Reviewer #2 (Public Review):

Schmit et al. analyze and compare different strategies for the allocation of funding for insecticide-treated nets (ITNs) to reduce the global burden of malaria. They use previously published models of Plasmodium falciparum and Plasmodium vivax malaria transmission to quantify the effect of ITN distribution on clinical malaria numbers and the population at risk. The impact of different resource allocation strategies on the reduction of malaria cases or a combination of malaria cases and achieving pre-elimination is considered to determine the optimal strategy to allocate global resources to achieve malaria eradication.

Strengths:<br /> Schmit et al. use previously published models and optimization for rigorous analysis and comparison of the global impact of different funding allocation strategies for ITN distribution. This provides evidence of the effect of three different approaches: the prioritization of high-transmission settings to reduce the disease burden, the prioritization of low-transmission settings to "shrink the malaria map", and a resource allocation proportional to the disease burden.

Weaknesses:<br /> The analysis and optimization which provide the evidence for the conclusions and are thus the central part of this manuscript necessitate some simplifying assumptions which may have important practical implications for the allocation of resources to reduce the malaria burden. For example, seasonality, mosquito species-specific properties, stochasticity in low transmission settings, and changing population sizes were not included. Other challenges to the reduction or elimination of malaria such as resistance of parasites and mosquitoes or the spread of different mosquito species as well as other beneficial interventions such as indoor residual spraying, seasonal malaria chemoprevention, vaccinations, combinations of different interventions, or setting-specific interventions were also not included. Schmit et al. clearly state these limitations throughout their manuscript.

The focus of this work is on ITN distribution strategies, other interventions are not considered. It also provides a global perspective and analysis of the specific local setting (as also noted by Schmit et al.) and different interventions as well as combinations of interventions should also be taken into account for any decisions. Nonetheless, the rigorous analysis supports the authors' conclusions and provides evidence that supports the prioritization of funding of ITNs for settings with high Plasmodium falciparum transmission. Overall, this work may contribute to making evidence-based decisions regarding the optimal prioritization of funding and resources to achieve a reduction in the malaria burden.

Reviewer #1 (Public Review):

In this manuscript, Marmor and colleagues reanalyze a previously published dataset of chronic widefield Ca2+ imaging from the dorsal cortex of mice as they learn a go/no-go somatosensory discrimination task. Comparing hit trials that have a distinct history (i.e. are preceded by distinct trial types), the authors find that hit trials preceded by correct rejections of the non-target stimulus are associated with larger subsequent neural responses than trials precede by other hits, across the cortex. The authors analyze the time course over which this effect emerges in the barrel cortex (BC) and the rostrolateral visual area (RL), and find that its magnitude increases as the animals become expert task performers. Although the findings are potentially interesting, I, unfortunately, believe that there are important methodological concerns that could put them into question. I also disagree with the rationale that singles out BC and RL as being especially important for the emergence of trial history effects on neural responses during decision-making. I detail these points below.

1) The authors did not perform correction for hemodynamic contamination of GCaMP fluorescence. In widefield imaging, blood vessels divisively decrease neural signals because they absorb green-wavelength photons, which could lead to crucial confounds in the interpretation of the main results because of neurovascular coupling, which lags neural activity by seconds. For example, if a reward response from the previous trial is associated with a lagged hemodynamic contamination that artificially decreases the signal in the following trial, one could get artificially higher activity in trials that were not preceded by a reward (i.e. CR), which is what the authors observed. Ideally, the experiments would be repeated with proper hemodynamic correction, but at the very least the authors should try to address this with control analyses. For example, what is the time course of reward-related responses in BC and elsewhere? Do hemodynamics artifacts have a trial-by-trial correlation with the subsequent trial history effect? What is the learning time course of reward responses? Note that I don't believe the FA-Hit condition analysis that the authors have already presented provides adequate control, as punishment responses are also pervasive in the cortex and therefore suffer from the same interpretational caveat. Unfortunately, I believe this is a serious methodological issue given the above. However, I will proceed to take the reported results at face value.

2) The statistics used to assess the effect of trial history over learning are inadequate (e.g., Fig 2b). The existence of a significant effect in one condition (e.g., CR-Hit vs. Hit-Hit in expert) but not in another (e.g., same comparison in naive) does not imply that these two conditions are different. This needs to be tested directly. Moreover, the present analysis does not account for the fact that measures across learning stages are taken from the same animals. Thus, the appropriate analysis for these cases would be to first use a two-way ANOVA with repeated measures with factors of trial history and learning stage (or equivalent non-parametric test) and then derive conclusions based on post hoc pairwise tests, corrected for multiple comparisons.

3) I am not convinced that BC and RL are especially important for trial-history-dependent effects. Figures 4 and 5 suggest that this modulation is present across the cortex, and in fact, the difference between CR-Hit and Hit-Hit in some learning stages appears stronger in other areas. BC and RL do have the highest absolute activity during the epochs in Figs 4 and 5, but I would argue that this is likely due to other aspects of the task (e.g., touch) and therefore is not necessarily relevant to the issue of trial history.

4) Because of similar arguments to the above, and because this was not directly assessed, I do not believe the conclusion that history information emerges in RL and is transferred to BC is warranted. For instance, there is no direct comparison between areas, but inspection of the ROC plots in Fig 6b suggests that history information emerges concomitantly across cortical areas. I suggest directly comparing the time course between these (and other areas).

5) How much is task performance itself modulated by trial history? How does this change over the course of learning? These behavioral analyses would greatly help interpret the neural findings and how this trial history might be used behaviorally.

Reviewer #2 (Public Review):

Marmor et al. mine a previously published dataset to examine whether recent reward/stimulus history influences responses in sensory (and other) cortices. Bulk L2/3 calcium activity is imaged across all of the dorsal cortex in transgenic mice trained to discriminate between two textures in a go/no-go behavior. The authors primarily focus on comparing responses to a specific stimulus given that the preceding trial was or was not rewarded. There are clear differences in activity during stimulus presentation in the barrel cortex along with other areas, as well as differences even before the second stimulus is presented. These differences only emerge after task learning. The data are of high quality and the paper is clear and easy to follow. My only major criticism is that I am not completely convinced that the observed difference in response is not due to differences in movement by the animal on the two trial types. That said, the demonstration of differences in sensory cortices is relatively novel, as most of the existing literature on trial history effect demonstrates such differences only in higher-order areas.

Major:

1a. The claim that body movements do not account for the results is in my view the greatest weakness of the paper - if the difference in response simply reflects a difference in movement, perhaps due to "excitement" in anticipation of reward after not receiving one on CR-H vs. H-H trials, then this should show up in movement analysis. The authors do a little bit of this, but to me, more is needed.

First, given the small sample size and use of non-parametric tests, you will only get p<.05 if at least 6 of the 7 mice perform in the same way. So getting p>.05 is not surprising even if there is an underlying effect. This makes it especially important to do analyses that are likely to reveal any differences; using whisker angle and overall body movement, which is poorly explained, is in my opinion insufficient. An alternative approach would be to compare movements within animals; small as the dataset is, it is feasible to do an animal-by-animal analysis, and then one could leverage the large trial count to get much greater statistical power, foregoing summary analyses that pool over only n=7.

The authors only consider a simple parametrization of movement (correlation across successive frames), and given the high variability in movement across animals, it is likely that different mice adopt different movements during the task, perhaps altering movement in specific ways. Aggregating movement across different body parts after an analysis where body parts are treated separately seems like an odd choice - perhaps it is fine, but again, supporting evidence for this is needed. As it stands, it is not clear if real differences were averaged out by combining all body parts, or what averaging actually entails.

If at all possible, I would recommend examining curvature and not just the whisker angle, since the angle being the same is not too surprising given that the stimulus is in the same place. If the animal is pressing more vigorously on CR-H trials, this should result in larger curvature changes.

Finally, the authors presumably have access to lick data. Are reaction times shorter on CR-H trials? Is lick count or lick frequency shorter?

If movement differs across trial types, it is entirely plausible that at least barrel cortex activity differences reflect differences in sensory input due to differences in whisker position/posture/etc. This would mitigate the novelty of the present results.

1b. Given the importance of this control to the story, both whisker and body movement tracking frames should be explicitly shown either in the primary paper or as a supplement. Moreover, in the methods, please elaborate on how both whisker and body tracking were performed.

2. Did streak length impact the response? For instance, in Fig. 1f "Learning", there is a 6-trial "no-go" streak; if the data are there, it would be useful to plot CR-H responses as a function of preceding unrewarded trials.

Reviewer #1 (Public Review):

In their manuscript titled "A human mitofusion 2 mutation causes mitophagic cardiomyopathy", Franco et al suggest that a rare mutation in MFN2 (R400Q) is over-represented in patients with cardiomyopathy, causes loss of conformational malleability, leading to mitochondrial fusion defects, impaired Parkin recruitment to mitochondria, and suppressed MFN2-Parkin mediated mitophagy. This work is an extension of previous work from the same group that found the MFN2 R400Q mutation is loss of function in a Drosophila model. Unlike MFN2 R94Q and T105M that cause Charcot-Marie-Tooth disease type 2 A, the MFN2 R400Q mutant has normal GTPase activity and mitochondrial electrochemical integrity, motility, and respiration. MFN2 R400Q knock-in mice exhibit cardiac-specific phenotypes.

Strengths include detailed characterization of the MFN2 R400Q variant in variety of models, including cell models and novel knock-in mouse model.<br /> However, there are some weaknesses. The central claim that the R400Q mutation causes cardiomyopathy in humans and the claim that the pathogenetic mechanism is decreased mitophagy require additional support.

First, the claim of an association between the R400Q variant (identified in three individuals) and cardiomyopathy has some limitations based on the data presented. The initial association is suggested by comparing the frequency of the mutation in three small cohorts to that in a large database gnomAD, which aggregates whole exome and whole genome data from many other studies including those from specific disease populations. Having a matched control population is critical in these association studies. For instance, according to gnomAD the MFN2 Q400P variant, while not observed in those of European ancestry, has a 10-fold higher frequency in the African/African American and South Asian populations (0.0004004 and 0.0003266, respectively). If the authors data in table one is compared to the gnomAD African/African American population the p-value drops to 0.029262, which would not likely survive correction for multiple comparison (e.g., Bonferroni). (The source and characteristics of the subjects used by the authors in Table 1 is not clear from the methods.)

Relatedly, evaluation in a knock-in mouse model is offered as a way of bolstering the claim for an association with cardiomyopathy. Some caution should be offered here. Certain mutations have caused a cardiomyopathy in mice when knocked in have not been observed in humans with the same mutation. A recent example is the p.S59L variant in the mitochondrial protein CHCHD10, which causes cardiomyopathy in mice but not in humans (PMID: 30874923). While phenocopy is suggestive there are differences in humans and mice, which makes the correlation imperfect.

Additionally, the argument that the Mfn2 R400Q variant causes a dominant cardiomyopathy in humans would be better supported by observing of a cardiomyopathy in the heterozygous Mfn2 R400Q mice and not just in the homozygous Mfn2 R400Q mice. Relatedly, it is not clear what the studies in the KI mouse prove over what was already known. Mfn2 function is known to be essential during the neonatal period and the authors have previously shown that the Mfn2 R400Q disrupts the ability of Mfn2 to mediate mitochondrial fusion, which is its core function. The results in the KI mouse seem consistent with those two observations, but it's not clear how they allow further conclusions to be drawn.

Additionally, the authors conclude that the effect of R400Q on the transcriptome and metabolome in a subset of animals cannot be explained by its effect on OXPHOS (based on the findings in Figure 4H). However, an alternative explanation is that the R400Q is a loss of function variant but does not act in a dominant negative fashion. According to this view, mice homozygous for R400Q (and have no wildtype copies of Mfn2) lack Mfn2 function and consequently have an OXPHOS defect giving rise to the observed transcriptomic and metabolomic changes. But in the rat heart cell line with endogenous rat Mfn2, exogenous of the MFN2 R400Q has no effect as it is loss of function and is not dominant negative. Additionally, as the authors have shown MFN2 R400Q loses its ability to promote mitochondrial fusion, and this is the central function of MFN2, it is not clear why this can't be the explanation for the mouse phenotype rather than the mitophagy mechanism the authors propose.

Finally, it is asserted that the MFN2 R400Q variant disrupts Parkin activation, by interfering with MFN2 acting a receptor for Parkin. The support for this in cell culture however is limited. Additionally, there is no assessment of mitophagy in the hearts of the KI mouse model.

Reviewer #2 (Public Review):

In this manuscript, Franco et al show that the mitofusin 2 mutation MFN2 Q400 impaires mitochondrial fusion with normal GTPase activity. MFN2 Q400 fails to recruit Parkin and further disrupts Parkin-mediated mitophagy in cultured cardiac cells. They also generated MFN2 Q400 knock-in mice to show the development of lethal perinatal cardiomyopathy, which had an impairment in multiple metabolic pathways.

The major strength of this manuscript is the in vitro study that provides a thorough understanding in the characteristics of the MFN2 Q400 mutant in function of MFN2, and the effect on mitochondrial function. However, the in vivo MFN2 Q/Q400 knock-in mice are more troubling given the split phenotype of MFN2 Q/Q400a vs MFN2 Q/Q400n subtypes. Their main findings towards impaired metabolism in mutant hearts fail to distinguish between the two subtypes.

While the data support the conclusion that MFN2 Q400 causes cardiomyopathy, several experiments are needed to further understand mechanism. This manuscript will likely impact the field of MFN2 mutation-related diseases and show how MFN2 mutation leads to perinatal cardiomyopathy in support of previous literature.

Reviewer #2 (Public Review):

MCM8 and MCM9 together form a hexameric DNA helicase that is involved in homologous recombination (HR) for repairing DNA double-strand breaks. The authors have previously reported on the winged-helix structure of the MCM8 (Zeng et al. BBRC, 2020) and the N-terminal structure of MCM8/9 hexametric complex (MCM8/9-NTD) (Li et al. Structure, 2021). This manuscript reports the structure of a near-complete MCM8/9 complex and the conformational change of MCM8/9-NTD in the presence of its binding protein, HROB, as well as the residues important for its helicase activity.

The presented data might potentially explain how MCM8/9 works as a helicase. However, additional studies are required to conclude this point because the presented MCM8/9 structure is not a DNA-bound form and HROB is not visible in the presented structural data. Taking into these accounts, this work will be of interest to biologists studying DNA transactions.

A strength of this paper is that the authors revealed the near-complete MCM8/9 structure with 3.66A and 5.21A for the NTD and CTD, respectively (Figure 1). Additionally, the authors discovered a conformational change in the MCM8/9-NTD when HROB was included (Figure 4) and a flexible nature of MCM8/9-CTD (Figure S6 and Movie 1).

The revised version of "Structural and mechanistic insights into the MCM8/9 helicase complex" by Weng et al. includes only very minor changes in the text and incorporates two additional supplementary figures (S8 and S11) illustrating the size of MCM8/9 mutants.

In the previous version, I raised two important concerns that required addressing. 1) The presented structures exclusively depicted the unbound forms of DNA. It is crucial to elucidate the structure of a DNA-bound form. 2) The MCM8/9 activator, HROB, was not visible in the structural data. Although HROB induced a conformational change in MCM8/9-NTD, it is essential to visualize the structure of an MCM8/9-HROB complex.

The authors neither addressed nor provided new data in response to these issues. Consequently, I maintain my initial stance and have no further comments on the revised version.

Reviewer #1 (Public Review):

MCM8 and MCM9 are paralogues of the eukaryotic MCM2-7 proteins. MCM2-7 form a heterohexameric complex to function as a replicative helicase while MCM8-9 form another hexameric helicase complex that may function in homologous recombination-mediated long-tract gene conversion and/or break-induced replication. MCM2-7 complex is loaded during the low Cdk period by ORC, CDC6, and Cdt1, when the origin DNA may intrude into the central channel via the MCM2-MCM5 entry "gate". In the S phase, MCM2-7 complex is activated as CMG helicase with the help of CDC45 and GINS complex. On the other hand, it still remains unclear how MCM8-9 complex is loaded onto DNA and then activated.

In this study, the authors first investigated the cryo-EM structure of chicken MCM8-9 (gMCM8-9) complex. Based on the data obtained, they suggest that the observed gMCM8-9 structure might represent the structure of a loading state with possible DNA entry "gate". The authors further investigated the cryo-EM structure of human MCM8-9 (hMCM8-9) complex in the presence of the activator protein, HROB, and compared the structure with that obtained without HROB1, which the authors published previously. As a result, they suggest that MCM8-9 complex may change the conformation upon HROB binding, leading to helicase activation. Furthermore, based on the structural analyses, they identified some important residues and motifs in MCM8-9 complex, mutations of which actually impaired the MCM8-9 activity in vitro and in vivo.

Overall, the data presented would support the authors' conclusions and would be of wide interest for those working in the fields of DNA replication and repair. One caveat is that most of the structural data are shown only as ribbon model without showing the density map data obtained by cryo-EM, which makes accurate evaluation of the data somewhat difficult.

Addition after review of the revised manuscript: The authors have made a reasonable attempt to address the points raised by the reviewers, by which the paper is significantly improved.

Reviewer #1 (Public Review):

Detection of early-stage colorectal cancer is of great importance. Recently, both laboratory scientists and clinicians have reported different exosomal biomarkers to identify colorectal cancer patients.

Here, the authors exhibited a full RNA landscape for plasma exosomes of 60 individuals, including 31 colorectal cancer (CRC) patients, 19 advanced adenoma (AA) patients, and 10 noncancerous controls. RNAs with high fold change, high absolute abundance, and various module attribution were used to construct RT-qPCR-based RNA models for CRC and AA detection.

Overall, this is a well-performed proof-of-concept study to highlight exosomal RNAs as potential biomarkers of early-stage colorectal cancer and its precancerous lesions.

Depicting the full RNA landscape of circulating exosomes is still quite challenging. The authors annotated 58,333 RNA species in exosomes, most of which were lncRNAs, but the authors do not explain how they characterized those RNAs.

The authors tested their models in a medium size population of 124 individuals, which is not enough to obtain an accurate evaluation of the specificity and sensitivity of the biomarkers proposed here. External validation would be required.

Reviewer #2 (Public Review):

The authors present an important study on the potential of small extracellular vesicle (sEV)-derived RNAs as biomarkers for the early detection of colorectal cancer (CRC) and precancerous adenoma (AA). The authors provide a detailed analysis of the RNA landscape of sEVs isolated from participants, identifying differentially expressed sEV-RNAs associated with T1a stage CRC and AA compared to normal controls. The paper further categorises these sEV-RNAs into modules and constructs a 60-gene model that successfully distinguishes CRC/AA from NC samples. The authors also validate their findings using RT-qPCR and propose an optimised classifier with high specificity and sensitivity. Additionally, the authors discuss the potential of sEV-RNAs in understanding CRC carcinogenesis and suggest that a comprehensive biomarker panel combining sEV-RNAs and proteins could be promising for identifying both early and advanced CRC patients. Overall, the study provides valuable insights into the potential clinical application of sEV-RNAs in liquid biopsy for the early detection of CRC and AA.

Major strengths:<br /> 1. Comprehensive sEV RNA profiling: The study provides a valuable dataset of the whole-transcriptomic profile of circulating sEVs, including miRNA, mRNA, and lncRNA. This approach adds to the understanding of sEV-RNAs' role in CRC carcinogenesis and facilitates the discovery of potential biomarkers.

2. Detection of early-stage CRC and AA: The developed 60-gene t-SNE model successfully differentiated T1a stage CRC/AA from normal controls with high specificity and sensitivity, indicating the potential of sEV-RNAs as diagnostic markers for early-stage colorectal lesions.

3. Independent validation cohort: The study combines RNA-seq, RT-qPCR, and modelling algorithms to select and validate candidate sEV-RNAs, maximising the performance of the developed RNA signature. The comparison of different algorithms and consideration of other factors enhance the robustness of the findings.

Major weaknesses:<br /> 1. Lack of analysis on T1-only patients in the validation cohort: While the study identifies key sEV-RNAs associated with T1a stage CRC and AA, the validation cohort is only half of the patients in T1(25 out of 49). It would be better to do an analysis using only the T1 patients in the validation cohort, so the conclusion is not affected by the T2-T3 patients.

2. Lack of performance analysis across different demographic and tumor pathology factors listed in Supplementary Table 12. It's important to know if the sEV-RNAs identified in the study work better/worse in different age/sex/tumor size/Yamada subtypes etc.

Reviewer #1 (Public Review):

In this manuscript, the authors investigated the roles of the target of rapamycin (TOR) pathway in various pathobiological processes of Aspergillus flavus. They found that rapamycin treatment affects the growth, sporulation, sclerotia, and aflatoxin synthesis of A. flavus. The authors identified four immunophilin genes (FKBP1 -4), among which FKBP3 is involved in both rapamycin and FK506 resistances, with K19 residue being essential for succinylation. The authors identified a single Tor kinase and characterized its function. Subsequently, the authors analyzed a series of downstream effectors of the TOR pathway, including Sch9, TapA, SitA, Ppg1, and Spot7/Nem1, in terms of vegetative growth, sexual development, stress responses, and aflatoxin production.

While the authors provided a large amount of data regarding the genes involved in the TOR pathway, it is highly descriptive and mostly confirmative data, as numerous papers have already shown that the TOR pathway plays essential roles in a myriad of biological processes in multiple fungi. The authors seemed to perform a series of parallel studies in several genes involved in the TOR pathway in other fungi. However, their data are not properly interconnected to understand the TOR signaling pathway in this fungal pathogen. The authors frequently drew premature conclusions from basic phenotypic observations. For instance, based on their finding that sch9 mutant showed high calcium stress sensitivity, they concluded that Sch9 is the element of the calcineurin-CrzA pathway. Furthermore, based on their finding that the sch9 mutant show weak rapamycin sensitivity and increased Hog1 phosphorylation, they concluded that Sch9 is involved in TOR and HOG pathways. To make such conclusions, the authors should provide more detailed mechanistic data.

In the section "Tor kinase plays important roles in A. flavus", some parts of their data are confusing. The authors said they identified a single Tor kinase ortholog, which is orthologous to S. cerevisiae Tor2. And then, they said failed to obtain a null mutant, but constructed a single copy deletion strain delta Tor1+/Tor2-. What does this mean? Does this mean A. flavus diploid strain? So is this heterozygous TOR/tor mutant? Otherwise, does the haploid A. flavus strain they used contain multiple copies of the TOR gene within its genome? What is the real name of A. flavus Tor kinase (Tor1 or Tor2?). "tor1+/tor2-" is the wrong genetic nomenclature. What is the identity of detalTor1+/Tor2-? Please provide detailed information on how all these mutants were generated. A similar issue was found in the analysis of TapA, which is speculated to be essential (what is the deltaTapA1+/TapA2-?). I couldn't find any detailed information even in Materials and Methods. The authors should provide southern blot data to validate all their mutants.

