6 might be involved in IPAN-IPAN synapses (line 247ff.). However, Cdh6 does not only interact in synapses and is expressed by non-neuronal cells as well (see e.g., expression in the proximal tubuli of the kidney). Moreover, cdh6 does not only build homodimers, but also heterodimers with Chd9 as well as Cdh7, -10, and -14 (see e.g., Shimoyama et al. 2000, DOI: 10.1042/0264-6021:3490159). It would therefore be interesting to assess the expression pattern of cdh6-proteins using immunostainings in combination with synaptic markers to substantiate the authors' claim or at least add the possibility of cell-cell-interactions other than synapses to the discussion. Additionally, an immunostaining of cdh6 would confirm if the expression of tdTomato in smooth muscle cells of the cdh6-creERT model is valid or a leaky expression (false positive).

Reviewer #2 (Public review):

Summary:

Intrinsic primary afferent neurons are an interesting population of enteric neurons that transduce stimuli from the mucosa, initiate reflexive neurocircuitry involved in motor and secretory functions, and modulate gut immune responses. The morphology, neurochemical coding, and electrophysiological properties of these cells have been relatively well described in a long literature dating back to the late 1800's but questions remain regarding their roles in enteric neurocircuitry, potential subsets with unique functions, and contributions to disease. Here, the authors provide RNAscope, immunolabeling, electrophysiological, and organ function data characterizing IPANs in mice and suggest that Cdh6 is an additional marker of these cells.

Strengths:

This paper would likely be of interest to a focused enteric neuroscience audience and increase information regarding the properties of IPANs in mice. These data are useful and suggest that prior data from studies of IPANs in other species are likely translatable to mice.

Weaknesses:

The advance presented here beyond what is already known is minimal. Some of the core conclusions are overstated and there are multiple other major issues that limit enthusiasm. Key control experiments are lacking and data do not specifically address the properties of the proposed Cdh6+ population.

Major weaknesses:

(1) The novelty of this study is relatively low. The main point of novelty suggests an additional marker of IPANs (Cdh6) that would add to the known list of markers for these cells. How useful this would be is unclear. Other main findings basically confirm that IPANs in mice display the same classical characteristics that have been known for many years from studies in guinea pigs, rats, mice and humans.

(2) Some of the main conclusions of this study are overstated and claims of priority are made that are not true. For example, the authors state in lines 27-28 of the abstract that their findings provide the "first demonstration of selective activation of a single neurochemical and functional class of enteric neurons". This is certainly not true since Gould et al (AJP-GIL 2019) expressed ChR2 in nitrergic enteric neurons and showed that activating those cells disrupted CMC activity. In fact, prior work by the authors themselves (Hibberd et al Gastro 2018) showed that activating calretinin neurons with ChR2 evoked motor responses. Work by other groups has used chemogenetics and optogenetics to show the effects of activating multiple other classes of neurons in the gut.

(3) Critical controls are needed to support the optogenetic experiments. Control experiments are needed to show that ChR2 expression a) does not change the baseline properties of the neurons, b) that stimulation with the chosen intensity of light elicits physiologically relevant responses in those neurons, and c) that stimulation via ChR2 elicits comparable responses in IPANs in the different gut regions focused on here.

(4) The electrophysiological characterization of mouse IPANs is useful but this is a basic characterization of any IPAN and really says nothing specifically about Cdh6+ neurons. The electrophysiological characterization was also only done in a small fraction of colonic IPANs, and it is not clear if these represent cell properties in the distal colon or proximal colon, and whether these properties might be extrapolated to IPANs in the different regions. Similarly, blocking IH with ZD7288 affects all IPANs and does not add specific information regarding the role of the proposed Cdh6+ subtype.

(5) Why SMP IPANs were not included in the analysis of Cdh6 expression is a little puzzling. IPANs are present in the SMP of the small intestine and colon, and it would be useful to know if this proposed marker is also present in these cells.

(6) The emphasis on IH being a rhythmicity indicator seems a bit premature. There is no evidence to suggest that IH and IT are rhythm-generating currents in the ENS.

(7) As the authors point out in the introduction and discuss later on, Type II Cadherins such as Cdh6 bind homophillically to the same cadherin at both pre- and post-synapse. The apparent enrichment of Cdh6 in IPANs would suggest extensive expression in synaptic terminals that would also suggest extensive IPAN-IPAN connections unless other subtypes of neurons express this protein. Such synaptic connections are not typical of IPANs and raise the question of whether or not IPANs actually express the functional protein and if so, what might be its role. Not having this information limits the usefulness of this as a proposed marker.

(8) Experiments shown in Figures 6J and K use a tethered pellet to drive motor responses. By definition, these are not CMCs as stated by the authors.

(9) The data from the optogenetic experiments are difficult to understand. How would stimulating IPANs in the distal colon generate retrograde CMCs and stimulating IPANs in the proximal colon do nothing? Additional characterization of the Cdh6+ population of cells is needed to understand the mechanisms underlying these effects.

Reviewer #1 (Public review):

Summary:

In this manuscript the Treisman and colleagues address the question of how protein phosphatase 1 (PP1) regulatory subunits (or PP1-interacting protein (PIPs)) confer specificity on the PP1 catalytic subunit which by itself possesses little substrate specificity. In prior work the authors showed that the PIP Phactrs confers specificity by remodelling a hydrophobic groove immediately adjacent to the PP1 catalytic site through residues within the RVxF- ø ø -R-W string of Phactrs. Specifically, the residues proximal and including the 'W' of the RVxF- ø ø -R-W string remodel the hydrophobic groove. Other residues of the RVxF- ø ø -R-W string (i.e. the RVxF- ø ø -R) are not involved in this remodelling.

The authors suggest that the RVxF- ø ø -R-W string is a conserved feature of many PIPs including PNUTS, Neurabin/spinophilin and R15A. However, from a sequence and structural perspective, only the RVxF- ø ø -R- is conserved. The W is not conserved in most and in the R15A structure (PDB:7NZM) the Trp side chain points away from the hydrophobic channel - this could be a questionable interpretation due to model-building into the low-resolution cryo-EM map (4 A).

In this paper, the authors convincingly show that Neurabin confers substrate specificity through interactions of its PDZ domain with the PDZ domain-binding motif (PBM) of 4E-BP. They show the PBM motif is required for Neurabin to increase PP1 activity towards 4E-BP and a synthetic peptide modelled on 4E-BP and also a synthetic peptide based on IRSp53 with a PBM added. The PBM of 4E-BP1 confers high affinity binding to the Neurabin PDZ domain. A crystal structure of a PP1-4E-BP1 fusion with Neurabin shows that the PBM of 4E-BP interacts with the PDZ domain of Neurabin. No interactions of 4E-BP and the catalytic site of PP1 are observed. Cell biology work showed that Neurabin-PP1 regulates the TOR signalling pathway by dephosphorylating 4E-BPs.

Strengths:

This work demonstrates convincingly using a variety of cell biology, proteomics, biophysics and structural biology that the PP1 interacting protein Neurabin confers specificity on PP1 through an interaction of its PDZ domain with a PDZ-binding motif of 4E-BP1 proteins. Remodelling of the hydrophobic groove of the PP1 catalytic subunit is not involved in Neurabin-dependent substrate specificity, in contrast to how Phactrs confers specificity on PP1. The active site of the Neurabin/PP1 complex does not recognise residues in the vicinity of the phospho-residue, thus allowing for multiple phospho-sites on 4E-BP to be dephosphorylated by Neurabin/PP1. This contrasts with substrate specificity conferred by the Phactrs PIP that confers specificity of Phactrs/PP1 towards its substrates in a sequence-specific context by remodelling the hydrophobic groove immediately adjacent to the catalytic. The structural and biochemical insights are used to explore the role of Neurabin/PP1 in dephosphorylation 4E-BPs in vivo, showing that Neurabin/PP1 regulates the TOR signalling pathway, specifically mTORC1-dependent translational control.

Weaknesses:

The only weakness is the suggestion that a conserved RVxF- ø ø -R-W string exists in PIPs. The 'W' is not conserved in sequence and 3 dimensions in most of the PIPs discussed in this manuscript. The lack of conservation of the W would be consistent with the finding based on multiple PP1-PIP structures that apart from Phactrs, no other PIP appears to remodel the PP1 hydrophobic channel.

Reviewer #2 (Public review):

This manuscript explores the molecular mechanisms that are involved in substrate recognition by the PP1 phosphatase. The authors previously showed that the PP1 interacting protein (PPI), PhactrI, conferred substrate specificity by remodelling the PP1 hydrophobic substrate groove. In this work, the authors aimed to understand the key determinant of how other PIPs, Neurabin and Spinophilin, mediate substrate recognition.

The authors generated a few PP1-PIP fusion constructs, undertook TMT phosphoproteomics and validated their method using PP1-Phactr1/2/3/4 fusion constructs. Using this method, the authors identified phsophorylation sites controlled by PP1-Neurabin and focussed their work on 4E-BP1, thereby linking PP1-Neurabin to mTORC1 signalling. Upon validating that PP1-Neurabin dephosphorylates 4E-BP1, they determined that 4E-BP1 PBM binds to the PDZ domain of Neurabin with an affinity that was greater than 30-fold as compared to other substrates. PP1-Neurabin dephosphorylated 4E-BP1WT and IRSp53WT with a catalytic efficiency much greater than PP1 alone. However, PP1-Neurabin bound to 4E-BP1 and IRSp53 mutants lacking the Neurabin PDZ domain with a catalytic efficiency lesser than that observed with 4E-BP1WT. These results indicate the involvement of the PDZ domain in facilitating substrate recruitment by PP1-Neurabin. Interestingly, PP1-Phactr1 dephosphorylation of 4E-BP1 phenocopies PP1 alone, while PP1-Phactr1 dephosphorylates IRSp53 to a much higher extent than PP1 alone. These results highlight the importance of the PDZ domain and also shed light on how different PP1-PIP holoenzymes mediate substrate recognition using distinct mechanisms. The authors also show that the remodelling of the hydrophobic PP1 substrate groove which is essential for substrate recognition by PP1-Phactr1, was not required by PP1-Neurabin. Additionally, the authors also resolved the structure of a PP1-4E-BP1 fusion with the PDZ-containing C-terminal of Neurabin and observed that the Neurabin/PP1-4E-BP1 complex structure was oriented at 21{degree sign} to that in the unliganded Spinophilin/PP1 complex (resolved by Ragusa et al., 2010) owing to a slight bend in the C-terminal section that connects it to the RVxF-ΦΦ-R-W string. Since no interaction was observed with the remodelled PP1-Neurabin hydrophobic groove, the authors utilised AlphaFold3 to further answer this. They observed a high confidence of interaction between the groove and phosphorylated substrate and a low confidence of interaction between the groove and unphosphorylated substrate, thereby suggesting that the hydrophobic groove remodelling is not involved in PP1-Neurabin recognition and dephosphorylation of 4E-BP1.

In this work, the authors provide novel insights into how Neurabin depends on the interaction between its PDZ domain and PBM domains of potential substrates to mediate its recruitment by PP1. Additionally, they uncover a novel PP1-Neurabin substrate, 4E-BP1. They systematically employ phosphoproteomics, biochemical, and structural methods to investigate substrate specificity in a robust fashion. Furthermore, the authors also compare the interactions between PP1-Neurabin to 4E-BP1 and IRSp53 (PP1-Phactr1 substrate) with PP1-Phactr1, to showcase the specificity of the mode of action employed by these complexes in mediating substrate specificity. The authors employ an innovative PP1-PIP fusion strategy previously explored by Oberoi et al., 2016 and the authors themselves in Fedoryshchak et al., 2020. Although this method, allows for a more controlled investigation of the interactions between PP1-PIPs and its substrates, this methodology may not fully recapitulate the interactions that may occur in a physiological setting. This could potentially be overcome by studying the interactions of the full proteins using classical biochemical approaches in cell lines. Furthermore, the authors have substantially characterised the importance of the PDZ domain using their fusion constructs, however, I believe that further exploration into either structural or AlphaFold3 modelling of PBM domain substrate mutants, or a Neurabin PDZ-domain mutant might further strengthen this claim. Overall, the paper makes a substantial contribution to understanding substrate recognition and specificity in PP1-PIP complexes. The study's innovative methods, biological relevance, and mechanistic insights are strengths, but whether this mechanism occurs in a physiological context is unclear.

Reviewer #3 (Public review):

Protein Phosphatase 1 (PP1), a vital member of the PPP superfamily, drives most cellular serine/threonine dephosphorylation. Despite PP1's low intrinsic sequence preference, its substrate specificity is finely tuned by over 200 PP1-interacting proteins (PIPs), which employ short linear motifs (SLIMs) to bind specific PP1 surface regions. By targeting PP1 to cellular sites, modifying substrate grooves, or altering surface electrostatics, PIPs influence substrate specificity. Although many PIP-PP1-substrate interactions remain uncharacterized, the Phactr family of PIPs uniquely imposes sequence specificity at dephosphorylation sites through a conserved "RVxF-ΦΦ-R-W" motif. In Phactr1-PP1, this motif forms a hydrophobic pocket that favors substrates with hydrophobic residues at +4/+5 in acidic contexts (the "LLD motif"), a specificity that endures even in PP1-Phactr1 fusions. Neurabin/Spinophilin remodel PP1's hydrophobic groove in distinct ways, creating unique holoenzyme surfaces, though the impact on substrate specificity remains underexplored. This study investigates Neurabin/Spinophilin specificity via PDZ domain-driven interactions, showing that Neurabin/PP1 specificity is governed more by PDZ domain interactions than by substrate sequence, unlike Phactr1/PP1.

A significant strength of this work is the use of PP1-PIP fusion proteins to effectively model intact PP1•PIP holoenzymes by replicating the interactions that remodel the PP1 interface and confer site-specific substrate specificity. When combined with proteomic analyses to assess phospho-site depletion in mammalian cells, these fusions offer critical insights into holoenzyme specificity, revealing new candidate substrates for Neurabin and Spinophilin. The studies present compelling evidence that the PDZ domain of PP1-Neurabin directs its specificity, with the remodelled PP1 hydrophobic groove interactions having minimal impact. This mechanism is supported by structural analysis of the PP1-4E-BP1 substrate fusion bound to a Neurabin construct, highlighting the 4E-BP1/PDZ interaction. This work delivers crucial insights into PP1-PIP holoenzyme function, combining biochemical, proteomic, and structural approaches. It validates the PP1-PIP fusion protein model as a powerful tool, suggesting it may extend to studying additional holoenzymes. While an extremely useful model, it must be considered unlikely the PP1-PIP fusions fully recapitulate the specificity and regulation of the holoenzyme.

Reviewer #1 (Public review):

The authors of this study developed a method to quantify calvarial bone marrow from MRI head scans, enabling the study of its composition in large datasets of adults, usually collected to study the brain. Bone marrow intensity can be semi-quantitatively measured in T1-weighted MRI scans due to the greater signal intensity of fat than watery red marrow. This is an ingenious use of the MRI-produced information for other important phenotypes, such as bone structure and marrow content. Different head types were tested for complying with the model, which is notable.

The model was also successfully validated using several publicly available MRI resources - real data - in (1) a dataset consisting of 30 individuals that were scanned 10 times each at 3-day intervals, and (2) the monozygotic (MZ) twin data from the Human Connectome Project cohort. Then the authors applied this validated method to head-MRI scans from the UK Biobank (n=33,042) to extract information on the spatial distribution of bone marrow adiposity (BMA) in the calvaria, allowing a GWAS to identify associated genes.

The authors revealed high heritability and identified 41 genetic loci significantly associated with the BMA trait, including six sex-specific loci. Of note, statistics estimate that 99% of BMA trait-influencing variants are shared with BMD (497 of 500 variants), which may mean these results demonstrate the biological relevance to bone health. Some of the BMA genes were found related to the Wnt pathway, including WNT16, WNT4, NXN; this is a "positive control", since the Wnt/β-catenin signaling pathway was suggested as an important determinant of BMA. Also, associations in genes (BMP4, DLX5, LGR4, LRP4, SFRP4) that are known to specifically influence adiposity, are encouraging. Integrating mapped genes with bone marrow single-cell RNA-seq data revealed patterns of adipogenic lineage differentiation and lipid loading.

The study also investigated the genetic overlap between BMA and twelve (or 13) "brain and body" traits and identified significant genetic correlations with BMI, cognitive ability, and Parkinson's disease.

In sum, since MRI head scans present a hitherto unexplored opportunity to address unresolved aspects of bone marrow biology, this study is both timely and innovative.

There are, however, some assumptions, findings, and their interpretation, which require more critical focus.

Sex-specificity is well described and studied here. Men have higher BMA than women, but post-menopausal women catch up in the BMA values. The authors believe that calvarial marrow has a number of features that make it particularly well-suited to the study of BMA process - which is clinically important in other bone sites. It has a simple "sandwiched" structure that they are able to model. This is true only to some extent: a condition called "Hyperostosis frontalis interna", of unknown etiology (described by Smith & Hemphill in 1956) - is characterized by irregular overgrowth of the inner table of the frontal bone (symmetric/bilateral). Although not of clinical significance, typically benign, studies report a prevalence of 12%; However, it's most common in postmenopausal women - where prevalences up to 49% in women over the age of 65 - have been reported. Thus, sexual dimorphism is obvious and the effect of estrogen is likely shared with whichever bone - and marrow - age-related pathology. So, for women not using HRT, this new layer of the bone might interfere with the calvarial BMA readings and in turn, affect the BMA-related analyses. The authors suspect that the effect of BMA on BMD may be biased in women; they should comment on those "with low BMD and high BMA" given that hyperostosis frontalis might be an issue. A strong effect of SNPs in the ESR1 chromosomal region might be akin to the above concern.

Then, there is a perfect overlap of the BMA SNPs that are shared with BMD (497 of 500 variants), which may prove a "face validity" of the MRI-derived BMA. However, the BMD in the study was heel-derived eBMD - which is a good proxy for osteoporosis and is mostly driven by trabecular bone. Thus, there might be a concern that the BMA metrics capture some trabecular BMD.

Next, integrating mapped genes with existing bone marrow single-cell RNA-sequencing data revealed patterns of adipogenic lineage differentiation and lipid loading. The problem here is that the scRNAseq studies of the Bone Marrow niche are overwhelmingly mouse. The authors might wish to justify why they are relevant to humans (in the absence of the human-specific scRNAseq).

For genetic correlation analysis, the authors selected 7 body and 6 brain traits. The latter traits reflect cognition (general cognitive ability and educational attainment) and brain-related disorders. This selection might seem arbitrary. The interpretation of genetic correlation with cognitive ability, education, and Parkinson's disease was attributed to the recently discovered vascular channels that link calvarial bone marrow to the meninges. This is a fascinating hypothesis, which requires functional proof. However, there might be simpler explanations. Thus, the diploe and the inner table of the calvarium are drained by the same veins as the dura. From the anatomy textbook, we know that diploic veins connect the pericranial and endocranial venous system through the skull.

Reviewer #2 (Public review):

Summary:

This study develops a new artificial intelligence method for high-throughput analysis of skull bone marrow from MRI data, which may be useful for large-scale biological analyses. Using this method, the authors then attempt to estimate skull bone marrow adiposity (BMA) using T1-weighted signal intensity from MRI scans of ~33,000 people, followed by genome-wide association analysis; however, the approach is inadequate because T1-weighted signal intensity is not validated for measurement of bone marrow adiposity. If it could be validated, the study would be an important advance in understanding of bone marrow adiposity and skeletal biology.

Strengths:

This paper is well-written, and the figures are nicely presented. The neural network method used for analysing skull bone marrow is innovative, and the authors validate this through several approaches. Therefore, the authors have achieved the aim of developing a method for large-scale analysis of skull bone marrow from MRI data.

The GWAS is reasonably well-powered and addresses potential ethnicity differences, with one GWAS done across white males and females, and a separate GWAS in non-white participants. The methodology also conforms to common GWAS standards, including for mapping genetic variants to candidate genes. Moreover, the study further investigates the biological roles of these genes by analysing their expression in single-cell RNA sequencing data.

Weaknesses:

The fundamental weakness is that T1-weighted MRI signal intensity (T1W) is used as an estimate of BMA, but it has never been validated for this. The authors show that this T1W parameter measures something that is heritable and can be compared between subjects, but they don't show that it actually measures (or even estimates) calvarial BMA. There is an attempt to do so by comparing the T1W parameter with data from quantitative T1 images: the authors show a reasonable correlation with some of the quantitative T1 image data. However, this still does not show that the parameter is measuring BMA; it could be measuring some other biological characteristic, but this remains unclear. So, there is a need to validate the T1W parameter against an established measure of BMA, such as the bone marrow fat-fraction or proton density fat fraction measured from multi-echo MRI analysis.

Without validating this BMA measurement method, it is not possible to interpret the GWAS or other findings reported in the study.

A less critical weakness is that the GWAS has been done only on a single cohort, without replicating the findings in a follow-up cohort. For example, the authors could repeat their analysis on the remaining ~50,000 UK Biobank imaging participants for whom MRI data is now available. However, this would be pointless without knowing what biological characteristic(s) the T1W parameter is actually reflecting.

Reviewer #3 (Public review):

Summary:

This manuscript, "Estimating bone marrow adiposity from head MRI and identifying its genetic 2 architecture", brings together the groups of Drs. Kaufmann and Hughes in a tour de force work to develop an artificial neural network that localizes calvaria bone marrow in T1-weighted MRI head scans, with the goal of studying its composition in several large MRI datasets, and to model sex-dimorphic age trajectories, including the effect of menopause.

Strengths:

Bone marrow adiposity is a very active tissue with far-reaching implications for tissue crosstalk and human health than we had initially recognized. Although MRI has been used to measure BM, studies such as the one by these two groups are still lacking whereas very large datasets are analyzed using advanced AI machine learning tools coupled with genetic studies and a specific pathology. The groups had to develop new methods and new AI machine-learning tools for the imaging analyses.

Weaknesses:

Some aspects of the work that authors could add additional clarification.

(1) Imaging Limitations: The authors provide an excellent overview and references supporting the use of MRI as a method for assessing marrow fat, particularly with some specific modifications. However, MRI images can be affected by various factors, including the presence of other tissues as well as specific MRI settings, which are much harder to precisely control when using different datasets.

(2) The specific density of cranial bones as it relates to the types of bone marrow: Cranial bones are extremely dense structures, which naturally interfere with MRI imaging. While it is thought that cranial bones have mostly "red bone marrow", this is only true for a short time in humans. How sensitive is their system in differentiating between red and yellow BM?

(3) Both items above are further complicated by aging, but aging is not a linear event as we have learned. There are specific bursts of aging in humans around the age of 45 and early 60s. How do the system and model predict or incorporate these peaks of aging? It seems from the data shown that aging is reflected more as a linear phenomenon. Is this because additional aging datasets are needed?

(4) The authors describe in richness of detail their AI learning programming and how it extracted the data from datasets. The authors also show some important correlations with specific genes, SNPs. What is not clear is how conditions such as anemia for example. An expected finding would be that patients with chronic anemia have lower bone marrow (BM) signal intensity on MRI scans than healthy people. This is because the signal intensity of BM depends on the fat-to-cell ratio in the tissue. Furthermore, patients with a host of musculoskeletal disorders ranging from osteopenia to osteoporosis, sarcopenia, and osteosarcopenia will also have altered MRI scans. When using such large datasets how did the authors control or exclude these pathological conditions, or were all these conditions likely present?

(5) Some of the genes and SNPs although significant showed very small correlations. What is their likely physiological significance?

(6) The authors could use this excellent manuscript to expand their discussion to include the need for studies like theirs to be also complemented by multi-OMICS studies that will include proteomics and lipidomics of BM, bones, and muscles.

Reviewer #1 (Public review):

Summary:

Ma, Yang et al. report a new investigation aimed at elucidating one of the key nutrients S. Typhimurium (STM) utilizes with the nutrient-poor intracellular niche within the macrophage, focusing on the amino acid beta-alanine. From these data, the authors report that beta-alanine plays an important role in mediating STM infection and virulence. The authors employ a multidisciplinary approach that includes some mouse studies and ultimately propose a mechanism by which panD, involved in B-Ala synthesis, mediates the regulation of zinc homeostasis in Salmonella. The impact of this work is questionable. There are already many studies reporting Salmonella-effector interactions, and while this adds to that knowledge it is not a significant advance over previous studies. While the authors are investigating an interesting question, the work has two important weaknesses; if addressed, the conclusions of this work and broader relevance to bacterial pathogenesis would be enhanced.

Strengths:

This reviewer appreciates the multidisciplinary nature of the work. The overall presentation of the figure graphics are clear and organized.

Weaknesses:

First, this study is very light on mechanistic investigations, even though a mechanism is proposed. Zinc homeostasis in cells, and roles in bacteria infections, are complex processes with many players. The authors have not thoroughly investigated the mechanisms underlying the roles of B-Ala and panD in impacting STM infection such that other factors cannot be ruled out. Defining the cellular content of Zn2+ STM in vivo would be one such route. With further mechanistic studies, the possibility cannot be ruled out that the authors have simply deleted two important genes and seen an infection defect - this may not relate directly to Zn2+ acquisition.

Second, the authors hint at their newly described mechanism/pathway being important for disease and possibly a target for therapeutics. This claim is not justified given that they have employed a single STM strain, which was isolated from chickens and is not even a clinical isolate. The authors could enhance the impact of their findings and relevance to human disease by demonstrating it occurs in human clinical isolates and possibly other serovars. Further, the use of mouse macrophage as a model, and mice, have limited translatability to human STM infections.

Reviewer #2 (Public review):

Summary:

Salmonella exploits host- and bacteria-derived β-alanine to efficiently replicate in host macrophages and cause systemic disease. β-alanine executes this by increasing the expression of zinc transporter genes and therefore the uptake of zinc by intracellular Salmonella.

Strengths:

The experiments designed are thorough and the claims made are directly related to the outcome of the experiments. No overreaching claims were made.

Weaknesses:

A little deeper insight was expected, particularly towards the mechanistic aspects. For example, zinc transport was found to be the cause of the b-alanine-mediated effect on Salmonella intracellular replication. It would have been very interesting to see which are the governing factors that may get activated or inhibited due to Zn accumulation that supports such intracellular replication.

Reviewer #3 (Public review):

Summary:

Salmonella is interesting due to its life within a compact compartment, which we call SCV or Salmonella containing vacuole in the field of Salmonella. SCV is a tight-fitting vacuole where the acquisition of nutrients is a key factor by Salmonella. The authors among many nutrients, focussed on beta-alanine. It is also known from many other studies that Salmonella requires beta-alanine. The authors have done in vitro RAW macrophage infection assays and In vivo mouse infection assays to see the life of Salmonella in the presence of beta-alanine. They concluded by comprehending that beta-alanine modulates the expression of many genes including zinc transporters which are required for pathogenesis.

Strengths:

This study made a couple of knockouts in Salmonella and did a transcriptomic investigation to understand the global gene expression pattern.

Weaknesses:

The following questions are unanswered:

(1) It is not clear how the exogenous beta-alanine is taken up by macrophages.

(2) It is not clear how the Beta-alanine from the cytosol of the macrophage enters the SCV.

(3) It is not clear how the beta-alanine from SCV enters the bacterial cytosol.

(4) There is no clarity on the utilization of exogenous beta-alanine of the host and the de novo synthesis of beta-alanine by panD of Salmonella.

Reviewer #1 (Public review):

Summary:

The paper by Tolossa et al. presents classification studies that aim to predict the anatomical location of a neuron from the statistics of its in-vivo firing pattern. They study two types of statistics (ISI distribution, PSTH) and try to predict the location at different resolutions (region, subregion, cortical layer).