How were the FRB domain deletion mutants constructed? If the FKBP12-rapamycin binding (FRB) domain is specifically deleted in the Tor kinase allele, should it be insensitive and resistant to rapamycin? However, the authors showed that the FRB domain deleted TOR allele was indeed non-functional.

In Figure 4C, the authors should monitor Hog1 phosphorylation patterns under stressed conditions, such as NaCl treatment, and provide quantitative measurements. Similar issues were found in the western blot analysis of Slt2 (Fig. 8D).

For all the deletion mutants generated in this study, the authors should generate complemented strains to validate their data.

Reviewer #2 (Public Review):

In this study, the authors identified the complex TOR, HOG, and CWI signaling networks-involved genes that relatively modulate the development, aflatoxin biosynthesis and pathogenicity of A. flavus by gene deletions combined with phenotypic observation.

They also analyzed the specific regulatory process and proposed that the TOR signaling pathway interacts with other signaling pathways (MAPK, CWI, calcineurin-CrzA pathway) to regulate the responses to various environmental stresses. Notably, they found that FKBP3 is involved in sclerotia and aflatoxin biosynthesis and rapamycin resistance in A. flavus, and that the conserved site K19 of FKBP3 plays a key role in regulating the aflatoxin biosynthesis. In general, there is a heavy workload task carried in this study and the findings are interesting and important for understanding or controlling aflatoxin biosynthesis. However, findings have not been deeply explored and conclusions mostly are based on parallel phenotypic observations. In addition, there are some concerns that exist surrounding the conclusions.

Reviewer #3 (Public Review):

The paper by Li et al. describes the role of the TOR pathway in Aspergillus flavus. The authors tested the effect of rapamycin in WT and different deletion strains. This paper is based on a lot of experiments and work but remains rather descriptive and confirms the results obtained in other fungi. It shows that the TOR pathway is involved in conidiation, aflatoxin production, pathogenicity, and hyphal growth. This is inferred from rapamycin treatment and TOR1/2 deletions. Rapamycin treatment also causes lipid accumulation in hyphae. The phenotypes are not surprising as they have been shown already for several fungi. In addition, one caveat is in my opinion that the strains grow very slowly and this could cause many downstream effects. Several kinases and phosphatases are involved in the TOR pathway. They were known from S. cerevisiae or filamentous fungi. The authors characterized them as well with knock-out approaches.

As for many results, I miss the re-complementation of the created mutants throughout the manuscript. This is standard praxis.

Fig. 1: cultures were grown for 48 h before measuring the transcript level. The authors show that brlA, abaA, and some sexual regulators are less expressed. In my opinion, this does not allow the conclusion that there is a direct control through rapamycin. Since the colonies grow very slowly in the presence of rapamycin, the authors should add rapamycin and follow gene expression after 15, 30, 60, 90 min. The figure legend needs to be more detailed. Which type of cultures were used, liquid, solid medium? Etc.

Why in chapter one Fig. 9 is already cited? Those data should then be included in Fig. 1 for the general phenotype.

The authors wrote that radial growth and conidiation were gradually reduced with increasing rapamycin concentrations. This is not true. There is no gradient! However, it should be tested if there is a gradient if lower concentrations are used. The current data imply that there is a threshold concentration, so either there is 100 % growth or a reduction to 25 %. This looks strange.

Reviewer #1 (Public Review):

In this study, the authors strive to characterize the role of protein arginine methyltransferase 5 (Prmt5) in chromatin organization and transcriptional regulation during adipogenesis. Their main aim is to delve into Prmt5's function during the differentiation of preadipocytes by conducting genome-wide analyses, including ChIP-Seq, RNA-Seq, and Hi-C experiments. They hypothesize and present evidence for Prmt5's broad regulatory effect on gene expression and its role in maintaining topologically associating domain (TAD) boundaries and overall chromatin organization.

Strengths of the study include its genome-wide approach, which provides a comprehensive perspective on Prmt5's potential involvement in adipogenesis. These methods yield a large dataset and offer novel insights into Prmt5's possible function in adipogenesis.

However, there are a few areas where the methodology and interpretation of results fall short. One noticeable gap is the absence of a comprehensive control in the ChIP-Seq experiments. Specifically, a control such as ChIP in Prmt5 knockdown cells would have been valuable to account for potential non-specific binding. This lack of a robust control raises questions about the confidence in the detected Prmt5 peaks.

Moreover, the knockdown experiments predominantly employ a single siRNA. Given the well-known off-target effects of siRNA-mediated RNA interference, this approach might cast doubts on the reliability of the results derived from these experiments. Therefore, the inclusion of multiple siRNAs in each assay would greatly strengthen the data.

Lastly, the authors' assertion of Prmt5's influence on the weakening of TAD boundaries and transcriptional dysregulation could benefit from further experimentation. As it stands, this finding is merely correlative, not causative, and represents a very minor fraction of Prmt5 occupied sites. Hence, the evidence provided does not overwhelmingly support the authors' conclusions regarding Prmt5's role in chromatin organization.

This research is potentially important for our understanding of adipogenesis and the role of Prmt5, which is already known for its diverse roles in cellular processes. However, while the authors have taken on an ambitious research question, there are some areas where the study falls short in substantiating the broad conclusions it draws.

Reviewer #2 (Public Review):

This study by Syed et al identifies Prmt5 as a novel and broad modulator of gene expression and genome architecture during the early stages of adipogenesis. Specifically, Prmt5 is reported to be required to maintain strong insulation at TAD boundaries.

This is a logically and clearly conducted study that relies on the integration of public datasets (PCHi-C) to identify chromatin loops, with its own new genomics datasets, including Prmt5 ChIPseq and Hi-C data in control and Prmt5 kd cells. Despite showing relatively model effects of Prmt5 kd on genome architecture, the results are informative and contribute to advancing our knowledge of chromatin-linked processes during early adipogenesis.

The manuscript would benefit from incorporating ATACseq data (public or own) to better appreciate binding profiles of Prmt5 at H3K27ac sites. A more detailed analysis of these relative enrichments would also be useful, particularly if linked to a transcription factor footprint from ATAC data.

Reviewer #1 (Public Review):

The regulation of motor autoinhibition and activation is essential for efficient intracellular transport. This manuscript used biochemical approaches to explore two members in the kinesin-3 family. They found that releasing UNC-104 autoinhibition triggered its dimerization whereas unlocking KLP-6 autoinhibition is insufficient to activate its processive movement, which suggests that KLP-6 requires additional factors for activation, highlighting the common and diverse mechanisms underlying motor activation. They also identified a coiled-coil domain crucial for the dimerization and processive movement of UNC-104. Overall, these biochemical and single-molecule assays were well performed, and their data support their statements. The manuscript is also clearly written, and these results will be valuable to the field.

Ideally, the authors can add some in vivo studies to test the physiological relevance of their in vitro findings, given that the lab is very good at worm genetic manipulations. Otherwise, the authors should speculate the in vivo phenotypes in their Discussion, including E412K mutation in UNC-104, CC2 deletion of UNC-104, D458A in KLP-6.

While beyond the scope of this study, can the author speculate on the candidate for an additional regulator to activate KLP-6 in C. elegans?

The authors discussed the differences between their porcine brain MTs and chlamydonomas axonemes in UNC-104 assays. However, the authors did not really retest UNC-104 on axonemes after more than two decades, thereby not excluding other possibilities.

Reviewer #2 (Public Review):

The Kinesin superfamily motors mediate the transport of a wide variety of cargos which are crucial for cells to develop into unique shapes and polarities. Kinesin-3 subfamily motors are among the most conserved and critical classes of kinesin motors which were shown to be self-inhibited in a monomeric state and dimerized to activate motility along microtubules. Recent studies have shown that different members of this family are uniquely activated to undergo a transition from monomers to dimers.

Niwa and colleagues study two well-described members of the kinesin-3 superfamily, unc104 and KLP6, to uncover the mechanism of monomer to dimer transition upon activation. Their studies reveal that although both Unc104 and KLP6 are both self-inhibited monomers, their propensities for forming dimers are quite different. The authors relate this difference to a region in the molecules called CC2 which has a higher propensity for forming homodimers. Unc104 readily forms homodimers if its self-inhibited state is disabled while KLP6 does not.

The work suggests that although mechanisms for self-inhibited monomeric states are similar, variations in the kinesin-3 dimerization may present a unique form of kinesin-3 motor regulation with implications on the forms of motility functions carried out by these unique kinesin-3 motors.

Reviewer #3 (Public Review):

In this work, Kita et al., aim to understand the activation mechanisms of the kinesin-3 motors KLP-6 and UNC-104 from C. elegans. As with many other motor proteins involved in intracellular transport processes, KLP-6 and UNC-104 motors suppress their ATPase activities in the absence of cargo molecules. Relieving the autoinhibition is thus a crucial step that initiates the directional transport of intracellular cargo. To investigate the activation mechanisms, the authors make use of mass photometry to determine the oligomeric states of the full-length KLP-6 and the truncated UNC-104(1-653) motors at sub-micromolar concentrations. While full-length KLP-6 remains monomeric, the truncated UNC-104(1-653) displays a sub-population of dimeric motors that is much more pronounced at high concentrations, suggesting a monomer-to-dimer conversion. The authors push this equilibrium towards dimeric UNC-104(1-653) motors solely by introducing a point mutation into the coiled-coil domain and ultimately unleashing a robust processivity of the UNC-104 dimer. The authors find that the same mechanistic concept does not apply to the KLP-6 kinesin-3 motor, suggesting an alternative activation mechanism of the KLP-6 that remains to be resolved. The present study encourages further dissection of the kinesin-3 motors with the goal of uncovering the main factors needed to overcome the 'self-inflicted' deactivation.

Reviewer #2 (Public Review):

Yu et al. investigated the structural landscape of 'secreted in xylem' (SIX) effector (virulence and avirulence) proteins from the plant-pathogenic fungus, Fusarium oxysporum f. sp. lycopersici (Fol), with the goal of better understanding effector function and recognition by host (tomato) immune receptors. In recent years, several experimental and computational studies have shown that many effector proteins of plant-associated fungi can be assigned to one of a few major structural families. In the study by Yu et al., X-ray crystallography was used to show that two avirulence effectors of Fol, Avr1 (SIX4) and Avr3 (SIX1), which are recognized by the tomato immune receptors I and I-3, respectively, form part of a new structural family, the Fol dual-domain (FOLD) family, found across three fungal divisions. Using AlphaFold2, an ab initio structural prediction tool, the authors then predicted the structures of all proteins within the Fol SIX effector repertoire (and other effector candidates) and provided evidence that two other effectors, SIX6 and SIX13, also belong to this family.

In addition to identifying members of the FOLD family, structural prediction revealed that proteins of the Fol effector repertoire can largely be classified into a reduced set of structural families. Examples included four members of the ToxA-like family (including Avr2 (SIX3) and SIX8), as well as four members of a new family, Family 4 (including SIX5 and PSL1). Given previous studies had demonstrated that Avr2 (ToxA-like) and SIX5 (Family 4) interact and function together and that the genes encoding these proteins are divergently transcribed, and because homologues of SIX8 (ToxA-like) and PSL1 (Family 4) from another Fusarium pathogen are functionally dependent on each other and, in the case of Fol, are encoded by genes that are next to each other in the genome, the authors hypothesized that SIX8 and PSL1 may also physically interact. In line with this, co-incubation of the SIX8 and PSL1 proteins, followed by size exclusion chromatography (SEC), gave elution and gel migration profiles consistent with interaction in the form of a heterodimer. AlphaFold2-Multimer modelling then suggested that this interaction was mediated through an intermolecular disulfide bond. Such a prediction was subsequently confirmed through mutational analysis of the relevant cysteine residue in each protein in conjunction with SEC.

Finally, using a variant (homologue) of Avr1 from another Fusarium pathogen, as well as chimeric forms of this protein that integrated regions of Avr1 from Fol, Yu et al. determined through co-expression assays in Nicotiana benthamiana with the I immune receptor, as well as subsequent ion leakage assays, that the C-domain of Avr1 is recognized by the I immune receptor. Furthermore, through these assays, the authors were also able to show that surface-exposed residues in the C-domain enable Avr1 to evade recognition by a variant of the I receptor in Moneymaker tomato that does not provide resistance to Fol.

Overall, the manuscript presents a large body of work that is well supported by the data. A key strength of the manuscript is the validation (benchmarking) of protein structures predicted using AlphaFold2, which is a first for largescale effector structure prediction papers published to date. Another key strength is the use of largescale effector structure predictions to make hypotheses about functional relationships or interactions that are then tested (i.e. the SIX8-PSL1 protein interaction and recognition of Avr1 by the I immune receptor). This testing again goes above and beyond the large scale effector structure prediction papers published to date. Taken together, this showcases how experimental and computational experiments can be effectively combined to provide biologically relevant data for the plant protection and molecular plant-microbe interactions fields.

In terms of weaknesses, the manuscript could have validated the SIX8-PSL1 protein interaction with in planta experiments, such as co-immunoprecipitation assays or co-localization experiments in conjunction with confocal microscopy, to provide support for the interaction in a plant setting. However, given what is already known about the Avr2-SIX5 interaction, these additional experiments are not crucial and could instead form part of a follow-up study. With regards to the Agrobacterium tumefaciens-mediated transient expression assays involving co-expression of the Avr1 effector and I immune receptor, the authors need to make clear how many biological replicates were performed as this information is only provided for the ion leakage assay.

Reviewer #1 (Public Review):

Yu et al. investigated Fusarium oxysporum f. sp. lycopersici SIX effectors structure using experimental and computational approaches, and while doing so, the authors identified several SIX effectors as member of the FOLD family, and expanded the known diversity of the SIX effectors. A very interesting and novel finding is the presence of FOLD putative effectors in other Ascomycetes secretome, sharing structural similarities with SIX effectors Avr1, Avr3 and SIX6.

By performing technically sound predictions and experimental confirmation, the authors also confirmed co-operative interactions between Fol effectors, something that was previously known for different pairs of proteins, expanding this observation for new SIX effectors. In addition, the authors contributed to the understanding of the interaction Fol effectors, specifically FOLD and LARS effectors, - I receptors to suppress immunity by structurally similar effectors.

The conclusions of this paper are supported by data and I think it is a pioneer study analyzing the correspondence between AlphaFold predictions and experimentally derived structures, highlighting that models can answer the scientific questions in some cases but could not be enough in others.

Reviewer #3 (Public Review):

In this work, the authors shed light onto the structures of Fusarium oxysporum f.sp. lycopersici proteins involved in the infection of tomato. They unravelled several new secreted effector protein structures and additionally used computational approaches to structurally classify the remaining effectors known from this pathogen. Through this they uncovered a new and unique structural class of proteins which they found to be present and widely distributed in fungal plant pathogens and plant symbiotic fungi. The authors further predicted structural models for the full SIX effector set revealing that genome-proximal effector pairs share similar structural classes. Building on their Avr1 structure, the authors also define the C-terminal domain and specific amino acid residues that are essential to Avr1 detection by its cognate immune receptor.

A major strength of this work is a portfolio of several (Avr1, Avr3, SIX6, SIX8) new structurally resolved proteins which led to the discovery that several of them fall into the same structural class. These findings are supported by strong results.

The experiments addressing the structure-function relationship of Avr1's avirulence activity are highly relevant to our understanding of disease resistance mechanisms against Fusarium, but will require additional controls to allow for solid conclusions to be drawn. In particular, it needs to be demonstrated that specific I gene alleles are at all required for FonSIX4's cell death activity in N.benthamiana leaves or whether FonSIX4 and those of some chimeric proteins is independent of the tomato I receptor. Complementary work in Fusarium mutants lacking Avr1 and expressing chimeric versions would document that the observations from transient expressions in Nicotiana benthamiana are relevant in the biological context of a Fusarium/tomato interaction.

The discovered solvent-exposed residues conditioning Avr1 recognition by the I receptor seem to be positioned in an area of the protein which had previously been highlighted as being highly variable in FOLD proteins of symbiotic fungi but it is not clear from the work whether this is indeed the case or whether Avr1 differs significantly in its structure from FOLD proteins found in other fungi.<br /> It also remains to be addressed whether the residues conditioning avirulence activity is also crucial for virulence activity in Fusarium?

This work uncovered a new structural class of proteins with critical roles in plant-pathogen interactions. Structure-based predictions and genome-wide comparisons have emerged as a new approach enabling the identification of similar proteins with divergent sequences. The work undertaken by the authors adds to a growing body of work in plant-microbe research, predominantly from pathogenic organisms, and more recently in symbiotic fungi.

Reviewer #3 (Public Review):

This is an interesting and carefully done study that will be of considerable value to the field of cortical interneurons. The main result is the development of a novel intersectional genetic strategy to identify and manipulate neurogliaform cells (NGFCs), an interneuron subtype that has been somewhat under-explored to date (but perhaps not quite as enigmatic as implied by the authors). The new strategy, using Id2-CreER transgenic mice crossed with a pan-interneuronal Flp line, appears to label all interneurons which do not express PV, Sst, or VIP, and thus defines a fourth subclass of interneurons. The main members of this subclass are NPY-expressing NGFCs. The strategy allows the targeting of NGFCs in all cortical layers, in contrast to previous strategies using the NDNF-Cre mice which target mostly Layer 1 NGFCs (and possibly also other Layer 1 subtypes). The same strategy also labels a relatively small population of non-NGF Id2 cells belonging to the CCK-expressing subtype(s).

In the first stage of the study, the authors characterize the labeled neurons by their expression of protein markers (most notably NPY and CCK), by their dendritic and axonal morphology, and by their electrophysiological properties. This characterization is detailed and rigorous and the observed characteristics are consistent with what is already known about the properties of NGFCs. The weaknesses here are that the morphological features are not analyzed quantitatively, the definition of electrophysiological subtypes remains somewhat subjective, and the authors do not attempt a multivariate analysis that could provide a data-driven parcellation into subtypes.

The authors then go two steps further. First, they use ex-vivo recordings to demonstrate that presumed CCK+ neurons (identified by their firing pattern as "non-late-spiking), but not NGFCs (identified by their "late-spiking" phenotype), are sensitive to endocannabinoids released from postsynaptic pyramidal cells upon depolarization of the latter. This DSI ("depolarization suppression of inhibition") is a well-studied property of hippocampal CCK+ basket cells, so its demonstration adds to the validation of the intersectional strategy in targeting this subtype in the neocortex. Somewhat surprisingly, the authors do not attempt to demonstrate in their ex-vivo experiments what may be the best-known property of NGFCs - their propensity to preferentially activate GABAB receptors.

The authors then perform in-vivo silicon probe recordings in which Id2 cells are "optotagged" with ChR2 and can thus be identified in extracellular recordings. These in-vivo recordings are probably the first ever from identified NGFCs below layer 1, although some uncertainty remains about the identification of optotagged cells as NGFCs vs CCK-expressing interneurons. They find several differences between firing patterns of NGFCs and other interneurons or pyramidal cells (identified by their extracellular spike waveforms), the most dramatic being a pronounced "rebound" of NGFC firing during slow-wave sleep immediately after a DOWN-to-UP state transition. While the functional significance of these findings is not clear, these experiments provide proof of concept that this fourth (and last?) interneuron subclass can be identified, recorded, and manipulated in freely behaving animals.

In summary, while adding only modestly to our knowledge of NGFCs and CCK-expressing interneurons per se, this work provides an important new tool that will no doubt be used in future studies to target cortical NGFCs and CCK interneurons for in-vivo and ex-vivo recordings, for optogenetic manipulations and for calcium or voltage imaging using genetically-encoded probes. In this sense, the current study is a breakthrough into what may truly be "the last frontier" of cortical interneurons.

Reviewer #1 (Public Review):

The authors have previously suggested that virtually all cortical interneurons are positive for one of three markers: Pvalb, Sst, or Htr3. Here they present convincing evidence that the Htr3a group, includes a significant fraction that does not in fact express this marker in the adult and has distinct properties. By mining existing single-cell RNAseq results, the authors conclude that these cells express the marker Id2. Because some excitatory neurons and non-neuronal cells also express this marker, they selectively target these Id2+ interneurons by combining a pan-interneuron driver and an Id2-creER driver. They show that these neurons are present across cortical layers and account for about 18% of all interneurons. Based on morphological and electrophysiological analyses, this group includes non-VIP neurons in layer 1, neurogliaform cells in layers 2-6, and a small population of CCK basket cells. These in vitro characterizations are well done and will make it straightforward for others to adopt the same or related genetic strategies to target these cells for other functional or mechanistic studies. Since it was not previously possible to genetically target most of these cells, they have been less studied than the other populations of interneurons, so the present study fills an important gap in the field.

The authors also use optogenetics and silicon probe multielectrode recordings to characterize the state-dependent firing and impact of these neurons in vivo. By monitoring sleep, and wake state, the authors show convincingly that these cells reduce their firing during NREM sleep and show a rebound increase in firing after this reduction. The authors then stimulate these neurons optogenetically and show that the firing of other neurons is more likely to be reduced than increased, as expected if these neurons provide broad inhibitory output. The magnitude of this effect is a bit difficult to assess from the current presentation of these data and so it is not clear whether the authors' suggestion that recruitment of these neurons "might drive a widespread switch in the activity of all other cortical neurons" is supported, or whether the effects on circuit activity are more subtle. Regardless of this concern, this is an important study for our understanding of the properties and functions of cortical interneurons.

Reviewer #2 (Public Review):

Machold and colleagues develop and describe an intersectional genetic mouse (Id2Cre:Dlx5/6FlpE) that allows for the targeting of a cortical interneuron subpopulation predominantly consisting of the neurogliaform cell subtype (NGFCs). The strategy is a modification of that previously published by the authors (Id2cre:Nkx2-1Flpo; Valero et al., 2021) in which a subset of deep layer 6 NGFCs with distinct embryonic origins were targeted. Conversely, using the NDNF transgenic mouse lines previous studies, including those from the Rudy laboratory, have clearly shown the prevalence of NGFCs in the outermost cortical Layer 1 region. Thus, the Id2Cre:Dlx5/6FlpE mouse poses an advantage over these previous approaches permitting the targeting of NGFCs in Layers 2-5. NGFCs in these regions have been hitherto difficult to study in an expedited manner.

The manuscript is of the resource/toolbox type and the authors are thorough in their description of the distribution and molecular characteristics of the ID2 neurons labelled by this intersectional approach. Furthermore, the authors perform a series of in vivo experiments. These entail the identification of NGFCs, the assessment of their influence on other neuronal populations, and the ability to delineate their activity during various network and behavioral states. Indeed, the authors reveal an activity pattern that is unique to NGFCs across epochs of specific network states. Therefore, this clearly demonstrates the applicability of the ID2Cre:Dlx5/6Flpe mouse to study the role of L2-5 NGFCs in a whole brain setting and these in vivo experiments constitute a major strength of the current study.

However, as with many transgenic mice, they are not always perfect, and the authors are very transparent regarding the additional, albeit a relatively smaller number of reported non-NGFCs particularly those of the CCK IN subtype. Indeed, clear morpho-functional divergence is revealed by the authors between these ID2 IN subpopulations. Furthermore, it is possible that this variability may differ across varying cortical regions. Thus, careful consideration of this caveat is necessary when using this mouse for future in vitro and in vivo studies. Related to this matter is a concern regarding the framing of the manuscript. The authors term the ID2 mixed population as the "4th group" since they do not express PV, SST, and VIP. One could argue this is a matter of semantics but to combine IN types that display distinct morphological and physiological properties into a single "group" based on one molecular feature is not consistent with that proposed by the widely accepted Petilla terminology (Ascoli et al., 2008).

Of interest to many who investigate cortical INs is the ability to genetically target specific subtypes during development. To this end, a potential and welcome addition to the manuscript would be an analysis (perhaps restricted to distribution/molecular characterization) highlighting whether the Id2cre:Dlx5/6Flpe strategy allows genetic access to layer 2-5 NGFCs during postnatal development following maternal tamoxifen administration.

Regardless, the experiments in the current study are, in general, well performed and clearly presented with the authors' conclusions supported by the results. Thus, it is clear that further refinements to genetic strategies are obviously required to exclusively target NGFCs throughout the cortical depth. Nevertheless, in the interim, the approach described in this current manuscript will be of use to the neuroscience community and help to further unravel the physiological role of this relatively understudied neuronal subtype.

Reviewer #1 (Public Review):

This manuscript explores physiological properties of Purkinje-to-nuclear synapses. The report provides largely incremental advances over what has already been discovered about this synaptic relationship. The main findings, as articulated by the authors, are that Purkinje-to-nuclear synaptic strength is variable, with a few very strong inputs to the cerebellar nuclei. They show that single inputs effectively inhibit nuclear firing and that the diversity of synaptic strength influences nuclear neuron responsivity to inputs by enhancing synaptic variance. In addition, while not necessarily surprising, it's nice to see that stronger inputs would have a stronger influence on a postsynaptic cell, both in terms of rates and temporal coding transfer. Overall, as it stands, the manuscript is not very scholarly, overstates the novelty of findings, and frames a straw-man. That said, buried in here are some potentially interesting observations.

Reviewer #2 (Public Review):

In this manuscript, the authors address how cerebellar Purkinje cells (PC) control the firing of nuclear cells (CbN), the output stage of the cerebellar. They used patch-clamp recordings in acute cerebellar slices, and combined dynamic clamp with simulations of nuclear cell firing rate.

This article addresses one of the most fundamental unresolved question of the cerebellar physiology: how inhibitory PCs control the output stage of the cerebellum?<br /> They first described a developmental evolution of the that PC-CbN synapses. Inhibitory synaptic weights become highly variable after three weeks of age, with a group of very large PC inputs. They used dynamic clamp to examine the influence of these variable inputs on CbN firing rate. They demonstrate that while all input size affect CbN discharge, larger ones can stop them for a few milliseconds. Using a distribution of variable input size, they showed that increasing the variability of PC inputs favor CbN discharge, while increasing the magnitude of a constant inhibitory conductance decrease their firing rate. By varying the frequency of PC inputs, they suggest that CbNs faithfully transmit rate code, but larger inputs are more effective to decrease their firing rate. Finally, addressing how synchrony of variable PC inputs influence CbN discharge, dynamic clamp studies and simulations showed that input synchronization enhance firing, but driven by the total charge of the inhibitory input.

The keystone observations that PC inputs are highly variable is very interesting and convincing and open new questions about PC-CbN plasticity. More importantly the combination of dynamic clamp and simulations is a real strength of the study, allowing the authors to test many combinations of inputs in real cells and extrapolating their hypotheses in silico. Weaknesses result from the assumptions made on the construction of the distribution of inputs and the many different conditions explored. The organization of the article could be difficult to read for a non-specialist of cerebellar physiology.

Reviewer #1 (Public Review):

The authors have employed a digital twin approach to show that depending on the underlying disease mechanism, a digital replica constructed from human data can both recapitulate clinical findings, but also provide important insights into the fundamental disease state by revealing underlying contributing mechanisms. Moreover, the authors are able to show that a disease state caused by two different underlying genetic anomalies exhibit different electrical and morphological profiles.

This is important information as it allows for potential stratification of treatment approaches in future cases based on underlying phenotype by linking it to specific genotype properties. One of the most innovative aspects of the study is the mismatch switching between personalized structure, remodelling and genotype specific electrophysiological properties. The approach is elegant and allows for further exposure of the key mechanisms that contribute to the development of ventricular tachycardia circuits. One addition that could add more insight is to predict the effect of structural remodelling alone well, considering only normal electrophysiological models. Another interesting approach would be a sensitivity analysis, to determine how sensitive the VT circuits are to the specific geometry of the patient and remodelling that occurs during the disease, such an approach could also be used to determine how sensitive the outputs are to electrophysiological model inputs.

Reviewer #2 (Public Review):

The authors of this paper use a "digital twin" computational model of electrophysiology to investigate the pathology of Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) in several patients undergoing Electro-Physiological Studies (EPS) to treat Ventricular Tachycardias (VTs). The digital twin computational models are customised to the individual patient in two ways. Firstly, information on the patient's heart geometry and muscle/fibrous structure is extracted from Late Gadolium-Enhanced Magnetic Resonance Image (LGE-MRI) scans. Secondly, information from the patient's genotype is used to decide the particular electrophysiological cell model to use in the computational model. The two patient genotypes investigated include a Gene Ellusive (GE) group characterised by abnormal fibrous but normal cell electrical physiology and a palakophilin-2 (PKP2) group in which patients have abnormal fibrotic remodelling and distorted electrical conduction. The computational model predicts the locations and pathways of re-entrant circuits that cause VT. The model results are compared to previous recordings of induced VTs obtained from EPS studies.

The paper is very well written, and the modelling study is well thought out and thorough and represents an exemplar in the field. The major strengths of the paper are the use of a personalised patient model (geometry, fibrous structure and genotype) in a clinically relevant setting. Such a comprehensive personal model puts this paper at the forefront of such models in the field. The main weaknesses of the paper are more of a reflection on what is required for creating such models than on the study itself. As the authors acknowledge, the number of patients in each group is small. Additional patients would allow for statistical significance to be investigated.

The paper's authors set out to demonstrate the use of a "digital twin" computational model in the clinical setting of ARVC. The main findings of the paper were threefold. Firstly, the locations of VTs could be accurately predicted. There was a difference in the abnormal fibrous structure between the two genotype groups. Finally, there was an interplay between the fibrous structure of the heart and the cellular electrophysiology in that the fibrous remodelling was responsible for VTs in the GE group, but in the PKP2 group VTs were caused by slowed electrical conduction and altered restitution. The study successfully met the aims of the paper.

The major impact of the paper will be in demonstrating that a personalised computational model can a) be developed from available measurements (albeit at the high end of what would normally be measured clinically) and b) generate accurate results that may prove helpful in a clinical setting. Another impact is the finding in the paper that the cause of VTs may be different for the two genotypes investigated. The different interplay between fibrous and electrophysiology suggested by the modelling results may provide insights into different treatments for the different genotypes of the pathology. The authors use open-source software and have deposited all non-confidential data in publically available repositories.

Reviewer #3 (Public Review):

Overview: The authors propose a personalized ventricular computational model (Geno-DT) that incorporates the patient's structural remodeling (fibrosis and scar locations based on LGE-CMR scans) as well as genotyping (cell membrane kinetics based on genetic testing results) to predict VT locations and morphologies in ARVC setting.<br /> To test the model, the authors conducted a retrospective study using 16 ARVC patient data with two genotypes (PKP2, GE) and reported high degree of sensitivity, specificity, and accuracy. In addition, the authors determined that in GE patients, VT was driven by fibrotic remodeling, whereas, in PKP2 patients, VT was associated with a combination of structural and electrical remodeling (slowed conduction and altered restitution).<br /> Based on the findings, the authors recommend using Geno-DT approach to augment therapeutic accuracy in treatment of ARVC patients.

Critiques:

1. The small sample size is a limitation but has already been acknowledged and documented by the authors.

2. Another limitation is the consideration of only two of the possible genotypes in developing the cell membrane kinetics, but again has been acknowledged by the authors.

Final Thoughts: The authors have done a commendable job in targeting a disease phenotype that is relatively rare, which constrains the type of data that can be collected for research. Their personalized computational model approach makes a valuable contribution to furthering our understanding of ARVC mechanisms.

Reviewer #1 (Public Review):

Establishing direct links between the neuronal connectivity information of connectomics datasets with circuit physiology and behavior and exciting current research area in neurobiology. Until recently, studies of aggression in Drosophila had been conducted largely in males, and many of the neurons involved in this behavior are male-specific clusters. Since the currently available fly brain connectomes come from female brains, their applicability for the study of the circuitry underlying aggressive behavior is very limited.

The authors have previously used the Janelia hemibrain connectome paired with behavior analysis to show that activating either the aIPg or pC1d cell types can induce short-term aggression in females, while activation of other PC1 clusters (a-c and e) does not. Here they expand on those findings, showing that optogenetic stimulation of aIPg neurons was sufficient to promote an aggressive internal state lasting at least 10 minutes following a 30-second activation. In addition, the authors show that while stimulation of PC1d alone is not sufficient to induce this persistent aggressive state, simultaneous activation of PC1d + PC1e is, suggesting a synergistic effect. Connectomics analysis performed in the authors' previous study had shown that PC1d and aIPg are interconnected. However, silencing pC1d neuronal activity did not reduce aIPg-evoked persistent aggression, indicating that the aggressive state did not depend on pC1d-aIPg recurrent connectivity.

The conclusions are well supported by the data, and the results presented in this manuscript represent an important contribution to our understanding of the neuronal circuitry underlying female aggression.

Reviewer #2 (Public Review):

The mechanisms that mediate female aggression remain poorly understood. Chiu, Schretter, and colleagues, employed circuit dissection techniques to tease apart the specific roles of particular doublesex and fruitless expressing neurons in the fly Drosophila in generating a persistent aggressive state. They find that activating the fruitless positive alPg neurons, generated an aggressive state that persisted for >10min after the stimulation ended. Similarly, activating the doublesex positive pC1de neurons also generated a persistent state. Activating pC1d or pC1e individually did not induce a persistent state. Interestingly, while neural activation of alPGs and pC1d+e neurons induced persistent behavioural states it did not induce persistent activity in the neurons being activated.

The conclusions of this paper are well supported by the data, there were only a few points where clarification might help:

1) Figure 3 is a little confusing. This is a circuit behavioural epistasis experiment where the authors activate alPg with CsChrimson while inhibiting pC1d with Kir2.1. In Fig. 2 flies were separated for 10 min following stimulation which allowed for identification of a persistent state. However, in Fig 3 it appears as if flies were allowed to freely interact during and immediately post-stimulation. It is unclear why flies were not separated as in Fig. 2, which makes it difficult to compare the two results. Some discussion of this point would help. Also, from the rasters it appears as if inhibition of pC1d reduced aggression induced by alPg during the stimulation period. Is this true?

2) pC1e neurons also have recurrent connectivity with alPg neurons. It might help to also discuss the potential role of this arm of the microcircuit.

Reviewer #3 (Public Review):

Two studies published in 2020 independently identified the alPg, pC1d, and pC1e neurons to be involved in initiating and maintaining a state of aggression in female Drosophila. Both studies combined behavioural analyses, optogenitic manipulation of neurons, and connectomics. One of these studies proposed that the extensive interconnections seen between the alPg and pC1d+e neurons might represent a recurrent motif known to support persistent behvioural states in other systems. In this manuscript, the authors test this idea and report that their data do not support it. Specifically, they report that alPg or pC1d+e (but not pC1d alone) can initiate a persistent state of aggression. But they find that the persistent aggressive state is maintained even when the pC1d neurons are inactivated. Finally, they show that neither of these neurons themselves sustains neuronal activity upon stimulation, nor do either of them induce a persistent activity in the other. Together, their data suggest that the recurrent connection between alPg and pC1d is not what supports the persistent state. The data underlying these claims are convincing. A possibility to explore before ruling out recurrent motifs (at this circuit level) in maintaining aggression is that the connections between alPg and pC1e can compensate for the loss of pC1e. Overall, the study is important and will be of interest to those who study the circuit basis of persistent behavioural states, but also to neuroscientists in general.

Reviewer #1 (Public Review):

In this study, Clark et. al. uncovered an association between the positional encoding of grid cell activity with good performance in spatial navigation tasks that requires path integration, highlighting the contribution of grid firing to behavior. Using electrophysiology approaches, the authors measured MEC neuron activity while mice performed a spatial memory task in one-dimensional (1D) virtual tracks, where the mice must stop in a specific reward zone for a reward. Individual trials either had a visual cue at the reward location (beaconed trials), no cue at the reward location (non-beaconed trials), or no cue and no reward regardless of stopping (probe trials). The authors identified that grid cells could encode track position or distance traveled, which were distinguished on a per-session or per-trial basis by calculating whether cell firing was periodic with respect to track length (firing at the same location on each trial) or periodic with respect to distance traveled (firing locations drift across trials), respectively. While some behavioral sessions had stable coding of either position or distances, other sessions exhibited coding schemes that switched between these two modes. The behavioral performance in beaconed trials was comparable when grid cells showed position or distance coding. In contrast, mice perform better on non-beaconed trials when grid cells showed position coding. The authors concluded that position coding in grid cells may enhance performance when tasks require path integration (non-beaconed condition).

The conclusions of this paper are mostly well supported by data, the finding about the association between grid cell encoding and behavior in spatial memory tasks is important. However, some aspects of the analysis need to be clarified or extended.

1. While the current dataset aims to demonstrate a "correlation" between grid cell encoding and task performance, the other variables that could confound this correlation should be carefully examined.<br /> (1) The exact breakdown of the fraction of beaconed/non-beaconed/probe trials is never shown. if the session makeup has a significant effect on the coding scheme or other results, this variable should be accounted for.<br /> (2) The manuscript did not provide information about whether individual mice experienced sessions with different combinations of the three trial types, and whether they show different preferences in position or distance encoding even in comparable sessions. This leads to the question of whether different behavior and activity encoding were dominated by experimental or natural differences between individual mice. Presenting the data per mouse will be helpful.<br /> (3) Related to the above point, in Figure 5, the mice appeared to behave worse in probe trials than non-beaconed trials. If the mouse did not know if a trial is a probe or a non-beacon trial, they should behave equivalently until the reward location and thus should stop an equal amount. If this difference is because multiple probe trials are placed consecutively, did the mouse learn that it will not get a reward and then stop trying to get rewards? Did this affect switching between position and distance coding?<br /> (4) It is not shown how the behaviors (e.g., running speed away from the reward zone, licking for reward) in beaconed/non-beaconed/probe trials were different and whether the difference in behaviors led to the different encoding schemes.<br /> 2. Regarding the behavior and activity encoding on a trial-by-trial basis, did the behavioral change occur first, or did the encoding switch occur first, or did they happen within the same trial? This analysis will potentially determine whether the encoding is causal for the behavior, or the other way around.<br /> 3. The author determined that the grid cell coding schemes were limited to distance encoding and position encoding. However, there could be other schemes, such as switching between different position encodings (with clear spatial fields but at different locations), as indicated by Low et. al., 2021, and switching between different distant encodings (with different distance periods). If these other schemes indeed existed in the data, they might contribute to the variation of the behaviors.<br /> 4. The percentage of neurons categorized in each coding scheme was similar between non-grid and grid cells. This implies that non-grid cells might switch coding schemes in sync with grid cells, which would mean the whole MEC network was switching between distance and position coding. This raises the question of whether the grid cell coding scheme was important per se, or just the MEC network coding scheme.<br /> 5. In Figure 2 there are several cell examples that are categorized as distance or position coding but have a high fraction of the other coding scheme on a per-trial basis. Given this variation, the full session data in F should be interpreted carefully, since this included all cells and not just "stable" coding cells. It will be cleaner to show the activity comparison only between the stable cells.<br /> 6. The manuscript is not well written. Throughout the manuscript, there are many unexplained concepts (especially in the introduction) and methods, mis-referenced figures, and unclear labels.

Reviewer #2 (Public Review):

Clark and Nolan's study aims to test whether the stability of grid cell firing fields is associated with better spatial behavior performance on a virtual task. Mice were trained to stop at a rewarded location along a virtual linear track. The rewarded location could be marked by distinct visual stimuli or be unmarked. When the rewarded location was unmarked, the animal had to estimate its distance run from the beginning of the trial to know where to stop. When the mouse reached the end of the virtual track, it was teleported back to the start of the virtual track.

The authors found that grid cells could fire in at least two modes. In the "virtual position" mode, grid firing fields had stable positions relative to the virtual track. In the "distance run" mode, grid fields were decoupled from the virtual cues and appeared to be located as a function of distance run on the running wheel. Importantly, on trials in which the rewarded location was unmarked, the behavioral performance of mice was better when grid cells fired in the "virtual position" mode.

This study is very timely as there is a pressing need to identify/delimitate the contribution of grid cells to spatial behaviors. More studies in which grid cell activity can be associated with navigational abilities are needed. The link proposed by Clark and Nolan between "virtual position" coding by grid cells and navigational performance is a significant step toward better understanding how grid cell activity might support behavior. It should be noted that the study by Clark and Nolan is correlative. Therefore, the effect of selective manipulations of grid cell activity on the virtual task will be needed to evaluate whether the activity of grid cells is causally linked to the behavioral performance on this task. In a previous study by the same research group, it was shown that inactivating the synaptic output of stellate cells of the medial entorhinal cortex affected mice's performance of the same virtual task (Tennant et al., 2018). Although this manipulation likely affects non-grid cells, it is still one of the most selective manipulations of grid cells that are currently available.

When interpreting the "position" and "distance" firing mode of grid cells, it is important to appreciate that the "position" code likely involves estimating distance. The visual cues on the virtual track appear to provide mainly optic flow to the animal. Thus, the animal has to estimate its position on the virtual track by estimating the distance run from the beginning of the track (or any other point in the virtual world).

It is also interesting to consider how grid cells could remain anchored to virtual cues. Recent work shows that grid cell activity spans the surface of a torus (Gardner et al., 2022). A run on the track can be mapped to a trajectory on the torus. Assuming that grid cell activity is updated primarily from self-motion cues on the track and that the grid cell period is unlikely to be an integer of the virtual track length, having stable firing fields on the virtual track likely requires a resetting mechanism taking place on each trial. The resetting means that a specific virtual track position is mapped to a constant position on the torus. Thus, the "virtual position" mode of grid cells may involve 1) a trial-by-trial resetting process anchoring the grid pattern to the virtual cues and 2) a path integration mechanism. Just like the "virtual position" mode of grid cell activity, successful behavioral performance on non-beaconed trials requires the animal to anchor its spatial behavior to VR cues.

One main conclusion of this study is that better performance on the VR task was observed when the grid cells were anchored to the reference frame that was the most behaviorally relevant.

Reviewer #3 (Public Review):

This study addresses the major question of 'whether and when grid cells contribute to behavior'. There is no doubt that this is a very important question. My major concern is that I'm not convinced that this study gives a significant contribution to this question, although this study is well-performed and potentially interesting. This is mainly due to the fact that the relation between grid cell properties and behavior is exclusively correlative and entirely based on single cell activity, although the introduction mentions quite often the grid cell network properties and dynamics. In general, this study gives the impression that grid cells exclusively support the cognitive processes involved in this task. This problem is in part related to the text. However, it would be interesting to look at the population level (even beyond grid cells) to test whether at the network level, the link between behavioral performance and neural activity is more straightforward compared to the single-cell level. This approach could reconcile the present results with those obtained in their previous study following MEC inactivation.

The authors used a statistical method based on the computation of the frequency spectrum of the spatial periodicity of the neural firing to classify grid cells as 'position-coding' (with fields anchored to the virtual track) and 'distance-coding' (with fields repeating at regular intervals across trials). This is an interesting approach that has nonetheless the default to be based exclusively on autocorelograms. It would be interesting to compare with a different method based on the similarities between raw maps. Beyond this minor point, cell categorization is performed using all trial types. Each trial type (i.e. beacon or non-beacon) is supposed to force mice to use different strategies and should induce different spatial representations within the entorhinal-hippocampal circuit (and not only in the grid cell system). In that context, since all trials are mixed, it is difficult to extrapolate general information. On page 5 the authors state that 'Since only position representations should reliably predict the reward location, ..., we reasoned that the presence of positional coding could be used to assess whether grid firing contributes to the ongoing behaviour'.

I do not agree with this statement. First of all, position coding should be more informative only in a cue-guided trial. Second, distance coding could be as informative as position coding since at the network level may provide information relevant to the task (such as distance from the reward). This possibility is not tested here. Third, position-coding is interpreted as more relevant because it predominates in correct trials. However, this does not imply that this coding scheme is indeed used to perform correct trials. It could be more informative to push forward the correlative analysis by looking at whether behavioral performance can be predicted by the coding scheme on a trial-by-trial basis. This analysis would not provide a causal relation between cell activity and behavior, but could strengthen the correlation between the two.