Strengths:

This paper provides a systematic quantification of the single-neuron firing vs location relationship.

The quality of the classification setup seems high.

The paper uncovers that, at the single neuron level, the firing pattern of a neuron carries some information on the neuron's anatomical location, although the predictive accuracy is not high enough to rely on this relationship in most cases.

Weaknesses:

As the authors mention in the Discussion, it is not clear whether the observed differences in firing are epiphenomenal. If the anatomical location information is useful to the neuron, to what extent can this be inferred from the vicinity of the synaptic site, based on the neurotransmitter and neuromodulator identities? Why would the neuron need to dynamically update its prediction of the anatomical location of its pre-synaptic partner based on activity when that location is static, and if that information is genetically encoded in synaptic proteins, etc (e.g., the type of the synaptic site)? Note that the neuron does not need to classify all possible locations to guess the location of its pre-synaptic partner because it may only receive input from a subset of locations. If an argument on activity-based estimation being more advantageous to the neuron than synaptic site-based estimation cannot be made, I believe limiting the scope of the paper (e.g., in the Introduction) to an epiphenomenal observation and its quantification will improve the scientific quality.Life Assessment

This article reports a useful set of findings on how electrophysiological response properties of neurons correlate with their position in the brain. The evidence currently remains incomplete, with reviewers making specific suggestions for how clustering needs to be redone. The manuscript would also benefit from a more focused presentation of results and the removal of incorrect claims about recording biases.

Reviewer #2 (Public review):

Summary:

In this manuscript, Tolossa et al. analyze Inter-spike intervals from various freely available datasets from the Allen Institute and from a dataset from Steinmetz et al. They show that they can modestly decode between gross brain regions (Visual vs. Hippocampus vs. Thalamus), and modestly separate sub-areas within brain regions (DG vs. CA1 or various visual brain areas).

Strengths:

The paper is reasonably well written, and the definitions are quite well done. For example, the authors clearly explained transductive vs. inductive inference in their decoders. E.g., transductive learning allows the decoder to learn features from each animal, whereas inductive inference focuses on withheld animals and prioritizes the learning of generalizable features.

Weaknesses:

However, even with some of these positive aspects, I still found the manuscript to be a laundry list of results, where some results are overly explained and not particularly compelling or interesting, whereas interesting results are not strongly described or emphasized. The overall problem is that the study is not cohesive, and the authors need to either come up with a tool or demonstrate a scientific finding. The current version attempts to split the middle and thus is not as impactful as it could be.

Reviewer #1 (Public review):

Summary:

Mitotic kinesins carry out crucial roles in intracellular motility and mitotic spindle organization. Although many mitotic kinesins have been extensively studied, a few conserved mitotic motors remain poorly explored, including chromosome-associated kinesins. Here, Furusaki et al reconstitute recombinant chromosome-associated kinesin or chromokinesin (Kid) and reveal processive plus-end motility along microtubules. The authors purify multiple versions of Kid, revealing dimeric organization and their processive microtubule plus-ended motility which depends on their conserved motor domains, neck linkers, and coiled-coil regions. The study reveals for the first time that KID can recruit and transport duplex DNA along microtubules using its conserved C-terminal DNA binding domain. The work provides crucial revised thinking about the mechanisms of Chromokinesins mitosis as physical processive motors that mobilize chromosomes towards the microtubule plus ends in early metaphase.

Strengths:

The authors reconstitute multiple chromosome-associated kinesin (KID) orthologs from Xenopus and humans with microtubules and determine their oligomerization. The study shows how coiled-coil and neck linker regions of KID are essential for its function as its deletion leads to non-processive motility. CHimeras placing the KID coiled-coil and neck linker on the KIF1A motor domain led to the production of a processive recombinant motor supporting the compatibility of their motility mechanisms. The KID c-terminal tail binds and transports only double-stranded DNA and its deletion or single-stranded DNA leads to defects in this activity.

Weaknesses:

A minor weakness in the studies is that they do not resolve the mechanisms of KID in binding large duplex DNA molecules or condensed chromatin. The authors suggest a model in which KID forms multimers along large chromosomes that lead to their transport, but this model was not directly tested.

Reviewer #2 (Public review):

Summary:

Previous work in the field highlighted the role of the kinesin-10 motor protein Kid (KIF22) in the polar ejection force during prometaphase. However, the biochemical and biophysical properties of Kid that enabled it to serve in this role were unclear. The authors demonstrate that human and xenopus Kid proteins are processive kinesins that function as homodimeric molecules. The data are solid and support the findings although the text could use some editing to improve clarity.

Strengths:

A highlight of the work is the reconstitution of DNA transport in vitro.

A second highlight is the demonstration that the monomer vs dimer state is dependent on protein concentration.

Weaknesses:

The authors make several assumptions of the monomer vs dimer state of various Kid constructs without verifying the protein state using e.g. size exclusion chromatography and/or nanophotometry. They also make statements about monomer-to-dimer transitions on the microtubule without showing or quantifying the data.

The discussion needs to better put the work into context regarding the ability of non-processive motors to work in teams (formerly thought to be the case for Kid) and how their findings on Kid change this prevailing view in the case of polar ejection force.

The authors also do not mention previous work on kinesins with non-conventional neck linker/neck coil regions that have been shown to move processively. Their work on Kid needs to be put into this context.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Weaknesses:

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

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

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

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

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

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

Weaknesses:

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

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

Reviewer #3 (Public review):

Summary:

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

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

Strengths:

(1) Rigorous anatomical analysis<br /> (2) Novel insights on the wiring logic of the neurosecretory cells.

Weaknesses:

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

Reviewer #1 (Public review):

Summary:

PROTACs are heterobifunctional molecules that utilize the Ubiquitin Proteasome System to selectively degrade target proteins within cells. Upon introduction to the cells, PROTACs capture the activity of the E3 ubiquitin ligases for ubiquitination of the targeted protein, leading to its subsequent degradation by the proteasome. The main benefit of PROTAC technology is that it expands the "druggable proteome" and provides numerous possibilities for therapeutic use. However, there are also some difficulties, including the one addressed in this manuscript: identifying suitable target-E3 ligase pairs for successful degradation. Currently, only a few out of about 600 E3 ligases are used to develop PROTAC compounds, which creates the need to identify other E3 ligases that could be used in PROTAC synthesis. Testing the efficacy of PROTAC compounds has been limited to empirical tests, leading to lengthy and often failure-prone processes. This manuscript addressed the need for faster and more reliable assays to identify the compatible pairs of E3 ligases-target proteins. The authors propose using the RiPA assay, which depends on rapamycin-induced dimerization of FKBP12 protein with FRB domain. The PROTAC technology is advancing rapidly, making this manuscript both timely and essential. The RiPA assay might be useful in identifying novel E3 ligases that could be utilized in PROTAC technology. Additionally, it could be used at the initial stages of PROTAC development, looking for the best E3 ligase for the specific target.

The authors described an elegant assay that is scalable, easy-to-use and applicable to a wide range of cellular models. This method allows for the quantitative validation of the degradation efficacy of a given pair of E3 ligase-target protein, using luciferase activity as a measure. Importantly, the assay also enables the measurement of kinetics in living cells, enhancing its practicality.

Strengths:

(1) The authors have addressed the crucial needs that arise during PROTAC development. In the introduction, they nicely describe the advantages and disadvantages of the PROTAC technology and explain why such an assay is needed.

(2) The study includes essential controls in experiments (important for generating new assay), such as using the FRB vector without E3 ligase as a negative control, testing different linkers (which may influence the efficacy of the degradation), and creating and testing K-less vectors to exclude the possibility of luciferase or FKBP12 ubiquitination instead of WDR5 (the target protein). Additionally, the position of the luc in the FKBP12 vector and the position of VHL in the FRB vector are tested. Different E3 ligases are tested using previously identified target proteins, confirming the assay's utility and accuracy.

(3) The study identified a "new" E3 ligase that is suitable for PROTAC technology (FBXL).

Weaknesses:

It is not clear how feasible it would be to adapt the assay for high-throughput screens.

Comments on revisions:

The authors have addressed my previous concerns and made changes to the manuscript, resulting in a well-written paper.

Reviewer #2 (Public review):

Summary:

Adhikari and colleagues developed a new technique, rapamycin-induced proximity assay (RiPA), to identify E3-ubiquitin (ub) ligases of a protein target, aiming at identifying additional E3 ligases that could be targeted for PROTAC generation or ligases that may degrade a protein target. The study is timely, as expanding the landscape of E3-ub ligases for developing targeted degraders is a primary direction in the field.

Strengths:

(1) The study's strength lies in its practical application of the FRB:FKBP12 system. This system is used to identify E3-ub ligases that would degrade a target of interest, as evidenced by the reduction in luminescence upon the addition of rapamycin. This approach effectively mimics the potential action of a PROTAC.

Weaknesses:

(1) While the technique shows promise, its application in a discovery setting, particularly for high-throughput or unbiased E3-ub ligase identification, may pose challenges. The authors now discuss these potential difficulties providing a more comprehensive understanding of RiPA's limitations.

(2) While RiPA will help identify E3 ligases, PROTAC design would still be empirical. The authors provide some discussion of this limitation.

Comments on revisions:

I thank the authors for addressing my prior concerns. I would recommend that individual replicate values are plotted in all the mean -/+ s.d or sem graphs.

Reviewer #1 (Public review):

The study investigates light chains (LCs) using three distinct approaches, with a focus on identifying a conformational fingerprint to differentiate amyloidogenic light chains from multiple myeloma light chains. The study's major contribution is the identification of a low-populated "H state," which the authors propose as a unique marker for AL-LCs. While this finding is promising, the review highlights several strengths and weaknesses. Strengths include the valuable contribution of identifying the H state and the use of multiple approaches, which provide a comprehensive understanding of LC structural dynamics. However, the study suffers from weaknesses, particularly in the interpretation of SAXS data, lack of clarity in presentation, and methodological inconsistencies. Critical concerns include high error margins between SAXS profiles and MD fits, unclear validation of oligomeric species in SAXS measurements, and insufficient quantitative cross-validation between experimental (HDX) and computational data (MD). This reviewer calls for major revisions including clearer definitions, improved methodology, and additional validation, to strengthen the conclusions.

Reviewer #2 (Public review):

Summary:

This well-written manuscript addresses an important but recalcitrant problem - the molecular mechanism of protein misfolding in Ig light chain (LC) amyloidosis (AL), a major life-threatening form of systemic human amyloidosis. The authors use expertly recorded and analyzed small-angle X-ray scattering (SAXS) data as a restraint for molecular dynamics simulations (called M&M) and to explore six patient-based LC proteins. The authors report that a highly populated "H-state" determined computationally, wherein the two domains in an LC molecule acquire a straight rather than bent conformation, is what distinguishes AL from non-AL LCs. They then use H-D exchange mass spectrometry to verify this conclusion. If confirmed, this is a novel and interesting finding with potentially important translational implications.

Strengths:

Expertly recorded and analyzed SAXS data combined with clever M&M simulations lead to a novel and interesting conclusion.

Regardless of whether or not the CL-CL domain interface is destabilized in AL LCs explored in this (Figure 6) and other studies, stabilization of this interface is an excellent idea that may help protect at least a subset of AL LCs from misfolding in amyloid. This idea increases the potential impact of this interesting study.

Weaknesses:

The HDX analysis could be strengthened.

Reviewer #3 (Public review):

Summary:

This study identifies confirmational fingerprints of amylodogenic light chains, that set them apart from the non-amylodogenic ones.

Strengths:

The research employs a comprehensive combination of structural and dynamic analysis techniques, providing evidence that conformational dynamics at VL-CL interface and structural expansion are distinguished features of amylodogenic LCs.

Weaknesses:

The sample size is limited, which may affect the generalizability of the findings. Additionally, the study could benefit from deeper analysis of specific mutations driving this unique conformation to further strengthen therapeutic relevance.

Reviewer #1 (Public review):

Summary:

This study investigated the heterogeneous responses to Mycobacterium tuberculosis (Mtb) in 19 wild-derived inbred mouse strains collected from various geographic locations. The goal of this study is to identify novel mechanisms that regulate host susceptibility to Mtb infection. Using the genetically resistant C57BL/6 mouse strain as the control, they successfully identified a few mouse strains that revealed higher bacterial burdens in the lung, implicating increased susceptibility in those mouse strains. Furthermore, using flow cytometry analysis, they discovered strong correlations between CFU and various immune cell types, including T cells and B cells. The higher neutrophil numbers correlated with significantly higher CFU in some of the newly identified susceptible mouse strains. Interestingly, MANB and MANC mice exhibited comparable numbers of neutrophils but showed drastically different bacterial burdens. The authors then focused on the neutrophil heterogeneity and utilized a single-cell RNA-seq approach, which led to identifying distinct neutrophil subsets in various mouse strains, including C57BL/6, MANA, MANB, and MANC. Pathway analysis on neutrophils in susceptible MANC strain revealed a highly activated and glycolytic phenotype, implicating a possible mechanism that may contribute to the susceptible phenotype. Lastly, the authors found that a small group of neutrophil-specific genes are expressed across many other cell types in the MANC strain.

Strengths:

This manuscript has many strengths.

(1) Utilizing and characterizing novel mouse strains that complement the current widely used mouse models in the field of TB. Many of those mouse strains will be novel tools for studying host responses to Mtb infection.

(2) The study revealed very unique biology of neutrophils during Mtb infection. It has been well-established that high numbers of neutrophils correlate with high bacterial burden in mice. However, this work uncovered that some mouse strains could be resistant to infection even with high numbers of neutrophils in the lung, indicating the diverse functions of neutrophils. This information is important.

Weaknesses:

The weaknesses of the manuscript are that the work is relatively descriptive. It is unclear whether the neutrophil subsets are indeed functionally different. While single-cell RNA seq did provide some clues at transcription levels, functional and mechanistic investigations are lacking. Similarly, it is unclear how highly activated and glycolytic neutrophils in MANC strain contribute to its susceptibility.

Reviewer #2 (Public review):

Summary:

These studies investigate the phenotypic variability and roles of neutrophils in tuberculosis (TB) susceptibility by using a diverse collection of wild-derived inbred mouse lines. The authors aimed to identify new phenotypes during Mycobacterium tuberculosis infection by developing, infecting, and phenotyping 19 genetically diverse wild-derived inbred mouse lines originating from different geographic regions in North America and South America. The investigators achieved their main goals, which were to show that increasing genetic diversity increases the phenotypic spectrum observed in response to aerosolized M. tuberculosis, and further to provide insights into immune and/or inflammatory correlates of pulmonary TB. Briefly, investigators infected wild-derived mice with aerosolized M. tuberculosis and assessed early infection control at 21 days post-infection. The time point was specifically selected to correspond to the period after infection when acquired immunity and antigen-specific responses manifest strongly, and also early susceptibility (morbidity and mortality) due to M. tuberculosis infection has been observed in other highly susceptible wild-derived mouse strains, some Collaborative Cross inbred strains, and approximately 30% of individuals in the Diversity Outbred mouse population. Here, the investigators normalized bacterial burden across mice based on inoculum dose and determined the percent of immune cells using flow cytometry, primarily focused on macrophages, neutrophils, CD4 T cells, CD8 T cells, and B cells in the lungs. They also used single-cell RNA sequencing to identify neutrophil subpopulations and immune phenotypes, elegantly supplemented with in vitro macrophage infections and antibody depletion assays to confirm immune cell contributions to susceptibility. The main results from this study confirm that mouse strains show considerable variability to M. tuberculosis susceptibility. Authors observed that enhanced infection control correlated with higher percentages of CD4 and CD8 T cells, and B cells, but not necessarily with the percentage of interferon-gamma (IFN-γ) producing cells. High levels of neutrophils and immature neutrophils (band cells) were associated with increased susceptibility, and the mouse strain with the most neutrophils, the MANC line, exhibited a transcriptional signature indicative of a highly activated state, and containing potentially tissue-destructive, mediators that could contribute to the strain's increased susceptibility and be leveraged to understand how neutrophils drive lung tissue damage, cavitation, and granuloma necrosis in pulmonary TB.

Strengths:

The strengths are addressing a critically important consideration in the tuberculosis field - mouse model(s) of the human disease, and taking advantage of the novel phenotypes observed to determine potential mechanisms. Notable strengths include,

(1) Innovative generation and use of mouse models: Developing wild-derived inbred mice from diverse geographic locations is innovative, and this approach expands the range of phenotypic responses observed during M. tuberculosis infection. Additionally, the authors have deposited strains at The Jackson Laboratory making these valuable resources available to the scientific community.

(2) Potential for translational research: The findings have implications for human pulmonary TB, particularly the discovery of neutrophil-associated susceptibility in primary infection and/or neutrophil-mediated disease progression that could both inform the development of therapeutic targets and also be used to test the effectiveness of such therapies.

(3) Comprehensive experimental design: The investigators use many complementary approaches including in vivo M. tuberculosis infection, in vitro macrophage studies, neutrophil depletion experiments, flow cytometry, and a number of data mining, machine learning, and imaging to produce robust and comprehensive analyses of the wild-derives d strains and neutrophil subpopulations in 3 weeks after M. tuberculosis infection.

Weaknesses:

The manuscript and studies have considerable strengths and very few weaknesses. One minor consideration is that phenotyping is limited to a single limited-time point; however, this time point was carefully selected and has a strong biological rationale provided by investigators. This potential weakness does not diminish the overall findings, exciting results, or conclusions.

Reviewer #1 (Public review):

Summary:

In this manuscript, Guo and colleagues used a cell rounding assay to screen a library of compounds for inhibition of TcdB, an important toxin produced by Clostridioides difficile. Caffeic acid and derivatives were identified as promising leads, and caffeic acid phenethyl ester (CAPE) was further investigated.

Strengths:

Considering the high morbidity rate associated with C. difficile infections (CDI), this manuscript presents valuable research in the investigation of novel therapeutics to combat this pressing issue. Given the rising antibiotic resistance in CDI, the significance of this work is particularly noteworthy. The authors employed a robust set of methods and confirmatory tests, which strengthened the validity of the findings. The explanations provided are clear, and the scientific rationale behind the results is well-articulated. The manuscript is extremely well-written and organized. There is a clear flow in the description of the experiments performed. Also, the authors have investigated the effects of CAPE on TcdB in careful detail and reported compelling evidence that this is a meaningful and potentially useful metabolite for further studies.

Weaknesses:

This is really a manuscript about CAPE, not caffeic acid, and the title should reflect that. Also, a few details are missing from the description of the experiments. The authors should carefully revise the manuscript to ascertain that all details that could affect the interpretation of their results are presented clearly. Just as an example, the authors state in the results section that TcdB was incubated with compounds and then added to cells. Was there a wash step in between? Could compound carryover affect how the cells reacted independently from TcdB? This is just an example of how the authors should be careful with descriptions of their experimental procedures. Lastly, authors should be careful when drawing conclusions from the analysis of microbiota composition data. Ascribing causality to correlational relationships is a recurring issue in the microbiome field. Therefore, I suggest authors carefully revise the manuscript and tone down some statements about the impact of CAPE treatment on the gut microbiota.

Reviewer #2 (Public review):

Summary:

This work is towards the development of nonantibiotic treatment for C. difficile. The authors screened a chemical library for activity against the C. difficile toxin TcdB, and found a group of compounds with antitoxin activity. Caffeic acid derivatives were highly represented within this group of antitoxin compounds, and the remaining portion of this work involves defining the mechanism of action of caffeic acid phenethyl ester (CAPE) and testing CAPE in mouse C. difficile infection model. The authors conclude CAPE attenuates C. difficile disease by limiting toxin activity and increasing microbial diversity during C. difficile infection.

Strengths/ Weaknesses:

The strategy employed by the authors is sound although not necessarily novel. A compound that can target multiple steps in the pathogenies of C. difficile would be an exciting finding. However, the data presented does not convincingly demonstrate that CAPE attenuates C. difficile disease and the mechanism of action of CAPE is not convincingly defined. The following points highlight the rationale for my evaluation.

(1) The toxin exposure in tissue culture seems brief (Figure 1). Do longer incubation times between the toxin and cells still show CAPE prevents toxin activity?

(2) The conclusion that CAPE has antitoxin activity during infection would be strengthened if the mouse was pretreated with CAPE before toxin injections (Figure 1D).

(3) CAPE does not bind to TcdB with high affinity as shown by SPR (Figure 4). A higher affinity may be necessary to inhibit TcdB during infection. The GTD binds with millimolar affinity and does not show saturable binding. Is the GTD the binding site for CAPE? Autoprocessing is also affected by CAPE indicating CAPE is binding non-GTD sites on TcdB.

(4) In the infection model, CAPE does not statistically significantly attenuate weight loss during C. difficile infection (Figure 6). I recognize that weight loss is an indirect measure of C. difficile disease but histopathology also does not show substantial disease alleviation (see below).

(5) In the infection model (Figure 6), the histopathology analysis shows substantial improvement in edema but limited improvement in cellular infiltration and epithelial damage. Histopathology is probably the most critical parameter in this model and a compound with disease-modifying effects should provide substantial improvements.

(6) The reduction in C. difficile colonization is interesting. It is unclear if this is due to antitoxin activity and/or due to CAPE modifying the gut microbiota and metabolites (Figure 6). To interpret these data, a control is needed that has CAPE treatment without C. difficile infection or infection with an atoxicogenic strain.

(7) Similar to the CAPE data, the melatonin data does not display potent antitoxin activity and the mouse model experiment shows marginal improvement in the histopathological analysis (Figure 9). Using 100 µg/ml of melatonin (~ 400 micromolar) to inactivate TcdB in cell culture seems high. Can that level be achieved in the gut?

(8) The following parameters should be considered and would aid in the interpretation of this work. Does CAPE directly affect the growth of C. difficile? Does CAPE affect the secretion of TcdB from C. difficile? Does CAPE alter the sporulation and germination of C. diffcile?

Reviewer #3 (Public review):

Summary:

The study is well written, and the results are solid and well demonstrated. It shows a field that can be explored for the treatment of CDI

Strengths:

The results are really good, and the CAPE shows a good and promising alternative for treating CDI. The methodology and results are well presented, with tables and figures that corroborate them. It is solid work and very promising.

Weaknesses:

Some references are too old or missing.

Reviewer #1 (Public review):

Summary:

The present study aims to associate reproduction with age-related disease as support of the antagonistic pleiotorpy hypothesis of ageing, predominantly using Mendelian Randomization. The authors found evidence that early-life reproductive success is associated with advanced ageing.

Strengths:

Large sample size. Many analyses.

Weaknesses:

There are some errors in the methodology, that require revisions.

In particular, the main conclusions drawn by the authors refer to the Mendelian Randomization analyses. However, the authors made a few errors here that need to be reconsidered:

(1) Many of the outcomes investigated by the authors are continuous outcomes, while the authors report odds ratios. This is not correct and should be revised.

(2) Some of the odds ratios (for example the one for osteoporosis) are really small, while still reaching the level of statistical significance. After some checking, I found the GWAS data used to generate these MR estimates were processed by the program BOLT-LLM. This program is a linear mixed model program, which requires the transformation of the beta estimates to be useful for dichotomous outcomes. The authors should check the manual of BOLT-LLM and recalculate the beta estimates of the SNP-outcome associations prior to the Mendelian Randomization analyses. This should be checked for all outcomes as it doesn't apply to all.

(3) The authors should follow the MR-Strobe guidelines for presentation.

(4) The authors should report data in the text with a 95% confidence interval.

(5) The authors should consider correction for multiple testing.

Reviewer #2 (Public review):

Summary:

The authors present an interesting paper where they test the antagonistic pleiotropy theory. Based on this theory they hypothesize that genetic variants associated with later onset of age at menarche and age at first birth have a positive causal effect on a multitude of health outcomes later in life, such as epigenetic aging and prevalence of chronic diseases. Using a mendelian randomization and colocalization approach, the authors show that SNPs associated with later age at menarche are associated with delayed aging measurements, such as slower epigenetic aging and reduced facial aging, and a lower risk of chronic diseases, such as type 2 diabetes and hypertension. Moreover, they identified 128 fertility-related SNPs that are associated with age-related outcomes and they identified BMI as a mediating factor for disease risk, discussing this finding in the context of evolutionary theory.

Strengths:

The major strength of this manuscript is that it addresses the antagonistic pleiotropy theory in aging. Aging theories are not frequently empirically tested although this is highly necessary. The work is therefore relevant for the aging field as well as beyond this field, as the antagonistic pleiotropy theory addresses the link between fitness (early life health and reproduction) and aging.

Points that have to be clarified/addressed:

(1) The antagonistic pleiotropy is an evolutionary theory pointing to the possibility that mutations that are beneficial for fitness (early life health and reproduction) may be detrimental later in life. As it concerns an evolutionary process and the authors focus on contemporary data from a single generation, more context is necessary on how this theory is accurately testable. For example, why and how much natural variation is there for fitness outcomes in humans? How do genetic risk score distributions of the exposure data look like? Also, how can the authors distinguish in their data between the antagonistic pleiotropy theory and the disposable soma theory, which considers a trade-off between investment in reproduction and somatic maintenance and can be used to derive similar hypotheses? There is just a very brief mention of the disposable soma theory in lines 196-198.

(2) The antagonistic pleiotropy theory, used to derive the hypothesis, does not necessarily distinguish between male and female fitness. Would the authors expect that their results extrapolate to males as well? And can they test that?

(3) There is no statistical analyses section providing the exact equations that are tested. Hence it's not clear how many tests were performed and if correction for multiple testing is necessary. It is also not clear what type of analyses have been done and why they have been done. For example in the section starting at line 47, Odds Ratios are presented, indicating that logistic regression analyses have been performed. As it's not clear how the outcomes are defined (genotype or phenotype, cross-sectional or longitudinal, etc.) it's also not clear why logistic regression analysis was used for the analyses.

(4) Mendelian Randomization is an important part of the analyses done in the manuscript. It is not clear to what extent the MR assumptions are met, how the assumptions were tested, and if/what sensitivity analyses are performed; e.g. reverse MR, biological knowledge of the studied traits, etc. Can the authors explain to what extent the genetic instruments represent their targets (applicable expression/protein levels) well?

(5) It is not clear what reference genome is used and if or what imputation panel is used. It is also not clear what QC steps are applied to the genotype data in order to construct the genetic instruments of MR.

(6) A code availability statement is missing. It is understandable that data cannot always be shared, but code should be openly accessible.

Reviewer #1 (Public review):

Summary:

The characteristics of endometrium health are an increasing topic in women's health issues, especially in the context of endometriosis. In this respect, having access to information is hampered by the inaccessibility of the uterine tissue. The authors propose here using the menstrual fluid (easily accessible by non-invasive methods) as an access door towards getting relevant information.

Overall, the paper is divided into two parts:<br /> (1) The comparison between menstrual fluid samples and biopsies of the endometrium.<br /> 2) As a proof of concept, the authors then compared 11 controls and 7 endometriosis cases in this way, from different severity stages.

Strengths:

In Figure 1, general features of the 15 samples are presented (volume/number of cells/hematopoietic cells - cd45 labeling). The authors then used single-cell RNA-seq to characterize the different samples. Through having access to endometrium biopsies, they were able to compare the profiles obtained.

In the MF samples from the second part of the paper - aiming at comparing endometriosis and controls - one question is raised about the effect of culture. The authors compared freshly isolated and cultured tissues (ex vivo vs in vitro) by bulk RNA seq. Biases induced by the culture procedure were identified. Deconvolution was applied to strengthen this observation, with an important increase of seemingly stromal and unknown cells, especially in the unsorted cells and the CD45+ cells.