Reviewer #1 (Public Review):

Aging is associated with a number of physiologic changes including perturbed circadian rhythms. However, mechanisms by which rhythms are altered remain unknown. Here authors tested the hypothesis that age-dependent factors in the sera affect the core clock or outputs of the core clock in cultured fibroblasts. They find that both sera from young and old donors are equally potent at driving robust ~24h oscillations in gene expression, and report the surprising finding that the cyclic transcriptome after stimulation by young or old sera differs markedly. In particular, genes involved in the cell cycle and transcription/translation remain rhythmic in both conditions, while genes associated with oxidative phosphorylation and Alzheimer's Disease lose rhythmicity in the aged condition. Also, the expression of cycling genes associated with cholesterol biosynthesis increases in the cells entrained with old serum. Together, the findings suggest that age-dependent blood-borne factors, yet to be identified, affect circadian rhythms in the periphery. The most interesting aspect of the paper is that the data suggest that the same system (BJ-5TA), may significantly change its rhythmic transcriptome depending on how the cells are synchronized. While there is a succinct discussion point on this, it should be expanded and described whether there are parallels with previous works, as well as what would be possible mechanisms for such an effect.

Major points:<br /> Fig 1 and Table S1. Serum composition and levels of relevant blood-borne factors probably change in function of time. At what time of the day were the serum samples from the old and young groups collected? This important information should be provided in the text and added to Table S1.

Fig 2A. Luminescence traces: the manuscript would greatly benefit from inclusion of raw luminescence traces.

Fig 2. Of the many genes that change their rhythms after stimulation with young and old sera, what are the typical fold changes? For example, it would be useful to show histograms for the two groups. Does one group tend to have transcript rhythms of higher or lower fold changes?

Fig. 2 Gene expression. Also here, the presentation would benefit from showing a few key examples for different types of responses.

What was the rationale to use these cells over the more common U2OS cells? Are there similarities between the rhythmic transcriptomes of the BJ-5TA cells and that of U2OS cells or other human cells? This could easily be assessed using published datasets.

For the rhythmic cell cycle genes, could this be the consequence of the serum which synchronizes also the cell cycle, or is it rather an effect of the circadian oscillator driving rhythms of cell cycle genes?

While the reduction of rhythmicity in the old serum for oxidative phosphorylation transcripts is very interesting and fits with the general theme that metabolic function decreases with age, it is puzzling that the recipient cells are the same, but it is only the synchronization by the old and young serum that changes. Are the authors thus suggesting that decrease of metabolic rhythms is primarily a non cell-autonomous and systemic phenomenon? What would be a potential mechanism?

The delayed shifts after aged serum for clock transcripts (but not for Bmal1) are interesting and indicate that there may be a decoupling of Bmal1 transcript levels from the other clock gene phases. How do the authors interpret this? could it be related to altered chronotypes in the elderly?

Reviewer #2 (Public Review):

Schwarz et al. have presented a study aiming to investigate whether circulating factors in sera of subjects are able to synchronize depending on age, circadian rhythms of fibroblast. The authors used human serum taken from either old (age 70-76) or young (age 25-30) individuals to synchronise cultured fibroblasts containing a clock gene promoter driven luciferase reporter, followed by RNA sequencing to investigate whole gene expression.

This study has the potential to be very interesting, as evidence of circulating factors in sera that mediate peripheral rhythms has long been sought after. Moreover, the possibility that those factors are affected by age which could contribute to the weaken circadian rhythmicity observed with aging.

Here, the authors concluded that both old and young sera are equally competent at driving robust 24 hour oscillations, in particular for clock genes, although the cycling behaviour and nature of different genes is altered between the two groups, which is attributed to the age of the individuals. This conclusion could however be influenced by individual variabilities within and between the two age groups. The groups are relatively small, only four individual two females and two males, per group. And in addition, factors such as food intake and exercise prior to blood drawn, or/and chronotype, known to affect systemic signals, are not taken into consideration. As seen in figure 4, traces from different individuals vary heavily in terms of their patterns, which is not addressed in the text. Only analysing the summary average curve of the entire group may be masking the true data. More focus should be attributed to investigating the effects of serum from each individual and observing common patterns. Additionally, there are many potential causes of variability, instead or in addition to age, that may be contributing to the variation both, between the groups and between individuals within groups. All of this should be addressed by the authors and commented appropriately in the text.

The authors also note in the introduction that rhythms in different peripheral tissues vary in different ways with age, however the entire study is performed on only fibroblast, classified as peripheral tissue by the authors. It would be very interesting to investigate if the observed changes in fibroblast are extended or not to other cell lines from diverse organ origin. This could provide information about whether circulating circadian synchronising factors could exert their function systemically or on specific tissues. At the very least, this hypothesis should be addressed within the discussion.

In addition to the limitations indicated above I consider that the data of the study is an insufficiently analysis beyond the rhythmicity analysis. Results from the STRING and IPA analysis were merely descriptive and a more comprehensive bioinformatic analysis would provide additional information about potential molecular mechanism explaining the differential gene expression. For example, enrichment of transcription factors binding sites in those genes with different patters to pinpoint chromatin regulatory pathways.

Reviewer #1 (Public Review):

Warnhoff et al present a genetic investigation of the response of C. elegans to high dietary cysteine. Using a Pcdo-1::CDO-1::GFP reporter (for a cysteine dioxygenase gene) and unbiased mutagenesis, they identify multiple alleles, including nonsense alleles, in egl-9 and rhy-1, which they validate with reference alleles. Further mutational analysis shows that hif-1 and cysl-1, components of the same established genetic regulatory pathway, also act in cdo-1 regulation. High dietary levels of cysteine activate cdo-1 expression, but loss of cdo-1 does not cause sensitivity to excess dietary cysteine, whereas cysl-1 and hif-1 are completely inviable in these conditions. Using sulfite oxidase suox-1 mutant and double and triple mutant analysis the authors show that the defects caused by suox-1 deletion (which causes sulfite accumulation) are exacerbated by loss of egl-9, which is alleviated by concomitant loss of enzymes cdo-1 / cth-2 or regulators rhy-1 / hif-1, demonstrating that the key issue is cysteine derived sulfites. Further genetic analysis shows that although egl-9 is required for cdo-1 induction, this is only partially dependent on its hydroxylase activity and the egl-9 partner vhl-1 is also only partially involved.

The significance of the findings is that they describe a regulatory pathway by which organisms might respond to high levels of cysteine in vivo.

Strengths<br /> - The genetic analysis is generally well done and convincing, with multiple alleles identified for each gene, several reporters used for cdo-2, etc.<br /> - Genetic analysis using site-directed mutagenesis of egl-9 and cdo-1 with point mtuations is especially nice.<br /> - The data are analyzed and represented properly, and microscopy data have been quantified.<br /> - The paper is also written quite clearly and the figures are easy to understand.

Weaknesses<br /> - The relevance is somewhat unclear. High cysteine levels can be achieved in the laboratory, but, is this relevant in the life of C. elegans? Or is there physiological relevance in humans, e.g. a disease? The authors state "cells and animals fed excess cysteine and methionine", but is this more than a laboratory excess condition? SUOX nonfunctional conditions in humans don't appear to tie into this, since, in that context, the goal is to inactivate CDO or CTH to prevent sulfite production. The authors also mention cancer, but the link to cysteine levels is unclear. In that sense, then, the conditions studied here may not carry much physiological relevance.<br /> - The pathway is described as important for cysteine detoxification, which is described to act via H2S (Figure 6). Much of that pathway has already been previously established by the Roth, Miller, and Horvitz labs as critical for the H2S response. While the present manuscript adds some additional insight such as the additional role of RHY-1 downstream on HIF-1 in promoting toxicity, this study therefore mainly confirms the importance of a previously described signalling pathway, essentially adding a new downstream target rhy-1 -> cysl-1 -> egl-9 -> hif-1 -> sqrd-1/cdo-1. The impact of this finding is reduced by the fact that cdo-1 itself isn't actually required for survival in high cysteine, suggesting it is merely a maker of the activity of this previously described pathway.

Reviewer #2 (Public Review):

The authors investigate the transcriptional regulation of cysteine dioxygenase (CDO-1) in C. elegans and its role in maintaining cysteine homeostasis. They show that high cysteine levels activate cdo-1 transcription through the hypoxia-inducible transcription factor HIF-1. Using transcriptional and translational reporters for CDO-1, the authors propose a negative feedback pathway involving RHY-1, CYSL-1, EGL-9, and HIF-1 in regulating cysteine homeostasis.

Genetics is a notable strength of this study. The forward genetic screen, gene interaction, and epistasis analyses are beautifully designed and rigorously conducted, yielding solid and unambiguous conclusions on the genetic pathway regulating CDO-1. The writing is clear and accessible, contributing to the overall high quality of the manuscript.

Addressing the specifics of cysteine supplementation and interpretation regarding the cysteine homeostasis pathway would further clarify the paper and strengthen the study's conclusions.

First, the authors show that the supplementation of exogenous cysteine activates cdo-1p::GFP. Rather than showing data for one dose, the author may consider presenting dose-dependency results and whether cysteine activation of cdo-1 also requires HIF-1 or CYSL-1, which would be important data given the focus and major novelty of the paper in cysteine homeostasis, not the cdo-1 regulatory gene pathway. While the genetic manipulation of cdo-1 regulators yields much more striking results, the effect size of exogenous cysteine is rather small. Does this reflect a lack of extensive condition optimization or robust buffering of exogenous/dietary cysteine? Would genetic manipulation to alter intracellular cysteine or its precursors yield similar or stronger effect sizes?

Second, there remain several major questions regarding the interpretation of the cysteine homeostasis pathway. How much specificity is involved for the RHY-1/CYSL-1/EGL-9/HIF-1 pathway to control cysteine homeostasis? Is the pathway able to sense cysteine directly or indirectly through its metabolites or redox status in general? Given the very low and high physiological concentrations of intracellular cysteine and glutathione (GSH, a major reserve for cysteine), respectively, there is a surprising lack of mention and testing of GSH metabolism. In addition, what are the major similarities and differences of cysteine homeostasis pathways between C. elegans and other systems (HIF dependency, transcription vs post-transcriptional control)? These questions could be better discussed and noted with novel findings of the current study that are likely C. elegans specific or broadly conserved.

Reviewer #3 (Public Review):

There has been a long-standing link between the biology of sulfur-containing molecules (e.g., hydrogen sulfide gas, the amino acid cysteine, and its close relative cystine, et cetera) and the biology of hypoxia, yet we have a poor understanding of how and why these two biological processes and are co-regulated. Here, the authors use C. elegans to explore the relationship between sulfur metabolism and hypoxia, examining the regulation of cysteine dioxygenase (CDO1 in humans, CDO-1 in C. elegans), which is critical to cysteine catabolism, by the hypoxia inducible factor (HIF1 alpha in humans, HIF-1 in C. elegans), which is the key terminal effector of the hypoxia response pathway that maintains oxygen homeostasis. The authors are trying to demonstrate that (1) the hypoxia response pathway is a key regulator of cysteine homeostasis, specifically through the regulation of cysteine dioxygenase, and (2) that the pathway responds to changes in cysteine homeostasis in a mechanistically distinct way from how it responds to hypoxic stress.

Briefly summarized here, the authors initiated this study by generating transgenic animals expressing a CDO-1::GFP protein chimera from the cdo-1 promoter so that they could identify regulators of CDO-1 expression through a forward genetic screen. This screen identified mutants with elevated CDO-1::GFP expression in two genes, egl-9 and rhy-1, whose wild-type products are negative regulators of HIF-1, raising the possibility that cdo-1 is a HIF-1 transcriptional target. Indeed, the authors provide data showing that cdo-1 regulation by EGL-9 and RHY-1 is dependent on HIF-1 and that regulation by RHY-1 is dependent on CYSL-1, as expected from other published findings of this pathway. The authors show that exogenous cysteine activates cdo-1 expression, reflective of what is known to occur in other systems. Moreover, they find that exogenous cysteine is toxic to worms lacking CYSL-1 or HIF-1 activity, but not CDO-1 activity, suggesting that HIF-1 mediates a survival response to toxic levels of cysteine and that this response requires more than just the regulation of CDO-1. The authors validate their expression studies using a GFP knockin at the cdo-1 locus, and they demonstrate that a key site of action for CDO-1 is the hypodermis. They present genetic epistasis analysis supporting a role for RHY-1, both as a regulator of HIF-1 and as a transcriptional target of HIF-1, in offsetting toxicity from aberrant sulfur metabolism. The authors use CRISPR/Cas9 editing to mutate a key amino acid in the prolyl hydroxylase domain of EGL-9, arguing that EGL-9 inhibits CDO-1 expression through a mechanism that is largely independent of the prolyl hydroxylase activity.

Overall, the data seem rigorous, and the conclusions drawn from the data seem appropriate. The experiments test the hypothesis using logical and clever molecular genetic tools and design. The sample size is a bit lower than is typical for C. elegans papers; however, the experiments are clearly not underpowered, so this is not an issue. The paper is likely to drive many in the field (including the authors themselves) into deeper experiments on (1) how the pathway senses hypoxia and sulfur/cysteine/H2S using these distinct mechanisms/modalities, (2) how oxygen and sulfur/cysteine/H2S homeostasis influence one another, and (3) how this single pathway evolved to sense and respond to both of these stress modalities.

Major strengths of the paper include (1) the use of the powerful whole animal C. elegans model to reveal results that have meaning in vivo, (2) the careful demonstration through mutant rescue experiments that key transgenes have functional activity, (3) the use of CRISPR/Cas9 editing to mutate a critical residue in the catalytic domain of the EGL-9 prolyl hydroxylase, (4) transgenic rescue experiments that show that CDO-1 operates in the hypodermis with regard to the larval arrest phenotype, and (5) the thorough epistatic analysis of different pathway mutants.

Major weaknesses of the paper include (1) the over-reliance on genetic approaches, (2) the lack of novelty regarding prolyl hydroxylase-independent activities of EGL-9, and (3) the lack of biochemical approaches to probe the underlying mechanism of the prolyl hydroxylase-independent activity of EGL-9.

Major Issues We Feel the Authors Should Address:

1. One particularly glaring concern is that the authors really do not know the extent to which the prolyl hydroxylase activity is (or is not) impacted by the H487A mutation in egl-9(rae276). If there is a fair amount of enzymatic activity left in this mutant, then it complicates interpretation. The paper would be strengthened if the authors could show that the egl-9(rae276) eliminates most if not all prolyl hydroxylase activity. In addition, the authors may want to consider doing RNAi for egl-9 in the egl-9(rae276) mutant as a control, as this would support the claim that whatever non-hydroxylase activity EGL-9 may have is indeed the causative agent for the elevation of CDO-1::GFP. Without such experiments, readers are left with the nagging concern that this allele is simply a hypomorph for the single biochemical activity of EGL-9 (i.e., the prolyl hydroxylase activity) rather than the more interesting, hypothesized scenario that EGL-9 has multiple biochemical activities, only one of which is the prolyl hydroxylase activity.

2. The authors observed that EGL-9 can inhibit HIF-1 and the expression of the HIF-1 target cdo-1 through a combination of activities that are (1) dependent on its prolyl hydroxylase activity (and subsequent VHL-1 activity that acts on the resulting hydroxylated prolines on HIF-1), and (2) independent of that activity. This is not a novel finding, as the authors themselves carefully note in their Discussion section, as this odd phenomenon has been observed for many HIF-1 target genes in multiple publications. While this manuscript adds to the description of this phenomenon, it does not really probe the underlying mechanism or shed light on how EGL-9 has these dual activities. This limits the overall impact and novelty of the paper.

3. Cysteine dioxygenases like CDO-1 operate in an oxygen-dependent manner to generate sulfites from cysteine. CDO-1 activity is dependent upon availability of molecular oxygen; this is an unexpected characteristic of a HIF-1 target, as its very activation is dependent on low molecular oxygen. Authors neither address this in the text nor experimentally, and it seems a glaring omission.

4. The authors determined that the hypodermis is the site of the most prominent CDO-1::GFP expression, relevant to Figure 4. This claim would be strengthened if a negative control tissue, in the animal with the knockin allele, were shown. The hypodermal specific expression is a highlight of this paper, so it would make this article even stronger if they could further substantiate this claim.

Minor issues to note:

Mutants for hif-1 and cysl-1 are sensitive to exogenous cysteine levels, yet loss of CDO-1 expression is not sufficient to explain this phenomenon, suggesting other targets of HIF-1 are involved. Given the findings the authors (and others) have had showing a role for RHY-1 in sulfur amino acid metabolism, shouldn't the authors consider testing rhy-1 mutants for sensitivity to exogenous cysteine?

The cysteine exposure assay was performed by incubating nematodes overnight in liquid M9 media containing OP50 culture. The liquid culture approach adds two complications: (1) the worms are arguably starving or at least undernourished compared to animals grown on NGM plates, and (2) the worms are probably mildly hypoxic in the liquid cultures, which complicates the interpretation.

An easily addressable concern is the wording of one of the main conclusions: that cdo-1 transcription is independent of the canonical prolyl hydroxylase function of EGL-9 and is instead dependent on one of EGL-9's non-canonical, non-characterized functions. There are several points in which the wording suggests that CDO-1 toxicity is independent of EGL-9. In their defense, the authors try to avoid this by saying, "EGL-9 PHD," to indicate that it is the prolyl hydroxylase function of EGL-9 that is not required for CDO-1 toxicity. However, this becomes confusing because much of the field uses PHD and EGL-9/EGLN as interchangeable protein names. The authors need to be clear about when they are describing the prolyl hydroxylase activity of EGL-9 rather than other (hypothesized) activities of EGL-9 that are independent of the prolyl hydroxylase activity.

The authors state in the text, "the egl-9; suox-1 double mutants are extremely sick and slow growing." We appreciate that their "health" assay, based on the exhaustion of food from the plate, is qualitative. We also appreciate that it is a functional measure of many factors that contribute to how fast a population of worms can grow, reproduce, and consume that lawn of food. However, unless they do a lifespan assay and/or measure developmental timing and specifically determine that the double mutant animals themselves are developing and/or growing more slowly, we do not think it is appropriate to use the words "slow growing" to describe the population. As they point out, the rate of consumption of food on the plate in their health assay is determined by a multitude and indeed a confluence of factors; the growth rate is one specific one that is commonly measured and has an established meaning.

Reviewer #1 (Public Review):

The mutation rate and spectrum have been found to differ between populations as well as across individuals within the same population. Hypothesizing that some of the observed variation has a genetic basis, the authors of this paper have made important contributions in the past few years in identifying genetic variants that modify mutation rate or spectrum in natural populations. This paper makes one significant step further by developing a new method for mapping genetic variants associated with the mutation spectrum, which reveals new biological insights.

Using traditional quantitative trait locus (QTL) mapping in the BXD mouse recombinant inbred lines (RILs), the authors of this paper previously identified a genetic locus associated with C>A mutation rate. However, this approach has limited power, as it suffers from multiple testing burden as well as noise in the "observed mutation rate/spectrum phenotype" due to rarity and randomness of mutation events. To overcome these limitations, the authors developed a new method that they named "inter-haplotype distance" (IHD), which in short measures the difference in the aggregate mutation spectrum between two groups of individuals with distinct genotypes at a specific genomic locus. With this new approach, they recover the previously reported candidate mutator locus (near Mutyh gene) and identify a new candidate variant that modifies the C>A mutation rate on only one genetic background. Using more rigorous statistical testing, the authors show convincingly synergistic epistatic effects between the mutator alleles at the two loci.

Overall, the analyses presented are well done and provide convincing evidence for the major findings, including the new candidate mutator locus and its epistatic interaction with the Mutyh locus. The new IHD method introduced is innovative and outperforms traditional QTL mapping under certain conditions, but some of its statistical properties and limitations are not fully described. The part that describes how the method works is a little hard to follow (partially due to the confusing name; see comments below), but the rest of the paper is very well written. Below are my comments and suggestions on how to improve, but I identify no major issues.

The name of the new method "inter-haplotype distance" is more confusing than helpful, as the haplotype information is not critical for implementing this method. First, the mutation spectrum is aggregated genome-wide regardless of the haplotypes where the mutations are found. Second, the only critical haplotype information is that at the focal site (i.e., the locus that is tested for association): individuals are aggregated together when they belong to the same "haplotype group" at the focal site. However, for the classification step, haplotype information is not really necessary: individuals can be grouped based on their genotypes at the given locus (e.g., AA vs AB). As the authors mentioned, this method can be potentially applied to other mutation datasets, where haplotype information may well be unavailable. I hope the authors can reconsider the name and remove the term "haplotype" (perhaps something like "inter-genotype distance"?) to avoid giving the wrong impression that haplotype information is critical for applying this method.

The biggest advantage of the IHD method over QTL mapping is alleviation of the multiple testing burden, as one comparison tests for any changes in the mutation spectrum, including simultaneous, small changes in the relative abundance of multiple mutation types. Based on this, the authors claim that IHD is more powerful to detect a mutator allele that affects multiple mutation types. Although logically plausible, it is unclear under what quantitative conditions IHD can actually have greater power over QTL. It will be helpful to support this claim by providing some simulation results.

The flip side of this advantage of IHD is that, when a significant association is detected, it is not immediately clear which mutation type is driving the signal. Related to this, it is unclear how the authors reached the point that "...the C>A mutator phenotype associated with the locus on chromosome 6", when they only detected significant IHD signal at rs46276051 (on Chr6), when conditioning on D genotypes at the rs27509845 (on Chr4) and no significant signal for any 1-mer mutation type by traditional mapping. The authors need to explain how they deduced that C>A mutation is the major source of the signal. In addition, beyond C>A mutations, can mutation types other than C>A contribute to the IHD signal at rs46276051? More generally, I hope the authors can provide some guidelines on how to narrow a significant IHD signal to specific candidate mutation type(s) affected, which will make the method more useful to other researchers.

To account for differential relatedness between the inbred lines, the authors regressed the cosine distance between the two aggregate mutation spectra on the genome-wide genetic similarity and take the residual as the adjusted test metric. What is the value of the slope from this regression? If significantly non-zero, this would support a polygenic architecture of the mutation spectrum phenotype, which could be interesting. If not, is this adjustment really necessary? In addition, is the intercept assumed to be zero for this regression, and does such an assumption matter? I would appreciate seeing a supplemental figure on this regression.

Reviewer #2 (Public Review):

In this paper Sasani, Quinlan and Harris present a new method for identifying genetic factors affecting germline mutation, which is particularly applicable to genome sequence data from mutation accumulation experiments using recombinant inbred lines. These are experiments where laboratory organisms are crossed and repeatedly inbred for many generations, to build up a substantial number of identifiable germline mutations. The authors apply their method to such data from mice, and identify two genetic factors at two separate genetic loci. Clear evidence of such factors has been difficult to obtain, so this is an important finding. They further show evidence of an epistatic interaction between these factors (meaning that they do not act independently in their effects on the germline mutation process). This is exciting because such interactions are difficult to detect and few if any other examples have been studied.