Interestingly, since the authors got successive samples from the same donor, they could evaluate the consistency of the samples and reveal indeed an overall stability of the molecular profile of the samples in a given patient.

The authors then attempted - quite originally - to characterize biomarkers in two major cell compartments that they studied - CD45- (stromal-like) and CD45+ (immune cells).

Weaknesses:

A potential problem is the justification of the a priori mix of cell types of three different phenotypes (CD45+, CD45- EPCAM+, and CD45- EPCAM-) from each patient before moving to the scRNAseq. It is not clear to me why this has been done, I guess that using directly the samples would supposedly bias the result. But in this case, why is it supposed that three categories are enough (immune cells, epithelial cells, and stromal cells)? I suppose that other markers could characterize other subtypes of the cells, and take into account the possibility of other cell types, for instance, connected to pain sensitivity, such as neuron precursor. Hence, the justification of the organized mixes should be much more detailed in my opinion.

It is a bit unclear to me when the biopsies were collected in the cycle of the donor patients.

The description of these markers that are deregulated is presented as a list, and connected with existing publications, which could rather be presented in discussion than in the results. The authors do tend to demonstrate that the Menstrual Fluid is a good proxy to analyse the endometrium health status of the women affected with endometriosis.

The identification of MTRNR2L1 seems to be a major discovery of the paper, as well as in a lesser measure HBG2, and it is a bit strange why these putative markers were not emphasized in the abstract. HBG2 was certainly identified previously in endometriosis endothelial cells but seems extremely variable from one sample to another - Geo profile (GDS3060, GDS3060 / 213515_x_at (inist.fr)).

Overall, the transcriptome analysis is a bit shallow, with no effort made to try to find potential transcription factors or miRNA that could activate/inhibit a series of modified genes; it could be relevant to identify such master genes or master regulators through bioinformatics analyses and wet-lab validations, to understand better the cascade of events.

Another issue that was overlooked is the presence of 'stem-cells' in the MF obtained. Since endometriosis is supposed to occur from the implantation of uterine stem cells, this category could be a major topic of scrutiny, in terms of quantity in the MF, as well as in terms of their specific molecular properties.

Reviewer #2 (Public review):

Summary:

The authors provided further evidence that menstrual fluid (MF) can be used as a non-invasive source of endometrial tissue for studying its normal physiological state and when it is abnormal such as in endometriosis. Single-cell RNA sequencing confirmed the presence of the major cell types -blood and tissue immune cells and endometrial stromal, epithelial, and vascular cells. The major new finding was that interindividual variation for the blood immune cells was minimal between multiple MF samples from an individual. A comparison between the ex vivo MF gene profile and cultured MF showed the expected attachment and culture of stromal (and a small number of epithelial) cells, but the immune cells failed to attach. Several differentially expressed genes between controls and endometriosis were suggested as potential biomarkers of the disease, however, these were a mitochondrial pseudogene and a hemoglobin subunit, both very unlikely related to endometriosis pathogenesis.

Strengths:

The Spearman correlation analysis between the control MF gene profiles of multiple samples from the same individual and its graphic presentation provided strong evidence that there is little variation between MF samples. Together with another study which showed similar findings for endometrial stem cells and a number of proteins in MF supernatant, this important data shows MF as a promising biofluid for pathology testing.

The bioinformatic analyses conducted by bioinformatic and computational experts are a major strength of the manuscript and in particular the comparison between MF and endometrial biopsy data obtained from published scRNAseq studies. This is an important finding, particularly if comparisons included late secretory and early proliferative stage biopsy tissue which would be most similar to shedding menstrual endometrium.

The inclusion of workflows in the Figures for the various studies and the use of symbols in the various panels is very helpful for the reader.

MF cell suspensions were enriched for stromal and epithelial cells to enable a detailed bioinformatic analysis of their respective gene profiles

Weaknesses:

Two patient cohorts from different institutions were used in the study and somewhat different methods were used to extract the cellular fraction from these cohorts for further study: (1) sample dilution and differential filtration to separate blood-derived immune cells from endometrial tissue then dissociated into single cells and separated into CD45+, CD45-EpCAM+ and CD45-EpCAM- cells, and (2) gradient density separation to generate unsorted, CD45+, CD45- and putative mesenchymal stem cells (MSC) CD45-CD105+ which were also cultured. In addition, questions on pelvic pain and proven fertility would have addressed the 2 key symptoms of endometriosis.

The use of CD105 to purify MSC from MF rather than well-characterised markers of clonogenic, self-renewing, and mesodermal differentiating endometrial MSC such as CD146+PDGFRB+ or SUSD2 (both mentioned in references 22 and 23) is a weakness. The ISCT markers are not specific and are also found on stromal fibroblasts of many tissues (Phinney and Sensebe Cytotherapy 2013; Demu et al Acta Haematologica 2016).<br /> The UMAPs generated from the scRNAseq were at low resolution and more individual immune and endometrial cell types have previously been identified and reported in MF. More comparisons with these studies would also have enhanced the Discussion.

It was not always possible to work out how the data was reported in the gene expression tables (Supplementary Tables 2, 4-10) as they were not in adjusted P value order and sometimes positive log2 fold change values appeared amongst the negative log2FC. In some comparisons described, the adj P values were not significant but were described as up or down-regulated in the text.

The 2 DEGs highlighted in the endometriosis and control arm of the study appear as poor choices from many others that could have been chosen as MTRNR2L1 is a mitochondrial pseudogene and HBG2 is a hemoglobin subunit. Neither are likely indicators of endometriosis pathogenesis.

The manuscript format and organisation could be improved by reducing the discussion in the Results section and providing a more in-depth Discussion. More references need to be included in the Discussion and other work in the MF analysis field that supports - or not - the authors' findings or at least puts them into context, and should be included and referenced.

The potential to use MF as a non-invasive source of endometrial tissue for potential diagnosis is a very important avenue of research that is currently in its infancy and could have a major impact in the endometriosis research arena.

Reviewer #1 (Public review):

The authors attempted to replicate previous work showing that counterconditioning leads to more persistent reduction of threat responses, relative to extinction. They also aimed to examine the neural mechanisms underlying counterconditioning and extinction. They achieved both of these aims and were able to provide some additional information, such as how counterconditioning impacts memory consolidation. Having a better understanding of which neural networks are engaged during counterconditioning may provide novel pharmacological targets to aid in therapies for traumatic memories. It will be interesting to follow up by examining the impact of varying amounts of time between acquisition and counterconditioning phases, to enhance replicability to real-world therapeutic settings.

Major strengths

• This paper is very well written and attempts to comprehensively assess multiple aspects of counterconditioning and extinction processes. For instance, the addition of memory retrieval tests is not core to the primary hypotheses but provides additional mechanistic information on how episodic memory is impacted by counterconditioning. This methodical approach is commonly seen in animal literature, but less so in human studies.

• The Group x Cs-type x Phase repeated measure statistical tests with 'differentials' as outcome variables are quite complex, however, the authors have generally done a good job of teasing out significant F test findings with post hoc tests and presenting the data well visually. It is reassuring that there is a convergence between self-report data on arousal and valence and the pupil dilation response. Skin conductance is a notoriously challenging modality, so it is not too concerning that this was placed in the supplementary materials. Neural responses also occurred in logical regions with regard to reward learning.

• Strong methodology with regards to neuroimaging analysis, and physiological measures.

• The authors are very clear on documenting where there were discrepancies from their pre-registration and providing valid rationales for why.

Major Weaknesses

• The statistics showing that counterconditioning prevents differential spontaneous recovery are the weakest p values of the paper (and using one-tailed tests, although this is valid due to directions being pre-hypothesised). This may be due to a relatively small number of participants and some variability in responses. It is difficult to see how many people were included in the final PDR and neuroimaging analyses, with exclusions not clearly documented. Based on Figure 3, there are relatively small numbers in the PDR analyses (n=14 and n=12 in counterconditioning and extinction, respectively). Of these, each group had 4 people with differential PDR results in the opposing direction to the group mean. This perhaps warrants mention as the reported effects may not hold in a subgroup of individuals, which could have clinical implications.

Reviewer #2 (Public review):

Summary:

The present study sets out to examine the impact of counterconditioning (CC) and extinction on conditioned threat responses in humans, particularly looking at neural mechanisms involved in threat memory suppression. By combining behavioral, physiological, and neuroimaging (fMRI) data, the authors aim to provide a clear picture of how CC might engage unique neural circuits and coding dynamics, potentially offering a more robust reduction in threat responses compared to traditional extinction.

Strengths:

One major strength of this work lies in its thoughtful and unique design - integrating subjective, physiological, and neuroimaging measures to capture the variouse aspects of counterconditioning (CC) in humans. Additionally, the study is centered on a well-motivated hypothesis and the findings have the potential to improve the current understanding of pathways associated with emotional and cognitive control.

The data presentation is systematic, and the results on behavioral and physiological measures fit well with the hypothesized outcomes. The neuroimaging results also provide strong support for distinct neural mechanisms underlying CC versus extinction.

Weaknesses:

Overall, this study is a well-conducted and thought-provoking investigation into counterconditioning, with strong potential to advance our understanding of threat modulation mechanisms. Two main weaknesses concern the scope and decisions regarding analysis choices. First, while the findings are solid, the topic of counterconditioning is relatively niche and may have limited appeal to a broader audience. Expanding the discussion to connect counterconditioning more explicitly to widely studied frameworks in emotional regulation or cognitive control would enhance the paper's accessibility and relevance to a wider range of readers. This broader framing could also underscore the generalizability and broader significance of the results. In addition, detailed steps in the statistical procedures and analysis parameters seem to be missing. This makes it challenging for readers to interpret the results in light of potential limitations given the data modality and/or analysis choices.

Reviewer #3 (Public review):

Summary:

In this manuscript, Wirz et al use neuroimaging (fMRI) to show that counterconditioning produces a longer lasting reduction in fear conditioning relative to extinction and appears to rely on the nucleus accumbens rather than the ventromedial prefrontal cortex. These important findings are supported by convincing evidence and will be of interest to researchers across multiple subfields, including neuroscientists, cognitive theory researchers, and clinicians.

In large part, the authors achieved their aims of giving a qualitative assessment of the behavioural mechanisms of counterconditioning versus extinction, as well as investigating the brain mechanisms. The results support their conclusions and give interesting insights into the psychological and neurobiological mechanisms of the processes that underlie the unlearning, or counteracting, of threat conditioning.

Strengths:

* Mostly clearly written with interesting psychological insights<br /> * Excellent behavioural design, well-controlled and tests for a number of different psychological phenomena (e.g. extinction, recovery, reinstatement, etc).<br /> * Very interesting results regarding the neural mechanisms of each process.<br /> * Good acknowledgement of the limitations of the study.

Weaknesses:

* I think the acquisition data belongs in the main figure, so the reader can discern whether or not there are directional differences prior to CC and extinction training that could account for the differences observed. This is particularly important for the valence data which appears to differ at baseline (supplemental figure 2C).<br /> * I was confused in several sections about the chronology of what was done and when. For instance, it appears that individuals went through re-extinction, but this is just called extinction in places.<br /> * I was also confused about the data in Figure 3. It appears that the CC group maintained differential pupil dilation during CC, whereas extinction participants didn't, and the authors suggest that this is indicative of the anticipation of reward. Do reward-associated cues typically cause pupil dilation? Is this a general arousal response? If so, does this mean that the CSs become equally arousing over time for the CC group whereas the opposite occurs for the extinction group (i.e. Figure 3, bottom graphs)? It is then further confusing as to why the CC group lose differential responding on the spontaneous recovery test. I'm not sure this was adequately addressed.<br /> * I am not sure that the memories tested were truly episodic<br /> * Twice as many female participants than males<br /> * No explanation as to why shocks were varied in intensity and how (psuedo-randomly?)

Reviewer #1 (Public review):

Summary:

In this work, the authors examine the activity and function of D1 and D2 MSNs in dorsomedial striatum (DMS) during an interval timing task. In this task, animals must first nosepoke into a cued port on the left or right; if not rewarded after 6 seconds, they must switch to the other port. Thus, this task requires animals to estimate if at least 6 seconds have passed after the first nosepoke. After verifying that animals estimate the passage of 6 seconds, the authors examine striatal activity during this interval. They report that D1-MSNs tend to decrease activity, while D2-MSNs increase activity, throughout this interval. They suggest that this activity follows a drift-diffusion model, in which activity increases (or decreases) to a threshold after which a decision is made. The authors next report that optogenetically inhibiting D1 or D2 MSNs, or pharmacologically blocking D1 and D2 receptors, increased the average wait time. This suggests that both D1 and D2 neurons contribute to the estimate of time, with a decrease in their activity corresponding to a decrease in the rate of 'drift' in their drift-diffusion model. Lastly, the authors examine MSN activity while pharmacologically inhibiting D1 or D2 receptors. The authors observe most recorded MSNs neurons decrease their activity over the interval, with the rate decreasing with D1/D2 receptor inhibition.

Major strengths:

The study employs a wide range of techniques - including animal behavioral training, electrophysiology, optogenetic manipulation, pharmacological manipulations, and computational modeling. The question posed by the authors - how striatal activity contributes to interval timing - is of importance to the field and has been the focus of many studies and labs. This paper contributes to that line of work by investigating whether D1 and D2 neurons have similar activity patterns during the timed interval, as might be expected based on prior work based on striatal manipulations. However, the authors find that D1 and D2 neurons have distinct activity patterns. They then provide a decision-making model that is consistent with all results. The data within the paper is presented very clearly, and the authors have done a nice job presenting the data in a transparent manner (e.g., showing individual cells and animals). Overall, the manuscript is relatively easy to read and clear, with sufficient detail given in most places regarding the experimental paradigm or analyses used.

Major weaknesses:

The results are based on a relatively small dataset (tens of cells).

Impact:

The task and data presented by the authors are very intriguing, and there are many groups interested in how striatal activity contributes to the neural perception of time. The authors perform a wide variety of experiments and analysis to examine how DMS activity influences time perception during an interval-timing task, allowing for insight into this process.

Reviewer #2 (Public review):

This study found that D1-MSNs and D2-MSNs have opposing dynamics during interval timing in a mouse-optimized interval timing task. Further optogenetic and pharmacologic inhibition of either D1 or D2 MSNs increased response time. This study provides useful experimental evidence in the coding of time in striatum. However, there are some major weaknesses in this study.

(1) Regarding the data in Figure S3, The variance within each mouse was too big, the authors need to figure out and explain what caused the large variance within the same mouse, or the authors need to increase the sample size.<br /> (2) Regarding the results in Figure 3 C and D, Figure 6 H and Figure 7 D, what is the sample size? From the single data points in the figures, it seems that the authors were using the number of cells to do statistical tests and plot the figures. For example, Figure 3 C, if the authors use n= 32 D2 MSNs and n= 41D1 MSNs to do the statistical test, it could make small difference to be statistically significant. The authors should use the number of mice to do the statistical tests.<br /> (3) Regarding the results in Figure 5, what is the reason for the increase in the response times? The authors should plot the position track during intervals (0-6 s) with or without optogenetic or pharmacologic inhibition. The authors can check Figure 3, 5, and 6 in paper https://doi.org/10.1016/j.cell.2016.06.032 for reference to analyze the data.

Reviewer #3 (Public review):

Summary:

The cognitive striatum, also known as the dorsomedial striatum, receives input from brain regions involved in high-level cognition and plays a crucial role in processing cognitive information. However, despite its importance, the extent to which different projection pathways of the striatum contribute to this information processing remains unclear. In this paper, Bruce et al. conducted a study using various causal and correlational techniques to investigate how these pathways collectively contribute to interval timing in mice. Their results were consistent with previous research, showing that the direct and indirect striatal pathways perform opposing roles in processing elapsed time. Based on their findings, the authors proposed a revised computational model in which two separate accumulators track evidence for elapsed time in opposing directions. These results have significant implications for understanding the neural mechanisms underlying cognitive impairment in neurological and psychiatric disorders, as disruptions in the balance between direct and indirect pathway activity are commonly observed in such conditions.

Strengths:

The authors employed a well-established approach to study interval timing and employed optogenetic tagging to observe the behavior of specific cell types in the striatum. Additionally, the authors utilized two complementary techniques to assess the impact of manipulating the activity of these pathways on behavior. Finally, the authors utilized their experimental findings to enhance the theoretical comprehension of interval timing using a computational model.

Weaknesses:

The behavioral task used in this study is best suited for investigating elapsed time perception rather than interval timing. Timing bisection tasks are often employed to study interval timing in humans and animals. Given the systemic delivery of pharmacological interventions, it is difficult to conclude that the effects are specific to the dorsomedial striatum. Future studies should use the local infusion of drugs into the dorsomedial striatum.

Reviewer #1 (Public review):

Mitochondria are essential organelles consisting in mammalian cells of about 1500 different proteins. Most of those are synthesized in the cytosol as precursor proteins, imported into mitochondria, and sorted into one of the four sub-mitochondrial compartments. The TIM23 complex, which is embedded in the mitochondrial inner membrane, facilitates the import of proteins that harbor Mitochondrial Targeting Sequence (MTS) at their N-terminus. Such proteins are sorted mainly to the mitochondrial matrix while some sub-groups are destined also to the inner membrane or the intermembrane space. TIMM50 (Tim50 in yeast) is an essential component of the TIM23 complex and mutations in this protein were reported to cause several diseases.

Summary:

In the current study, the authors analyzed the impact of TIMM50 mutations on the mitochondrial proteome in both patients' cells and mouse neurons. They provide compelling evidence for several surprising and highly interesting observations: (i) TIMM50 mutations affect the steady-state levels of only a portion of the putative TIMM50 substrates, (ii) such mutations result in increased electrical activity in mice neurons and in reduced levels of some potassium ion channels in the plasma membrane. These findings shed new light on mitochondrial biogenesis in mammalian cells and hint at an unexpected link between mitochondria and ion channels at the plasma membrane.

Strengths:

The authors used both cells from patients and neurons from mice to investigate the impact of mutations in TIMM50 on mitochondrial proteome and function.

Comments on revisions:

The authors addressed all my concerns regarding the original submission.

Reviewer #2 (Public review):

Summary:

Mitochondria import hundreds of precursor proteins from the cytosol. The TOM and TIM23 complexes facilitate the import on the matrix-targeting pathway of mitochondria. In yeast, Tim50 is a critical and essential subunit of the TIM23 complex that mediates the transition of precursors from the outer to the inner membrane. The human Tim50 homolog TIMM50 is highly similar in structure and a comparable function of Tim50 and TIMM50 was proven by several biochemical and genetic studies in the past.

In this study, the authors characterize human cells which express lower levels or mutated versions of TIMM50. They found that in these TIMM50-depletion cells, the levels of other TIM23 core subunits are also diminished but many mitochondrial proteins are unaffected. Moreover, they observed alterations in the electrical activity and the levels of potassium channels in neuronal cells of TIMM50-deficient mice. They propose that these changes explain the pathology of patients who often suffer from epilepsy.

Strengths:

The paper is written by experts in the field, and it is very clear. The experiments are of high quality and sufficiently well-controlled. The study is interesting for a broad readership.

Weaknesses:

The authors show that even upon low levels of Tim50, mitochondrial proteins are not considerably depleted. However, it remains somewhat unclear why this is. TIMM50 and the TIM23 complex might not be rate-limiting for the biogenesis of mitochondrial proteins. Alternatively, the import defect is compensated indirectly, for example by a reduced growth of cells. It will be interesting to study the physiological consequences of TIMM50-depletion in more depth in the future.

Reviewer #2 (Public review):

Summary

The authors address three primary questions:<br /> (1) how FGF13 variants confer seizure susceptibility,<br /> (2) the specific cell types involved, and<br /> (3) the underlying mechanisms, particularly regarding Nav dysfunction.

They use different Cre drivers to generate cell type-specific knockouts (KOs). First, using Nestin-Cre to create a whole-brain Fgf13 KO, they observed spontaneous seizures and premature death. While KO of Fgf13 in excitatory neurons does not lead to spontaneous seizures, KO in inhibitory neurons recapitulates the seizures and premature death observed in the Nestin-Cre KO. They further narrow down the critical cell type to MGE-derived interneurons (INs), demonstrating that MGE-neuron-specific KO partially reproduces the observed phenotypes. "All interneuron" KOs exhibit deficits in synaptic transmission and interneuron excitability, not seen in excitatory neuron-specific KOs. Finally, they rescue the defects in the interneuron-specific KO by expressing specific Fgf13 isoforms. This is an elegant and important study adding to our knowledge of mechanisms that contribute to seizures.

Strengths<br /> • The study provides much-needed cell type-specific KO models.<br /> • The authors use appropriate Cre lines and characterize the phenotypes of the different KOs.<br /> • The metabolomic analysis complements the rest of the data effectively.<br /> • The study confirms and extends previous research using improved approaches (KO lines vs. in vitro KD or antibody infusion).<br /> • The methods and analyses are robust and well-executed.

Weaknesses

• One weakness lies in the use of the Nkx2.1 line (instead of Nkx2.1CreER) in the paper. As a result, some answers to key questions are incomplete. For instance, it remains unclear whether the observed effects are due to Chandelier cells or NGFCs, potentially both MGE and CGE derived, explaining why Nkx2.1 alone does not fully replicate the overall inhibitory KO. Using Nkx2.1CreER could have helped address the cell specificity. With the Nkx2.1 line used in the paper, the answer is partial.<br /> • While the mechanism behind the reduced inhibitory drive in the IN-specific KO is suggested to be presynaptic, the chosen method does not allow them to exactly identify the mechanisms (spontaneous vs mEPSC/mIPSC), and whether it is a loss of inhibitory synapses (potentially axo-axonic) or release probability.

General Assessment

The general conclusions of this paper are supported by data. As it is, the claim that "these results enhance our understanding of the molecular mechanisms that drive the pathogenesis of Fgf13-related seizures" is partially supported. A more cautious term may be more appropriate, as the study shows the mechanism is not Nav-mediated and suggests alternative mechanisms without unambiguously identifying them. The conclusion that the findings "expand our understanding of FGF13 functions in different neuron subsets" is supported, although somewhat overstated, as the work is not conclusive about the exact neuron subtypes. However, it does indeed show differential functions for specific neuronal classes, which is a significant result.

Impact and Utility

This paper is undoubtedly valuable. Understanding that excitatory neurons are not the primary contributors to the observed phenotypes is crucial. The finding that the effects are not MGE-unique is also important. This work provides a solid foundation for further research and will be a useful resource for future studies.

Reviewer #3 (Public review):

Summary:

The authors aimed to determine the mechanism by which seizures emerge in Developmental and Epileptic Encephalopathies caused by variants in the gene FGF13. Loss of FGF13 in excitatory neurons had no effect on seizure phenotype as compared to loss of FGF13 in GABAergic interneurons, which in contrast caused a dramatic proseizure phenotype and early death in these animals. They were able to show that Fgf13 ablation and consequent loss of FGF13-S and FGF13-VY reduced overall inhibitory input from Fgf13-expressing interneurons onto hippocampal pyramidal neurons. This was shown to occur not via disruption to voltage gated sodium channels but rather by reducing potassium currents and action potential repolarisation in these interneurons.

Strengths:

The authors employed multiple well validated, novel mouse lines with FGF13 knocked out in specific cell types including all neurons, all excitatory cells, all GABAergic interneurons, or a subset of MGE-derived interneurons, including axo-axonic chandelier cells. The phenotypes of each of these four mouse lines were carefully characterised to reveal clear differences with the most fundamental being that Interneuron-targeted deletion of FGF13 led to perinatal mortality associated with extensive seizures and impaired the hippocampal inhibitory/excitatory balance while deletion of FGF13 in excitatory neurons caused no detectable seizures and no survival deficits.<br /> The authors made excellent use of western blotting and in situ hybridisation of the different FGF13 isoforms to determine which isoforms are expressed in which cell types, with FGF3-S predominantly in excitatory neurons and FGF13-VY and FGF13-V predominantly in GABAergic neurons.

The authors performed highly detailed electrophysiological analysis of excitatory neurons and GABAergic interneurons with FGF13 deficits using whole-cell patch clamp. This enabled them to show that FGF13 removal did not affect voltage-gated sodium channels in interneurons, but rather reduced the action of potassium channels, with the resultant effect of making it more likely that interneurons enter depolarisation block. These findings were strengthened by the demonstration that viral re-expression of different Fgf13 splice isoforms could partially rescue deficits in interneuron action potential output and restore K+ channel current size.

Additionally, the discussion was nuanced, and demonstrated how the current findings resolved previous apparent contradictions in the field involving the function of FGF13.

These findings will have a significant impact on our understanding of how FGF13 causes seizures and death in DEEs, and the action of different FGF13 isoforms within different neuronal cell types, particularly GABAergic interneurons.

Comments on revisions:

I appreciate the author's responses to the previous round of reviews. All my comments have been addressed. Congratulations on an excellent body of work.

Reviewer #1 (Public review):

Summary:

This paper investigates the neural population activity patterns of the medial frontal cortex in rats performing a nose poking timing task using in vivo calcium imaging. The results showed neurons that were active at the beginning and end of the nose poking and neurons that formed sequential patterns of activation that covaried with the timed interval during nose poking on a trial-by-trial basis. The former were not stable across sessions, while the latter tended to remain stable over weeks. The analysis of incorrect trials suggests the shorter non-rewarded intervals were due to errors in the scaling of the sequential pattern of activity.

Strengths:

This study measured stable signals using in vivo calcium imaging during experimental sessions that were separated by many days in animals performing a nose poking timing task. The correlation analysis on the activation profile to separate the cells in the three groups was effective and the functional dissociation between beginning and end, and duration cells was revealing. The analysis on the stability of decoding of both the nose poking state and poking time was very informative. Hence, this study dissected a neural population that formed sequential patterns of activation that encoded timed intervals.

Weaknesses:

It is not clear whether animals had enough simultaneously recorded cells to perform the analyzes of Figures 2-4. In fact, rat 3 had 18 responsive neurons which probably is not enough to get robust neural sequences for the trial-by-trial analysis and the correct and incorrect trial analysis. In addition, the analysis of behavioral errors could be improved. The analysis in Figure 4A could be replaced by a detailed analysis on the speed, and the geometry of neural population trajectories for correct and incorrect trials. In the case of Figure 4G is not clear why the density of errors formed two clusters instead of having a linear relation with the produce duration. I would be recommendable to compute the scaling factor on neuronal population trajectories and single cell activity or the computation of the center of mass to test the type III errors.

Due to the slow time resolution of calcium imaging, it is difficult to perform robust analysis on ramping activity. Therefore, I recommend downplaying the conclusion that: "Together, our data suggest that sequential activity might be a more relevant coding regime than the ramping activity in representing time under physiological conditions."

Comments on revisions:

The authors responded properly to my initial comments. However, I have three additional recommendations for the reviewed manuscript.

First, the paper urgently needs proofreading by a professional English editor. Second, Figure 4 must be divided in 2, it has too many panels and the resolution of the figure is low. Finally, please consider that what is called scaling factor in Figure 4G should be called something like neural sequence position index. A scaling factor in the timing literature implies that the pattern of activation of a cell contracts or expands according to the timed interval.

Reviewer #2 (Public review):

In this manuscript, Li and collaborators set out to investigate the neuronal mechanisms underlying "subjective time estimation" in rats. For this purpose, they conducted calcium imaging in the prefrontal cortex of water-restricted rats that were required to perform an action (nose-poking) for a short duration to obtain drops of water. The authors provided evidence that animals progressively improved in performing their task. They subsequently analyzed the calcium imaging activity of neurons and identify start, duration, and stop cells associated with the nose poke. Specifically, they focused on duration cells and demonstrated that these cells served as a good proxy for timing on a trial-by-trial basis, scaling their pattern of actvity in accordance with changes in behavioral performance. In summary, as stated in the title, the authors claim to provide mechanistic insights into subjective time estimation in rats, a function they deem important for various cognitive conditions.

This study aligns with a wide range of studies in system neuroscience that presume that rodents solve timing tasks through an explicit internal estimation of duration, underpinned by neuronal representations of time. Within this framework, the authors performed complex and challenging experiments, along with advanced data analysis, which undoubtedly merits acknowledgement. However, the question of time perception is a challenging one, and caution should be exercised when applying abstract ideas derived from human cognition to animals. Studying so-called time perception in rats has significant shortcomings because, whether acknowledged or not, rats do not passively estimate time in their heads. They are constantly in motion. Moreover, rats do not perform the task for the sake of estimating time but to obtain their rewards are they water restricted. Their behavior will therefore reflect their motivation and urgency to obtain rewards. Unfortunately, it appears that the authors are not aware of these shortcomings. These alternative processes (motivation, sensorimotor dynamics) that occur during task performance are likely to influence neuronal activity. Consequently, my review will be rather critical. It is not however intended to be dismissive. I acknowledge that the authors may have been influenced by numerous published studies that already draw similar conclusions. Unfortunately, all the data presented in this study can be explained without invoking the concept of time estimation. Therefore, I hope the authors will find my comments constructive and understand that as scientists, we cannot ignore alternative interpretations, even if they conflict with our a priori philosophical stance (e.g., duration can be explicitly estimated by reading neuronal representation of time) and anthropomorphic assumptions (e.g., rats estimate time as humans do). While space is limited in a review, if the authors are interested, they can refer to a lengthy review I recently published on this topic, which demonstrates that my criticism is supported by a wide range of timing experiments across species (Robbe, 2023). In addition to this major conceptual issue that casts doubt on most of the conclusions of the study, there are also several major statistical issues.

Main Concerns

(1) The authors used a task in which rats must poke for a minimal amount of time (300 ms and then 1500 ms) to be able to obtain a drop of water delivered a few centimeters right below the nosepoke. They claim that their task is a time estimation task. However, they forget that they work with thirsty rats that are eager to get water sooner than later (there is a reason why they start by a short duration!). This task is mainly probing the animals ability to wait (that is impulse control) rather than time estimation per se. Second, the task does not require to estimate precise time because there appear to be no penalties when the nosepokes are too short or when they exceed. So it will be unclear if the variation in nosepoke reflects motivational changes rather than time estimation changes. The fact that this behavioral task is a poor assay for time estimation and rather reflects impulse control is shown by the tendency of animals to perform nose-pokes that are too short, the very slow improvement in their performance (Figure 1, with most of the mice making short responses), and the huge variability. Not only do the behavioral data not support the claim of the authors in terms of what the animals are actually doing (estimating time), but this also completely annihilates the interpretation of the Ca++ imaging data, which can be explained by motivational factors (changes in neuronal activity occurring while the animals nose poke may reflect a growing sens of urgency to check if water is available).

(2) A second issue is that the authors seem to assume that rats are perfectly immobile and perform like some kind of robots that would initiate nose pokes, maintain them, and remove them in a very discretized manner. However, in this kind of task, rats are constantly moving from the reward magazine to the nose poke. They also move while nose-poking (either their body or their mouth), and when they come out of the nose poke, they immediately move toward the reward spout. Thus, there is a continuous stream of movements, including fidgeting, that will covary with timing. Numerous studies have shown that sensorimotor dynamics influence neural activity, even in the prefrontal cortex. Therefore, the authors cannot rule out that what the records reflect are movements (and the scaling of movement) rather than underlying processes of time estimation (some kind of timer). Concretely, start cells could represent the ending of the movement going from the water spout to the nosepoke, and end cells could be neurons that initiate (if one can really isolate any initiation, which I doubt) the movement from the nosepoke to the water spout. Duration cells could reflect fidgeting or orofacial movements combined with an increasing urgency to leave the nose pokes.

(3) The statistics should be rethought for both the behavioral and neuronal data. They should be conducted separately for all the rats, as there is likely interindividual variability in the impulsivity of the animals.

(4) The fact that neuronal activity reflects an integration of movement and motivational factors rather than some abstract timing appears to be well compatible with the analysis conducted on the error trials (Figure 4), considering that the sensorimotor and motivational dynamics will rescale with the durations of the nose poke.

(5) The authors should mention upfront in the main text (result section) the temporal resolution allowed by their Ca+ probe and discuss whether it is fast enough in regard of behavioral dynamics occurring in the task.

Comments on the revised version

I have read the revised version of the manuscript and the rebuttal letter. My major concern was that the task used is not a time estimation task but primarily taps into impulse control and that animals are not immobile during the nose-poking epoch. I provided factual evidence for this (the animal's timing performance is poor and, on average, animals struggle to wait long enough), and I pointed to a review that discusses the results of many studies congruent with the importance of movement/motivation, not only in constraining the timing of reward-oriented actions during so-called time estimation tasks but also in powerfully modulating neuronal activity.

The authors' responses to my comments are puzzling and unconvincing. First, on the one hand, they acknowledge in their rebuttal letter the difficulty of demonstrating a neuronal representation of explicit internal estimation of time. Then, they seem to imply that this issue is beyond the scope of their study and focus in the rebuttal on whether the neuronal activity they report shows signs of being sensitive to movement and motivation, which they claim is independent of movement and motivation. This leads the authors to make no major changes in their manuscript. Their title, abstract, introduction, and discussion are largely unchanged and do not reflect the possibility that there are major confounding factors in so-called time estimation (rodents are not disembodied passive information processors) that may well explain some of the neuronal patterns. Evidently, the dismissive treatment by the authors is not satisfying. I will briefly restate my comments and reply to their responses and their new figure, which not only is unconvincing but raises new questions.

My comments were primarily focused on the behavioral task. The authors replied: "Studying the neural representation of any internal state may suffer from the same ambiguity [by ambiguity they meant that it is difficult to know if animals are explicitly estimating time]. With all due respect, however, we would like to limit our response to the scope of our results. According to the reviewer, two alternative interpretations of the task-related sequential activity exist." The authors imply that my comments are beyond the scope of their study. That is not true. My comments were targeted at the behavior of the animals, behavior they rely on to title their study: "Stable sequential dynamics in prefrontal cortex represents a subjective estimation of time." When I question whether the task and behavioral data presented are congruent with "subjective estimation of time," my comments are not beyond the scope of the study-they directly tackle the main point of the authors. Other researchers will read the title and abstract of this manuscript and conclude: "Here is a paper that provides evidence of a mechanism for animals estimating duration internally (because subjective time perception is assumed to be different from using clocks)." Still, there is a large body of literature showing that the behavior of animals in such tasks can be entirely explained without invoking subjective time perception and internal representation. How can the authors acknowledge that they can't be sure that mice are estimating time and then have such an affirmative title and abstract?

In my opinion, science is not just about forcing ideas (often reflecting philosophical preconceptions) on data and dismissing those who disagree. It is about discussing alternative possibilities fairly and being humble. In their revised version, I see no effort by the authors to investigate the importance of movement and motivation during their task or seriously engage with this idea. It's much easier to dismiss my comments as being beyond the scope of their results. According to the authors, it seems that movements and motivations play no role in the task. Still, the animals are water-restricted, and during the task, they will display decreased motivation (due to increased satiety), and their history of rewarded vs. non-rewarded trials will affect their behavior. This is one of the most robust effects seen across all behavioral studies. Moreover, the animals are constantly moving. Maybe the authors used a special breed of mice that behave like some kind of robots? I acknowledge that this is not easy to investigate, but if the authors did not use high-quality video recording or an experimental paradigm that allows disentangling motivational confounds, then they should refrain from using big words such as subjective time estimation and discuss alternative representations by acknowledging the studies that do find that movement and motivation are present during reward-based timing tasks and do in fact modulate neuronal activity, even in associative brain regions.

To sustain their claim that what they reported is movement-independent, the authors provided a supplementary figure in which they correlated neuronal activity and head movement tracked using DeepLabCut. I have to say that I was particularly surprised by this figure. First, in the original manuscript, there was absolutely no mention of video recording. Now it appears in the methods section, but the description is very short. There is no information on how these video recordings were made. The quality of the images provided in Figure S2 is far from reassuring. It is unclear whether the temporal and spatial resolution would be good enough to make meaningful correlations. Fast head/orofacial movements that occur during nose-poking can be on the order of 20 Hz. To be tracked, this would require at least a 40 Hz sampling rate. But no sampling information is provided. The authors should explain how they synchronized behavioral and neuronal data acquisition. Could the authors share behavioral videos of the 5 sessions shown in Figure S2 so we can judge the behavior of the animals, the quality of the video, and the possibility of making correlations?

Figure S2A-F: I am not sure why the authors correlated nose-poking duration (time estimation) and the duration between upper and lower nose-pokes (reward-oriented movement). It is not relevant to the issue I raised. Without any information about video acquisition frame rate, the y-axis legend (frame) is not very informative. Still, in Figure S2A-F, Rat 5 shows a clear increase in nose-poke duration, which is congruent with decreased impulsivity. Is the time coding different in this rat compared to other rats? There are some similar trends in other animals (Rat 1 and maybe Rat 3), but what is surprising is the huge variability (big downward deflections in the nose-poke duration). I would not be surprised if those deflections occurred after a long pause in activity. Could the authors plot trial time instead of trial number? How do the authors explain such a huge deflection if the animals are estimating time?

Regarding Figure S2H: I don't see how it addresses my concern. My concern is that some of the Ca activity recorded during nose-poking reflects head movements. The authors need to show if they can detect head movement during nose-poking. Aligning the Ca data relative to head movement should give the same result as when aligning the data relative to the time at which the animals pull out of the upper nose-poke.

Minor comments:

In their introduction, the authors wrote: "While these findings [correlates of time perception] provide strong evidence for a neural mechanism of time coding in the brain, true causal evidence at single-cell resolution remains beyond reach due to technical limitations. Although inhibiting certain brain regions (such as medial prefrontal cortex, mPFC,22) led to disruption in the performance of the timing task, it is difficult to attribute the effect specifically to the ramping or sequential activity patterns seen in those regions as other processes may be involved. Lacking direct experimental evidence, one potential way of testing the causal involvement of 'time codes' in time estimation function is to examine their correlation at a finer resolution."<br /> This statement is inaccurate at two levels. First, very good causal evidence has been obtained on this topic (see Monteiro et al., 2023, Nature Neuroscience), and see my News & Views on the strengths and weaknesses of this paper. Second, their proposal is inaccurate. Looking at a finer correlation will still be a correlative approach, and the authors will not be able to disentangle motor/motivation confounds.

Reviewer #2 (Public review):

Summary

Lines et al investigate the integration of sensory-evoked calcium signals in astrocytes of the primary somatosensory cortex in anesthetized mice. More precisely, their goal is to better characterize the mechanisms that govern the emergence of whole-cell events in astrocytes, here referred to as calcium surges. As a single astrocyte communicates with hundreds of thousands of synapses simultaneously, understanding the spatial and temporal integration of calcium signals in astrocytes and the mechanisms governing these phenomena is of tremendous importance to deepen our understanding of signal processing in the central nervous system. In line with previous reports in the field, the authors find that most signals originate in the arborization of astrocytes, occasionally leading to somatic and whole-cell events. On average, the latter occur following domain activity closer to the soma, suggesting a centripetal propagation of signals leading to somatic events. Moreover, they observe that the distance from the soma to active domains increases with time after somatic events, suggesting a potential centrifugal propagation of signals post-somatic activity. The results suggest that most calcium surges depend on the expression of IP3R2, the main calcium channel in astrocytes, located at the membrane of the endoplasmic reticulum. Finally, they report a correlation between the percentage of active domains in the astrocyte "arbor", the emergence of a somatic event, and the frequency of slow inward currents from neighboring neurons. The main claim of this manuscript is that there would be a spatial threshold inherent to astrocytes of ~23% of domain activation above which a calcium surge is observed. Although the study provides data and concepts that are important for the glia field, the conclusions seem a little too assertive and general with respect to what can be deduced from the data and methods used.

Strengths

The major strength of this study is the experimental approach that allowed the authors to obtain numerous and informative calcium recordings in vivo in the somatosensory cortex in mice in response to sensory stimuli as well as in situ. Notably, they developed an interesting approach to modulate the percentage of active domains in the astrocyte arborization by varying the intensity of peripheral stimulation (its amplitude, frequency, or duration). The question investigated is important as the mechanisms governing signal integration in astrocytes and its effect on neighboring cells are poorly understood.

Weaknesses

The major weakness of the manuscript is the method used to analyze and quantify calcium activity, which mostly relies on the analysis of averaged data and overlooks the variability of the signals measured. As a result, the main claims from the manuscript seem to be incompletely supported by the data.

Although the revised version includes more discussion on the experiments that could be done to extend the results from this study, more discussion would be needed to clarify the limitations on what can be deduced from the proposed experimental and analytical design. Notably, the analysis pipeline seems biased by the assumption of the existence of a spatial threshold dictating the emergence of global calcium events in astrocytes. Although there is a clear linear correlation between the percentage of active somas and the percentage of active domains in the arborization (Figure 2 panel F), concluding on the existence of an inherent threshold of domain activity is not completely supported by the data (see e.g. Figure 2 panel F or Figure 4 panel E). It would probably be more accurate to report that most somatic events occur when the percentage of arbor domains being active is above 21-24% (95% confidence interval of the reported threshold). Thus, some of the conclusions from the manuscript, such as p.14 l.34-35 " spatial threshold of domains that needs to be reached in order to lead to soma activation", seem a bit too assertive as some astrocytes did display soma activation with a much smaller percentage of active domains or on the contrary, no somatic event despite domain activity way above the threshold. Similarly, as Figure 6 demonstrates a strong effect of IP3R2 knock-out on somatic activation but reports a non-zero probability of soma activity in IP3R2 -/- mice (panel F), the conclusion that IP3R2 are necessary to trigger an astrocytic calcium surge seems a bit too strong. Finally, the results reported in Figure 7 demonstrate the existence of a strong correlation between SICs, the percentage of active astrocyte domains on, and somatic activation, so that the conclusion "These results indicate that spatial threshold of the astrocyte calcium surge has a functional impact on gliotransmission" (l.4&-48 page 13) also seems a bit too assertive.

Reviewer #3 (Public Review):

Summary:

The study aims to elucidate the spatial dynamics of subcellular astrocytic calcium signaling. Specifically, they elucidate how subdomain activity above a certain spatial threshold (~23% of domains being active) heralds a calcium surge that also affects the astrocytic soma. Moreover, they demonstrate that processes on average are included earlier than the soma and that IP3R2 is necessary for calcium surges to occur. Finally, they associate calcium surges with slow inward currents.

The revised manuscript is improved compared to the first iteration. While some concerns have been addressed, my main critique pertaining to ROI approach/sampled area, statistical analyses and anesthesia are in my view still important caveats of the study that I think should have been even more clearly addressed in the manuscript.

Strengths:<br /> The study addresses an interesting topic that is only partially understood. The study uses multiple methods including in vivo two-photon microscopy, acute brain slices, electrophysiology, pharmacology, and knockout models. The conclusions are strengthened by the same findings in both in vivo anesthetized mice and in brain slices.

Weaknesses:

The method that has been used to quantify astrocytic calcium signals only analyzes what seems to be a small proportion of the total astrocytic domain on the example micrographs, where a structure is visible in the SR101 channel (see for instance Reeves et al. J. Neurosci. 2011, demonstrating to what extent SR101 outlines an astrocyte). This would potentially heavily bias the results: from the example illustrations presented it is clear that the calcium increases in what is putatively the same astrocyte goes well beyond what is outlined with automatically placed small ROIs. The smallest astrocytic processes are an order of magnitude smaller than the resolution of optical imaging and would not be outlined by either SR101 or with the segmentation method judged by the ROIs presented in the figures. Completely ignoring these very large parts of the spatial domain of an astrocyte, in particular when making claims about a spatial threshold, seems inappropriate. Several recent methods published use pixel-by-pixel event-based approaches to define calcium signals. The data should have been analyzed using such a method within a complete astrocyte spatial domain in addition to the analyses presented. Also, the authors do not discuss how two-dimensional sampling of calcium signals from an astrocyte that has processes in three dimensions (see Bindocci et al, Science 2017) may affect the results: if subdomain activation is not homogeneously distributed in the three-dimensional space within the astrocyte territory, the assumptions and findings between a correlation between subdomain activation and somatic activation may be affected.

Authors reply: In order to reduce noise from individual pixels, we chose to segment astrocyte arborizations into domains of several pixels. As pointed out previously, including pixels outside of the SR101-positive territory runs the risk of including a pixel that may be from a neighboring cell or mostly comprised of extracellular space, and we chose the conservative approach to avoid this source of error. We agree that the results have limitations from being acquired in 2D instead of 3D, but it is likely to assume the 3D astrocyte is homogeneously distributed and that the 2D plane is representative of the whole astrocyte. Indeed, no dimensional effects were reported in Bindocci et al, Science 2017. We have included a paragraph in the discussion to address this limitation in our study on P15, L23-27:<br /> "The investigation of the spatial threshold could be improved in the future in a number of ways. One being the use of state-of-the-art imaging in 3D(Bindocci et al., 2017). While the original publication using 3D imaging to study astrocyte physiology does not necessarily imply that there would be different calcium dynamics in one axis over another, the three-dimensional examination of the spatial threshold could refine the findings we present here.

Comments on revisions: It is good that 3D imaging aspects are mentioned as a limitation, and I agree that Bindocci et al. do not necessarily suggest that results in this manuscript would have been different if also the third spatial dimension was included in the analyses. However, the way I see it, the added analyses and text changes throughtout still do not adequately address my concern pertaining to basing a spatial threshold on a fraction of the astrocyte territory.

The study uses a heaviside step function to define a spatial 'threshold' for somata either being included or not in a calcium signal. However, Fig 4E and 5D showing how the method separates the signal provide little understanding for the reader. The most informative figure that could support the main finding of the study, namely a ~23% spatial threshold for astrocyte calcium surges reaching the soma, is Fig. 4G, showing the relationship between the percentage of arborizations active and the soma calcium signal. A similar plot should have been presented in Fig 5 as well. Looking at this distribution, though, it is not clear why ~23% would be a clear threshold to separate soma involvement, one can only speculate how the threshold for a soma event would influence this number. Even if the analyses in Fig. 4H and the fact that the same threshold appears in two experimental paradigms strengthen the case, the results would have been more convincing if several types of statistical modeling describing the continuous distribution of values presented in Fig. 4E (in addition to the heaviside step function) were presented.

Authors reply: We agree with the reviewer and have added to the paper a discussion for our justification on the use of the Heaviside step function, and have included this in the methods section. We chose the Heaviside step function to represent the on/off situation that we observed in the data that suggested a threshold in the biology. We agree with the reviewer that Fig. 4G is informative and demonstrates that under 23% most of the soma fluorescence values are clustered at baseline. We agree that a different statistical model describing the data would be more convincing and confirmed the spatial threshold with the use of a confidence interval in the text and supported the use of percent domains active for this threshold over other properties such as spatial or temporal clustering using a general linear model. P18-19, L34-2:<br /> "Heaviside step function<br /> The Heaviside step function below in equation 4 is used to mathematically model the transition from one state to the next and has been used in simple integrate and fire models (Bueno-Orovio et al., 2008; Gerstner, 2000).<br /> 𝐻(𝑎) ∶=<br /> 0, 𝑎 < 𝑎T<br /> {<br /> 1, 𝑎 {greater than or equal to} 𝑎T<br /> (4)<br /> The Heaviside step function 𝐻(𝑎) is zero everywhere before the threshold area (𝑎T) and one everywhere afterwards. From the data shown in Figure 4E where each point (𝑆(𝑎)) is an individual astrocyte response with its percent area (𝑎) domains active and if the soma was active or not denoted by a 1 or 0 respectively. To determine 𝑎T in our data we iteratively subtracted 𝐻(𝑎) from 𝑆(𝑎) for all possible values of 𝑎T to create an error term over 𝑎. The area of the minimum of that error term was denoted the threshold area.

Comments on revisions: Even with the added explanations, I am still not sure that the data show a specific threshold, or that the statistical model enforce a threshold onto the data. The data in Fig. 4G does not in my view clearly show a clear threshold as suggested. The analyses are strengthened with an added statistical modeling, however, the details of the modeling is not presented in the manuscript as far as I can see. As a bare minimum the statistical packages/tools used, the model details and goodness of fit as residual plots must be shown/commented.

The description of methods should have been considerably more thorough throughout. For instance which temperature the acute slice experiments were performed at, and whether slices were prepared in ice-cold solution, are crucial to know as these parameters heavily influence both astrocyte morphology and signaling. Moreover, no monitoring of physiological parameters (oxygen level, CO2, arterial blood gas analyses, temperature etc) of the in vivo anesthetized mice is mentioned. These aspects are critical to control for when working with acute in vivo two-photon microscopy of mice; the physiological parameters rapidly decay within a few hours with anesthesia and following surgery.

Authors reply: We have increased the thoroughness of our methods section. Especially including that body temperature and respiration were indeed monitored throughout anesthesia.

Comments on revisions: Bath temperature for slice experiments, or cutting conditions are still not reported. For the in vivo experiments, it must be commented that this level of physiological monitoring for acute in vivo brain physiology experiments (self breathing, no control of O2/CO2) is barely adequate and could represent a considerable caveat of the study.

Reviewer #1 (Public review):

Summary:

There is a long-standing idea that choices influence evaluation: options we choose are re-evaluated to be better than they were before the choice. There has been some debate about this finding, and the authors developed several novel methods for detecting these re-evaluations in task designs where options are repeatedly presented against several alternatives. Using these novel methods the authors clearly demonstrate this re-evaluation phenomenon in several existing datasets and show that estimations of dynamic valuation correlate with neural activity in prefrontal cortex.

Strengths:

The paper is well-written and figures are clear. The authors provided evidence for the behaviour effect using several techniques and generated surrogate data (where the ground truth is known) to demonstrate the robustness of their methods. The author avoid over-selling the work, with a lucid description of limitations, and potential for further exploration of the work, in the discussion.

Comments on revisions:

The authors did a good job responding to the comments.

Reviewer #2 (Public review):

Zylberberg and colleagues show that food choice outcomes and BOLD signal in the vmPFC are better explained by algorithms that update subjective values during the sequence of choices compared to algorithms based on static values acquired before the decision phase. This study presents a valuable means of reducing the apparent stochasticity of choices in common laboratory experiment designs. The evidence supporting the claims of the authors is solid, although currently limited to choices between food items because no other goods were examined. The work will be of interest to researchers examining decision making across various social and biological sciences.

Comments on revisions:

We thank the authors for carefully addressing our concerns about the first version of the manuscript. The manuscript text and contributions are now much more clear and convincing.

Reviewer #1 (Public review):

Summary:

The authors investigated if/how distractor suppression derived from statistical learning may be implemented in early visual cortex. While in a scanner, participants conducted a standard additional singleton task in which one location more frequently contained a salient distractor. The results showed that activity in EVC was suppressed for the location of the salient distractor as well as for neighbouring neutral locations. This suppression was not stimulus specific - meaning it occurred equally for distractors, targets and neutral items - and it was even present in trials in which the search display was omitted. Generally, the paper was clear, the experiment was well-designed, and the data are interesting. Nevertheless, I do have several concerns mostly regarding the interpretation of the results.

(1) My biggest concern with the study is regarding the interpretation of some of the results. Specifically, regarding the dynamics of the suppression. I appreciate that there are some limitations with what you might be able to say here given the method but I do feel as if you have committed to a single interpretation where others might still be at play. Below I've listed a few alternatives to consider.

(a) Sustained Suppression. I was wondering if there is anything in your results that would speak for or against the suppression being task specific. That is, is it possible that people are just suppressing the HPDL throughout the entire experiment (i.e., also through ITI, breaks, etc., rather than just before and during the search). Since the suppression does not seem volitional, I wonder if participants might apply a blanket suppression to HPDL until they learn otherwise. Since your localiser comes after the task you might be able to see hints of sustained suppression in the HPDL during these trials.

(b) Enhancement followed by suppression. Another alternative that wasn't discussed would be an initial transient enhancement of the HPDL which might be brought on by the placeholders followed by more sustained suppression through the search task. Of course, on the whole this would look like suppression, but this still seems like it would hold different implications compared to simply "proactive suppression". This would be something like search and destroy however could be on the location level before the actual onset of the search display.

(2) I was also considering whether your effects might be at least partially attributable to priming type effects. This would be on the spatial (not feature) level as it is clear that the distractors are switching colours. Basically, is it possible that on trial n participants see the HPDL with the distractor in it and then on trial n+1 they suppress that location. This would be something distinct from the statistical learning framework and from the repetition suppression discussion you have already included. To test for this, you could look at the trials that follow omission or trials. If there is no suppression or less suppression on these trials it would seem fair to conclude that the suppression is at least in part due to the previous trial.

Reviewer #2 (Public review):

The authors of this work set out to test ideas about how observers learn to ignore irrelevant visual information. Specifically, they used fMRI to scan participants who performed a visual search task. The task was designed in such a way that highly salient but irrelevant search items were more likely to appear at a given spatial location. With a region-of-interest approach, the authors found that activity in visual cortex that selectively responds to that location was generally suppressed, in response to all stimuli (search targets, salient distractors, or neutral items), as well as in the absence of an anticipated stimulus.

Strengths of the study include: A well-written and well-argued manuscript; clever application of a region of interest approach to fMRI design, which allows articulating clear tests of different hypotheses; careful application of follow-up analyses to rule out alternative, strategy-based accounts of the findings; tests of the robustness of the findings to detailed analysis parameters such as ROI size; and exclusion of the role of regional baseline differences in BOLD responses.

The report might be enhanced by analyses (perhaps in a surface space) that distinguish amongst the multiple "early" retinotopic visual areas that are analysed in the aggregate here. Furthermore, the study could benefit from an analysis that tests the correlation over observers between the magnitude of their behavioural effects and their neural responses.

The study provides an advance over previous studies, which identified enhancement or suppression in visual cortex as a function of search target/distractor predictability, but in less spatially-specific way. It also speaks to open questions about whether such suppression/enhancement is observed only in response to the arrival of visual information, or instead is preparatory, favouring the latter view. The theoretical advance is moderate, in that it is largely congruent with previous frameworks, rather than strongly excluding an opposing view or providing a major step change in our understanding of how distractor suppression unfolds.

Joint Public Review:

In the microglia research community, it is accepted that microglia change their shape both gradually and acutely along a continuum that is influenced by external factors both in their microenvironments and in circulation. Ideally, a given morphological state reflects a functional state that provides insight into a microglia's role in physiological and pathological conditions. The current manuscript introduces MorphoCellSorter, an open-source tool designed for automated morphometric analysis of microglia. This method adds to the many programs and platforms available to assess the characteristics of microglial morphology; however, MorphoCellSorter is unique in that it uses Andrew's plotting to rank populations of cells together (in control and experimental groups) and presents "big picture" views of how entire populations of microglia alter under different conditions. Notably, MorphoCellSorter is versatile, as it can be used across a wide array of imaging techniques and equipment. For example, the authors use MorphoCellSorter on images of fixed and live tissues representing different biological contexts such as embryonic stages, Alzheimer's disease models, stroke, and primary cell cultures.