The authors present a careful comparison of their method to another similar approach, quantitative trait locus (QTL) analysis, and demonstrate that in situations such as the one analysed it has greater power to detect genetic factors with a certain magnitude of effect. They also test the statistical properties of their method using simulated data and permutation tests. Overall the analysis is rigorous and well motivated, and the methods explained clearly.

The main limitation of the approach is that it is difficult to see how it might be applied beyond the context of mutation accumulation experiments using recombinant inbred lines. This is because the signal it detects, and hence its power, is based on the number of extra accumulated mutations linked to (i.e. on the same chromosome as) the mutator allele. In germline mutation studies of wild populations the number of generations involved (and hence the total number of mutations) is typically small, or else the mutator allele becomes unlinked from the mutations it has caused (due to recombination), or is lost from the population altogether (due to chance or perhaps selection against its deleterious consequences).

Nevertheless, accumulation lines are a common and well established experimental approach to studying mutation processes in many organisms, so the new method could have wide application and impact on our understanding of this fundamental biological process.

The evidence presented for an epistatic interaction is convincing, and the authors suggest some plausible potential mechanisms for how this interaction might arise, involving the DNA repair machinery and based on previous studies of the proteins implicated. However as with all such findings, given the higher degree of complexity of the proposed model it needs to be treated with greater caution, perhaps until replicated in a separate dataset or demonstrated in follow-up experiments exploring the pathway itself.

Reviewer #3 (Public Review):

Sasani et al. develop and implement a new method for mutator allele discovery in the BXD mouse population. This new "IHD" method carries several notable strengths, including the ability to aggregate de novo mutations across individuals to reduce data sparsity and to combine mutation rate frequencies across multiple nucleotide contexts into a single estimate. These advantages may render the IHD method better suited to mutator discovery under certain scenarios, as compared to conventional QTL or association mapping. Overall, the theoretical premise of the IHD method is judged to be both strong and innovative, and careful simulation studies benchmark its power.

The authors then apply their method to the BXD mouse recombinant inbred mapping population. As proof-of-principle, they first successfully re-identify a known mutator locus in this population on chr4. Next, to assess possible genetic interactions involving this known mutator, Sasani et al. condition on the chr4 mutator genotype and reimplement the IHD scan. This strategy led them to identify a second locus on chr6 that interacts epistatically with the chr4 locus; mice with "D" alleles at both loci exhibit a significantly increased burden of C>A de novo mutations, even though mice with the D allele at the chr6 locus alone show no appreciable increase in the C>A mutation fraction. This exciting discovery not only adds to the catalog of known mutator alleles, but also reveals key aspects of mutator biology. Notably, this finding reinforces the hypothesis that segregating variants in genes associated with DNA repair influence germline mutation spectra. Further, Sasani et al.'s findings suggest that some mutators may lie dormant until recombined onto a permissive genetic background. This discovery could have intriguing implications for the evolution of mutators in natural populations.

Despite a high level of overall enthusiasm for this work, some weaknesses are identified in the IHD method, approach for nominating candidate genes within the newly identified chr6 locus, and the authors' conclusions.

Under simulated scenarios, the authors' new IHD method is not appreciably more powerful than conventional QTL mapping methods. While this does not diminish the rigor or novelty of the authors findings, it does temper enthusiasm for the IHD method's potential to uncover new mutators in other populations or datasets. Further, adaptation of this methodology to other datasets, including human trios or multigenerational families, will require some modification, which could present a barrier to broader community uptake. Notably, BXD mice are (mostly) inbred, justifying the authors consideration of just two genotype states at each locus, but this decision prevents out-of-the-box application to outbred populations and human genomic datasets. Lastly, some details of the IHD method are not clearly spelled out in the paper. In particular, it is unclear whether differences in BXD strain relatedness due to the breeding epoch structure are fully accounted for in permutations. The method's name - inter-haplotype distance - is also somewhat misleading, as it seems to imply that de novo mutations are aggregated at the scale of sub-chromosomal haplotype blocks, rather than across the whole genome.

Nominating candidates within the chr6 mutator locus requires an approach for defining a credible interval and excluding/including specific genes within that interval as candidates. Sasani et al. delimit their focal window to 5Mb on either side of the SNP with the most extreme P-value in their IHD scan. This strategy suffers from several weaknesses. First, no justification for using 10 Mb window, as opposed to, e.g., a 5 Mb window or a window size delimited by a specific threshold of P-value drop, is given, rendering the approach rather ad hoc. Second, within their focal 10Mb window, the authors prioritize genes with annotated functions in DNA repair that harbor protein coding variants between the B6 and D2 founder strains. While the logic for focusing on known DNA repair genes is sensible, this locus also houses an appreciable number of genes that are not functionally annotated, but could, conceivably, perform relevant biological roles. These genes should not be excluded outright, especially if they are expressed in the germline. Further, the vast majority of functional SNPs are non-coding, (including the likely causal variant at the chr4 mutator previously identified in the BXD population). Thus, the author's decision to focus most heavily on coding variants is not well-justified. Sasani et al. dedicate considerable speculation in the manuscript to the likely identity of the causal variant, ultimately favoring the conclusion that the causal variant is a predicted deleterious missense variant in Mbd4. However, using a 5Mb window centered on the peak IHD scan SNP, rather than a 10Mb window, Mbd4 would be excluded. Further, SNP functional prediction accuracy is modest [e.g., PMID 28511696], and exclusion of the missense variant in Ogg1 due its benign prediction is potentially premature, especially given the wealth of functional data implicating Ogg1 in C>A mutations in house mice. Finally, the DNA repair gene closest to the peak IHD SNP is Rad18, which the authors largely exclude as a candidate.

Additionally, some claims in the paper are not well-supported by the author's data. For example, in the Discussion, the authors assert that "multiple mutator alleles have spontaneously arisen during the evolutionary history of inbred laboratory mice" and that "... mutational pressure can cause mutation rates to rise in just a few generations of relaxed selection in captivity". However, these statements are undercut by data in this paper and the authors' prior publication demonstrating that a number of candidate variants are segregating in natural mouse populations. These variants almost certainly did not emerge de novo in laboratory colonies, but were inherited from their wild mouse ancestors. Further, the wild mouse population genomic dataset used by the authors falls far short of comprehensively sampling wild mouse diversity; variants in laboratory populations could derive from unsampled wild populations.

Finally, the implications of a discovering a mutator whose expression is potentially conditional on the genotype at a second locus are not raised in the Discussion. While not a weakness per se, this omission is perceived to be a missed opportunity to emphasize what, to this reviewer, is one of the most exciting impacts of this work. The potential background dependence of mutator expression could partially shelter it from the action of selection, allowing the allele persist in populations. This finding bears on theoretical models of mutation rate evolution and may have important implications for efforts to map additional mutator loci. It seems unfortunate to not elevate these points.

Reviewer #1 (Public Review):

Solution state 15N backbone NMR relaxation from proteins reports on the reorientational properties of the N-H bonds distributed throughout the peptide chain. This information is crucial to understanding the motions of intrinsically disordered proteins and as such has focussed the attention of many researchers over the last 20-30 years, both experimentally, analytically and using numerical simulation.

This manuscript proposes an empirical approach to the prediction of transverse 15N relaxation rates, using a simple formula that is parameterised against a set of 45 proteins. Relaxation rates measured under a wide range of experimental conditions are combined to optimize residue-specific parameters such that they reproduce the overall shape of the relaxation profile. The purely empirical study essentially ignores NMR relaxation theory, which is unfortunate, because it is likely that more insight could have been derived if theoretical aspects had been considered at any level of detail.

Despite some novel aspects, in particular the diversity of the relaxation data sets, the residue-specific parameters do not provide much new insight beyond earlier work that has also noted that sidechain bulkiness correlated with the profile of R2 in disordered proteins.

Nevertheless, the manuscript provides an interesting statistical analysis of a diverse set of deposited transverse relaxation rates that could be useful to the community.<br /> Crucially, and somewhat in contradiction to the authors stated aims in the introduction, I do not feel that the article delivers real insight into the nature of IDP dynamics. Related to this, I have difficulty understanding how an approximate prediction of the overall trend of expected transverse relaxation rates will be of further use to scientists working on IDPs. We already know where the secondary structural elements are (from 13C chemical shifts which are essential for backbone assignment) and the necessary 'scaling' of the profile to match experimental data actually contains a lot of the information that researchers seek.

1. The introduction is confusing, mixing different contributions to R2 as if they emanated from the same physics, which is not necessarily true. 15N transverse relaxation is said to report on 'slower' dynamics from 10s of nanoseconds up to 1 microsecond. Semi-classical Redfield theory shows that transverse relaxation is sensitive to both adiabatic and non-adiabatic terms, due to spin state transitions induced by stochastic motions, and dephasing of coherence due to local field changes, again induced by stochastic motions. These are faster than the relaxation limit dictated by the angular correlation function. Beyond this, exchange effects can also contribute to measured R2. The extent and timescale limit of this contribution depends on the particular pulse sequence used to measure the relaxation. The differences in the pulse sequences used could be presented, and the implications of these differences for the accuracy of the predictive algorithm discussed.

2. Previous authors have noted the correlation between observed transverse relaxation rates and amino acid sidechain bulkiness. Apart from repeating this observation and optimizing an apparently bulkiness-related parameter on the basis of R2 profiles, I am not clear what more we learn, or what can be derived from such an analysis. If one can possibly identify a motif of secondary structure because raised R2 values in a helix, for example, are missed from the prediction, surely the authors would know about the helix anyway, because they will have assigned the 13C backbone resonances, from which helical propensity can be readily calculated.

3. Transverse relaxation rates in IDPs are often measured to a precision of 0.1s-1 or less. This level of precision is achieved because the line-shapes of the resonances are very narrow and high resolution and sensitivity are commonly measurable. The predictions of relaxation rates, even when applying uniform scaling to optimize best-agreement, is often different to experimental measurement by 10 or 20 times the measured accuracy. There are no experimental errors in the figures. These are essential and should be shown for ease of comparison between experiment and prediction.

4. The impact of structured elements on the dynamic properties of IDPs tethered to them is very well studied in the literature. Slower motions are also increased when, for example the unfolded domain binds a partner, because of the increased slow correlation time. The ad hoc 'helical boosting' proposed by the authors seems to have the opposite effect. When the helical rates are higher, the other rates are significantly reduced. I guess that this is simply a scaling problem. This highlights the limitation of scaling the rates in the secondary structural element by the same value as the rest of the protein, because the timescales of the motion are very different in these regions. In fact the scaling applied by the authors contains very important information. It is also not correct to compare the RMSD of the proposed method with MD, when MD has not applied a 'scaling'. This scaling contains all the information about relative importance of different components to the motion and their timescales, and here it is simply applied and not further analysed.

5. Generally, the uniform scaling of all values by the same number is serious oversimplification. Motions are happening on all timescales they are giving rise to different transverse relaxation. It is not possible to describe IDP relaxation in terms of one single motion. Detailed studies over more than 30 years, have demonstrated that more than one component to the autocorrelation function is essential in order to account for motions on different timescales in denatured, partially disordered or intrinsically unfolded states. If one could 'scale' everything by the same number, this would imply that only one timescale of motion were important and that all others could be neglected, and this at every site in the protein. This is not expected to be the case, and in fact in the examples shown by the authors it is also never the case. There are always regions where the predicted rates are very different from experiment (with respect to experimental error), presumably because local dynamics are occurring on different timescales to the majority of the molecule. These observations contain useful information, and the observation that a single scaling works quite well probably tells us that one component of the motion is dominant, but not universally. This could be discussed.

6. With respect to the accuracy of the prediction, discussion about molecular detail such as pi-pi interactions and phase separation propensity is possibly a little speculative.

7. The authors often declare that the prediction reproduces the experimental data. The comparisons with experimental data need to be presented in terms of the chi2 per residue, using the experimentally measured precision which as mentioned, is often very high.

Reviewer #2 (Public Review):

Qin, Sanbo and Zhou, Huan-Xiang created a model, SeqDYN, to predict nuclear magnetic resonance (NMR) spin relaxation spectra of intrinsically disordered proteins (IDPs), based primarily on amino acid sequence. To fit NMR data, SeqDYN uses 21 parameters, 20 that correspond to each amino acid, and a sequence correlation length for interactions. The model demonstrates that local sequence features impact the dynamics of the IDP, as SeqDYN performs better than a one residue predictor, despite having similar numbers of parameters. SeqDYN is trained using 45 IDP sequences and is retrained using both leave-one-out cross validation and five-fold cross validation, ensuring the model's robustness. While SeqDYN can provide reasonably accurate predictions in many cases, the authors note that improvements can be made by incorporating secondary structure predictions, especially for alpha-helices that exceed the correlation length of the model. The authors apply SeqDYN to study nine IDPs and a denatured ordered protein, demonstrating its predictive power. The model can be easily accessed via the website mentioned in the text.

While the conclusions of the paper are primarily supported by the data, there are some points that could be extended or clarified.

1. The authors state that the model includes 21 parameters. However, they exclude a free parameter that acts as a scaling factor and is necessary to fit the experimental data (lambda). As a result, SeqDYN does not predict the spectrum from the sequence de-novo, but requires a one parameter fitting. The authors mention that this factor is necessary due to non-sequence dependent factors such as the temperature and magnetic field strength used in the experiment. Given these considerations, would it be possible to predict what this scaling factor should be based on such factors?

2. The authors mention that the Lorentzian functional form fits the data better than a Gaussian functional form, but do not present these results.

3. The authors mention that they conducted five-fold cross validation to determine if differences between amino acid parameters are statistically significant. While two pairs are mentioned in the text, there are 190 possible pairs, and it would be informative to more rigorously examine the differences between all such pairs.

Reviewer #3 (Public Review):

The manuscript by Qin and Zhou presents an approach to predict dynamical properties of an intrinsically disordered protein (IDP) from sequence alone. In particular, the authors train a simple (but useful) machine learning model to predict (rescaled) NMR R2 values from sequence. Although these R2 rates only probe some aspects of IDR dynamics and the method does not provide insight into the molecular aspects of processes that lead to perturbed dynamics, the method can be useful to guide experiments.

A strength of the work is that the authors train their model on an observable that directly relates to protein dynamics. They also analyse a relatively broad set of proteins which means that one can see actual variation in accuracy across the proteins.

A weakness of the work is that it is not always clear what the measured R2 rates mean. In some cases, these may include both fast and slow motions (intrinsic R2 rates and exchange contributions). This in turn means that it is actually not clear what the authors are predicting. The work would also be strengthened by making the code available (in addition to the webservice), and by making it easier to compare the accuracy on the training and testing data.

Reviewer #1 (Public Review):

A subclass of inhibitory heterotrimeric guanine nucleotide-binding protein subunits, GNAI, has been implicated in sensory hair cell formation, namely the establishment of hair bundle (stereocilia) orientation and staircase formation. However, the former role of hair bundle orientation has only been demonstrated in mutants expressing pertussis toxin, which blocks all GNAI subunits, but not in mutants with a single knockout of any of the Gnai genes, suggesting that there is a redundancy among various GNAI proteins in this role. Using various conditional mutants, the authors concluded that GNAI3 is the primary GNAI proteins required for hair bundle morphogenesis, whereas hair bundle orientation requires both GNAI2 and GNAI3.

Strength<br /> Various compound mutants were generated to decipher the contribution of individual GNAI1, GNAI2, GNAI3 and GNAIO in the establishment of hair bundle orientation and morphogenesis. The study is thorough with detailed quantification of hair bundle orientation and morphogenesis, as well as auditory functions.

Weakness<br /> While the hair bundle orientation phenotype in the Foxg1-cre; Gnai2-/-; Gnai3 lox/lox (double mutants) appear more severe than those observed in Ptx cKO mutants, it may be an oversimplification to attribute the differences to more GNAI function in the Ptx cko mutants. The phenotypes between the double mutants and Ptx cko mutants appear qualitatively different. For example, assuming the milder phenotypes in the Ptx cKO is due to incomplete loss of GNAI function, one would expect the Ptx phenotype would be reproducible by some combination of compound mutants among various Gnai genes. Such information was not provided. Furthermore, of all the double mutant specimens analyzed for hair bundle orientation (Fig. 8), the hair bundle/kinocilium position started out normally in the lateral quadrant at E17.5 but failed to be maintained by P0. This does not appear to be the case for Ptx cKO, in which all affected hair cells showed inverted orientation by E17.5. It is not clear whether this is the end-stage of bundle orientation in Ptx cKO, and the kinocilium position started out normal, similar to the double mutants before the age of analysis at E17.5. Understanding these differences may reveal specific requirements of individual GNAI subunits or other factors are being affected in the Ptx mutants.

Reviewer #2 (Public Review):

Jarysta and colleagues set out to define how similar GNAI/O family members contribute to the shape and orientation of stereocilia bundles on auditory hair cells. Previous work demonstrated that loss of particular GNAI proteins, or inhibition of GNAIs by pertussis toxin, caused several defects in hair bundle morphogenesis, but open questions remained which the authors sought to address. Some of these questions include whether all phenotypes resulting from expression of pertussis toxin stemmed from GNAI inhibition; which GNAI family members are most critical for directing bundle development; whether GNAI proteins are needed for basal body movements that contribute to bundle patterning. These questions are important for understanding how tissue is patterned in response to planar cell polarity cues.

To address questions related to the GNAI family in auditory hair cell development, the authors assembled an impressive and nearly comprehensive collection of mouse models. This approach allowed for each Gnai and Gnao gene to be knocked out individually or in combination with each other. Notably, a new floxed allele was generated for Gnai3 because loss of this gene in combination with Gnai2 deletion was known to be embryonic lethal. Besides these lines, a new knockin mouse was made to conditionally express untagged pertussis toxin following cre induction from a strong promoter. The breadth and complexity involved in generating and collecting these strains makes this study unique, and likely the authoritative last word on which GNAI proteins are needed for which aspect of auditory hair bundle development.

Appropriate methods were employed by the authors to characterize auditory hair bundle morphology in each mouse line. Conclusions were carefully drawn from the data and largely based on excellent quantitative analysis. The main conclusions are that GNAI3 has the largest effect on hair bundle development. GNAI2 can compensate for GNAI3 loss in early development but incompletely in late development. The Gnai2 Gnai3 double mutant recapitulates nearly all the phenotypic effects associated with pertussis toxin expression and also reveals a role for GNAIs in early movement of the basal body. Although these results are not entirely unexpected based on earlier reports, the current results both uncover new functions and put putative functions on more solid ground.

Based on this study, loss of GNAI1 and GNAO show a slight shortening of the tallest row of stereocilia but no other significant changes to bundle shape. Antibody staining shows no change in GNAI localization in the Gnai1 knockout, suggesting that little to no protein is found in hair cells. One caveat to this interpretation is that the antibody, while proposed to cross-react with GNAI1, is not clearly shown to immunolabel GNAI1. More than anything, this reservation mostly serves to illustrate how challenging it is to nail down every last detail. In turn, the comprehensive nature of the current study seems all the more impressive.

Reviewer #1 (Public Review):

Aw et al. have proposed that utilizing stability analysis can be useful for fine-mapping of cross populations. In addition, the authors have performed extensive analyses to understand the cases where the top eQTL and stable eQTL are the same or different via functional data.

Major comments:

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

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

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

Reviewer #2 (Public Review):

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

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

Major:

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

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

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

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

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

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

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

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

Commentary: Causal Inference for Social Exposures

Reviewer #1 (Public Review):

The study by Hjaltelin, Novitski, and colleagues analyses clinical records of people with pancreatic cancer in the 5 years prior to their diagnosis, aiming to determine patterns of symptoms and disease trajectories that precede the pancreatic cancer diagnosis. The authors use established methodology to identify temporal disease patterns from the Danish National Patient Registry, covering >22,700 patients with pancreatic cancer and 8.1 million controls. They also apply a text-mining approach to extract potential symptoms from free-text clinical notes of a subset of individuals (>4,400 people with pancreatic cancer and >44,000 controls).

The large datasets used in this study present a very clear strength, and the results are presented quite clearly.

Weaknesses of the study include the relatively low sensitivity of the text-mining approach to identify symptoms (83.4%) and the comparison of findings from datasets including different individuals (rather than a comparison of findings based on free-text entries and diagnosis codes from specific entries for the same patients). It is also not clear which proportion of patients is captured by the symptom and disease trajectories catalogued in this work. The different average survival times associated with different trajectories are very interesting, and it would be helpful to examine whether these are due to differences in cancer stage at diagnosis.

Reviewer #2 (Public Review):

This manuscript reports on an important study that aims to identify symptom trajectories for the early detection of pancreatic cancer. The study's findings are based on the analysis of two complementary data sources: structured data obtained from the Danish National Patient Registry and unstructured information extracted from the free-text sections of patient notes. The researchers successfully identified various symptoms and disease trajectories that are strongly associated with pancreatic cancer, with compelling evidence from both data sources. Additionally, the study provides a detailed comparison and contrast of the results obtained from each data source, adding valuable insights into the strengths and limitations of each method.

Strengths:

The work is well motivated by the urgent need for early detection of pancreatic cancer, which is often difficult due to the lack of effective (computational) methods. The manuscript is generally well-written and includes relevant studies, providing a comprehensive overview of the current state of the field.

One of the unique contributions of this work is its use of both structured registry data and unstructured clinical notes to leverage complementary information. This approach enables a more nuanced and comprehensive understanding of the disease symptom trajectories, which is critical for improving early disease diagnosis and prognosis.

The methodology employed in this study is sound and robust, and the authors have candidly discussed its limitations. The results are significant and highlight previously unknown insights into symptom disease trajectories, which have important implications for the management of pancreatic cancer.

Overall, this is a well-designed and executed study that makes an important contribution to the field of cancer/informatics research, and it should be of great interest to both researchers and clinicians.

Weaknesses:

To complement the results in Figure 1, I'd also suggest that the authors compile a list of the most common (known) symptoms of pancreatic cancer as a reference. In other words, not only can you compare results found from the two sources but also compare them with existing knowledge. This is something you discussed partly in lines 245 but including this early as part of the results in Figure 1 would be more informative.

In terms of the text mining evaluation results, providing information on recall errors would be beneficial to better understand the performance of the method. Additionally, line 144 mentions 53 words, but it is still not clear to me what these words refer to. Could you please clarify this point or provide more context?