This manuscript outlines a strategy for efficiently ranking microglia beyond the classical homeostatic vs. active morphological states. The outcome offers only a minor improvement over the already available strategies that have the same challenge: how to interpret the ranking functionally.

Strengths and Weaknesses:

(1) The authors offer an alternative perspective on microglia morphology, exploring the option to rank microglia instead of categorizing them with means of clusterings like k-means, which should better reflect the concept of a microglia morphology continuum. They demonstrate that these ranked representations of morphology can be illustrated using histograms across the entire population, allowing the identification of potential shifts between experimental groups. Although the idea of using Andrews curves is innovative, the distance between ranked morphologies is challenging to measure, raising the question of whether the authors oversimplify the problem. Also, the discussion about the pipeline's uniqueness does not go into the details of alternative models. The introduction remains weak in outlining the limitations of current methods (L90). Acknowledging this limitation will be necessary.

(2) The manuscript suffers from several overstatements and simplifications, which need to be resolved. For example:

a) L40: The authors talk about "accurately ranked cells". Based on their results, the term "accuracy" is still unclear in this context.

b) L50: Microglial processes are not necessarily evenly distributed in the healthy brain. Depending on their embedded environment, they can have longer process extensions (e.g., frontal cortex versus cerebellum).

c) L69: The term "metabolic challenge" is very broad, ranging from glycolysis/FAO switches to ATP-mediated morphological adaptations, and it needs further clarification about the author's intended meaning.

d) L75: Is morphology truly "easy" to obtain?

e) L80: The sentence structure implies that clustering or artificial intelligence (AI) are parameters, which is incorrect. Furthermore, the authors should clarify the term "AI" in their intended context of morphological analysis.

f) L390f: An assumption is made that the contralateral hemisphere is a non-pathological condition. How confident are the authors about this statement? The brain is still exposed to a pathological condition, which does not stop at one brain hemisphere.

(3) Methodological questions:

a) L299: An inversion operation was applied to specific parameters. The description needs to clarify the necessity of this since the PCA does not require it.

b) Different biological samples have been collected across different species (rat, mouse) and disease conditions (stroke, Alzheimer's disease).<br /> Sex is a relevant component in microglia morphology. At first glance, information on sex is missing for several of the samples. The authors should always refer to Table 1 in their manuscript to avoid this confusion. Furthermore, how many biological animals have been analyzed? It would be beneficial for the study to compare different sexes and see how accurate Andrew's ranking would be in ranking differences between males and females. If they have a rationale for choosing one sex, this should be explained.<br /> In the methodology, the slice thickness has been given in a range. Is there a particular reason for this variability? Also, the slice thickness is inadequate to cover the entire microglia morphology. How do the authors include this limitation of their strategy? Did the authors define a cut-off for incomplete microglia?

c) The manuscript outlines that the authors have used different preprocessing pipelines, which is great for being transparent about this process. Yet, it would be relevant to provide a rationale for the different imaging processing and segmentation pipelines and platform usages (Supplementary Figure 7). For example, it is not clear why the Z maximum projection is performed at the end for the Alzheimer's Disease model, while it's done at the beginning of the others. The same holds through for cropping, filter values, etc. Would it be possible to analyze the images with the same pipelines and compare whether a specific pipeline should be preferable to others? On a note, Matlab is not open-access.<br /> This also includes combining the different animals to see which insights could be gained using the proposed pipelines.

d) L227: Performing manual thresholding isn't ideal because it implies the preprocessing could be improved. Additionally, it is important to consider that morphology may vary depending on the thresholding parameters. Comparing different acquisitions that have been binarized using different criteria could introduce biases.

e) Parameter choices:

L375: When using k-means clustering, it is good practice to determine the number of clusters (k) using silhouette or elbow scores. Simply selecting a value of k based on its previous usage in the literature is not rigorous, as the optimal number of clusters depends on the specific data structure. If they are seeking a more objective clustering approach, they could also consider employing other unsupervised techniques, (e.g. HDBSCAN) (L403f).

L373: A rationale for the choice of the 20 non-dimensional parameters as well as a detailed explanation of their computation such as the skeleton process ratio is missing. Also, how strongly correlated are those parameters, and how might this correlation bias the data outcomes? Differences between circularity and roundness factors are not coming across and require further clarification. One is applied to the soma and the other to the cell, but why is neither circularity nor loudness factor applied to both?

f) PCA analysis:

The authors spend a lot of text to describe the basic principles of PCA. PCA is mathematically well-described and does not require such depth in the description and would be sufficient with references. Furthermore, there are the following points that require attention:

L321: PC1 is the most important part of the data could be an incorrect statement because the highest dispersion could be noise, which would not be the most relevant part of the data. Therefore, the term "important" has to be clarified.

L323: As before, it's not given that the first two components hold all the information.

L327 and L331 contain mistakes in the nomenclature: Mix up of "wi" should be "wn" because "i" does not refer to anything. The same for "phi i = arctan(yn/wn)" should be "phi n".

L348: Spearman's correlation measures monotonic correlation, not linear correlation. Either the authors used Pearson Correlation for linearity or Spearman correlation for monotonic. This needs to be clarified to avoid misunderstandings.

g) If the authors find no morphological alteration, how can they ensure that the algorithm is sensitive enough to detect them? When morphologies are similar, it's harder to spot differences. In cases where morphological differences are more apparent, like stroke, classification is more straightforward.

h) Minor aspects:

{section sign} % notation requires to include (weight/volume) annotation.

{section sign} Citation/source of the different mouse lines should be included in the method sections (e.g. L117).

{section sign} L125: The length of the single housing should be specified to ensure no variability in this context.

{section sign} L673: Typo to the reference to the figure.

Reviewer #1 (Public review):

Summary:

The manuscript presents a significant and rigorous investigation into the role of CHMP5 in regulating bone formation and cellular senescence. The study provides compelling evidence that CHMP5 is essential for maintaining endolysosomal function and controlling mitochondrial ROS levels, thereby preventing the senescence of skeletal progenitor cells.

Strengths:

The authors demonstrate that the deletion of Chmp5 results in endolysosomal dysfunction, elevated mitochondrial ROS, and ultimately enhanced bone formation through both autonomous and paracrine mechanisms. The innovative use of senolytic drugs to ameliorate musculoskeletal abnormalities in Chmp5-deficient mice is a novel and critical finding, suggesting potential therapeutic strategies for musculoskeletal disorders linked to endolysosomal dysfunction.

Weaknesses:

The manuscript requires a deeper discussion or exploration of CHMP5's roles and a more refined analysis of senolytic drug specificity and effects. This would greatly enhance the comprehensiveness and clarity of the manuscript.

Reviewer #2 (Public review):

Summary:

The authors try to show the importance of CHMP5 for skeletal development.

Strengths:

The findings of this manuscript are interesting. The mouse phenotypes are well done and are of interest to a broader (bone) field.

Weaknesses:

The mechanistic insights are mediocre, and the cellular senescence aspect poor.

In total, it has not been shown that there are actual senescent cells that are reduced after D+Q-treatment. These statements need to be scaled back substantially.

Reviewer #3 (Public review):

Summary:

In this study, Zhang et al. reported that CHMP5 restricts bone formation by controlling endolysosome-mitochondrion-mediated cell senescence. The effects of CHMP5 on osteoclastic bone resorption and bone turnover have been reported previously (PMID: 26195726), in which study the aberrant bone phenotype was observed in the CHMP5-ctsk-CKO mouse model, using the same mouse model, Zhang et al., report a novel role of CHMP5 on osteogenesis through affecting cell senescence. Overall, it is an interesting study and provides new insights in the field of cell senescence and bone.

Strengths:

Analyzed the bone phenotype OF CHMP5-periskeletal progenitor-CKO mouse model and found the novel role of senescent cells on osteogenesis and migration.

Weaknesses:

(1) There are a lot of papers that have reported that senescence impairs osteogenesis of skeletal stem cells. In this study, the author claimed that Chmp5 deficiency induces skeletal progennitor cell senescence and enhanced osteogenesis. Can the authors explain the controversial results?

(2) Co-culture of Chmp5-KO periskeletal progenitors with WT ones should be conducted to detect the migration and osteogenesis of WT cells in response to Chmp5-KO-induced senescent cells. In addition, the co-culture of WT periskeletal progenitors with senescent cells induced by H2O2, radiation, or from aged mice would provide more information.

(3) Many EVs were secreted from Chmp5-deleted periskeletal progenitors, compared to the rarely detected EVs around WT cells. Since EVs of BMSCs or osteoprogenitors show strong effects of promoting osteogenesis, did the EVs contribute to the enhanced osteogenesis induced by Chmp5-defeciency?

(4) EVs secreted from senescent cells propagate senescence and impair osteogenesis, why do EVs secreted from senescent cells induced by Chmp5-defeciency have opposite effects on osteogenesis?

(5) The Chmp5-ctsk mice show accelerated aging-related phenotypes, such as hair loss and joint stiffness. Did Ctsk also label cells in hair follicles or joint tissue?

(6) Fifteen proteins were found to increase and five proteins to decrease in the cell supernatant of Chmp5Ctsk periskeletal progenitors. How about SASP factors in the secretory profile?

(7) D+Q treatment mitigates musculoskeletal pathologies in Chmp5 conditional knockout mice. In the previously published paper (CHMP5 controls bone turnover rates by dampening NF-κB activity in osteoclasts), inhibition of osteoclastic bone resorption rescues the aberrant bone phenotype of the Chmp5 conditional knockout mice. Whether the effects of D+Q on bone overgrowth is because of the inhibition of bone resorption?

(8) The role of VPS4A in cell senescence should be measured to support the conclusion that CHMP5 regulates osteogenesis by affecting cell senescence.

(9) Cell senescence with markers, such as p21 and H2AX, co-stained with GFP should be performed in the mouse models to indicate the effects of Chmp5 on cell senescence in vivo.

(10) ADTC5 cell as osteochondromas cells line, is not a good cell model of periskeletal progenitors. Maybe primary periskeletal progenitor cell is a better choice.

Reviewer #1 (Public review):

Summary:

This manuscript explores the role of the Evening Complex (EC), specifically focusing on ELF3, a disordered protein component of the EC, and its temperature-dependent phase behavior. The study highlights the role of polyQ tracts in modulating temperature-sensitive condensate formation and provides a combination of computational approaches, including REST2 simulations and coarse-grained Martini simulations, to investigate how polyQ tract length and sequence context influence this behavior.

Strengths:

The study addresses a key question in plant biology - how temperature influences circadian clock-mediated growth regulation through protein phase behavior. The manuscript introduces the novel finding that polyQ tract length modulates the temperature-dependent formation of helices and condensates.

Weaknesses:

(1) Coarse-Grained Simulation Results Not Supported by Data:<br /> The results presented in Figure 6A of the manuscript do not seem to show a clear trend in the number of clusters formed as a function of polyQ tract length. This is particularly evident in the comparison between 0Q and 7Q polyQ lengths, which display statistically similar values in terms of the number of clusters. The lack of distinction between these values raises questions about the sensitivity of the coarse-grained simulations to polyQ tract length, which the authors claim as a key modulator of condensate formation. This discrepancy weakens the argument that polyQ length directly impacts the clustering behavior in the simulations.<br /> Suggested Analysis:<br /> - A more detailed statistical analysis should be performed to assess whether the observed differences between polyQ lengths are significant. This could involve hypothesis testing or the use of error bars in the graphs to better communicate the variability in the data.<br /> - Additionally, the authors should examine whether there are other features, such as cluster shape or internal structure, that might differentiate between different polyQ lengths, even if the total number of clusters is similar.

(2) Inconsistency in Cluster Size Across Temperatures (Figure 6B):<br /> The results in Figure 6B show a striking difference in the size of the largest cluster between temperatures of 290K and 300K. This abrupt shift in behavior lacks a clear mechanistic explanation. Typically, phase transitions driven by temperature are more gradual, unless there is some underlying structural or chemical shift that the authors have not accounted for. Without a clear explanation, this sudden change in behavior reduces confidence in the simulation results.<br /> Suggested Analysis:<br /> - The authors should explore possible explanations for the dramatic difference in cluster size between 290K and 300K. For example, they could investigate whether specific interactions (such as the breaking or formation of hydrogen bonds or hydrophobic contacts) might explain the behavior at higher temperatures.<br /> - It is important to check whether the coarse-grained simulation model has been adequately parameterized and scaled for accurate temperature dependence. Atomistic simulations of monomers and dimers with varying polyQ tract lengths could be used to fine-tune the coarse-grained model, ensuring it accurately reflects molecular behavior. The gross estimate of a 10% scaling factor might be insufficient and could lead to inaccurate representations of cluster formation.

(3) Scaling of Coarse-Grained Model with Atomistic Simulations:<br /> As mentioned, the coarse-grained model used in the study may not have been properly scaled against atomistic data. A simple scaling factor of 10% may not be appropriate for accurately capturing the behavior of polyQ tracts across different lengths, especially considering their sensitivity to subtle changes in temperature. Without rigorous validation against atomistic simulations, the coarse-grained model's predictions could be skewed.<br /> Suggested Analysis:

(4) To address this, the authors should compare the coarse-grained model with atomistic simulations of monomeric and dimeric forms of ELF3 with different polyQ tract lengths. By comparing key structural parameters (e.g., radius of gyration, contact maps, and clustering propensity), the authors could adjust the coarse-grained model to more accurately reflect the atomistic behavior. The authors have wealth of atomistic simulation data that could afford such benchmarking and identification of scaling factor<br /> o Additionally, the authors should investigate whether the assumed scaling factor of 10% is appropriate for each polyQ length or whether it needs to be refined based on specific properties, such as the number of hydrophobic interactions or secondary structure stability.

(5) Lack of Analysis for Liquid-Like Behavior in Phase Separation:<br /> The simulations presented in the manuscript do not analyze the liquid-like behavior of ELF3 condensates, which is a key characteristic of liquid-liquid phase separation (LLPS). In LLPS systems, condensates are often dynamic, with chains exchanging between clusters, indicating liquid-like rather than solid-like behavior. The authors fail to probe this crucial aspect, which is necessary to support the claim that ELF3 undergoes phase separation.<br /> Suggested Analysis:<br /> - The authors should conduct additional analyses to probe the liquid-like nature of the clusters formed by ELF3. One approach would be to analyze the dynamics of chain exchange between clusters, measuring how frequently chains leave one cluster and join another over time. This analysis would reveal whether the condensates behave as liquid-like, dynamic structures or more static, solid-like aggregates.<br /> - Additionally, the temperature dependence of these exchange dynamics should be investigated. In true liquid-liquid phase separation, the rate of chain exchange is often sensitive to temperature. Observing how this rate changes between 290K and 300K, for instance, could help explain the abrupt shift in cluster size seen in Figure 6B.<br /> - The authors should also analyze whether the internal structures of the condensates are consistent with a liquid-like phase. For example, radial distribution functions and contact lifetimes could be calculated to reveal whether the clusters exhibit liquid-like organization.

(6) Lack of justification of polydispersity of polyQ:<br /> The authors don't provide any rationale for choice of different copies of polyQ used in the manuscript for their chain-growth simulation studies. It will be more apt if it can be motivated via some precedent experimental observations.

(7) Lack of initiative to connect to Experiments:<br /> While the computational models and simulations provide robust theoretical insights, the absence of direct experimental validation weakens the overall impact of the manuscript. For example, experimental data on how specific mutations in the polyQ tract influence ELF3 behavior in vivo would significantly bolster the authors' claims. The manuscript would benefit from either citing existing experimental studies that corroborate these findings or from suggesting future experimental directions.

Reviewer #2 (Public review):

Summary:

The authors aimed to explore how a key protein in the circadian clock of plants, ELF3, responds to temperature changes by forming molecular condensates. They focused on understanding the role of a specific region of the protein, a polyQ tract, in promoting temperature-sensitive structural changes and regulating the formation of condensates. Through a series of computational simulations, they sought to uncover the molecular basis for ELF3's temperature responsiveness and its broader implications for plant growth and adaptation to environmental conditions.

Strengths:

The study's strength lies in its focus on an important biological question: how plants sense and respond to temperature changes at the molecular level. The authors employed a variety of computational techniques, including coarse-grained simulations, to explore the role of specific molecular features in this process. These methods provide a multi-scale view of protein behavior and offer valuable insights into how molecular structures may influence biological function.

Weaknesses:

However, there are notable weaknesses in the evidence provided. While the authors present trends in molecular changes, such as shifts in helical propensity and the formation of condensates, these results seem subtle and are not strongly substantiated by statistical analysis. The lack of error bars in the figures makes it difficult to distinguish between meaningful signals and potential noise in the data. Furthermore, the temperature-sensitive behavior appears to be influenced more by chain length than by sequence-specific effects of the polyQ region, raising questions about whether the findings truly capture the molecular mechanisms responsible for temperature sensing. Additionally, some simulations, particularly those related to the formation of condensates, do not appear fully converged, which casts further doubt on the robustness of the results.

Additional Context for Readers:

Readers should interpret the results with caution, especially regarding the molecular mechanisms proposed for temperature sensing. While the study presents interesting trends, the evidence is not definitive, and the findings may be more reflective of general protein behavior (such as the effect of chain length on condensate formation) than specific sequence-driven responses to temperature. Further experimental studies and more converged simulations will be necessary to fully understand the role of ELF3 in temperature regulation.

Reviewer #1 (Public review):

(1) Summary of the Paper:

This paper by Chen et al. examines the cellular composition and gene expression of the hypothalamic medial preoptic area (MPOA) in two closely related deer mouse species (P. maniculatus and P. polionotus) that exhibit distinct social behaviors. Through single-nucleus RNA sequencing (snRNA-seq), Chen et al., identify sex- and species-specific neuronal cell types that likely contribute to differences in mating and parental care. By comparing monogamous and promiscuous species, the study provides insights into how neuronal diversity and gene expression changes in the MPOA might underlie the evolution of social behaviors.

(2) Strengths of the Paper:

The paper excels in several areas. First, the data presentation is clear and well-organized, making the complex findings easy to follow. The writing is straightforward and highly accessible, which enhances the overall readability. The experimental design is innovative, particularly in how they combined samples from different species into the same dataset and then used post-hoc identification to distinguish cell types by species. This dramatically controls for potential batch effects in my opinion. Additionally, the authors contextualize their findings within the framework of previously published studies on Mus musculus, providing a strong comparative analysis that enhances the significance of their work.

3) Weaknesses of the Paper:

The major limitation of the study is the absence of causal experiments linking the observed changes in MPOA cell types to species-specific social behaviors. While the study provides valuable correlational data, it lacks functional experiments that would demonstrate a direct relationship between the neuronal differences and behavior. For instance, manipulating these cell types or gene expressions in vivo and observing their effects on behavior would have strengthened the conclusions, although I certainly appreciate the difficulty in this, especially in non-musculus mice. Without such experiments, the study remains speculative about how these neuronal differences contribute to the evolution of social behaviors.

Reviewer #2 (Public review):

Summary:

The authors report several interesting species and sex differences in cell type expression that may relate to species differences in behavior. The differential cell type abundance findings build on previously observed species/sex differences in behavior and brain anatomy. These data will be a valuable resource for behavioral neuroscientists. These findings are important but the manuscript goes too far in attributing causal influences to differences in behavior. A second important problem is that dissections used for the sequencing data include other neuropeptide-rich areas of the hypothalamus like the PVN. Although histology is included, the results in the main manuscript often do not include the mPOA making it hard to know if species/sex differences are consistent across different hypothalamic regions. The manuscript would benefit from more precise language.

Strengths:

The data are novel because cell-type atlases are available for only a few species.

The authors have clearly defined appropriate steps taken to obtain trustworthy estimations of cell type abundance. Furthermore, the criteria for each cell type assignment were described in a way for readers to easily replicate. The rigor in comparing cell abundance provides convincing evidence that these species have differences in MPOA cellular composition.

The authors have a good explanation for why 19 of the 53 neuron clusters were not classified (possible Mus/Peromyscus anatomical differences, some cell types don't have well-defined transcriptional profiles).

Validated findings with histology

Weaknesses:

Some methodology could be further explained, like the decision of a 15% cutoff value for cell type assignment per cluster, or the necessity of a multi-step analysis pipeline for gene enrichment studies.

The authors should exercise strong caution in making inferences about these differences being the basis of parental behavior. It is possible, given connections to relevant research, but without direct intervention, direct claims should be avoided. There should be clear distinctions of what to conclude and what to propose as possibilities for future research.

Histology is not performed on all regions included in the sequencing analysis.

Reviewer #3 (Public review):

Summary:

The authors performed snRNA-seq in the pre-optic area (POA), a heterogeneous brain region implicated in multiple innate behaviors, comparing two species of Peromyscus mice that possess strikingly different parenting behaviors. P. polionotus shows high levels of parental care from both sexes of parent, and P. maniculatus shows lower levels of care, predominantly displayed by dams rather than sires. The overall goal of understanding the genomic basis of behavioral variation is significant and of broad interest and comparative studies in POA in these two species is an excellent approach to tackle this question. The authors correctly point out that existing studies largely compare species that are highly divergent, such as mice and humans, which confounds the association of specific neuronal populations or gene expression patterns with distinct behaviors. They identify neuronal populations with differential abundance between species and sexes and additionally report sex and species differences in gene expression within each transcriptomic cell type. Their cell type classification is aided by mapping their Peromyscus cells onto a previously existing POA single-cell dataset generated in lab mice. However, a significant fraction of the cells cannot be assigned to Mus types, which confounds their analysis. The detection and validation of previously observed sex differences in the Gal/Moxd1 cell type and species differences in Avp expression provide additional support that their data are solid. This study provides an important resource for comparative single-cell studies in the brain.

Strengths:

This is a pioneering comparative snRNA-seq study that provides a roadmap for similar approaches in non-traditional model organisms.

The authors have identified populations that may underlie sex- and species- differences in parenting behavior in rodents.

A significant strength of the manuscript is the histological validation of their most robust marker genes.

Weaknesses:

My primary concern is that the dataset is limited: 52,121 neuronal nuclei across 24 samples, which does not provide many cells per cluster to analyze comparatively across sex and species, particularly given the heterogeneity of the region dissected. The Supplementary table reports lower UMIs/genes per cell than is typically seen as well. Perhaps additional information could be obtained from the data by not restricting the analyses to cells that can be assigned to Mus types. A direct comparison of the two Peromyscus species could be valuable as would a more complete Peromyscus POA atlas.

In Supplement 7, it appears that most neurons can be assigned as excitatory or inhibitory, but then so many of these cells remain in the unassigned "gray blob" seen in panel 1E. Clustering of excitatory and inhibitory neurons separately, as in in prior cited work in Mus POA (refs 31 and 57) may boost statistical power to detect sex and species differences in cell types. Perhaps the cells that cannot be assigned to Mus contain too few reads to be useful, in which case they should be filtered out in the QC. The technical challenges of a comparative single-cell approach are considerable, so it benefits the scientific community to provide transparency about them.

The Calb1 dimorphism as observed by immunostaining, appears much more extensive in P. maniculatus compared to P. polionotus (Figures 3 E and F). This finding is not reflected in the counts of the i20:Gal/Moxd1 cluster. The use of Calb1 staining as a proxy for the Gal/Moxd1 cluster would be strengthened if the number of POA Calb1+ neurons that are found in each cluster was apparent. There may be additional Calb+ neurons in the cells that are not annotated to a Mus cluster. This clarification would add support to the overall conclusion that there is reduced sexual dimorphism in P. polionotus.

The relationship between the sex steroid receptor expression and the sex bias in gene expression would be improved if the sex bias in sex steroid receptor expression was included in Supplementary Figure 10.

There is no explanation for the finding that there is a female bias in gene expression across all cell types in P. polionotus.

Reviewer #1 (Public review):

Summary:

The researchers examined how individuals who were born blind or lost their vision early in life process information, specifically focusing on the decoding of Braille characters. They explored the transition of Braille character information from tactile sensory inputs, based on which hand was used for reading, to perceptual representations that are not dependent on the reading hand.

They identified tactile sensory representations in areas responsible for touch processing and perceptual representations in brain regions typically involved in visual reading, with the lateral occipital complex serving as a pivotal "hinge" region between them.

In terms of temporal information processing, they discovered that tactile sensory representations occur prior to cognitive perceptual representations. The researchers suggest that this pattern indicates that even in situations of significant brain adaptability, there is a consistent chronological progression from sensory to cognitive processing.

Strengths:

By combining fMRI and EEG, and focusing on the diagnostic case of Braille reading, the paper provides an integrated view of the transformation processing from sensation to perception in the visually deprived brain. Such a multimodal approach is still rare in the study of human brain plasticity and allows to discern the nature of information processing in blind people early visual cortex, as well as the timecourse of information processing in a situation of significant brain adaptability.

Weaknesses:

ROI and searchlight analyses are not completely overlapping, although this might be due to the specific limits and strengths of each approach. Moreover, the conclusions regarding the behavioral relevance of the sensory and perceptual representations in the putatively reorganized brain, although important, are limited due to the behavioral measurements adopted.

Reviewer #2 (Public review):

Summary:

Haupt and colleagues performed a well-designed study to test the spatial and temporal gradient of perceiving braille letters in blind individuals. Using cross-hand decoding of the read letters, and comparing it to the decoding of the read letter for each hand, they defined perceptual and sensory responses. Then they compared where (using fMRI) and when (using EEG) these were decodable. Using fMRI, they showed that low-level tactile responses specific to each hand are decodable from the primary and secondary somatosensory cortex as well as from IPS subregions, the insula and LOC. In contrast, more abstract representations of the braille letter independent from the reading hand were decodable from several visual ROIs, LOC, VWFA and surprisingly also EVC. Using a parallel EEG design, they showed that sensory hand-specific responses emerge in time before perceptual braille letter representations. Last, they used RSA to show that the behavioral similarity of the letter pairs correlates to the neural signal of both fMRI (for the perceptual decoding, in visual and ventral ROIs) and EEG (for both sensory and perceptual decoding).

Strengths:

This is a very well-designed study and it is analyzed well. The writing clearly describes the analyses and results. Overall, the study provides convincing evidence from EEG and fMRI that the decoding of letter identity across the reading hand occurs in the visual cortex in blindness. Further, it addresses important questions about the visual cortex hierarchy in blindness (whether it parallels that of the sighted brain or is inverted) and its link to braille reading.

Reviewer #1 (Public review):

Summary:

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancers. Compared to other types of breast cancer, TNBC exhibits highly aggressive clinical characteristics, a greater likelihood of metastasis, poorer clinical outcomes, and lower survival rates. Immunotherapy is an important treatment option for TNBC, but there is significant heterogeneity in treatment response. Therefore, it is crucial to accurately identify immunosuppressive patients before treatment and actively seek more effective therapeutic approaches for TNBC patients.

Strengths:

In this work, the authors collected and integrated data from single cells and large volumes of RNA sequencing and RNA-SEQ to analyze the TME landscape mediated by genes associated with iron death. On this basis, the prediction model of prognosis and treatment response of 131 patients was constructed using a machine learning algorithm, which is beneficial to provide individualized and precise treatment guidance for breast cancer patients.

Weaknesses:

However, there are still some issues that need to be clarified:

(1) The description of the research background is too brief and concise, and it is necessary to add some information about the limitations of existing methods and the differences and advantages of this study compared with other published relevant studies, so as to better highlight the necessity and research value of this study.

(2) This study is a retrospective analysis of a public data set and lacks experimental validation and prospective experiments to support the results of bioinformatics analysis. This should be added to the acknowledgment of limitations in the study.

Reviewer #2 (Public review):

Summary:

This study aims to explore the ferroptosis-related immune landscape of TNBC through the integration of single-cell and bulk RNA sequencing data, followed by the development of a risk prediction model for prognosis and drug response. The authors identified key subpopulations of immune cells within the TME, particularly focusing on T cells and macrophages. Using machine learning algorithms, the authors constructed a ferroptosis-related gene risk score that accurately predicts survival and the potential response to specific drugs in TNBC patients.

Strengths:

The study identifies distinct subpopulations of T cells and macrophages with differential expression of ferroptosis-related genes. The clustering of these subpopulations and their correlation with patient prognosis is highly insightful, especially the identification of the TREM2+ and FOLR2+ macrophage subtypes, which are linked to either favorable or poor prognoses. The risk model thus holds potential not only for prognosis but also for guiding treatment selection in personalized oncology.

Weaknesses:

The study has a relatively small sample size, with only 9 samples analyzed by scRNA-seq. Given the typically high heterogeneity of the tumor microenvironment (TME) in cancer patients, this may affect the accuracy of the conclusions. The scRNA-seq analysis focuses on the expression of ferroptosis-related genes in various cells within the TME. In contrast, bulk RNA sequencing uses data from tumor samples, and the results between the two analyses are not consistent. The bulk RNA sequencing results may not accurately capture the changes happening in the microenvironment.

Reviewer #3 (Public review):

Summary:

Juan Liu et al. investigated the interplay between habitat fragmentation and climate-driven thermophilization in birds in an island system in China. They used extensive bird monitoring data (9 surveys per year per island) across 36 islands of varying size and isolation from the mainland covering 10 years. The authors use extensive modeling frameworks to test a general increase of the occurrence and abundance of warm-dwelling species and vice versa for cold-dwelling species using the widely used Community Temperature Index (CTI), as well the relationship between island fragmentation in terms of island area and isolation from the mainland on extinction and colonization rates of cold- and warm-adapted species. They found that indeed there was thermophilization happening during the last 10 years, which was more pronounced for the CTI based on abundances and less clearly for the occurrence based metric. Generally, the authors show that this is driven by an increased colonization rate of warm-dwelling and an increased extinction rate of cold-dwelling species. Interestingly, they unravel some of the mechanisms behind this dynamic by showing that warm-adapted species increased while cold-dwelling decreased more strongly on smaller islands, which is - according to the authors - due to lowered thermal buffering on smaller islands (which was supported by air temperature monitoring done during the study period on small and large islands). They argue, that the increased extinction rate of cold-adapted species could also be due to lowered habitat heterogeneity on smaller islands. With regards to island isolation, they show that also both thermophilization processes (increase of warm and decrease of cold-adapted species) was stronger on islands closer to the mainland, due to closer sources to species populations of either group on the mainland as compared to limited dispersal (i.e. range shift potential) in more isolated islands.

The conclusions drawn in this study are sound, and mostly well supported by the results. Only few aspects leave open questions and could quite likely be further supported by the authors themselves thanks to their apparent extensive understanding of the study system.

Strengths:

The study questions and hypotheses are very well aligned with the methods used, ranging from field surveys to extensive modeling frameworks, as well as with the conclusions drawn from the results. The study addresses a complex question on the interplay between habitat fragmentation and climate-driven thermophilization which can naturally be affected by a multitude of additional factors than the ones included here. Nevertheless, the authors use a well balanced method of simplifying this to the most important factors in question (CTI change, extinction, colonization, together with habitat fragmentation metrics of isolation and island area). The interpretation of the results presents interesting mechanisms without being too bold on their findings and by providing important links to the existing literature as well as to additional data and analyses presented in the appendix.

Weaknesses:

The metric of island isolation based on distance to the mainland seems a bit too oversimplified as in real-life the study system rather represents an island network where the islands of different sizes are in varying distances to each other, such that smaller islands can potentially draw from the species pools from near-by larger islands too - rather than just from the mainland. Although the authors do explain the reason for this metric, backed up by earlier research, a network approach could be worthwhile exploring in future research done in this system. The fact, that the authors did find a signal of island isolation does support their method, but the variation in responses to this metric could hint on a more complex pattern going on in real-life than was assumed for this study.

Comments on revisions:

I'm happy with the revisions made by the authors.

Reviewer #1 (Public review):

Summary:

The Avrillon et al. explore the neural control of muscle by decomposing the firing activity of constituent motor units from the grid of surface electromyography (EMG) in the Tibialis (TA) Anterior and Vastus Lateralis (VL) during isometric contractions. The study involves extensive samples of motor units across the broadest range of voluntary contraction intensities up to 80% of MVC. The authors examine rate coding of the population of motor units, which describes the instantaneous firing rate of each motor unit as a function of muscle force. This relationship is characterized by a natural logarithm function that delineates two distinct phases: an initial phase with a steep acceleration in firing rate, particularly pronounced in low-threshold motor units, and a subsequent modest linear increase in firing rate, more significant in high-threshold motor units.

Strengths:

The study makes a significant contribution to the field of neuromuscular physiology by providing a detailed analysis of motor unit behavior during muscle contractions in a few ways.

(1) The significance lies in its comprehensive framework of motor unit activity during isometric contractions in the broad range of intensities, providing insights into the non-linear relationship between the firing rate and the muscle force. The extensive sample of motor units across the pool confirms the observation in animal studies in which the the spinal motoneuron exhibits a discharge consists of the distinct phases in response to synaptic currents, under the influence of persistent inward currents. As such, it is now reasonable to state the human motor units across the pool are also under control of gain modulation via some neuromodulatory effects in addition to synaptic inputs arising from ionotropic effects.<br /> (2) The firing scheme across in the entire motoneuron pool revealed in this study reconciles the discrepancy in firing organization under debate; i.e., whether it is 'onion skin' like or not (Heckman and Enoka 2012). The onion skin like model states that the low threshold motor units discharge higher than high threshold motor units and has been held for long time because the firing behaviors were examined in a partial range of contraction force range due to technical limitations. This reconciliation is crucial because it is fundamental to modelling the organization of motor unit recruitment and rate coding to achieve a desired force generation to advance our understanding of motor control.<br /> (3) The extensive data collection with a novel blind source separation algorithm on the expanded number of channel of surface EMG signal provides a robust dataset that enhances the reliability and validity of findings, setting a new standard for empirical studies in the field. \par<br /> Collectively, this study fills several knowledge gaps in the field and advances our understanding the mechanism underlying the isometric force generation.

Reviewer #2 (Public review):

Avrillon et al. provides a comprehensive assessment of firing rate parameters from a large percentage of the motor unit pool, in two muscles, during voluntary isometric contractions. The authors have used new quantitative methods to extract more unique motor units across contractions than prior studies. This was achieved by recording muscle fibre action potentials from four high density surface electromyogram (HDsEMG) arrays, quantifying residual EMG comparing the recorded and data-based simulation (Fig. 1A-B), and developing a metric to compare the spatial identification for each motor unit (Fig. 1D-E). From identified motor units, the authors have provided a detailed characterization of recruitment and firing rate responses during slow voluntary isometric contractions in the vastus lateralis and tibialis anterior muscles up to 75-80% of maximum intensity. In the lower limb it is interesting how lower threshold motor units have firing rate responses that saturate, whereas higher threshold units that presumably produce higher muscle contractile forces continue to increase their firing rate. Conceptually, the authors rightly focus on the literature of intrinsic motoneurone properties, but in vivo, other possibilities (that are difficult to measure in awake human participants) are that the form of descending supraspinal drive, spinal network dynamics and afferent inputs may have different effects across motor unit sizes, muscles and types of contractions. These results from single trail contractions and with a larger sample of motor units, supports the summary rate coding profiles of motor units in the extensor digitorum communis muscle (Monster and Chan, 1977).

Reviewer #3 (Public review):

Summary:

This is an interesting manuscript which uses state of the art experimental and simulation approaches to quantify motor unit discharge patterns in the human TA and VL. The non-linear profiles of motor unit discharge were calculated and found to have an initial acceleration phase followed by an attenuation phase. Lower threshold motor units had a larger gain of the initial acceleration whereas the higher threshold motor unit had a higher gain in the attenuation phase. These data represent a technical feat and are important for understanding how humans generate and control voluntary force.

Strengths:

The authors used rigorous, state-of-the art analyses to decompose and validate their motor unit data during a wide range of voluntary efforts.

Analyses are clearly presented, applied, and visualized.

The supplemental data provides important transparency.

Weaknesses:

Number of participants and muscles tested are relatively small - particularly given the constraints on yield. It is unclear if this will translate to other motor pools. The justification for TA and VL should be provided.

While in impressive effort was made to identify and track motor units across a range of contractions, it appears that a substantial portion of muscle force was not identified. Though high intensity contractions are challenging to decompose - the authors are commended in their technical ability in recording population motor unit discharge times with recruitment thresholds up to 75% a participant's maximal voluntary contractions. However previous groups have seen substantial recruitment motor units above 80% and even 90% maximum activation in the soleus. Given the innervation ratios of higher threshold motor units, if recruitment continued to 100%, the top quartile would likely represent a substantial portion of the traditional fast-fatigable motor units. It would be highly interesting to understand the recruitment and rate coding of the highest threshold motor units, at a minimum I would suggest using terms other than "entire range" or "full spectrum of recruitment thresholds"

The quantification of hysteresis using torque appears to make self-evident the observation that lower threshold motor units demonstrate less hysteresis with respect to torque - If there was motor unit discharge there will be force. I believe this limitation goes beyond the floor effects discussed in the manuscript. Traditionally individuals have used the discharge of a lower threshold unit as the measure on which to apply hysteresis analyses to infer ion channel function in human spinal motoneurons.

The main findings are not entirely novel. See Monster and Chan 1977 and Kanosue et al 1979

Comments on revisions:

I thank the authors for their thoughtful revision.

Just to confirm, the ranges for motor unit yield are for a single contraction. So, for example, in a participant there were 71 unique and concurrently active VL motor units able to be decomposed.

Reviewer #2 (Public review):

Summary:

In this manuscript, Otero-Coronel and colleagues use a combination of acoustic stimuli and electrical stimulation of the tectum to study MSI in the M-cells of adult goldfish. They first perform a necessary piece of groundwork in calibrating tectal stimulation for maximal M-cell MSI, and then characterize this MSI with slightly varying tectal and acoustic inputs. Next, they quantify the magnitude and timing of FFI that each type of input has on the M-cell, finding that both the tectum and the auditory system drive FFI, but that FFI decays more slowly for auditory signals. These are novel results that would be of interest to a broader sensory neuroscience community. By then providing pairs of stimuli separated by 50ms, they assess the ability of the first stimulus to suppress responses to the second, finding that acoustic stimuli strongly suppress subsequent acoustic responses in the M-cell, that they weakly suppress subsequent tectal stimulation, and that tectal stimulation does not appreciably inhibit subsequent stimuli of either type. Finally, they show that M-cell physiology mirrors previously reported behavioural data in which stronger stimuli underwent less integration.

The manuscript is generally well written and clear. The discussion of results is appropriately broad and open-ended. It's a good document. Our major concerns regarding the study's validity are captured in the individual comments below. In terms of impact, the most compelling new observation is the quantification of the FFI from the two sources and the logical extension of these FFI dynamics to M-cell physiology during MSI. It is also nice, but unsurprising, to see that the relationship between stimulus strength that MSI is similar for M-cell physiology to what has previously been shown for behavior. While we find the results interesting, we think that they will be of greatest interest to those specifically interested in M-cell physiology and function.

Strengths:

The methods applied are challenging and appropriate and appear to be well executed. Open questions about the physiological underpinnings of M-cell function are addressed using sound experimental design and methodology, and convincing results are provided that advance our understanding of how two streams of sensory information can interact to control behavior.

Weaknesses:

Our concerns about the manuscript are captured in the following specific comments, which we hope will provide a useful perspective for readers and actionable suggestions for the authors.

Comments relevant to the revised manuscript:

Our general assessment (above) stands unchanged from the original version. All of our comments and concerns about the original manuscript have been addressed except for two, one very minor and one quite important:

Original Comment 1 (Minor):<br /> "Line 124. Direct stimulation of the tectum to drive M-cell-projecting tectal neurons not only bypasses the retina, it also bypasses intra-tectal processing and inputs to the tectum from other sources (notably the thalamus). This is not an issue with the interpretation of the results, but this description gives the (false) impression that bypassing the retina is sufficient to prevent adaptation. Adding a sentence or two to accurately reflect the complexity of the upstream circuitry (beyond the retina) would be welcome."

The authors have replied:<br /> "The reviewer is right in that direct tectal stimulation bypasses all neural processing upstream, not only that produced in the retina and that the tectum does not exclusively process visual information. The revised version now acknowledges (lines 245-252, revised manuscript) the complexity of the system."

We think that this is sufficient to address our concern. Some citations may be in order to underpin the new text.

Original Comment 5 (Major):<br /> Figure 4C and lines 398-410.<br /> "These are beautiful examples of M-cell firing, but the text suggests that they occurred rarely and nowhere close to significantly above events observed from single modalities. We do not see this a valid result to report because there is insufficient evidence that the phenomenon shown is consistent or representative of your data."

The authors have replied:<br /> "Our experimental conditions required anesthesia and paralysis, conditions designed to reduce neuronal firing and suppress motor output. We think it is valuable to report that we still see that simultaneous presentation subthreshold unisensory stimuli can add up to become suprathreshold, paralleling behavioral observations. We do not claim and acknowledge that those examples are representative of our recording conditions, but are likely to be more representative of the multisensory integration process taking place in freely moving fish. The revised manuscript adds context to these example traces to justify their inclusion (lines 420-426)."

We do not feel that this important concern has been addressed. The stats are definitively negative. There is no statistical evidence from these data that multisensory integration is occurring in this assay. The aesthesia, paralysis, and low n may provide explanations for this negative result, but it is still a negative result (p=0.5269). To show two examples of multisensory integration for subthreshold stimuli fits the narrative, but this result is not supported. Examples where individual stimuli caused APs (and combined stimuli did not) also occurred, presumably, and at a rate that is statistically indistinguishable to the examples shown in Figure 5. As such, if results from this assay are going to be in the manuscript, acoustic-only and tectum-only examples should be shown as well, although they would not fit the narrative. To be meaningful, this experiment would have to show that multisensory integration is happening in this circuit. Frustrating though it must be, the experiment has given a negative result to that question.

Reviewer #1 (Public review):

Summary

A novel statistical model of neural population activity called the Random Projection model has been recently proposed. Not only is this model accurate, efficient, and scalable, but also is naturally implemented as a shallow neural network. This work proposes a new class of RP model called the reshaped RP model. Inheriting the virtue of the original RP model, the proposed model is more accurate in terms of data fitting and efficient in terms of lower firing rate than the original, as well as compatible with various biological constraints. In particular, the authors have demonstrated that normalizing the total synaptic input in the reshaped model has a homeostatic effect on the firing rates of the neurons, resulting in even more efficient representations with equivalent accuracy. These results suggest that synaptic normalization contributes to synaptic homeostasis as well as efficiency in neural encoding.

Strength

This paper demonstrates that the accuracy and efficiency of the random projection models can be improved by extending the model with reshaped projections. Furthermore, it broadens the applicability of the model under biological constraints of synaptic regularization. It also suggests the advantage of the sparse connectivity structure over the fully connected model for modeling spiking statistics. In summary, this work successfully integrates two different elements, statistical modeling of the spikes and synaptic homeostasis in a single biologically plausible neural network model. The authors logically demonstrate their arguments with clear visual presentations and well-structured text, facilitating an unambiguous understanding for readers.

Discussions

The authors have clearly responded to most of our questions in the revised manuscript and we are happy to recommend publishing the final version of the article as it is. Below, we would like to present some alternative interpretations of the results. These comments are not exclusive with the claims made in the articles; it is rather intended to enhance the understanding of readers by providing additional perspectives.

As summarized above, the main contribution of the work consists of two parts; (1) the reshaped RP model achieved higher performance in explaining the statistics of the spiking activity of cortical neurons with more efficient representations (=lower firing rate), (2) synaptic homeostatic normalization in the reshaped RP model yields even more efficient representations without impairing the fitting performance.

For part (1),<br /> Suppl. Fig. 1B compares reshaped RP models with RP and RP with pruning and replacement (R&P). The better performance of RP with P&R might imply the advantage of pruning over gradient descent in this setting, reflecting the non-convexities of the problem. Alternatively, it might suggest the benefit of the increased number of parameters, since pruning allows the network to explore the broader parameter space during the learning process. This latter view might partially explain the superiority of the reshaped RP model over the original RP model.<br /> It is interesting that the backprop model has higher firing rate than the reshaped model (Fig. 1D). This trend is unchanged when optimization of the neural threshold is also allowed (Supp. Fig. 2A). Since backprop model overperforms reshaped model slightly but robustly, high firing rates in the backprop model might be a genuine feature of the spike statistics.

For part (2),<br /> We note that λ regulates the average firing rate, since maximizing the entropy <-ln p(x)> with a regularization term -λ <\sum _i f(x_i)> results in λ_i = λ for all i in the Boltzmann distribution of eq. 2. Suppl. Fig. 2B could be understood as demonstrating this "homeostatic" effect of λ.<br /> Suppl. Fig. 3 could be interpreted as demonstrating the interaction of two different homeostatic mechanisms: one at the firing-rate level (as regulated by λ) and the other at the synaptic level (as regulated by φ). It shows that the range of synaptic constraints where the fitting performance is not impaired differs by the value of λ. For example, if lambda is small (\lambda = 0.25), synaptic constraint can easily deteriorate the performance; on the other hand, if lambda is large (\lambda = 4), performance remains unchanged under strict synaptic constraint. Considering that practically we are most interested in the regime where the model performs best (λ = 2.0), an advantageous feature of the homeostatic model is that homeostatic constraint is harmless at λ=2.0 for the wide range of constraints.

Reviewer #1 (Public review):

Summary:

The manuscript presents a short report investigating mismatch responses in the auditory cortex, following previous studies focused on visual cortex. By correlating mouse locomotion speed with acoustic feedback levels, the authors demonstrate excitatory responses in a subset of neurons to halts in expected acoustic feedback. They show a lack of responses to mismatch in he visual modality. A subset of neurons show enhanced mismatch responses when both auditory and visual modalities are coupled to the animal's locomotion.

While the study is well-designed and addresses a timely question, several concerns exist regarding the quantification of animal behavior, potential alternative explanations for recorded signals, correlation between excitatory responses and animal velocity, discrepancies in reported values, and clarity regarding the identity of certain neurons.

Strengths:

(1) Well-designed study addressing a timely question in the field.<br /> (2) Successful transition from previous work focused on visual cortex to auditory cortex, demonstrating generic principles in mismatch responses.<br /> (3) Correlation between mouse locomotion speed and acoustic feedback levels provides evidence for prediction signal in the auditory cortex.<br /> (4) Coupling of visual and auditory feedback show putative multimodal integration in auditory cortex.

Weaknesses:

(1) Lack of quantification of animal behavior upon mismatches, potentially leading to alternative interpretations of recorded signals.<br /> (2) Unclear correlation between excitatory responses and animal velocity during halts, particularly in closed-loop versus playback conditions.<br /> (3) Discrepancies in reported values in a few figure panels raise questions about data consistency and interpretation.<br /> (4) Ambiguity regarding the identity of the [AM+VM] MM neurons.

Comments on revisions:

I am satisfied with all clarifications and additional analyses performed by the authors.<br /> The only concern I have is about changes in running after [AM+VM] mismatches.<br /> The authors reported that they "found no evidence of a change in running speed or pupil diameter following [AM + VM] mismatch (Figures S5A)" (line 197).<br /> Nevertheless, it seems that there is a clear increase in running speed for the [AM+VM] condition (S5A). Could this be more specifically quantified? I am concerned that part of the [AM+VM] could stem from this change in running behavior. Could one factor out the running contribution?

Reviewer #2 (Public review):

In this study, Solyga and Keller use multimodal closed-loop paradigms in conjunction with multiphoton imaging of cortical responses to assess whether and how sensorimotor prediction errors in one modality influence the computation of prediction errors in another modality. Their work addresses an important open question pertaining to the relevance of non-hierarchical (lateral cortico-cortical) interactions in predictive processing within the neocortex.

Specifically, they monitor GCaMP6f responses of layer 2/3 neurons in the auditory cortex of head-fixed mice engaged in VR paradigms where running is coupled to auditory, visual, or audio-visual sensory feedback. The authors find strong auditory and motor responses in the auditory cortex, as well as weak responses to visual stimuli. Further, in agreement with previous work, they find that the auditory cortex responds to audiomotor mismatches in a manner similar to that observed in visual cortex for visuomotor mismatches. Most importantly, while visuomotor mismatches by themselves do not trigger significant responses in the auditory cortex, simultaneous coupling of audio-visual inputs to movement non-linearly enhances mismatch responses in the auditory cortex.

Their results thus suggest that prediction errors within a given sensory modality are non-trivially influenced by prediction errors from another modality. These findings are novel, interesting, and important, especially in the context of understanding the role of lateral cortico-cortical interactions and in outlining predictive processing as a general theory of cortical function.

Comments on revisions:

The authors thoroughly addressed the concerns raised. In my opinion, this has substantially strengthened the manuscript, enabling much clearer interpretation of the results reported. I commend the authors for the response to review. Overall, I find the experiments elegantly designed, and the results robust, providing compelling evidence for non-hierarchical interactions across neocortical areas and more specifically for the exchange of sensorimotor prediction error signals across modalities.

Reviewer #3 (Public review):

This study explores sensory prediction errors in sensory cortex. It focuses on the question of how these signals are shaped by non-hierarchical interactions, specifically multimodal signals arising from same level cortical areas. The authors used 2-photon imaging of mouse auditory cortex in head-fixed mice that were presented with sounds and/or visual stimuli while moving on a ball. First, responses to pure tones, visual stimuli and movement onset were characterized. Then, the authors made the running speed of the mouse predictive of sound intensity and/or visual flow (closed loop). Mismatches were created through the interruption of sound and/or visual flow for 1 second, disrupting the expected sensory signal. As a control, sensory stimuli recorded during the close loop phase were presented again decoupled from the movement (open loop). The authors suggest that auditory responses to the unpredicted interruption of the sound, which affected neither running speed nor pupil size, reflect mismatch responses. That these mismatch responses were enhanced when the visual flow was congruently interrupted, indicates cross-modal influence of prediction error signals.

This study's strengths are the relevance of the question and the design of the experiment. The authors are experts in the techniques used. The analysis explores neither the full power of the experimental design nor the population activity recorded with 2-photon, leaving open the question of to what extend what the authors call mismatch responses are not sensory responses to sound interruption (offset responses). The auditory system is sensitive to transitions and indeed responses to the interruption of the sound are similar in quality, if not quantity, in the predictive and the control situation.

Comments on revisions:

The incorporation of the analysis of the animal's running speed and the pupil size upon sound interruption improves the interpretation of the data. The authors can now conclude that responses to the mismatch are not due to behavioral effects.<br /> The issue of the relationship between mismatch responses and offset responses remains uncommented. The auditory system is sensitive to transitions, also to silence. See the work of the Linden or the Barkat labs (including the work of the first author of this manuscript) on offset responses, and also that of the Mesgarani lab (Khalighinejad et al., 2019) on responses to transitions 'to clean' (Figure 1c) in human auditory cortex. Offset responses, as the first author knows well, are modulated by intensity and stimulus length (after adaptation?). That responses to the interruption of the sound are similar in quality, if not quantity, in the closed and open loop conditions suggest that offset response might modulate the mismatch response. A mismatch response that reflects a break in predictability would presumably be less modulated by the exact details of the sensory input than an offset response. Therefore, what is the relationship between the mismatch response and the mean sound amplitude prior to the sound interruption (for example during the preceding 1 second)? And between the mismatch response and the mean firing rate over the same period?<br /> Finally, how do visual stimuli modulate sound responses in the absence of a mismatch? Is the multimodal response potentiation specific to a mismatch?

Reviewer #1 (Public review):

Summary:

This paper is focused on the role of Cadherin Flamingo (Fmi) in cell competition in developing Drosophila tissues. A primary genetic tool is monitoring tissue overgrowths caused by making clones in the eye disc that expression activated Ras (RasV12) and that are depleted for the polarity gene scribble (scrib). The main system that they use is ey-flp, which make continuous clones in the developing eye-antennal disc beginning at the earliest stages of disc development. It should be noted that RasV12, scrib-i (or lgl-i) clones only lead to tumors/overgrowths when generated by continuous clones, which presumably creates a privileged environment that insulates them from competition. Discrete (hs-flp) RasV12, lgl-i clones are in fact out-competed (PMID: 20679206), which is something to bear in mind. They assess the role of fmi in several kinds of winners, and their data support the conclusion that fmi is required for winner status. However, they make the claim that loss of fmi from Myc winners converts them to losers, and the data supporting this conclusion is not compelling.

Strengths:

Fmi has been studied for its role in planar cell polarity, and its potential role in competition is interesting.

Reviewer #2 (Public review):

Summary:

In this manuscript, Bosch et al. reveal Flamingo (Fmi), a planar cell polarity (PCP) protein, is essential for maintaining 'winner' cells in cell competition, using Drosophila imaginal epithelia as a model. They argue that tumor growth induced by scrib-RNAi and RasV12 competition is slowed by Fmi depletion. This effect is unique to Fmi, not seen with other PCP proteins. Additional cell competition models are applied to further confirm Fmi's role in 'winner' cells. The authors also show that Fmi's role in cell competition is separate from its function in PCP formation.

Strengths:

(1) The identification of Fmi as a potential regulator of cell competition under various conditions is interesting.<br /> (2) The authors demonstrate that the involvement of Fmi in cell competition is distinct from its role in planar cell polarity (PCP) development.

Reviewer #3 (Public review):

Summary:

In this manuscript, Bosch and colleagues describe an unexpected function of Flamingo, a core component of the planar cell polarity pathway, in cell competition in Drosophila wing and eye disc. While Flamingo depletion has no impact on tumour growth (upon induction of Ras and depletion of Scribble throughout the eye disc), and no impact when depleted in WT cells, it specifically tunes down winner clone expansion in various genetic contexts, including the overexpression of Myc, the combination of Scribble depletion with activation of Ras in clones or the early clonal depletion of Scribble in eye disc. Flamingo depletion reduces proliferation rate and increases the rate of apoptosis in the winner clones, hence reducing their competitiveness up to forcing their full elimination (hence becoming now "loser"). This function of Flamingo in cell competition is specific of Flamingo as it cannot be recapitulated with other components of the PCP pathway, does not rely on interaction of Flamingo in trans, nor on the presence of its cadherin domain. Thus, this function is likely to rely on a non-canonical function of Flamingo which may rely on downstream GPCR signaling.

This unexpected function of Flamingo is by itself very interesting. In the framework of cell competition, these results are also important as they describe, to my knowledge, one of the only genetic conditions that specifically affect the winner cells without any impact when depleted in the loser cells. Moreover, Flamingo do not just suppress the competitive advantage of winner clones, but even turn them in putative losers. This specificity, while not clearly understood at this stage, opens a lot of exciting mechanistic questions, but also a very interesting long term avenue for therapeutic purpose as targeting Flamingo should then affect very specifically the putative winner/oncogenic clones without any impact in WT cells.