The disparities between Figure 2A and 2B are noteworthy, from very different initial symptoms to the proportion of short median survival dates (<=90 days), with much more pronounced differences than those observed in Figure 1 comparing two data sources. The highlighted trajectories are almost completely different. Should this be expected? I was hoping to see at least some overlap between the two results.

All trajectories shown in Figure 2 include three symptoms. Is this by design? Could there be meaningful trajectories with different numbers of symptoms (e.g. 4 or more)?

Considering those patients with both clinical notes and registry data, it may be beneficial to merge their symptoms to generate more informative trajectories.

Given that results from two sources are being compared in Figures 1 and 2, have you considered calculating the top 20 most significant symptoms from the registry data as well?

While there is a discussion related to cardiovascular diseases, I noticed no mention of cataracts or gonarthrosis, which were found to be prevalent among patients with short survival in Figure 2.

Ultimately, the goal of this research is to improve the early detection and prognosis of pancreatic cancer, thus it is important to discuss how the findings of this work could be applied in practice towards this goal (e.g. used by disease prediction algorithms?)

Reviewer #1 (Public Review):

The paper describes a very interesting public health experience. The Danish breast cancer screening program is one of the few programs that never suspended its activity during the pandemic.

In general, in the discussion, I miss two of the main points that led to suspend screening programs in most countries during the pandemic: 1) protecting women from the risk of infection linked to attending a clinic during pandemic when health facilities were mostly attended by symptomatic people seeking care for Covid-19; 2) the of health professionals because they were mostly involved in covid related activities: lack of radiologists (addressed to the emergency department to assure diagnoses of pneumonia), lack of anesthesiologists (due to the expansion of intensive care), thus risking not having timely surgical treatment; lack of screening organization personal for invitations and phone calls (working on contact tracing). Lacking the rationale for suspending screening, it is not clear to the reader how the Danish program afforded these issues and was able to maintain open the program.

Reviewer #2 (Public Review):

The manuscript "Nation-wide mammography screening participation in Denmark during the COVID-19 pandemic: An observational study" aims at assessing the impact of COVID-19 on the participation to the breast cancer national screening program in Denmark.

Using a cohort of almost one million women, the authors used ageneralised linear model to estimate the prevalence ratios of participation to the screening program within 3, 6, and 12 months since the start of the pandemic.

The high quality of the data used represents the strongest point of the study, which provided a strong, reliable basis on which conduct the analysis. Some limitations are related to the way the date of invitation (to the screening program) is handled, the vaccination status of the cohort of interest (information not available) and the transferability of the study to other countries, for different countries handled the pandemic in different ways.

The authors show that there was an overall slight decrease in screening participation despite the screening program remained open throughout the pandemic and discuss likely reasons of why that may have happened. Further, they identified that groups of women who were already characterised by low participation rates, experienced a further reduction in attending screening. Those were mostly composed by immigrants and low income individuals. They also discuss the barrier that language may have posed in relation to the distribution of guidelines form the government, as those were delivered in Danish.

In conclusion, the study indicates that social iniquity, which usually relates to disparity in screening participation, has been slightly exacerbated during the pandemic. Although the authors do not discuss in detail what the consequences of those findings can be, it would be interesting to assess (through a follow-up study) whether they will have an impact on the cancer incidence and, in particular, the staging of cancers at detection for the interested groups.

Reviewer #1 (Public Review):

The authors set out to further probe how mTORC signaling can impact metabolism by modulating the function of the APA machinery. The major strength of the paper is the identification of a 'twin UGUA' motif that governs PAS selection as dictated by the CFIm complex. Further, the authors show that the twin UGUA motif is not just necessary but is sufficient to confer sensitivity to mTORC activity and the CFIm complex regulation. The weaknesses of the paper include a tenuous connection between mTORC signaling and CFIm as it was not rigorously established how CFIm gets activated/deactivated when mTORC is modulated.

Reviewer #2 (Public Review):

In this work, Herron et al. investigated the impact of mTORC1 and CFIm on the expression of the Trim9/TRIM9 isoforms in both mouse and human. They extend upon their cTAG-PAPERCLIP method and demonstrated that systemic AAV injection of cell type-specific Cre recombinases to cTag-PABP mice is a feasible method of APA profiling. From this they show that mTORC1 hyperactivation promotes a shift towards the long Trim9 isoform, Trim9-L. They further provide evidence that the mTORC1 signalling pathway controls Trim9/TRIM9 isoform usage in both human and mouse with high mTORC1 promoting usage of the long isoform and low mTORC1 favouring the short isoform. They also show that the CFIm subunits CPSF6 and NUDT21 play a crucial role in the use of the TRIM9-S/Trim9-S isoform and demonstrate the importance of a twin UGUA motif in this PAS for its regulation by CPSF6. Additionally, they find that this twin UGUA motif is functionally present in the human BMPR1B, MOB4 and BRD4 genes and that insertion of the twin UGUA motif into a heterologous PAS is enough to confer regulation by both CPSF6 and mTORC1. Critically, the position of the twin UGUA motif directs preferential cleavage and polyadenylation to generate an isoform, such that it's presence can result in the use of a short isoform (TRIM9) or a long isoform (BMPR1B, MOB4 and BRD4). The work expands upon the known cis-regulatory motifs for CPSF6 and provides further evidence of a connection between the mTORC1 signalling pathway and CPSF6-mediated alternative polyadenylation. The mechanistic connection between TORC1 signalling and CPSF6 function is, however, still opaque. An experiment probing the connection between TORC1 signalling and the nuclear-cytoplasmic shuttling of CPSF6 with its activity (regulating APA) would significantly strengthen the study. Most conclusions are well supported by the presented data.

Reviewer #3 (Public Review):

Alternative polyadenylation is an important aspect of RNA processing that can alter the type or amount of proteins that are produced from a gene, with consequences for many aspects of biology. Herron et al. set out to identify how the mTORC1 pathway, which regulates cellular metabolism, influences alternative polyadenylation in the mouse brain. They identified a novel mTORC1-regulated gene with alternative polyadenylation - TRIM9 - and convincingly demonstrate that its alternative polyadenylation is controlled by the CFIm complex of the cleavage and polyadenylation machinery. A major strength of these results is that the authors use multiple orthogonal methods - including PAPERCLIP, qPCR and western blotting, to demonstrate that TRIM9 is regulated by mTORC1 and CFIm. They also demonstrate that this regulation is conserved between mice and humans by using multiple different model systems, and use synthetic reporter constructs to identify the cis-regulatory elements that are responsible for TRIM9 regulation by CFIm. These results highlight the importance of alternative polyadenylation in controlling gene expression and are important for researchers wishing to understand how the mTORC1 pathway functions.

The authors also identify that a "twin" UGUA motif in the poly(A) site of the short form of TRIM9 is responsible for its regulation by CFIm. They show that this motif is conserved across mammals and suggest that the adjacent UGUA motifs are necessary for regulation by CFIm. The evidence supporting this aspect of the manuscript is incomplete because the authors only ever mutate both UGUA motifs of TRIM9, and so it is not possible to determine whether the full motif or only one of the UGUA motifs is necessary for regulation, nor whether the effect of the two UGUA motifs is simply additive. The only evidence for the necessity of the full twin motif comes from a synthetic JUNB reporter construct, where a single UGUA motif was insufficient to induce proximal polyadenylation. However, given that there is previous evidence that individual UGUA motifs can act as enhancers of polyadenylation, this may be due to context-specific issues with the JUNB reporter, and evidence from different contexts would make the authors conclusions more convincing.

Reviewer #1 (Public Review):

Hoang, Tsutsumi and colleagues use 2-photon calcium imaging to study the activity of Purkinje cells during a Go/No-go task and related this activity to their location in Aldolase-C bands. Tensor component analysis revealed that a substantial part of the calcium responses can be linked to four functional components. The manuscript addresses an important question with an elegant technical approach and careful analysis. There are a few points that I think could be addressed to further improve the quality of the manuscript.

1. The authors should be careful not to overstate the goal and results. For instance, in the abstract it is stated that dynamical functional organization is necessary for dimension reduction. However, the statement that the 4 TCs together account for about half of the variance (line 220) indicates that dimensionality may not be reduced that much. I would suggest revising the first and last sentence of the abstract accordingly.<br /> At the end of the introduction, the authors refer to "the first evidence supporting the two major theories of cerebellar function" but which two theories is referred to and how this manuscript support them is not very obvious. Similarly, they state that "This study unveiled the secret of cerebellar functional architecture", which I would consider to be an unnecessary overstatement of the impact of the work described.<br /> In the title, the authors use the word modular. In the consensus paper on cerebellar modules (Apps et al., 2018) an attempt is made to unify the terms used to describe cerebellar anatomical structures. Here "module" is used for the longitudinal zone of interconnected PCs, CN neurons and olivary neurons. As the authors only studied PC activity (and indirectly the IO), I would suggest using band, stripe or subpopulation instead.<br /> Finally, the term "CF firing" or "CF activity" is used when referring to the recorded signals. However, the authors measure postsynaptic calcium responses that are indeed likely driven by CF inputs, but could also be influenced by PF inputs. At the very least, because Purkinje cells and not climbing fibers are being imaged, "complex spike" should be used instead. It would be more accurate still to use the more general "calcium response" and make less of an assumption about the origin of the calcium response.

2. For some figure panels and statements in the manuscript error bars or confidence intervals and statistics are missing. This is the case for, for example, the changes in fraction correct, lick latency, fraction incorrect, etc. (Fig 1B, 2E-F, TC levels in 3, 4D-E and 5A-C). Including these is particularly relevant in Fig 4E as this is a key result, mentioned also in the abstract. Please indicate clearly if these plots are cumulative for all mice or per mouse and averaged. I advise the authors to statistically support the claim that the changes are significant and in opposite direction as this element of the study is referred to in the abstract and discussion (summary).

3. Data presentation sometimes does not do the work justice. For example, the data in Figure 6 are very interesting, but hard to read because of the design of the figure. It is clear how the components are mostly confined to Aldolase-C domains, but within the domains the distribution is not clear. I would advise to also more clearly indicate what the locations of the colors within the bands refers to. The spatial distribution of the selected top 300 cells for each TC could be added.

Reviewer #2 (Public Review):

Hoang, Tsutsumi et al provide a comprehensive functional mapping of cerebellar climbing fiber responses in Lobule Crus II. The study derives from analysis of a dataset originally published in Tsutsumi et al eLife 2019, using two photon Ca2+ imaging throughout the learning of a Go/No-go reward-driven licking behavior. Each recording session yielded data from a ~two-hundred micron patch of tissue, with neurons spatially localized relative the "zebrin" banding pattern of the cerebellar cortex as reported by an aldolaceC-tdTomato transgenic line. In the present work, complex spike times were extracted at higher temporal resolution using subframe raster line-scan timing information, and then decomposed at the trial-averaged population level using tensor component analysis.

The central conclusion is that the entirety of crus II climbing fiber responses decomposes into just a few patterns that capture key features of the behavior. Some of these patterns strengthen with learning, i.e., feature climbing fiber spiking that increases in frequency, while others decay with learning, i.e., feature climbing fiber responses that are prominent only in novice animals. These different climbing fiber activity components are in some cases associated with either positive or negative aldolace-C compartments of crus II. Finally, synchronization is concentrated among cells contributing to the same tensor components, and synchrony levels increase or decrease for different components over learning.

The analysis therefore suggests that distinct principles of climbing fiber function can be present simultaneously in distinct cerebellar modules (and, according to the TCA cell weightings, potentially simultaneously in individual climbing fibers). This conclusion is contrary to the implied dichotomy in the literature that climbing fibers either function as "error signals" or as "timing signals" in a particular behavioral context or cerebellar region. The authors speculate that resolution of this dichotomy could result from the biophysics of the inferior olive, in which flexibly coupled oscillators might self-organize into a low dimensional decomposition of task dynamics. Relatedly, the authors speculate that changes in synchronization that contrast between different components could serve to either regulate instructive signal dimensionality or climbing fiber timing functions, depending on each component's functional contribution. From a theoretical standpoint, this is a helpful new direction. The framework is more agnostic to the details of the activity profiles of any specific group of climbing fibers, but more attuned to the systems-level distribution of activity profiles and how these might collectively serve a behavior.

A valuable feature of the study is the simultaneous analysis of many imaging fields spanning 17 subjects and the entire dorsal surface of crus II. This bypasses some of the recurring interpretational issues with climbing fiber recordings that stem from their spatial organization across the cerebellar surface with often abrupt transitions at compartmental boundaries. By decomposing responses across many compartments simultaneously (at the trial-averaged level), the authors provide a quantitative estimate of the diversity of response patterns and their distribution across space and cells. It's worth noting that this approach is also a double-edged sword, as the trial-averaged decomposition does not depend on single-trial correlations between neurons, thus strictly speaking leaving it an open question whether apparently similar climbing fiber patterns present in distant imaging fields exhibit correlated variability either across trials or across learning.

The data convincingly show that several dominant tensor components explain a large amount of climbing fiber variance across crus II. The authors speculate that this reflects an olivary decomposition of task dynamics. Due to the nature of the analysis - TCA applied over an entire dataset - there is not a clear test of this hypothesis in the present manuscript.

The authors also present the interesting and compelling result that different CF response patterns undergo opposite learned changes in synchronization. They speculate that different trajectories of synchronization, specifically, increases for TC1 (hit) and decreases for TC2 (false alarm), could reflect different functional uses of TC1 and TC2, although it is difficult to assess the likelihood of this being true based on the data and analyses presented.

Reviewer #1 (Public Review):

The authors investigate the mechanistic underpinning of paradoxical activation (PA) of RAF by small molecule kinase inhibitors using mathematical modeling. The main novelty of the study is the consideration of RAF conformational autoinhibition by its N-terminal regulatory domains as a new determinant of PA. This mechanism has not been explicitly considered in previous theoretical studies, which are based on two other mechanisms: drug-induced RAF oligomerization into active dimers (dimer potentiation DP) and negative cooperativity (NC) of inhibitor binding by a second monomer in the inhibitor-induced RAF kinase dimerization. An important discovery of this study is that conformational autoinhibition is a critical determinant of PA and that in some cases, it can contribute to PA in the absence of DP and NC. Another novelty is the consideration of RAF interaction with 14-3-3 proteins, as a determinant of PA. The 14-3-3 dimeric scaffolds play an important role in the regulation of both autoinhibited and active states of RAF and thus understanding how their interaction with RAF influences PA by RAF inhibitors is important. Using mathematical modeling the authors show that 14-3-3 binding does indeed enhance PA in response to a spectrum of RAF inhibitors.

Strengths<br /> The overall strength of this study is that it increases the mechanistic understanding of how PA of RAF originates in response to its inhibitors. Consideration of the effect that the inhibitors play in breaking the autoinhibited conformation has been overlooked by previous mathematical analyses of PA, and this study bridges this gap. By doing so, the authors discover that breaking that autoinhibited state is in fact the biggest contribution to PAB by RAF inhibitors. In my opinion, this is the most impactful finding of this study, which additionally speaks to how important are the autoinhibitory mechanisms for constraining basal RAF signaling in cells. The presented analysis also shows that consideration of conformational autoinhibition can explain PA by all different types of RAF inhibitors (1, 1.5, and 2), which until now has been difficult to reconcile.

Another important contribution of this study is the investigation of how the 14-3-3 scaffold proteins can further contribute to PA. This is exciting, especially in light of recent elegant structural studies that unveiled complex regulation of RAF by 14-3-3 (which are both important for RAF inhibition and stabilization of the active dimers). The authors dissect these opposing roles of 14-3-3 in their model and show the autoinhibitory interaction with 14-3-3, but not the activating one, significantly increases the PA response. Their findings that an increase in the 14-3-3 levels amplifies PA is very interesting and somewhat provocative as it is unclear how much 14-3-3 levels in cells can oscillate. To this end, the authors show that elevated 14-3-3 levels are observed with increased time of RAF inhibitor treatment, which might point to a new mechanism of resistance to RAF inhibitors.

Weaknesses<br /> The main weakness of the study is the limited experimental analysis conducted to test the predictions that arise from the mathematical models. While some of these predictions might be challenging to test, the one which is tested is not tested rigorously. The experiments focus on 14-3-3-based regulation and are conducted in cells by observing the effect of 14-3-3 overexpression on the inhibition of RAF signaling by its different kinase inhibitors. While the authors acknowledge that too, 14-3-3 overexpression will have a multifaceted effect on signaling as these scaffold proteins participate in the regulation of almost all signaling events. Thus, the proposed experiments are not sufficient to conclude that the observed effects are in fact a result of 14-3-3/RAF interaction.

The authors consider conformational autoinhibition and 14-3-3 stabilization of autoinhibited RAF as two different mechanisms. While it is not a weakness, I am curious how accurate is the consideration of the autoinhibited state of RAF in the absence of 14-3-3. Is it known how the proportion of RAF in cells in its inactive state exists while not bound to 14-3-3?

Reviewer #2 (Public Review):

In this study, the authors set out to investigate factors that have been neglected in existing mathematical models for the paradoxical activation (PA) of RAF by pharmacological inhibitors. The PA phenomenon is well known and is thought to be an important factor in limiting the effectiveness of RAF inhibitors. The authors primarily use mathematical models, first to examine the importance of conformational autoinhibition of RAF monomers, and later to investigate the potential role played by binding of 14-3-3 proteins to either autoinhibited monomers or active dimers. The authors develop several model variants containing different candidate mechanisms and generate analytical solutions that demonstrate under which parameter conditions PA may occur within these models. The use of analytical solutions is a strong point of the paper, as it allows evaluation of the models independently of specific parameter values. This analysis suggests that conformational autoinhibition is a very strong contributor to paradoxical activation, as models that include this mechanism show substantially larger concentration ranges under which RAF is activated by inhibitors. Fitting the parameters of the model to a published dataset on multiple inhibitors further suggests that conformational activation is important, as models containing this mechanism can fit the dataset with significantly lower error. Another interesting observation is that the different types of RAF inhibitors (1, 1.5, 2) fit the data with parameter values that are reasonably similar within each type. A moderate weakness in this analysis is that all of these observations provide indirect evidence for the importance of conformational autoinhibition. A direct test of whether PA is reduced when conformational autoinhibition is removed would be more compelling, but such a test could be difficult to set up experimentally.

The authors then perform an analysis of how 14-3-3 binding to either autoinhibited monomers or active dimers might enhance PA. A new model is constructed that contains these binding events in the context of conformational activation, but without negative cooperativity or dimer potentiation included, for the sake of limiting complexity. These models implicate monomer binding, but not dimer binding as a contributor to PA. They follow up on this model result by overexpressing 14-3-3 proteins in two RAS-mutant cell lines, which leads to both higher baseline ERK phosphorylation and to a wider range of inhibitor-induced PA, as predicted by the model. A cell-based RAF dimerization assay also shows higher dimerization effects when 14-3-3 plasmids are transfected as well. This experimental evidence provides strong support for the model, although one drawback, which is noted by the authors in the discussion, is that 14-3-3 overexpression could potentially exert effects on RAF activity through pleiotropic effects other than the binding actions included in the model.

Overall, this study makes a strong contribution to understanding the paradoxical effects of RAF inhibitors on the RAS/ERK signaling pathway, which remains a significant problem in the use of targeted inhibitors for cancer. Demonstrating that both conformational activation and 14-3-3 binding strongly contribute to the PA effect is an important step forward, as it establishes that these mechanisms should not be overlooked when designing strategies to use Raf inhibitors.

Reviewer #3 (Public Review):

The authors describe a mathematical and computational modeling study of RAF paradoxical activation (PA), a phenomenon in which RAF inhibitors exhibit a bell-shaped dose-response curve of Erk phosphorylation - activating signaling through wild-type RAF at low drug concentrations before inhibiting it at higher concentrations. They explore three distinct mechanisms that may contribute to PA - conformational autoinhibition, negative cooperativity, and drug-induced dimerization - and conclude that all three are required to best fit published data that show the PA phenomenon. They explore the effect of 14-3-3 binding to RAF both computationally and experimentally and reach the conclusion that 14-3-3 can potentiate the PA phenomenon via stabilization of the autoinhibited conformation.

Strengths:

One key finding will be quite valuable to the field - that paradoxical activation can arise in the absence of negative cooperativity and without any effect of the inhibitor on the propensity of RAF to dimerize, provided that there exists a "conformationally autoinhibited" state that cannot dimerize and cannot bind inhibitor. This finding is important because negative cooperativity and dimer-induction have been a major focus - arguably the main focus - of prior studies of the phenomenon and also a source of considerable confusion. Inhibitors with very different chemical structures and binding properties - type 1.5 inhibitors that are dimer-breakers (and may or may not exhibit negative cooperativity) and type I and II inhibitors that can promote dimers (and almost certainly do not exhibit negative cooperativity) can nevertheless both exhibit PA. Thus the authors' modeling provides a unifying explanation - it is not dimer-induction or negative cooperativity that is at the root of PA, rather it is that there exists an autoinhibited state that can neither bind inhibitor nor dimerize. The authors further show that negative cooperativity and dimer-induction can act in concert with "conformational autoinhibition" to modify the PA response in a drug-specific manner.

Weaknesses:

Unfortunately, the authors don't really explain in a straightforward manner what is going on with the conformational autoinhibition model (Figure 2A). One has to read carefully and all the way to section 3 of appendix 1 to piece it together. In short, what the math shows is that at least for certain ranges of parameter values, the presence of an inhibitor can increase the concentration of dimers, even when it does not change the equilibrium constant for dimer formation, and some of those dimers will have an active, drug-free protomer. This is because the inhibitor effectively traps open monomers, which can then capture drug-free open monomers to form active dimers (active in one subunit, inactive and drug-bound in the other). As inhibitor concentration increases, the pool of autoinhibited RAF is diminished, and eventually, it is shifted completely to fully inhibited dimers. But at low concentrations of inhibitor, there is a net increase in dimerized (active) but drug-free protomers (see figure on page 27 of the appendix). Voila, paradoxical activation, with no need to invoke negative cooperativity.

Considering the potential for confusion around what is meant by "drug-induced dimerization" as an effect distinct from the effect of the drug in promoting RAF dimerization in their conformational autoinhibition model, it would have been helpful for the authors to explicitly address the distinction (drug-induced dimerization alters the equilibrium constant for dimerization; this is not a feature of the conformational autoinhibition model).

Also, I am confused by Figure 3C. The figure shows, and the authors state in the text, that for type II inhibitors an f > ~1 indicates a propensity to break dimers. But type 1.5 inhibitors should break dimers, and Type I and II inhibitors should promote dimers (at least some Type I and II drugs have been shown to promote kinase dimers). Seems that the predictions of the model are inconsistent with experimental data, at least for some inhibitors.