The data and the demonstration are very clean and compelling, with all the appropriate controls, proper quantifications and backed-up by observations in various tissues and genetic backgrounds. I don't see any weakness in the demonstration and all the points raised and claimed by the authors are all very well substantiated by the data. As such, I don't have any suggestions to reinforce the demonstration.

While not necessary for the demonstration, documenting the subcellular localisation and levels of Flamingo in these different competition scenarios may have been relevant and provide some hints on a putative mechanism (specifically by comparing its localisation in winner and loser cells).

Also, on a more interpretative note, the absence of impact of Flamingo depletion on JNK activation does not exclude some interesting genetic interactions. JNK output can be very contextual (for instance depending on Hippo pathway status), and it would be interesting in the future to check if Flamingo depletion could somehow alter the effect of JNK in the winner cells and promote downstream activation of apoptosis (which might normally be suppressed). It would be interesting to check if Flamingo depletion could have an impact in other contexts involving JNK activation or upon mild activation of JNK in clones.

Strengths:

- A clean and compelling demonstration of the function of Flamingo in winner cells during cell competition

- One of the rare genetic conditions that affects very specifically winner cells without any impact in losers, and then can completely switch the outcome of competition (which opens an interesting therapeutic perspective on the long term)

Reviewer #1 (Public review):

Summary:

Tobón and Moser reveal a remarkable amount of presynaptic diversity in the fundamental Ca dependent exocytosis of synaptic vesicles at the afferent fiber bouton synapse onto the pilar or mediolar sides of single inner hair cells of mice. These are landmark findings with profound implications for understanding acoustic signal encoding and presynaptic mechanisms of synaptic diversity at inner hair cell ribbon synapses. The paper will have an immediate and long-lasting impact in the field of auditory neuroscience.

Main findings: 1) Synaptic delays and jitter of masker responses are significantly shorter (synaptic delay: 1.19 ms) at high SR fibers (pilar) than at low SR fibers (mediolar; 2.57 ms). 2) Masked evoked EPSC are significantly larger in high SR than in low SR. 3) Quantal content and RRP size are 14 vesicles in both high and low SR fibers. 4) Depression is faster in high SR synapses suggesting they have a higher release probability and tighter Ca nanodomain coupling to docked vesicles. 5) Recovery of master-EPSCs from depletion is similar for high and low SR synapses, although there is a slightly faster rate for low SR synapses that have bigger synaptic ribbons, which is very interesting. 6) High SR synapses had larger and more compact (monophasic) sEPSCs, well suited to trigger rapidly and faithfully spikes. 7) High SR synapses exhibit lower voltage (~sound pressure in vivo) dependent thresholds of exocytosis.

Great care was taken to use physiological external pH buffers and physiological external Ca concentrations. Paired recordings were also performed at higher temperatures with IHCs at physiological resting membrane potentials and in more mature animals than previously done for paired recordings. This is extremely challenging because it becomes increasingly difficult to visualize bouton terminals when myelination becomes more prominent in the cochlear afferents. In addition, perforated patch recordings were used in the IHC to preserve its intracellular milieu intact and thus extend the viability of the IHCs. The experiments are tour-de-force and reveal several novel aspects of IHC ribbon synapses. The data set is rich and extensive. The analysis is detailed and compelling.

Reviewer #2 (Public review):

Summary:

The study by Jaime-Tobon & Moser is a truly major effort to bridge the gap between classical observations on how auditory neurons respond to sounds and the synaptic basis of these phenomena. The so-called spiral ganglion neurons (SGNs) are the primary auditory neurons connecting the brain with hair cells in the cochlea. They all respond to sounds increasing their firing rates, but also present multiple heterogeneities. For instance, some present a low threshold to sound intensity, whereas others have high threshold. This property inversely correlates with the spontaneous rate, i.e., the rate at which each neuron fires in the absence of any acoustic input. These characteristics, along with others, have been studied by many reports over years. However, the mechanisms that allow the hair cells-SGN synapses to drive these behaviors are not fully understood.

The level of experimental complexity described in this manuscript is unparalleled, producing data that is hardly found elsewhere. The authors provide strong proof for heterogeneity in transmitter release thresholds at individual synapses and they do so in an extremely complex experimental settings. In addition, the authors found other specific differences such as in synaptic latency and max EPSCs. A reasonable effort is put in bridging these observations with those extensively reported in in vivo SGNs recordings. Similarities are many and differences are not particularly worrying as experimental conditions cannot be perfectly matched, despite the authors' efforts in minimizing them.

Reviewer #3 (Public review):

Summary:

The manuscript by Jaime Tobon and Moser uses patch-clamp electrophysiology in cochlear preparations to probe the pre- and post-synaptic specializations that give rise to diverse activity of spiral ganglion afferent neurons (SGN). The experiments are quite an achievement! They use paired recordings from pre-synaptic cochlear inner hair cells (IHC) that allow precise control of voltage and therefore calcium influx, with post-synaptic recordings from type I SGN boutons directly opposed to the IHC for both presynaptic control of membrane voltage and post-synaptic measurement of synaptic function with great temporal resolution.

Any of these techniques by themselves are challenging, but the authors do them in pairs, at physiological temperatures, and in hearing animals, all of which combined make these experiments a real tour de force. The data is carefully analyzed and presented, and the results are convincing. In particular, the authors demonstrate that post-synaptic features that contribute to the spontaneous rate (SR) of predominantly monophasic post-synaptic currents (PSCs), shorter EPSC latency, and higher PSC rates are directly paired with pre-synaptic features such as a lower IHC voltage activation and tighter calcium channel coupling for release to give a higher probability of release and subsequent increase in synaptic depression. Importantly, IHCs paired with Low and High SR afferent fibers had the same total calcium currents, indicating that the same IHC can connect to both low and high SR fibers. These fibers also followed expected organizational patterns, with high SR fibers primarily contacting the pillar IHC face and low SR fibers primarily contacting the modiolar face. The authors also use in vivo-like stimulation paradigms to show different RRP and release dynamics that are similar to results from SGN in vivo recordings. Overall, this work systematically examines many features giving rise to specializations and diversity of SGN neurons.

Reviewer #1 (Public review):

Summary:

The study investigates protein-protein interactions (PPIs) within the nuage, a germline-specific organelle essential for piRNA biogenesis in Drosophila melanogaster, using AlphaFold2 to predict interactions among 20 nuage-localizing proteins. The authors identify five novel interaction candidates and experimentally validate three of them, including Spindle-E and Squash, through co-immunoprecipitation assays. They confirm the functional significance of these interactions by disrupting salt bridges at the Spn-E_Squ interface. The study further expands its scope to analyze approximately 430 oogenesis-related proteins, validating three additional interaction pairs. A comprehensive screen of around 12,000 Drosophila proteins for interactions with the key piRNA pathway player, Piwi, identifies 164 potential binding partners. Overall, the research demonstrates that in silico approaches using AlphaFold2 can link bioinformatics predictions with experimental validation, streamlining the identification of novel protein interactions and reducing the reliance on extensive experimental efforts. The manuscript is commendably clear and easy to follow; however, areas for improvement should be addressed to enhance its clarity and rigor.

Major Concerns:

(1) While AlphaFold2 was developed and trained primarily for predicting protein structures and their interactions, applying it to predict protein-protein interactions is an extrapolation of its intended use. This introduces several important considerations and risks. First, it assumes that AlphaFold's accuracy in structure prediction extends to interactions, despite not being explicitly trained for this task. Additionally, the assumption that high-scoring models with structural complementarity imply biologically relevant interactions is not always valid. Experimental validation is essential to address these uncertainties, as over-reliance on computational predictions without such validation can lead to false positives and inaccurate conclusions. The authors should expand on the assumptions, limitations, and risks associated with using AlphaFold2 for predicting protein-protein interactions.

(2) The authors experimentally validated three interactions, out of five predicted interactions, using co-immunoprecipitation (co-IP). They attributed the lack of validation for the other two predictions to the limitations of the co-IP method. However, further clarification on the potential limitations of the co-immunoprecipitation behind the negative results would strengthen the conclusions. While co-IP is a widely used technique, it may not detect weak or transient interactions, which could explain the failure to validate some predictions. Suggesting alternative validation methods such as FRET or mass spectrometry could further substantiate the results. On the other hand, AlphaFold2 predictions are not infallible and may generate false positives, particularly when dealing with structurally plausible but biologically irrelevant interactions. By acknowledging both the potential limitations of co-IP and the possibility of false positives from AlphaFold2, the authors can provide a more balanced interpretation of their findings.

(3) In line 143, the authors state that "This approach identified 13 pairs; seven of these were already known to form complexes, confirming the effectiveness of AlphaFold2 in predicting complex formations (Table 2). The highest pcScore pair was the Zuc homodimer, possibly because AlphaFold2 had learned from Zuc homodimer's crystal structure registered in the database." While the authors mentioned the presence of the Zuc homodimer's crystal structure, they do not provide a systematic bioinformatics analysis to evaluate pairwise sequence identity or check for the presence of existing structures for all the proteins or protein pairs (or their homologs) in databases such as the Protein Data Bank (PDB) or Swiss-Model. Conducting such an analysis is critical, as it significantly impacts the novelty and reliability of AlphaFold2 predictions. For instance, high sequence identity between the query proteins could lead to high-scoring models for biologically irrelevant interactions. Including this information would strengthen the conclusions regarding the accuracy and utility of the predictions.

(4) While the manuscript successfully identifies novel protein interactions, the broader biological significance of these interactions remains underexplored. The manuscript could benefit from elaborating on how these findings may contribute to understanding the piRNA pathway and its implications on germline development, transposon repression, and oogenesis.

Reviewer #2 (Public review):

Summary:

In this paper, the authors use AlphaFold2 to identify potential binding partners of nuage localizing proteins.

Strengths:

The main strength of the paper is that the authors experimentally verify a subset of the predicted interactions.

Many studies have been performed to predict protein-protein interactions in various subsets of proteins. The interesting story here is that the authors (i) focus on an organelle that contains quite some intrinsically disordered proteins and (ii) experimentally verify some (but not all) predictions.

Weaknesses:

Identification of pairwise interactions is only a first step towards understanding complex interactions. It is pretty clear from the predictions that some (but certainly not all) of the pairs could be used to build larger complexes. AlphaFold easily handles proteins up to 4-5000 residues, so this should be possible. I suggest that the authors do this to provide more biological insights.

Another weakness is the use of a non-standard name for "ranking confidence" - the author calls it the pcScore - while the name used in AlphaFold (and many other publications) is ranking confidence.

Reviewer #1 (Public review):

Summary:

Through a series of CRISPR-Cas9 screens, the GPX4 antioxidant pathway was identified as a critical suppressor of cold-induced cell death in hibernator-derived cells. Hamster BHK-21 cells exposed to repeated cold and rewarming cycles revealed five genes (Gpx4, Eefsec, Pstk, Secisbp2, and Sepsecs) as critical components of the GPX4 pathway, which protects against cold-induced ferroptosis. A second screen with continuous cold exposure confirmed the essential role of GPX4 in prolonged cold tolerance. GPX4 knockout lines exhibited complete cell death within four days of cold exposure, and pharmacological inhibition of GPX4 further increased cell death, underscoring the necessity of GPX4's catalytic activity in cold conditions.

An additional CRISPR screen in human cold-sensitive K562 cells identified 176 genes for cold survival. The GPX4 pathway was found to confer significant resistance to cold in hibernators and human cells, with GPX4 loss significantly increasing cold-induced cell death.

Comparing hamster and human GPX4, overexpression of GPX4 in human K562 cells, whether hamster or human GPX4, dramatically improved cold tolerance, while catalytically dead mutants showed no such effect. These findings suggest that GPX4 abundance is a key limiting factor for cold tolerance in human cells, and primary cell types show strong sensitivity to GPX4 loss, highlighting that differences in cold tolerance across species may be due to varying GPX4-mediated protection.

Strengths:

(1) Innovative Approach: The study employs a series of unbiased genome-wide CRISPR-Cas9 screens in both hibernator- and non-hibernator-derived cells to investigate the mechanisms controlling cellular cold tolerance. Notably, this is the first genome-scale CRISPR-Cas9 screen conducted in cells derived from a hibernator, the Syrian hamster.

(2) Identification of the GPX4 Pathway: Identifying glutathione peroxidase 4 (GPX4) as a critical suppressor of cold-induced cell death significantly contributes to the field. Recently, GPX4 was also reported as a potent regulator of cold tolerance through overexpression screening (Sone et al.) in hamsters, which further supports this finding.

(3) Improved Cold Viability Assessment: The study identifies an important technical artifact in using trypan blue to assess cell viability following cold exposure. It reveals that cells stained immediately after cold exposure retain the dye, inaccurately indicating cell death. By introducing a brief rewarming period before viability assessment, the authors significantly improve the accuracy of detecting cold-induced cell death. This refinement in methodology ensures more reliable results and sets a new standard for future research on cold stress in cells.

Weaknesses:

(1) Mechanisms Regulating GPX4 Levels: While the study highlights GPX4 levels as a major determinant of cellular cold tolerance, it does not discuss how these levels are regulated or why they differ between hibernators and non-hibernators. This omission leaves an important aspect of GPX4's role in cold tolerance unexplored.

(2) Generalizability Across Species: Although the study demonstrates the role of GPX4 in several mammalian species, it does not investigate whether this mechanism extends to other vertebrates (e.g., fish and amphibians) that also face cold challenges. This limitation could restrict the broader evolutionary claims made by the study.

(3) Variability in Cold Sensitivity Across Human Cell Lines: The study observes significant variability in cold tolerance among different human cell lines but does not explain these differences clearly. This leaves a key aspect of human cell cold sensitivity insufficiently addressed.

Reviewer #2 (Public review):

Summary:

Lam et al., present a very intriguing whole genome CRISPR screen in Syrian Hamster cells as well as K562 cells to identify key genes involved in hypothermia-rewarming tolerance. Survival screens were performed by exposing cells to 4C in a cooled CO2 incubator followed by a rewarming period of 30 minutes prior to survival analysis. In this paradigm, Syrian hamster-derived cell lines exhibit more robust survival than human cell lines (BHK-21 and HaK vs HT1080, HeLa, RPE1, and K562). A genome-wide Syrian hamster CRISPR library was created targeting all annotated genes with 10 guides/gene. LV transduction of the library was performed in BHK-21 cells and the survival screen procedures involved 3 cycles of 4C cold exposure x4 days followed by 2 days of re-warming.

When compared to controls maintained at 37C, 9 genes were required for BHK-21 survival of cold cycling conditions and 5 of these 9 are known components of the GPX4 antioxidant pathway. GPX4 KO BHK-21 cells had reduced cell growth at 37C and profoundly worse cold tolerance which could be reduced by GPX4 expression. GPX4 inhibitors also reduced survival in cold. CRISPR KO screens and GPX4 KO in K562 cells revealed comparable results (though intriguingly glutathione biosynthesis genes were more critical to K562 cells than BHK-21 cells). Human or Syrian hamster GPX4 overexpression improved cold tolerance.

Strengths:

This is a very nicely written paper that clearly communicates in figures and text complicated experimental manipulations and in vitro genetic screening and cell survival data. The focus on GPX4 is interesting and relatively novel. The converging pharmacologic, loss-of-function, and gain-of-function experiments are also a strength.

Weaknesses:

A recently published article (Reference 43, Sone et al.) also independently explored the role of GPX4 in Syrian hamster cold tolerance through gain-of-function screening. Further exploration of the GPX4 species-specific mechanisms would be of great interest, but this is considered a minor weakness given the already very comprehensive and compelling data presented.

Reviewer #3 (Public review):

Summary:

This work aims to address a fundamental biological question: how do mammalian cells achieve/lose tolerance to cold exposure? The authors first tried to establish an experimental system for cell cold exposure and evaluation of cell death and then performed genome-scale CRISPR-Cas9 screening on immortalized cell lines from Syrian Hamster (BHK-21) and human (K562) for key genes that are associated with cell survival during prolonged cold exposure. From these screenings, they focused on glutathione peroxidase 4 (GPX4). Using genetic modifications or pharmacological interventions, and multiple cell models including primary cells from various mammalian species, they showed that GPX4 proteins are likely to retain their activities at 4 {degree sign}C, functioning to prevent cold-induced cell ferroptosis.

Strengths:

(1) This paper is neatly written and hence easy to follow.

(2) Experiments are well designed.

(3) The data showing the overall good cell survival after a prolonged cold exposure or repeated cold-warm cycles are helpful to show the advantages of the experimental instruments and methods the authors used, and hence the validity of their results.

(4) The CRISPR-Cas9 screening is a great attempt.

(5) Multiple cell types from hibernating mammals (cold tolerant) and cold-intolerant species are used to test their findings.

(6) Although some may argue that other labs have published works with different approaches that have pointed out the importance of GPX4 and ferroptosis in hamster cell survival from anoxia-reoxygenation or cold exposure models, hence hurting the novelty of this work, this reviewer thinks that it is highly valuable to have independent research groups and different methods/systems to validate an important concept.

Weaknesses:

(1) Only cell death was robustly surveyed; though cell proliferation was evaluated too in some experiments, other cellular functions, such as mitochondrial ATP production vs. glycolysis, and the extent of lipid peroxidation, could have been measured to reflect cellular physiology.

Validations on complex tissues or in vivo systems would have further strengthened the work and its impact.

CRISPR-Cas9 screening may have technical limitations as knock-out of some essential genes/pathways may lead to cell lethality during screening, and hence the relevance of these genes/pathways to cell cold tolerance may not be noted. From the data presented in this study, this reviewer thinks that the GPX4 pathway is likely a conserved mechanism for long-term cold survival, but not for cold sensitivity or acute cell death from cold exposure. In line with my such speculation, their CRISPR-Cas9 screening revealed genes in the GPX4 pathway from a relatively cold-sensitive human cell line, but the endogenous GPX4 pathway is seemingly operational in this cold-sensitive cell line. Also, these cells are viable after GPX4 knock-out. Dead cells from the acute cold exposure phase may detached, or their genomic DNAs have been severely damaged by the time of sample collection, hence not giving any meaningful sequencing reads. Crippling other factors/pathways such as FOXO1 (PMID: 38570500) or 5-aminolevulinic acid (ALA) metabolism (PMID: 35401816) have been shown to severely aggravate cold-induced cell death, including TUNEL-revealed DNA damage, within a much shorter time scale, whilst loss-function knockouts of FOXO1 or ALA Synthase 1 (ALAS1) are usually cell lethal. Thus, they and other possible essential genes may not be screenable from the current experimental protocol. These important points need to be taken into consideration by the authors.

Reviewer #1 (Public review):

Summary:

This is an important and very well-presented set of experiments following up on prior work from the lab investigating knock-down (KD) of EMC10 in the restoration of neuronal and cognitive deficits in 22q11.2 Del models, including now both human iPSCs and a mouse model in vivo now with ASOs.

The valuable progress in this current manuscript is the development of ASOs, and the proof of efficacy in vivo in mice of the ASO in knock-down of EMC10 and amelioration of in vivo behavioral phenotypes.

The experiments include iPSC studies demonstrating elevations of EMC10 in a solid collection of paired iPSC lines. These studies also provide evidence of manipulation of EMC10 by overexpression and inhibition of miRNAs that exist in the 22q11 interval. The iPSC studies also nicely demonstrate the rescue of impairments with KD of EMC10 in neuronal arborization as well as KCl-induced neuronal activity. The major in vivo contributions reflect an impressive demonstration of the efficacy of two ASOs in vivo on both KD of EMC10 in vivo and through improvement in behavioral abnormalities in the 22q11 mouse in a range of different behaviors, including social behavior and learning behaviors.

Overall, there are many strengths reflected in this study, including in particular the synergy between in vitro studies in human cell models and in vivo studies in the well-characterized mouse model. The experiments are generally rigorously performed, well-powered, and nicely presented. The claims with regard to the mechanisms of EMC10 elevations and the importance of restoration of EMC10 expression to neuronal morphology and behavior are well supported by the data. The work may be further supported in future studies, by investigation of rescue by ASOs of circuit dysfunction in vivo or ex vivo through electrophysiology in the mouse model. Also, in future studies, investigation of the mechanism by which EMC10, an ER protein involved in protein processing, may function in the observed neuronal abnormalities; however, these studies are clearly for future investigations.

The potential impact of the work is found in the potential value of the ASO approach to the treatment of 22q11, or the pre-clinical evidence that knock-down of this protein may lead to some amelioration of cognitive symptoms. Overall, a very convincing and complementary set of experiments to support EMC10 KD as a therapeutic strategy.

Reviewer #2 (Public review):

Summary:

The manuscript by Thakur et. al seeks to establish a novel ASO-based approach to treat 22q11.2 deletion syndrome. Central to this thesis is that an ER membrane complex member called EMC10 is significantly increased in the disorder, which is largely attributed to the loss of miRNA-mediated repression. The authors generated three new iPSC cell lines for the disorder and showed that deletion of EMC10 rescues morphology and Ca-flux deficits. They go on to show that post-symptomatic deletion of Emc10 in mice using a conditional-off tamoxifen allele reverses social memory phenotypes. Finally, in collaboration with Ionis, they developed two new ASOs to knock down EMC10 and show that social and spatial memory phenotypes are rescued, even two months after injection.

Strengths:

In general, this represents a substantial undertaking and an impressive body of work. The experiments follow a logical progression and in most cases are well-controlled. The isolation of EMC10 effects relative to the broader miRNA disruption is viewed as impactful. The use of both genetic and ASO approaches to validate the therapeutic strategy is also viewed as highly positive. The authors' contention that EMC10 can be targeted at post-symptomatic time points to reverse 22q11.2 deletion syndrome is supported by the data. Further, they have provided a therapeutic mechanism to do so. These findings are likely to be impactful and lead to further development efforts.

Weaknesses:

The primary weaknesses of the manuscript lie in incomplete or inappropriate data analysis, as well as a failure to validate key experiments. For example, both genetic and ASO-mediated EMC10-mediated reductions are assessed at the level of mRNA, but only one experiment, in one brain region, is validated at the protein level. This brain region is the PFC, which is problematic when many of the phenotypes used have a strong hippocampal component. Likewise, the iPSC experiments make the case that excitatory neurons are central to the phenotype, but no effort is made to show that the ASOs are entering that type of neuron, or even any quantification of what percentage of cells in the target brain regions (HPC, PFC, etc.) are positive for the ASO. There is only a single image provided of staining with a phosphorothioate antibody and a claim of robust uptake, which cannot be assumed. The iPSC transcriptomics work would also benefit from a more comprehensive comparison between the EMC10 knockout lines and their parent 22q11 deletion lines. Further, there are other examples where the statistics used are either wrong (Figure 3 t-test vs ANOVA) or missing (Figure S2). These technical and analytical shortcomings make it challenging to fully interpret the data and detract from an otherwise exciting manuscript.

Reviewer #1 (Public review):

Summary:

Chang and colleagues used tetrode recordings in behaving rats to study how learning an audiovisual discrimination task shapes multisensory interactions in the auditory cortex. They found that a significant fraction of neurons in the auditory cortex responded to visual (crossmodal) and audiovisual stimuli. Both auditory-responsive and visually-responsive neurons preferentially responded to the cue signaling the contralateral choice in the two-alternative forced choice task. Importantly, multisensory interactions were similarly specific for the congruent audiovisual pairing for the contralateral side.

Strengths:

The experiments were conducted in a rigorous manner. Particularly thorough are the comparisons across cohorts of rats trained in a control task, in a unisensory auditory discrimination task, and the multisensory task, while also varying the recording hemisphere and behavioral state (engaged vs. anesthesia). The resulting contrasts strengthen the authors' findings and rule out important alternative explanations. Through the comparisons, they show that the enhancements of multisensory responses in the auditory cortex are specific to the paired audiovisual stimulus and specific to contralateral choices in correct trials and thus dependent on learned associations in a task-engaged state.

Weaknesses:

The main result is that multisensory interactions are specific for contralateral paired audiovisual stimuli, which is consistent across experiments and interpretable as a learned task-dependent effect. However, the alternative interpretation of behavioral signals is crucial to rule out, which would also be specific to contralateral, correct trials in trained animals. Although the authors focus on the first 150 ms after cue onset, some of the temporal profiles of activity suggest that choice-related activity could confound some of the results.

The auditory stimuli appear to be encoded by short transient activity (in line with much of what we know about the auditory system), likely with onset latencies (not reported) of 15-30 ms. Stimulus identity can be decoded (Figure 2j) apparently with an onset latency around 50-75 ms (only the difference between A and AV groups is reported) and can be decoded near perfectly for an extended time window, without a dip in decoding performance that is observed in the mean activity Figure 2e. The dynamics of the response of the example neurons presented in Figures 2c and d and the average in 2e therefore do not entirely match the population decoding profile in 2j. Population decoding uses the population activity distribution, rather than the mean, so this is not inherently problematic. It suggests however that the stimulus identity can be decoded from later (choice-related?) activity. The dynamics of the population decoding accuracy are in line with the dynamics one could expect based on choice-related activity. Also the results in Figures S2e,f suggest differences between the two learned stimuli can be in the late phase of the response, not in the early phase.

First, it would help to have the same time axis across panels 2,c,d,e,j,k. Second, a careful temporal dissociation of when the central result of multisensory enhancements occurs in time would discriminate better early sensory processing-related effects versus later decision-related modulations.

In the abstract, the authors mention "a unique integration model", "selective multisensory enhancement for specific auditory-visual pairings", and "using this distinct integrative mechanisms". I would strongly recommend that the authors try to phrase their results more concretely, which I believe would benefit many readers, i.e. selective how (which neurons) and specific for which pairings?

Reviewer #2 (Public review):

Summary

In this study, rats were trained to discriminate auditory frequency and visual form/orientation for both unisensory and coherently presented AV stimuli. Recordings were made in the auditory cortex during behaviour and compared to those obtained in various control animals/conditions. The central finding is that AC neurons preferentially represent the contralateral-conditioned stimulus - for the main animal cohort this was a 10k tone and a vertically oriented bar. Over 1/3rd of neurons in AC were either AV/V/A+V and while a variety of multisensory neurons were recorded, the dominant response was excitation by the correctly oriented visual stimulus (interestingly this preference was absent in the visual-only neurons). Animals performing a simple version of the task in which responses were contingent on the presence of a stimulus rather than its identity showed a smaller proportion of AV stimuli and did not exhibit a preference for contralateral conditioned stimuli. The contralateral conditioned dominance was substantially less under anesthesia in the trained animals and was present in a cohort of animals trained with the reverse left/right contingency. Population decoding showed that visual cues did not increase the performance of the decoder but accelerated the rate at which it saturated. Rats trained on auditory and then visual stimuli (rather than simultaneously with A/V/AV) showed many fewer integrative neurons.

Strengths

There is a lot that I like about this paper - the study is well-powered with multiple groups (free choice, reversed contingency, unisensory trained, anesthesia) which provides a lot of strength to their conclusions and there are many interesting details within the paper itself. Surprisingly few studies have attempted to address whether multisensory responses in the unisensory cortex contribute to behaviour - and the main one that attempted to address this question (Lemus et al., 2010, uncited by this study) showed that while present in AC, somatosensory responses did not appear to contribute to perception. The present manuscript suggests otherwise and critically does so in the context of a task in which animals exhibit a multisensory advantage (this was lacking in Lemus et al.,). The behaviour is robust, with AV stimuli eliciting superior performance to either auditory or visual unisensory stimuli (visual were slightly worse than auditory but both were well above chance).