A large part of the paper deals with the effect of 14-3-3 binding. In my view, this part of the manuscript is not particularly helpful. There is no evidence (that I am aware of) that 14-3-3 concentrations vary significantly, or that their variation affects RAF activity/signaling. Considering their abundance relative to RAF, and relatively high affinity for RAF, it is likely that both autoinhibited and active RAF are saturated with 14-3-3. (RAF that is not 14-3-3-bound is likely mostly bound to chaperones and not active). That said, the authors' conclusion (based on modeling) that 14-3-3 can increase the extent of paradoxical activation by stabilizing the autoinhibited state seems sensible, but hard to reconcile with their experimental result where they find increased basal signaling with 14-3-3 over-expression. It is also difficult to understand how increased 14-3-3 binding to RAF could lead to active RAF dimers that are not inhibited at 10-100 uM concentrations of potent RAF dimer inhibitors like LY3009120 (Fig. 5C). It seems more likely that 14-3-3 overexpression is promoting Erk phosphorylation in a manner that is (at least partially) Raf-independent. To their credit, the authors acknowledge this concern.

Finally, one comment regarding the presentation. The authors discuss conformational inhibition and 14-3-3 binding as if they are promoting and/or inducing paradoxical activation. This is pervasive in the paper, including in the title, and is distracting and potentially will mislead some readers. Obviously, it is RAF inhibitor that induces or promotes paradoxical activation. Conformational autoinhibition - mediated by 14-3-3 - is a feature of the system that makes paradoxical activation possible.

Reviewer #1 (Public Review):

This manuscript reports a study to investigate the reporting practices in three top cardiovascular research journals for articles published in 2019. The study was preregistered, which makes the intent and methodology transparent, and the authors also make their materials, data, and code open. While the preregistration and sample strategy is a strength, it suffers from a higher than expected number of non-empirical articles decreasing the sample size and thus inference that can be drawn. The author's focus was mainly on transparency of reporting and not on the actual reproducibility or replicability of the articles; however, the accessibility of data, code, materials, and methods is a prerequisite. While the authors were still able to draw inferences to their main objectives, they could not perform some of their proposed analyses because of a small sample size (due partly to the less than half empirical articles in their sample as well as the low number of papers with accessible information to code). One of the descriptive analyses they performed, the country level scores (Figure 6), in particular suffers from the small sample size and while the authors state indicates this in their manuscript I do not think it would be reasonable to include as it has the potential to be misinterpreted since so many are based on an n=1. Overall, I found the authors presentation and discussion clear and concise; however, a lack of a more in-depth discussion is an area to improve the current manuscript. The manuscript outlines opportunities for researchers, journals, funders, and institutions to improve the way cardiovascular research is reported to enable discovery, reuse, and reproducibility.

Reviewer #2 (Public Review):

This is a descriptive paper in the field of metascience, which documents levels of accessibility and reproducible research practices in the field of cardiovascular science. As such, it does not make a theoretical contribution, but it argues, first, that there is a problem for this field, and second, it provides a baseline against which the impact of future initiatives to improve reproducibility can be assessed. The study was pre-registered and the methods and data are clearly documented. This kind of study is extremely labour-intensive and represents a great deal of work.

I have a major concern about the analysis. It is stated that to be fully reproducible, publications must include sufficient resources (materials, methods, data and analysis scripts). But how about cases where materials are not required to reproduce the work? In line 128-129 it is noted that the materials criterion was omitted for meta-analyses, but what about other types of study where materials may be either described adequately in the text, readily available (eg published questionnaires), or impossible to share (e.g. experimental animals).

To see how valid these concerns might be, I looked at the first 4 papers in the deposited 'EmpricalResearchOnly.csv' file. Two had been coded as 'No Materials availability statement' and for two the value was blank.<br /> Study 1 used registry data and was coded as missing a Materials statement. The only materials that I could think might be useful to have might be 'standardized case report forms' that were referred to. But the authors did note that the Registry methods were fully documented elsewhere (I am not sure if that is the case).<br /> Study 2 was a short surgical case report - for this one the Materials field was left blank by the coder.<br /> Study 3 was a meta-analysis; the Materials field was blank by the coder<br /> Study 4 was again coded as lacking a Material statement. It presented a model predicting outcome for cardiac arrhythmias. The definitions of the predictor variables were provided in supplementary materials. I am not clear what other materials might be needed.<br /> These four cases suggest to me that it is rather misleading to treat lack of a Materials statement as contributing to an index of irreproducibility. Certainly, there are many studies where this is the case, but it will vary from study to study depending on the nature of the research. Indeed, this may also be true for other components of the irreproducibility index: for instance, in a case study, there may be no analysis script because no statistical analysis was done. And in some papers, the raw data may all be present in the text already - that may be less common, but it is likely to be so for case studies, for instance.

A related point concerns the criteria for selecting papers for screening: it was surprising that the requirement for studies to have empirical data was not imposed at the outset: it should be possible to screen these out early on by specifying 'publication type'; instead, they were included and that means that the numbers used for the actual analysis are well below 400. The large number of non-empirical papers is not of particular relevance for the research questions considered here. In the Discussion, the authors expressed surprise at the large number of non-emprical papers they found; I felt it would have been reasonable for them to depart from their preregistered plan on discovering this, and to review further papers to bring the number up to 400, restricting consideration to empirical papers only - also excluding case reports, which pose their own problems in this kind of analysis.

A more minor point is that some of the analyses could be dropped. The analysis of authorship by country had too few cases for many countries to allow for sensible analysis.

Overall, my concern is that the analysis presented here may create a backlash against metascientific analyses like this because it appears unfair on authors to use a metric based on criteria that may not apply to their study. I am strongly in favour of open, reproducible science, and agree it is important to document the state of the science for different disciplines. But what this study demonstrates to me is that if you are going to evaluate papers as to whether they include things like materials/data/ availability statements, then you need to have a N/A option. Unfortunately, I suspect it may not be possible to rely on authors' self-evaluation of N/A and that means that metascientists doing an evaluation would need to read enough of the paper to judge whether such a statement should apply.

Reviewer #1 (Public Review):

The COVID-19 pandemic strained population-level mammography screening programs, but to what quantitative degree is unclear. Through a rapid review, the authors quantified the changes in breast screening volume and uptake during the first year of the COVID-19 pandemic, compared to a prior year.

A major strength of this rapid review is that the detail provided by the authors makes this rapid review easily replicated. The detail provided in the time frames used as comparison and the added rigor of using grey literature make this a strong study. The authors nonetheless note that a limitation of this review is that the production of articles is rapid and that newly published articles relevant to the topic could have been missed. However, the authors lay out well how to replicate and strengthen this rapid review to replicate the findings.

The authors found evidence supporting the concern that the COVID-19 pandemic disrupted breast mammography screening on a global scale. They conclude that overall, there were global volume and uptake reductions in breast cancer screening. The volume and uptake reductions varied regionally and there was compelling evidence that these reductions were in part due to health care coverage.

What I found most compelling about this rapid review is the thorough assessment of the included articles and the detailed accounting of the limitations of these articles. This rapid review revealed major deficits in the evidence quality in global assessments of breast mammography screening uptake and volume and future studies that include common and rigorous measures are needed.

In conclusion, the implications of the findings suggest that monitoring patient volume and uptake could be early warning signs to determine if prevention services need strengthening. Especially for those with public vs private insurance and additional markers of social determinants of health.

Reviewer #2 (Public Review):

The authors undertook a review of studies describing the effects of the COVID-19 pandemic on breast cancer screening in countries across the world. The major strengths of the study are its breadth and the rigour of the literature search and review. The volume of studies included, and their different contexts and designs, make it challenging to summarize succinctly and the authors have done a good job. The weakness of this review, or any like it, is that we have limited data to explain the findings which a likely a complex mix of societal, structural, and personal reasons. The importance of the findings lies in the consistency of the overall trend and what the implications of potential delayed/missed breast cancer screening are and how far into the future these implications will reach.

Reviewer #1 (Public Review):

TP73 is a member of the p53 family of tumor suppressors and is expressed as TAp73 and DNp73 and multiple C-terminal isoforms as a result of alternative splicing. In this study, the authors used isoform-specific disruption of the TP73 gene to investigate the physiological functions of p53 C-terminal isoforms, focussing on p73a and p73g. They identify an oncogenic role of TAp73-γ in tumorigenesis via regulating the expression of a novel target Leptin. Furthermore, they generated and characterized a mouse mode that expresses the TAp73 isoform γ but not α and shows how this splicing switch has oncogenic effects and causes metabolic defects. Overall, this is an important and well-done study uncovering a key role of TAp63-g in tumorigenesis via regulating Leptin expression.

Reviewer #2 (Public Review):

The submitted manuscript deals with the intricate and complex network among different members of the p53 family with a specific focus on TAp73alpha and TAp73 gamma. The authors provide in vitro and in vivo evidence on the oncogenic role of TAp73 gamma which opposes the tumor suppressor activity of TAp73 alpha. Mice carrying exon 11 loss which is the molecular event leading to the switch to TAp73 gamma, are obese when compared to their counterparts. Interestingly, the authors propose that obesity in E11 mice relies on TAp73 gamma-induced aberrant expression of Leptin. The strength of the reported findings resides mainly in the combination of in vitro and in vivo approaches, while its weaknesses are related to the validation of reported findings in human tumoral contexts.

Reviewer #3 (Public Review):

The authors employed a set of cell-based and animal studies with tumor model systems that harbor a genetically deleted specific isoform of p73 to identify a novel function of this isoform in the regulation of lipid metabolism and obese disorder, which are associated with tumorigenesis. Interestingly, this new function was found to be through the increase in leptin expression. This is probably the first study showing the connection of the p73 family members with leptin, a molecule that has been shown to play a critical role in obesity and metabolism. Overall, their findings are novel, interesting, and important.

Reviewer #1 (Public Review):

In their manuscript entitled "Scavenger receptor endocytosis controls apical membrane morphogenesis in the Drosophila airways," Pinheiro and colleagues identify a requirement for Epithelial membrane protein (Emp), a Drosophila CD36 homolog, in embryonic viability and show that mutant embryos display tracheal tube elongation and gas-filling defects. The authors first generate a null allele of emp. The authors validate gene-specific defects by the transgenic rescue of a deletion allele of emp, and further show partial rescuing activity of human CD36. The authors generate and validate an Emp antibody. In mutant trachea, the authors determine there is defective internalization of Serpentine and Vermiform from the tube lumens. Endocytic defects appear selective as GASP internalization does not appear affected. Crb is also found to accumulate to higher levels. The affected process is not clathrin-dependent, as disruption of clathrin function blocks endocytosis of GASP but not Serp or Verm. Emp localizes to apical/adherens junction membranes in epithelia. Actin bundles regulate endocytosis and affect Emp internalization as seen by disruption of actin bundles with Ptp mutants or expression of DAAM dominant negative. Some Emp-GFP colocalized with Rab and Rab7 endosomal markers. A fraction of Srp-GFP co-localized with early and late endosomes and that colocalization was decreased in emp mutants. The LDLr domain was identified as responsible for Emp-dependent endocytosis. In Srp-GFP overexpression, Emp and Crb accumulate on the apical membrane; in serp, verm double mutants Emp and Crb levels on the apical membrane are decreased. These data are consistent with ligand clustering driving Emp and Crb apical localization. Overall Emp and Crb protein levels did not change, arguing for a role in subcellular distribution rather than protein stability. In emp; SerpGFP embryos, DT length was decreased relative to SerpGFP alone; authors suggest this implies that SerpGFP increase in length is partially dependent upon Emp.

In emp mutants, DE-Cad and Crb accumulate along longitudinal junctions, whereas only DE-Cad shows increased accumulation at transverse junctions. Western blots indicate no change in overall protein levels for DE-Cad or Crb. alpha-Catenin (adherens junction component) levels were indistinguishable from wt.

MoeGFP distribution in emp embryos is altered compared to wild-type, with a diffuse appearance. The formin, DAAM, accumulates apically in emp mutant embryos as compared to wt.

A yeast 2 hybrid screen revealed a physical interaction with beta-heavy spectrin. Co-IP experiments in S2 cells support this interaction. kst mutants show tube elongation defects suggesting that the two proteins function in the same process. Kst levels were reduced near the apical membrane in emp mutants. Emp localization was not notably altered in kst mutant embryos.

In emp mutants, pSrc levels are higher. Also seen in western blot. Embryos mutants for src have a shorter dorsal trunk. Double mutant embryos (emp; src42A) showed significant suppression of the emp phenotype. Crb and DE-Cad accumulation could be suppressed by the expression of an srcDN transgene.

The authors propose that Emp affects pSrc levels to regulate tube size and possibly other morphogenetic processes.

The manuscript makes excellent use of genetic and cell biological approaches to provide insight into the regulation of tube length during embryonic tracheal development. Many genes and pathways have been implicated in this process and this study begins to make some connections. A weakness of the manuscript is the lack of a molecular mechanism linking Emp to pSrc distribution.

Reviewer #2 (Public Review):

Pinheiro et al unravel the role of a new scavenger receptor in tubular morphogenesis. To do so they use the Drosophila respiratory network, the tracheal system. Here, the apical extracellular matrix (aECM) and the apical cytoskeleton are essential players in tube length regulation. A few years ago, a feedback mechanism between the aECM and the underlying cells was proposed (Ozturk-Çolak et al., eLife 2016), by which the aECM and the apical F-actin could regulate levels of phosphorylated Src protein to ensure proper tube morphogenesis. However, the connection between the aECM and the cells had not been found. In this manuscript, that Emp, a Drosophila scavenger receptor homologous to human CD36, could fulfill such a role. The authors show that Emp localizes in apical epithelial membranes and shows cargo selectivity for LDLr-domain-containing proteins. They show that emp mutant embryos fail to internalize the luminal chitin deacetylases Serp and Verm at the final stages of airway maturation and die at hatching with liquid-filled airways and over-elongated tracheal tubes with increased levels of the apical proteins Crb, DE-cad and phosphorylated Src (p-Src). Overexpression or loss of the Emp cargo protein Serp leads to abnormal apical accumulations of Emp and perturbations in p-Src levels. They propose a model linking aECM with cell elongation and open new lines of research in downstream signalling effectors.

Strengths:<br /> The finding of a novel receptor involved in the modulation of aECM-cellular homeostasis. A solid genetic and cellular analysis was provided. The implications for a scavenger receptor function during morphogenesis and overall implications in ECM to cell interactions and downstream signalling.

Weaknesses:<br /> The authors fail to clearly show the localization of Emp at the apical membrane and its connection to apical actin structures and chitinous aECM.

Reviewer #3 (Public Review):

Controlling the shape of biological tubes (blood vessels, lungs, etc) is essential for optimizing the traffick of liquid and gas in organisms. Tracheal tubulogenesis of Drosophila embryos is regulated separately in two dimensions, width, and length. Molecules controlling the tracheal tube length function at three levels of location, luminal ECM, plasma membrane mediating cell-apical ECM interaction, and the signaling at the membrane/cell junction. In this paper, Pinheiro et al. reported a novel function of a scavenger receptor family molecule, Emp, which mediates endocytosis of a subset of luminal proteins including chitin deacetylates Serp and Verm that are required for restricting the tube length. It was previously shown that endocytosis and recycling of Serp and Verm maintain the level of luminal chitin deacetylates for keeping the property of the apical ECM to restrict the tube length (10.1016/j.celrep.2014.03.066).

This work is novel in two ways. First, Emp was shown to mediate the endocytosis of Serp and Verm by most likely interacting with the LDLr domains of cargo molecules and acting in parallel with the clathrin-mediated endocytosis to clear luminal materials. Second and surprisingly, the Emp-mediate endocytosis is coupled with the widespread alteration of the apical plasma membrane, including reduction of junctional E-cadherin and Crumbs, apical membrane protein DAAM1, and the cortical membrane skeleton component beta heavy spectrin (Kst). The elevation of junctional Crumbs protein in Emp mutants is noteworthy because the authors showed Crumbs was selectively upregulated in the longitudinal cell junctions. This change in Crumbs polarity may be related to the axial over-elongation of the trachea in Emp mutants. Furthermore, the authors showed that Src42A, which was previously shown to promote tube elongation, is also regulated negatively by Emp.

Overall, the information provided in this work supports a model of endocytic coupling of luminal materials and the axial polarity of the tracheal tube. This leads to a new idea distinct (but none-exclusive) from the previously proposed mechanical coupling model (10.1016/j.celrep.2014.03.066) and would advance a fundamental understanding of biological tube shape regulation. One critical point of linking endocytosis to the axial polarity is the selective enrichment of Crumbs to the longitudinal cell boundaries (10.1371/journal.pgen.1007824), which is shown to be enhanced in Emp mutants (Fig. 5D-F). Discussing how the junction-enriched Crmbs contribute to selective axial cell elongation will be desirable to expand the scope of this work. This point is essential, given that the expression of the dominant-active form of Crumbs lacking the extracellular domain (Crumbs-intra) is mislocalized in the cytoplasmic puncta promotes axial cell elongation (Laprise et al., 2010).

Reviewer #1 (Public Review):

The authors have developed a new method to measure brain activity in the developing chick embryo. Thereby they have provided convincing evidence of asymmetry in the chick embryo and shown how it is influenced by exposure of the embryo to light. This is an important step forward in understanding the development of visual lateralization of behaviour and asymmetry of the thalamofugal visual pathway. Although asymmetry of the thalamofugal visual projections to the Wulst in newly hatched chicks has been well-documented previously, until now, it has not been possible to obtain such direct evidence of lateralized neural activity in the embryo.

The method that the authors have developed has potential for future research. It could now be applied at other times during embryonic development and to other species. In fact, since the tectofugal system is asymmetrical in the pigeon, it would be interesting to use the technique in pigeon embryos, as a comparison.

Reviewer #2 (Public Review):

The study was highly interesting personally as it tries to address a very important question of light induced brain development. The study uses a very efficient model system of birds. Using in-vivo MRI and a contrast agent increases the confidence on the results but also makes the experiments more challenging. I feel that the protocol will help fellow researchers interested in such questions a lot.

Reviewer #3 (Public Review):

Because of the position of pigeon embryos in eggs, light exposure will only stimulate the right eye, leading to lateralisation of brain responses and behaviour. Lorenzi and colleagues injected manganese chloride into pigeon eggs, to assess neuronal activation in the embryonic brain. While the eggs were placed in the light or dark, manganese ions accumulated in neurons that were activated (in cell bodies and axons), which was then visualized with MRI of the embryos before hatching. The authors report lateralisation of neuronal activity in three brain regions, which could potentially be important for our understanding of experience-dependent development of lateralised neural activation.

The tectofugal pathway in pigeons projects from the retina to the optical tectum, then to the nucleus rotundus in the thalamus, and then to the entopallium. The thalamofugal pathway projects from the retina to the GLd in the thalamus, and then to the wulst in the hyperpallium. The two pathways involve different thalamic nuclei (e.g., Deng 2006). In the methods and throughout the manuscript it should be specified which thalamic region is used as ROI.

This manuscript only describes neural activity, but the MEMRI technique should also be used to assess the effect of experimental manipulations on axonal connectivity. It is important to learn about the asymmetry of contralateral projections in the light vs dark groups for answering the research question.

There is an overinterpretation of post-hoc statistics that are reported without correction for multiple testing. The wulst light group lateralization is probably not actually different from zero (uncorrected p=0.04).

The first line in the discussion states that there is thalamofugal lateralization, but no lateralization in the tectofugal pathway. To my understanding, previous literature reported it the other way around: in altricial pigeons, light exposure in the egg mainly affected the tectofugal pathway (Deng & Rogers 2002), while the thalamofugal pathway in pigeons was not lateralized (Strockens et al., 2013). The manuscript should compare the current findings with the literature and discuss differences.

Moreover, the tectum is the only region shown here from the tectofugal pathway. However, lateralization of contralateral connections is expected from tectum to the nucleus rotundus in the thalamus, and thus lateralization of activation may only arise in downstream brain regions from the optical tectum. Therefore, the conclusion that there is no lateralization in the tectofugal pathway is not supported by the data.

In conclusion, I think it is interesting and worthwhile that the authors assessed neural activity in response to visual stimulation in the embryo prior to hatching, but multiple methodological weaknesses and unclarities should be addressed.

Reviewer #1 (Public Review):

This study aims to compare the impact on KCNQ1/ KCNE1 channel complexes of localizing PKA components in distinct ways: by targeting of PKA domains to the C-terminus of KCNQ1 or KCNE1 or overexpression of an untargeted catalytic domain. The evidence is compelling that targeting PKA domains to the C-terminus of KCNQ1 causes distinct phosphorylation as well as decreasing channel conductance and channel protein at the cell surface when compared to overexpression of PKA subunits with other constructs. The study effectively deploys a symbiotic combination of techniques to link electrophysiology, surface expression, and phosphorylation changes responding to targeted recruitment. Support seems incomplete for the minor claims of retention specifically to the ER/Golgi, and that targeted recruitment of PKA domains to KCNE1 was successful and distinct from untargeted overexpression. This study demonstrates the potential for engineering submolecularly-targeted phosphorylation to post-translationally modify a single protein in multiple ways with the same kinase. That distinct intramolecular patterns of phosphorylation can be encoded by the recruitment point of a kinase is very interesting and expected to be of value to the studies of ion channel modulation, kinase activity, and the development of related biotechnology.

Reviewer #2 (Public Review):

This is a well-conceived and interesting study that investigates how a targeted protein phosphorylation (TPP) approach could be implemented to reconstitute PKA regulation of the cardiac KCNQ1/KCNE1 (IKs) potassium channel in the absence of an A-kinase anchoring protein (AKAP9). Using a genetically encoded GFP/YFP nanobody-based system they showed distinctive modulation of cAMP-mediated IKs activity. To that aim, they used an anti-GFP nanobody to recruit either the PKA holoenzyme RIIα or Cα subunits to YFP-tagged Q1 or YFP-E1 of reconstituted IKs channel complexes in CHO and HEK cells. They showed that targeted recruitment of endogenous Cα to E1-YFP using nano-RIIα modestly enhanced PKA-mediated IKs activity, whereas tethering of either nano-RIIα or nano-Cα to Q1-YFP retained KCNQ1 in the ER and Golgi thereby reducing IKs function. Using (LC-MS/MS), they further demonstrated that compared to free Cα, Cα targeted to Q1-YFP phosphorylated KCNQ1 subunit in multiple sites. Overall, the experiments are nicely done and yield sound data. The contribution of the paper is significant because it provides knowledge about the distinctive regulation of IKs by PKA, which could be used in the future to develop potential new drugs to prevent exercise-induced sudden cardiac death.