Weaknesses

I have a number of points that in my opinion require clarification and I have suggestions for ways in which the paper could be strengthened. In addition to these points, I admit to being slightly baffled by the response latencies; while I am not an expert in the rat, usually in the early sensory cortex auditory responses are significantly faster than visual ones (mirroring the relative first spike latencies of A1 and V1 and the different transduction mechanisms in the cochlea and retina). Yet here, the latencies look identical - if I draw a line down the pdf on the population level responses the peak of the visual and auditory is indistinguishable. This makes me wonder whether these are not sensory responses - yet, they look sensory (very tightly stimulus-locked). Are these latencies a consequence of this being AuD and not A1, or ... ? Have the authors performed movement-triggered analysis to illustrate that these responses are not related to movement out of the central port, or is it possible that both sounds and visual stimuli elicit characteristic whisking movements? Lastly, has the latency of the signals been measured (i.e. you generate and play them out synchronously, but is it possible that there is a delay on the audio channel introduced by the amp, which in turn makes it appear as if the neural signals are synchronous? If the latter were the case I wouldn't see it as a problem as many studies use a temporal offset in order to give the best chance of aligning signals in the brain, but this is such an obvious difference from what we would expect in other species that it requires some sort of explanation.

Reaction times were faster in the AV condition - it would be of interest to know whether this acceleration is sufficient to violate a race model, given the arbitrary pairing of these stimuli. This would give some insight into whether the animals are really integrating the sensory information. It would also be good to clarify whether the reaction time is the time taken to leave the center port or respond at the peripheral one.

The manuscript is very vague about the origin or responses - are these in AuD, A1, AuV... ? Some attempts to separate out responses if possible by laminar depth and certainly by field are necessary. It is known from other species that multisensory responses are more numerous, and show greater behavioural modulation in non-primary areas (e.g. Atilgan et al., 2018).

Reviewer #3 (Public review):

Summary:

The manuscript by Chang et al. aims to investigate how the behavioral relevance of auditory and visual stimuli influences the way in which the primary auditory cortex encodes auditory, visual, and audiovisual information. The main result is that behavioral training induces an increase in the encoding of auditory and visual information and in multisensory enhancement that is mainly related to the choice located contralaterally with respect to the recorded hemisphere.

Strengths:

The manuscript reports the results of an elegant and well-planned experiment meant to investigate if the auditory cortex encodes visual information and how learning shapes visual responsiveness in the auditory cortex. Analyses are typically well done and properly address the questions raised

Weaknesses:

Major

(1) The authors apparently primarily focus their analyses of sensory-evoked responses in approximately the first 100 ms following stimulus onset. Even if I could not find an indication of which precise temporal range the authors used for analysis in the manuscript, this is the range where sensory-evoked responses are shown to occur in the manuscript figures. While this is a reasonable range for auditory evoked responses, the same cannot be said for visual responses, which commonly peak around 100-120 ms, in V1. In fact, the latency and overall shape of visual responses are quite different from typical visual responses, that are commonly shown to display a delay of up to 100 ms with respect to auditory responses. All traces that the authors show, instead, display visual responses strikingly overlapping with auditory ones, which is not in line with what one would expect based on our physiological understanding of cortical visually-evoked responses. Similarly, the fact that the onset of decoding accuracy (Figure 2j) anticipates during multisensory compared to auditory-only trials is hard to reconcile with the fact that visual responses have a later onset latency compared to auditory ones. The authors thus need to provide unequivocal evidence that the results they observe are truly visual in origin. This is especially important in view of the ever-growing literature showing that sensory cortices encode signals representing spontaneous motor actions, but also other forms of non-sensory information that can be taken prima facie to be of sensory origin. This is a problem that only now we realize has affected a lot of early literature, especially - but not only - in the field of multisensory processing. It is thus imperative that the authors provide evidence supporting the true visual nature of the activity reported during auditory and multisensory conditions, in both trained, free-choice, and anesthetised conditions. This could for example be achieved causally (e.g. via optogenetics) to provide the strongest evidence about the visual nature of the reported results, but it's up to the authors to identify a viable solution. This also applies to the enhancement of matched stimuli, that could potentially be explained in terms of spontaneous motor activity and/or pre-motor influences. In the absence of this evidence, I would discourage the author from drawing any conclusion about the visual nature of the observed activity in the auditory cortex.

(2) The finding that AC neurons in trained mice preferentially respond - and enhance - auditory and visual responses pertaining to the contralateral choice is interesting, but the study does not show evidence for the functional relevance of this phenomenon. As has become more and more evident over the past few years (see e.g. the literature on mouse PPC), correlated neural activity is not an indication of functional role. Therefore, in the absence of causal evidence, the functional role of the reported AC correlates should not be overstated by the authors. My opinion is that, starting from the title, the authors need to much more carefully discuss the implications of their findings.

MINOR:

(1) The manuscript is lacking what pertains to the revised interpretation of most studies about audiovisual interactions in primary sensory cortices following the recent studies revealing that most of what was considered to be crossmodal actually reflects motor aspects. In particular, recent evidence suggests that sensory-induced spontaneous motor responses may have a surprisingly fast latency (within 40 ms; Clayton et al. 2024). Such responses might also underlie the contralaterally-tuned responses observed by the authors if one assumes that mice learn a stereotypical response that is primed by the upcoming goal-directed, learned response. Given that a full exploration of this issue would require high-speed tracking of orofacial and body motions, the authors should at least revise the discussion and the possible interpretation of their results not just on the basis of the literature, but after carefully revising the literature in view of the most recent findings, that challenge earlier interpretations of experimental results.

(2) The methods section is a bit lacking in details. For instance, information about the temporal window of analysis for sensory-evoked responses is lacking. Another example: for the spike sorting procedure, limited details are given about inclusion/exclusion criteria. This makes it hard to navigate the manuscript and fully understand the experimental paradigm. I would recommend critically revising and expanding the methods section.

Reviewer #1 (Public Review):

The authors investigate the role of chirping in a species of weakly electric fish. They subject the fish to various scenarios and correlate the production of chirps with many different factors. They find major correlations between the background beat signals (continuously present during any social interactions) or some aspects of social and environmental conditions with the propensity to produce different types of chirps. By analyzing more specifically different aspects of these correlations they conclude that chirping patterns are related to navigation purposes and the need to localize the source of the beat signal (i.e. the location of the conspecific).

The study provides a wealth of interesting observations of behavior and much of this data constitutes a useful dataset to document the patterns of social interactions in these fish. Some data, in particular the high propensity to chirp in cluttered environments, raises interesting questions. Their main hypothesis is a useful addition to the debate on the function of these chirps and is worth being considered and explored further.

After the initial reviewers' comments, the authors performed a welcome revision of the way the results are presented. Overall the study has been improved by the revisions.

Reviewer #2 (Public Review):

Studying Apteronotus leptorhynchus (the weakly electric brown ghost knifefish), the authors provide evidence that 'chirps' (brief modulations in the frequency and amplitude of the ongoing wave-like electric signal) function in active sensing (specifically homeoactive sensing) rather than communication. Chirping is a behavior that has been well studied, including numerous studies on the sensory coding of chirps and the neural mechanisms for chirp generation. Chirps are largely thought to function in communication behavior, so this alternative function is a very exciting possibility that should have a great impact on the field.

The authors provide convincing evidence that chirps may function in homeoactive sensing. In particular, the evidence showing increased chirping in more cluttered environments and a relationship between chirping and movement are especially strong and suggestive. Their evidence arguing against a role for chirps in communication is not as strong. However, based on an extensive review of the literature, the authors conclude, I think fairly, that the evidence arguing in favor of a communication function is limited and inconclusive. Thus, the real strength of this study is not that it conclusively refutes the communication hypothesis, but that it calls this hypothesis into question while also providing compelling evidence in favor of an alternative function.

In summary, although the evidence against a role for chirps in communication is not as strong as the evidence for a role in active sensing, this study presents very interesting data that is sure to stimulate discussion and follow-up studies. The authors acknowledge that chirps could function as both a communication and homeactive sensing signal, and the language arguing against a communication function is appropriately measured. A given electrical behavior could serve both communication and homeoactive sensing. I suspect this is quite common in electric fish (not just in gymnotiforms such as the species studied here, but also in the distantly related mormyrids), and perhaps in other actively sensing species such as echolocating animals.

Reviewer #3 (Public Review):

Summary:

This important paper provides the best-to-date characterization of chirping in weakly electric fish using a large number of variables. These include environment (free vs divided fish, with or without clutter), breeding state, gender, intruder vs resident, social status, locomotion state and social and environmental experience, without and with playback experiments. It applies state-of-the-art methods for reducing the dimensionality of the data and finding patterns of correlation between different kinds of variables (factor analysis, K-means). The strength of the evidence, collated from a large number of trials with many controls, leads to the conclusion that the traditionally assumed communication function of chirps may be secondary to its role in environmental assessment and exploration that takes social context into account. Based on their extensive analyses, the authors suggest that chirps are mainly used as probes that help detect beats caused by other fish as well as objects.

Strengths:

The work is based on completely novel recordings using interaction chambers. The amount of new data and associated analyses is simply staggering, and yet, well organized in presentation. The study further evaluates the electric field strength around a fish (via modelling with the boundary element method) and how its decay parallels the chirp rate, thereby relating the above variables to electric field geometry. The BEM modelling also convincingly predicts how the electric image of a receiver conspecific on a sending fish is enhanced by a chirp.

The main conclusions are that the lack of any significant behavioural correlates for chirping, and the lack of temporal patterning in chirp time series, cast doubt on a primary communication goal for most chirps. Rather, the key determinants of chirping are the difference in frequency between two interacting conspecifics as well as individual subjects' environmental and social experience. The paper concludes that there is a lack of evidence for stereotyped temporal patterning of chirp time series, as well as of sender-receiver chirp transitions beyond the known increase in chirp frequency during an interaction. The authors carefully submit that the new putative echolocation function of chirps is not mutually exclusive with a possible communication function.

These conclusions by themselves will be very useful to the field. They will also allow scientists working on other "communication" systems to perhaps reconsider and expand the goals of the probes used in those senses. A lot of data are summarized in this paper, with thorough referencing to past work.

The alternative hypotheses that arise from the work are that chirps are mainly used as environmental probes for better beat detection and processing and object localization, and in this sense are self-directed signals. This led to their prediction that environmental complexity ("clutter") should increase chirp rate, which is fact was revealed by their new experiments. The authors also argue that waveform EODs have less power across high spatial frequencies compared to pulse-type fish, with a resulting relatively impoverished power of resolution. Chirping in wave-type fish could temporarily compensate for the lower frequency resolution while still being able to resolve EOD perturbations with a good temporal definition (which pulse-type fish lack due to low pulse rates).

The authors also advance the interesting idea that the sinusoidal frequency modulations caused by chirps are the electric fish's solution to the minute (and undetectable by neural wetware) echo-delays available to it, due to the propagation of electric fields at the speed of light in water. The paper provides a number of experimental avenues to pursue in order to validate the non-communication role of chirps.

Reviewer #1 (Public review):

Summary:

The manuscript the authors have tried to dissect the functions of Proteasome activator 28γ (PA28γ) which is known to activate proteosomal function in an ATP independent manner. Although there are multiple works that have highlighted the role of this protein in tumour, this study specifically tried to develop a correlate with Complement C1q binding protein (C1QBp) that is associated with immune response and energy homeostasis.

Strengths:

The observations of the authors hint that beyond PA28y association with proteasome, it might also stabilize certain proteins such as C1QBP which influences the energy metabolism.

Weaknesses:

The strength of the work also becomes its main drawback. That is, how PA28y stabilizes C1QBP or how C1QBP elicits its pro-tumourigenic role under PA28y OE.

In most of the experiments the authors have been dependent on the parallel changes in the expression of both the proteins to justify their stabilizing interaction. However, this approach is indirect at best and does not confirm the direct stabilizing effect of this interaction. IP experiments do not indicate direct interaction and have some quality issues. The upregulation of C1QBP might be indirect at best. It is quite possible that PA28y might be degrading some secondary protein/complex which is responsible for C1QBP expression. Since the core idea of the work is PA28y direct interaction with C1QBP stabilizing it, the same should be demonstrated in more convincing manner.

In all of the assays C1QBP has been detected as doublet. However, the expression pattern of the two bands vary depending on the experiment. In some cases the upper band is intensely stained and in some the lower bands. Does C1QBP isoforms exist and whether they are differentially regulated depending on experiment conditions/tissue types?

Problems with the background of the work: Line 76. This statement is far-fetched. There are presently a number of literatures that have dealt with metabolic programming of OSCC including identification of specific metabolites. Moreover, beyond estimation of OCR, the authors have not conducted any experiments related to metabolism. In the Introduction, significance of this study and how it will extend our understanding of OSCC needs to be elaborated.

Review of Revised Version:

Although the authors have partly corrected the manuscript by removing the mislabeling in their Co-IP experiments, my primary concern on the actual functional connotations and direct interaction between PA28y and C1QBP still remains unaddressed. As already mentioned in my previous review, since the core idea of the work is PA28y's direct interaction with C1QBP, stabilizing it, the same should be demonstrated in a more convincing manner.

My other observation on the detection of C1QBP as a doublet has been addressed by usage of anti-C1QBP Monoclonal antibody against the polyclonal one used before. C1QBP doublets have not been observed in the present case.

The authors have also worked on the presentation of the background by suitably modifying the statements and incorporating appropriate citations.

However, the authors are requested to follow the recommendations provided to them by the reviewers to address the major concerns.

Reviewer #2 (Public review):

Summary:

The authors tried to determine how PA28g functions in oral squamous cell carcinoma (OSCC) cells. They hypothesized it may act through metabolic reprogramming in the mitochondria.

Strengths:

They found that the genes of PA28g and C1QBP are in an overlapping interaction network after an analysis of a genome database. They also found that the two proteins interact in coimmunoprecipitation and pull-down assays using the lysate from OSCC cells with or without expression of the exogenous genes. They used truncated C1QBP proteins to map the interaction site to the N-terminal 167 residues of C1QBP protein. They observed the levels of the two proteins are positively correlated in the cells. They provided evidence for the colocalization of the two proteins in the mitochondria and the effect on mitochondrial form and function in vitro and in vivo OSCC models, and the correlation of the protein expression with the prognosis of cancer patients.

Weaknesses:

Many data sets are shown in figures that cannot be understood without more descriptions either in the text or the legend, e.g., Fig. 1A. Similarly, many abbreviations are not defined.

The revision addressed these issues.

Some of the pull-down and coimmunoprecipitation data do not support the conclusion about the PA28g-C1QBP interaction. For example, in Appendix Fig. 1B the Flag-C1QBP was detected in the Myc beads pull-down when the protein was expressed in the 293T cells without the Myc-PA28g, suggesting that the pull-down was not due to the interaction of the C1QBP and PA28g proteins. In Appendix Fig. 1C, assume the SFB stands for a biotin tag, then the SFB-PA28g should be detected in the cells expressing this protein after pull-down by streptavidin; however, it was not. The Western blot data in Fig. 1E and many other figures must be quantified before any conclusions about the levels of proteins can be drawn.

The revision addressed these problems.

The immunoprecipitation method is flawed as it is described. The antigen (PA28g or C1QBP) should bind to the respective antibody that in turn should binds to Protein G beads. The resulting immunocomplex should end up in the pellet fraction after centrifugation, and analyzed further by Western blot for coprecipitates. However, the method in the Appendix states that the supernatant was used for the Western blot.

The revision corrected this method.

To conclude that PA28g stabilizes C1QBP through their physical interaction in the cells, one must show whether a protease inhibitor can substitute PA28q and prevent C1QBP degradation, and also show whether a mutation that disrupt the PA28g-C1QBP interaction can reduce the stability of C1QBP. In Fig. 1F, all cells expressed Myc-PA28g. Therefore, the conclusion that PA28g prevented C1QBP degradation cannot be reached. Instead, since more Myc-PA28g was detected in the cells expressing Flag-C1QBP compared to the cells not expressing this protein, a conclusion would be that the C1QBP stabilized the PA28g. Fig. 1G is a quantification of a Western blot data that should be shown.

The binding site for PA28g in C1QBP was mapped to the N-terminal 167 residues using truncated proteins. One caveat would be that some truncated proteins did not fold correctly in the absence of the sequence that was removed. Thus, the C-terminal region of the C1QBP with residues 168-283 may still bind to the PA29g in the context of full-length protein. In Fig. 1I, more Flag-C1QBP 1-167 was pull-down by Myc-PA28g than the full-length protein or the Flag-C1QBP 1-213. Why?

The interaction site in PA28g for C1QBP was not mapped, which prevents further analysis of the interaction. Also, if the interaction domain can be determined, structural modeling of the complex would be feasible using AlphaFold2 or other programs. Then, it is possible to test point mutations that may disrupt the interaction and if so, the functional effect.

The revision added AlphaFold models for the protein interaction. However, the models were not analyzed and potential mutations that would disrupt the interact were not predicted, made and tested. The revision did not addressed the request for the protease inhibitor.

Reviewer #1 (Public Review):

Summary:

The manuscript by Poltavski and colleagues describes the discovery of previously unreported enteric neural crest-derived cells (ENCDC) which are marked by Pax2 and originating from the Placodes. By creating multiple conditional mouse mutants, the authors demonstrate these cells are a distinct population from the previously reported ENCDCs which originate from the Vagal neural crest cells and express Wnt1.

These Pax2-positive ENCDCs are affected due to the loss of both Ret and Ednrb highlighting that these cells are also ultimately part of the canonical processes governing ENCDC and enteric nervous system (ENS) development. The authors also make explant cultures from the mouse GI tract to detect how Ednrb signaling is important for Ret signaling pathways in these cells and rediscovers the interactions between these 2 pathways. One important observation the authors make is that CGRP-positive neurons in the adult distal colon seem to be primarily derived from these Pax2-positive ENCDCs, which are significantly reduced in the Ednrb mutants, thus highlighting the role of Ednrb in maintaining this neuronal type.

Comments on latest version:

Author response: We disagree that the datasets from previous studies provide additional insights that are relevant to the current study. It must be appreciated that Wnt1Cre and Pax2Cre are genetic lineage tracers and that migratory ENS progenitor cells labeled with these reagents do not maintain expression of Wnt1 and Pax2 mRNA or protein. The Wnt1 and Pax2 genes are only transiently expressed within their distinct regions of the ectoderm, and their expression turns off as cells delaminate and begin migration. Thus, Pax2Cre-labeled ENS progenitor cells are not Pax2-positive thereafter. The single cell RNA-Seq studies suggested by the reviewer were collected from older embryos and postnatal mice, and do not represent the E10.5-E11.5 period that accounts for genesis of Ret-mediated and Ednrb-mediated Hirschsprung disease pathology. Even with the most recent work by Zhou et al (Dev Cell, 2024) that included E10.5 cells, this analysis only evaluated neural crest-derived Sox10Cre lineage cells, which does not include the placode-derived Pax2Cre lineage (as we show explicitly in Fig. 2-figure supplement 2). Consequently, it would not be possible to find the "Pax2-positive cells" in these datasets. Performing a new transcriptomic analysis by isolating Pax2Cre-lineage and Wnt1Cre-lineage cells at the appropriate developmental time points could be the basis of future studies, but we think these are beyond the scope of the present paper.

Reviewer comment: Since these cells are a completely new discovery, additional validation would be beneficial. Whole early GI tract datasets are available, such as human 6-week fetal gut data (PMID: 29802404) and whole mouse embryo studies spanning development that include ENS (PMID: 38355799). If the authors believe that none of these existing datasets can detect these cells in their developmental state and that targeted cell studies with specific Cre drivers would be required, they should make this explicitly clear.

A key advantage of discovering a new cell type, particularly in the relatively understudied field of ENS, is the opportunity for the broader community to leverage this finding to inform their own research. If these cells are absent from current datasets, even those covering the whole GI tract, this should be clearly communicated.

I aim to support the authors here. New discoveries in science require robust validation to enhance their impact. The authors have generated an important reagent with great potential for broader use, and addressing these straightforward requests would strengthen the study and make it more valuable to the scientific community.

Author response: The observation that human mutations in RET and EDNRB both cause Hirschsprung disease is decades old, and of course numerous studies in human, mouse, and cells have addressed the relation between the two signaling pathways. We did not mean to imply that we were the first to discover that Ret and Ednrb signaling pathways interact. The reviewer cites a number of papers all from the Chakravarti lab that address this phenomenon; while these are a valuable contribution to the field, there is still more to be learned. The model elaborated in PMID: 31313802, in which Ret and Ednrb are both enmeshed in a common gene regulatory network, does not readily explain why each has a different phenotypic manifestation and doesn't take into account the importance of the placodal lineage. The main new contributions of our paper are the existence of a new cell lineage that contributes to the ENS, and that the placodal and neural crest lineages utilize Ret and Ednrb signaling differently. The clarification of how these elements are differentially used by the two lineages explains long-segment and short-segment Hirschsprung disease (Ret and Ednrb mutants, respectively) far better than in past studies. The reviewer unfortunately dismisses these insights and seems to feel that a biochemical exploration of one specific component of the signaling interaction (Y1015 phosphorylation) would be more relevant. This should be the basis of future studies and are beyond the scope of the new findings reported in the present paper

Reviewer comment: The authors completely miss the point. There is no association between phenotypic severity (L-HSCR, S-HSCR, or TCA) and mutations in a given gene in HSCR. EDNRB, for example, has a syndromic association with Waardenburg-Shah syndrome (WS4-A), which includes pigmentation anomalies due to EDNRB expression in neural crest cells that give rise to pigment cells.

The authors' discovery reinforces the current paradigm that nearly all HSCR is mediated by mutations in genes within the GRN, accounting for 72% of the population attributable risk. This is valuable; reinforcing established paradigms with new data is crucial, and the authors should appreciate the significance of this contribution.

The discovery of the signaling interaction is particularly important, as it offers a potential explanation for disease severity and provides a basis for classifying patients in future sequencing studies. It is surprising that the authors seem reluctant to highlight this novel finding, as it could greatly benefit future research, including the development of specific mouse mutants and advancing human genetics studies.

Author response: The reviewer overlooked that one of the review articles that we cited (Chen, Hsu, & Hung, 2020) has a dedicated paragraph for RET (section 3.14), which summarizes the work by Barheri-Yarmand et al (PMID: 25795775) which is the very paper noted by the reviewer in the comment above. The reviewer also somewhat misstated the results of the Barheri-Yarmand et al study. By immunostaining, this paper showed nuclear localization of endogenous Ret, albeit a version of Ret with a disease-associated mutation that makes it constitutively active by constitutive autophosphorylation. Nonetheless, this was endogenous Ret. The paper also used overexpression of GFP-tagged RET in HEK293 cells to show that wildtype RET can behave in a similar manner, at least under these circumstances. Our point is simply that Ret (and other receptor tyrosine kinases) can be found in the nucleus in certain biological contexts, and our observations are consistent with this precedent. The reviewer also suggests a biochemical follow-up analysis related to this observation, which we agree would be of interest. Such an investigation however is beyond the scope of the present study.

Reviewer comment: As the authors themselves now highlight from the cited paper that any evidence of RET entering the nucleus is of a mutant RET protein, How does this align with their discovery for wildtype protein?

This finding of nuclear localization of RET is both intriguing and unprecedented. Despite extensive biochemical studies on RET, given its role as an oncogene, this feature has not been identified before. If validated, this discovery could significantly advance the field and improve interpretation of future studies. I reiterate my previous point: a novel finding that challenges the current paradigm requires additional evidence.

Reviewer #2 (Public review):

Summary:

This manuscript by Poltavski and colleagues explores the relative contributions of Pax2- and Wnt1- lineage derived cells in the enteric nervous system (ENS) and how they are each affected by disruptions in Ret and Endrb signaling. The current understanding of ENS development in mice is that vagal neural crest progenitors derived from a Wnt1+ lineage migrate into and colonize the developing gut. The sacral neural crest was thought to make a small contribution to the hindgut in addition but recent work has questioned that contribution and shown that the ENS is entirely populated by vagal crest (PMID: 38452824). GDNF-Ret and Endothelin3-Ednrb signaling are both known to be essential for normal ENS development and loss of function mutations are associated with a congenital disorder called Hirschsprung's disease. The transcription factor Pax2 has been studied in CNS and cranial placode development but has not been previously implicated in ENS development. In this work, the authors begin with the unexpected observation that conditional knockout of Ednrb in Pax2-expressing cells causes a similar aganglionosis, growth retardation, and obstructed defecation as conditional knockout of Ednrb in Wnt1-expressing cells. The investigators then use the Pax2 and Wnt1 Cre transgenic lines to lineage-trace ENS derivatives and assess the effects of loss of Ret or Ednrb during embryonic development in these lineages. Finally, they use explants from the corresponding embryos to examine the effects of GDNF on progenitor outgrowth and differentiation.

Strengths:

- The manuscript is overall very well illustrated with high resolution images and figures. Extensive data are presented.

- The identification of Pax2 expression as a lineage marker that distinguishes a subset of cells in the ENS that may be distinct from cells derived from Wnt1+ progenitors is an interesting new observation that challenges current understanding of ENS development

- Pax2 has not been previously implicated in ENS development - this manuscript does not directly test that role but hints at the possibility

- Interrogation of two distinct signaling pathways involved in ENS development and their relative effects on the two purported lineages

Weaknesses:

- The major challenge with interpreting this work is the use of two transgenic lines, Wnt1-Cre and Pax2-Cre, which are not well characterized in terms of fidelity to native gene expression and recombination efficiency in the ENS. If 100% of cells that express Wnt1 do not express Cre or if the Pax2 transgene is expressed in cells that do not normally express Pax2, then these observations would have very different interpretations and would not support the conclusions made. The two lineages are never compared in the same embryo, which also makes it difficult to assess relative contributions and renders the evidence more circumstantial than definitive.

- Visualization of the Pax2-Cre and Wnt-1Cre induced recombination in cross-sections at postnatal ages would help with data interpretation. If there is recombination evident in the mesenchyme, this would particularly alter interpretation of Ednrb mutant experiments, since that pathway has been shown to alter gut mesenchyme and ECM, which could indirectly alter ENS colonization.

- The data on distinct lineages in Fig 3 is somewhat weak and the description in the Results section tends to over-interpretation. For example, "A minimum number (approx. 3%) of CGRP+ neurons were labeled by Wnt1Cre ... which indicates that Wnt1Cre-derived cells have little or no commitment to a mechanosensory fate in the distal colon." The data panel in Fig 3f shows that most of the CGRP-IR cells in Wnt1-Cre-Tomato mice are tdTomato+ though their tdTomato fluorescence is less intense than in neighboring smaller, likely glial cells. This suggests that CGRP+/Tomato+ neurons were likely undercounted. IHC for tdTomato to ensure detection of low levels of Tomato expression and quantification of observations would strengthen the authors' claim. CGRP+ enteric neurons have been visualized and functionally described by several investigators in the field using Wnt1-Cre-GCaMP mice, which also challenges the authors' conclusions. Finally, quantification of CGRP+ enteric neurons by measuring CGRP mucosal fiber immunoreactivity is not accurate because it would reflect both ENS CGRP-expressing neurons and visceral afferents from DRG. Moreover, it is not known if all CGRP+ enteric neurons project to the mucosa or if all mucosal-projecting neurons are mechanosensory. Finally, most of the signal seems to be non-specific background staining in the mucosa and quantification of mucosal signal in this context does not seem meaningful.

- No consideration of glia - are these derived from both lineages?

- No discussion of how these observations may fit in with recent work that suggests a mesenchymal contribution of enteric neurons (PMID: 38108810)