Reviewer #3 (Public Review):

In this latest installment of a growing body of work from Henry Colecraft's lab in which native enzymes, ion channels, and other machinery are hijacked for therapeutic potential, cells can be made to respond to beta-adrenergic signals even when lacking the critical adaptor protein AKAP9. Normally, the cardiac repolarizing current IKs is enhanced in the face of beta-adrenergic signaling when increased cAMP activates PKA anchored to the channel protein by AKAP9. PKA phosphorylates the channel, increasing function or density in the membrane, especially during exercise or fright. Under these circumstances, when AKAP9 is missing in patients, the action potential fails to repolarize in a timely manner and arrhythmias can result. In this study, targeting the PKA catalytic or regulatory subunit to the E1 auxiliary channel subunit via a targeting nanobody restores at least some of the normal modulation in the presence of cAMP. This primary finding demonstrates a potential therapeutic approach when mutations disrupt its interaction with the channel complex.

A secondary finding of the study is that, contrary to expectation, targeting the enzyme to the Q1 alpha subunit C-terminus does not restore modulation but rather inhibits current by tying up the protein in the ER. Retention apparently depends on phosphorylation because a kinase-dead PKA catalytic subunit exhibits normal current. These findings demonstrate that the efficacy of correction is critically dependent on the site of recruitment. The results represent a starting point whereby kinase-based signaling can be synthetically harnessed to restore normal function in a disease setting.

The strengths of the study are the therapeutic potential of its principal finding and the clever approach to redirecting cellular components. Controls for the constructs are carefully designed and executed. Most of the conclusions are supported by the data presented. The weaknesses are minor and include providing more than an exemplar to support findings of enhanced phosphorylation and an accounting of how the findings from immunofluorescent images were quantitatively established. The study represents a major contribution to an emerging field of study in which modulation is induced by the proximity of enzymes to otherwise undruggable targets.

Reviewer #1 (Public Review):

An interesting combination of simultaneous broadband NIRS and EEG was acquired in 5-month-old infants (N=42) while they watched social and non-social videos. This substantial undertaking yielded a valuable dataset. The analysis was well developed, including a metabolic measure (COO) as well as haemoglobin measures; localisation of the NIRS signal; and an investigation of the EEG frequency bands correlated with the NIRS. The results, that the temporoparietal junction is engaged by social stimuli, are consistent and reassuring.

The contributions of the manuscript seem largely methodological, which is valuable, but in places the authors oversell the implications of the work - both theoretically and methodologically.

Reviewer #2 (Public Review):

This work uses broadband NIRS to investigate metabolic and hemodynamic changes in the brains of infants watching social or non-social stimuli, with simultaneous EEG providing the reference for specialization. The authors postulate that metabolic changes and neurovascular coupling will correlate better with power in the high-frequency beta and gamma band, but this is only justified by references to adult work. I suggest to justify better this assumption at the end of the introduction line 115 and discussing why this should be the case in infants as well.

The authors test the hypothesis that metabolic, hemodynamic, and high-frequency EEG activity will show similar spatial localization. The results support the claim. The methods are sound and thoroughly described, graphics are excellent.

At the moment though, the GitHub repository for code is empty and could not be used (sentence "All code used to analyse the NIRS data and the integration of the NIRS and EEG data is available on GitHub (https://github.com/maheensiddiqui91/NIRS-EEG)" line 346.

The Discussion is appropriate, although limitations could be more elaborate, particularly concerning spatial coverage issues and the methodological improvements required for improved fNIRS spatial resolution.

Reviewer #3 (Public Review):

This manuscript proposes to tackle a very interesting and methodologically challenging topic: the mechanistic underpinnings of neural specialization in the infant brain. The authors presented 4- to 7-month-old infants with social and non-social stimuli while their neural, hemodynamic, and metabolic activity was monitored, and they report a complex pattern of relationships between neural and metabolic or hemodynamic responses during social processing on the one hand, and during non-social processing on the other hand.

The approach described in this manuscript is very interesting and the combined use of EEG and bNIRS data appears very promising. However, there is some confusion between the initial aims of the study, and the analyses performed, which jeopardizes the clarity and the impact of this manuscript. Besides, the predictions of the authors are often underspecified which complexifies the interpretation of the results.

Based on its abstract, the goal of this work is to "combine simultaneous measures of coordinated neural activity metabolic rate and oxygenated blood supply to measure emerging specialization in the infant brain". The introduction nicely elaborates on the "interactive specialization theory" and the potential role of the interplay between brain energy consumption and neural activity in the emergence of functionally specialized brain regions during development. The authors present a novel multimodal approach, with potentially important implications for the study of brain specialization as a function of experience or maturation. Yet the experimental procedure presented in this manuscript only assesses specialized brain activity in response to social processing in 4- to 7-month-old infants, using multimodal neuroimaging.<br /> Indeed, the authors presented 4- to 7-month-old infants with social and non-social stimuli while their neural, hemodynamic, and metabolic activity was monitored. The authors report significant differences between the two conditions in terms of neural activity in the delta, alpha, beta, and gamma bands; as well as in the pattern of hemodynamic to metabolic coupling. Using a GLM approach, the authors report on fNIRS channels and EEG sensors showing significant relationships between the evoked neural activity in the beta and gamma frequency bands, and each of the bNIRS signals (HbO, HbR & CCO), in the social and in the non-social conditions. The authors identify a particular fNIRS channel overlaying posterior STS, showing a positive relationship between Pz EEG beta activity and HbO, as well as CCO, together with a negative relationship between that same neural activity and HbR, in the social condition. This pattern of activity was not observed in the non-social condition.<br /> Overall, these results indicate differential neural responses to social and non-social stimuli, coupled metabolic and hemodynamic activity in response to social as well as nonsocial stimuli. These results additionally indicate coordinated metabolic, hemodynamic, and neural responses in brain regions selective for social processing, but it does not allow us to conclude that this coordinated activity is actually related to the functional specialization process (e.g. last sentence of the abstract).

Another weakness of this manuscript relates to the unclear or underspecified motivations behind some of the performed analyses. For example, the authors contrast brain responses to social vs. baseline, non-social vs. baseline, and social vs. non-social. For clarity in the manuscript, the authors should specify the motivation behind each of these contrasts and their predictions.

Another example is in the analysis of the hemodynamic and metabolic coupling analysis, here the authors analyze only the social vs. baseline and non-social vs. baseline contrast, and they do not analyze the social vs non-social contrast. It would be useful for the reader to understand why only these two contrasts are performed and not the social vs. non-social, and what are the predictions of the authors.

Finally, the core result of this work derives from the final GLM analysis which relates EEG activity to hemodynamic or metabolic responses. This analysis implies the inspection of interactions between 3 neuroimaging modalities, with 4 EEG measures, 2 hemodynamic measures, and 1 metabolic measure, which represents a very rich and relatively complex analytic approach. Unfortunately, the predictions are not clearly specified, which makes results interpretation difficult.

Based on the results (L160-162) and discussion (L233-235) sections, it appears that the authors aim at identifying brain regions showing a precise pattern of activity, with a positive relationship between EEG activity and HbO/CCO responses together with a concurrent negative relationship between EEG and HbR responses in response to social events, but not in response to non-social events. Importantly, the social vs. non-social contrast seems crucial to assess the selectivity of the response. Yet, the authors analyze the 3 chromophores separately, and they do not contrast the two conditions (figure 3). As a result, the authors are limited to reporting a descriptive pattern of relationships between EEG and HbO/HbR/CCO activations for the social condition. And another one for the non-social condition. Overall, the authors conclude that channel 14, overlaying the right TPJ, shows the expected pattern of activity, specifically in response to social stimuli. Yet, this statement is only supported by visual inspection/comparison of the results between the social vs baseline and non-social vs baseline conditions. The authors do not assess analytically the differential patterns of activations between the two conditions. Instead, a GLM including all 3 chromophores and contrasting the two experimental conditions would allow us to directly test the predicted pattern of activity, and the selectivity of the activity for social stimuli.

Reviewer #1 (Public Review):

Qing et al. hypothesize that CD8+ tissue-resident memory T (Trm) cells contribute to the pathogenesis of oral lichen planus. They compare oral mucosal lesions from patients with non-erosive oral lichen planus (NEOLP; n=3) and erosive oral lichen planus (EOLP; n=1) using single-cell RNA-sequencing and spatial transcriptomics and report that CD8+ Trm is enriched and more functionally active in EOLP. Their principal findings are 1) increased proportion of CD8+ Trm in EOLP (vs NEOLP), 2) CD8 Trm in OLP lesions produce TNF, IFNg, and IL-17, and 3) CD8 Trm exist in the healthy epithelium and in lamina propria adjacent to the damaged epithelium in NEOLP/EOLP. The strength of evidence for findings reported in the manuscript is weak.

Strengths:<br /> The pathogenic CD8+ T cell response in lichen planus is a relatively unexplored topic and oral lichen planus is a debilitating disease, thus advancements in its understanding are impactful. The authors' approach is innovative; the manuscript's spatial transcriptomics data are completely novel. The logistical regression analyses that tie CD8+ T cell transcriptional signatures to clinical outcomes are compelling.

Weaknesses:<br /> The authors' data do not firmly support their conclusions. The methods section and figures/figure legends lack important details and labels which makes it difficult to interpret the data. For instance, it is unclear to me how the authors have defined CD8+ tissue-resident memory T (Trm) cells. Human CD8 Trm expresses specific surface markers (CD69, CD103, and CD49a) and a core transcriptional signature (Kumar et al Cell Rep 2017; Cheuk et al Immunity 2017; Fonseca et al Nat Immunol 2022) that are not described herein. In Figures 1-2, the authors do not describe how they annotated their scRNA-seq data, what samples they are including/comparing, criteria used to identify relevant gene expression changes, and they report T cell phenotypes that are inconsistent with published reports and make me question the validity of their T cell/NK cell cluster annotation. Double positive (CD4+CD8+) T cells are not thought to exist outside of the thymus. Human CD8+ Trm has been described to express Itga1 (CD49a), Itgae (CD103), and granzymes yet the authors' CD8 Trm cluster (Fig 2B) exhibits little to no expression of these genes. Also, the authors report il23a expression by CD8 Trm when T cells are not a recognized source of IL-23.

Impact<br /> In my opinion, the main comparison made (NEOLP vs EOLP) is not meaningful. The authors' main conclusion is that there is more CD8+ Trm-mediated inflammation occurring in erosive OLP compared to non-erosive OLP. This is in line with what one would predict, as erosive OLP is a more severe form of the disease. Thus, I don't believe this manuscript significantly advances the understanding of lichen planus immunopathogenesis. The utility of exploring the pathogenesis of human disease is it may identify new targets of intervention and lead to better treatments. The methods used within the manuscript (scRNA-seq, spatial transcriptomics) have the potential to yield significant insights into OLP however in their present form, the authors' analyses do not support the premise that CD8 Trm is the pathogenic cell type in OLP. Thus I do not feel that the authors achieved their central aim.

Reviewer #2 (Public Review):

Qing et al conducted high-resolution single-cell RNA sequencing and spatial transcriptomic profiling to characterize the immunological state of oral mucosa tissue from non-erosive OLP and erosive OLP patients. They find that tissue from erosive OLP patients possessed greater numbers and displayed enhanced activation of CD8+ tissue-resident memory T (CD8+ Trm) cells when compared to non-erosive OLP patients. The authors also designed a cohort study that demonstrated that tissues from patients with recent bouts of erosion displayed a more activated immunological state assessed by transcriptional profiling. Finally, the authors conducted immunological assays to demonstrate greater recovery and higher activation of CD8+ Trm cells from erosive OLP patients.

The sequencing data presented in the study are of high quality and demonstrate key immunological differences between patients with non-erosive OLP and erosive OLP. The authors focused on T cells due to their strong correlation with OLP pathogenesis, but they also observe significant changes to B cell and mast cell levels in erosive OLP compared to non-erosive OLP. Further commentary on the contribution(s) of B cells and mast cells to OLP pathogenesis would be helpful to fully capture the importance of the sequencing dataset.

My major criticism of the study is that the authors argue for CD8+ Trm activity as a key mechanism for OLP pathogenesis but have presented mostly descriptive datasets. The data strongly argue for CD8+ Trm cells as a defining feature of erosive OLP, but there is no data to support their involvement in disease pathogenesis. The authors note the lack of a mouse model for OLP which represents a significant technical barrier to interrogating the role of CD8+ Trm cells in OLP pathogenesis.

Another criticism is the lack of strong findings in the analysis of CD8+ Trm cells isolated from non-erosive and erosive OLP tissues. The authors note increases in CD8+ Trm cell recovery, however, they only observe minor changes in CD8+ Trm activity upon restimulation. Analyzing the activation status or proliferative capacity of CD8+ Trm cells from non-erosive and erosive OLP could be informative and more robust measures of functional changes.

A minor criticism is the formatting of the data presented in Figure 4. The authors should clearly label each marker used in the flow cytometry experiments as well as clearly labeling y-axes for graphs 4H and 4I.

Reviewer #1 (Public Review):

Romagosa, Nieukirk et al. present an interesting approach and interpretation to what is assumed to be a learned animal behavior. In this case, the observed behavior is fin whale (Balaenoptera physalus) singing and the analyses provide results indicating spatio-temporal variation in three fin whale song features at distinct locations within the Central and Northeast North Atlantic Ocean (ONA) region within a two-decade time period. The data set is a non-standardized collection of acoustic recordings obtained from multiple research scientists. Most of the acoustic recording samples are very sparse, with the majority of data coming from an area around the Azores and collected by Okeanos scientists. The senior author undertook the enormously demanding task of analyzing the acoustic data using non-automatic, standardized techniques and protocols. Songs from individual periods of singing on any given day were selected for analysis based on song quality. Song measurements included interval of time between successive 20-Hz song notes (INI), peak frequencies of those 20-Hz notes and peak frequencies of higher frequency notes (HF note). The resultant units of analysis are daily measures of INI (average and s.d.), 20-Hz note peak frequencies (average and s.d.), and HF note peak frequencies (average and s.d.). Several of the figures are confused by not representing the time axis in a typical, uniformly linear way (Fig. 2A and Fig. 3). This form of dynamic time warping smooths and distorts the time-varying features of the results and obscures the inherent sparseness of and high variability in the durations and locations of recordings in available data set. This fundamental characteristic of the available data (see Fig. S1), represents a form of sample aliasing, is not adequately addressed in the paper in terms of how it influences or restricts interpretation of the results. Another possible over-interpretation of results involves misrepresentation of the actual areas sampled. For example, data were collected on Dec 2007-Feb 2008 and Oct 2015 March from a recorder location off the southwest of the Iberian Peninsula. The acoustic sampling detection space is restricted to the ocean within some tens of kilometers of a single sensor, a very small dot on the maps in the manuscript, yet the data from this recorder are assigned to the relatively very large region referred to as the "Bay of Biscay & Iberian Coast". Within the two-decade period of the study (ca. 120 months), recordings were collected at this site (E in Figure 1) for 9 months (7.5%), and the two sampling periods occurred within the December 2007 through March 2018 time span (see Fig S1). It is scientifically inappropriate to translate this as data representing the Bay of Biscay & Iberian Coast as this kind of misrepresentation can lead to misinterpretation of the results.

Despite these spatial and temporal sampling issues, the analyses reveal several important features (Fig. 2 and Fig. 3) about fin whale song in the ONA. The import of the analytical results is that the time span and spatial scale over which recordings were collected provide a unique opportunity to observe whether or not there were variations in fin whale song features within a large ocean region, across a span of two decades. One can consider these spatial and temporal scales appropriately matched to the known scales of fin whale natural history and ecology. Thus, the study results, although confronted by some sampling issues, are not biased by inappropriately sized spatial and temporal scales.

This MS joins a small but growing list of papers documenting variability in baleen whale acoustic behaviors over ecologically appropriate spatial and temporal scales. These papers are primarily focused on singing, an acoustically obvious male reproductive display. As with several recent papers, the author takes advantage of a growing body of data collected during previous studies. The actual measurements utilized several established acoustic analysis software tools. The interpretation of the results focuses on evidence of vocal learning in fin whale singers (i.e. males performing reproductive displays) and wisely remains tangential to interpreting fin whale song through a cultural lens.

Reviewer #2 (Public Review):

This research brings togethor an impressively long timescale dataset of fin whale song vocalisations in the North Atlantic, measuring the note frequency content and inter-note intervals and thereby tracking shifts in both over time. Different time periods are covered in different regions of the north Atlantic during the course of the study. There are two principal results - the study documents a shift in the inter-note interval (INI) in an ICES eco-region termed 'Oceanic Northeast Atlantic' (although the relevance of this to fin whale populations is unclear) occuring relatively rapidly in the years 2000-2001. This shift is discontuous and appears to show an abrupt change in note intervals in most (though not all) of the songs recorded. The second key result is that this INI measure and also the peak frequency of song element termed the 'HF note' both show consistent directional change over timescales of 12 years. The INI measure begins to change back toward the value it held prior to the 2000/2001 shift, suggestive of a cyclical process of change coupled with resets. The average HF note peak frequency descended by about 5Hz during the study period but there was no evidence of abrupt shifts.

The research significance is largely in the description of these processes in a new area, similar changes in rorqual song have been examined in the Southern Ocean and Mediterranean, and the argued interpretation of these changes as evidence for cultural learning processes in song change - the debate over whether these changes have environmental causation or are due to learning processes similar to song change in humpbacks is ongoing and this study therefore contributes interesting evidence from a newly covered population.

I think the methods and analyses broadly support the claims but also that there are weaknesses in interpretation and presentation that should be addressed. I think perhaps the degree to which this is evidence of vocal learning may be a bit overplayed. Definitely there is change, but it is tricky to compare this to e.g. experimental demonstrations. For example, age-related changes in a changing post-whaling demographic scenario should at least be considered? Is there also any possibility for large-scale oceanographic variations to be included in some way - temperature shifts, for example? This could help understand the different roles of environment and learning in these processes. I think it is also important that these results be placed in a more detailed context of current knowledge of fin whale population structure in the north Atlantic - could population range shifts be a factor? The INI data show an interesting variation in the recordings from the Barents Sea and this could be discussed in the light of population structure knowledge also. It is unclear from the presentation whether the INI shift in 2000/2001 was coupled with any frequency shifts - if not, it suggests different trajectories and processes affecting these two aspects of the acoustic display.

I am not convinced the main story here is about conformity, and I think it would be a mistake to too easily reach for the humpback comparison but there are certainly questions to be asked about the 2000/2001 shift in terms of the processes that led to it.

Reviewer #3 (Public Review):

The authors used passive acoustic monitoring over a vast range of the North Atlantic to study the call rates of fin whales. They found a 'take over' of a new rythm (inter call intervals) during their study period. This was interpreted as a change in song production.

I am not completely convinced the authors are correct in describing this change in rate as a change in the song. Even though fin whale calls are evidently a male mating ground display, little is known about its function. Compared to humpback whales with their impressive repertoire of vocalizations, repeating themselves on the breeding grounds after some tens of minutes and therefore qualifying as a very slow 'song' similar to bird song, fin whale only emit a single type of call, which is remaining the same throughout the study period. It can be contested, I would assume, that a ,erely change the repetition rate of calls, even though seemingly done here in an 'overtake' fasion, can qualify as a change and learning of song,

Reviewer #1 (Public Review):

The hippocampus is a structure in the cerebral cortex known to be compartmentalised into regions with different functions. Dorsal hippocampus is involved in cognitive functions such as declarative memory and spatial navigation and interconnects chiefly with the neocortex. Ventral hippocampus interconnects with limbic structures such as amygdala and hypothalamus and is involved in affective states and anxiety. What specifies this functional regionalisation during development is not well understood. The present study focuses on the role of transcription factors COUPTFI and COUPTFII, confirming a previously observed dorsal to ventral gradient of expression of COUPTFI in both embryonic and adult mouse hippocampus, and reporting that expression of COUPTFII is strongest in ventral hippocampus. The aim of the authors was then to probe the role of these transcription factors with the use of conditional knockout of one or both factors using RxCre+ mice (sometimes Emx1Cre+ for comparison). As predicted, COUPTFI insufficiency resulted in failure of the CA1 subregion of the dorsal hippocampus to develop properly (with concomitant loss of performance in a spatial memory task) COUPTFII knockdown had even more marked effects upon the ventral hippocampus with ectopic CA1/CA3 domains forming, while a double knockout lead to a drastic reduction in size of the hippocampus with subsequent effects upon the appearance of hippocampal synaptic circuitry and the capacity for adult neurogenesis (a feature of rodent hippocampus). In order to help explain the role of COUPTFI/II a role in regulating expression of two transcription factors LHX2 and LHX5, known to be crucial to hippocampal development, was tested by examining gene and protein expression. Changes in LHX2 and LHX5 was observed and a role for COUPTFI/II in regulating expression of these genes was postulated.

I believe the authors have largely achieved their aims and the results mostly support the conclusions, but, as discussed further below, there are some weaknesses in the data and some areas that could be expanded upon and improved. The methods are mostly appropriate. The use of the transgenic mice and the application of histological methods, especially tyramide amplified immunohistochemistry, is exemplary. However, I'm not sure a wide enough range of tests to explore the phenotype of the transgenic mice was employed to back the conclusions drawn by the authors. The introduction and discussion are nicely written and explain the general concepts and conclusions well. The work makes an important contribution to our understanding of brain development in general and hippocampal development in particular.

Turning to more specific comments, I must first point out that specification of the ventral hippocampus by expression of COUPTFII is not an entirely original finding, as it was suggested for the developing human hippocampus following immunohistochemical experiments illustrating COUPTFII expression to be confined to the ventral hippocampal structures of the medial temporal cortex (doi: 10.1093/cercor/bhx185). Of course, this study, unlike the present study, was restricted to fetal cortex, not adult, and also reported expression of COUP-TFI throughout dorsal and ventral hippocampal structures but without observing any dorsal to ventral gradient, however I feel its contribution to the field has been overlooked by the present study, and should be incorporated into the introduction and/or discussion.

More information about Rx-cre mice would be informative and could help explain the different phenotype observed when EMX1-cre mice were used to conditionally knock down COUPTFI/II expression.

The demonstration of antagonistic gradients of COUP-TFI and -TFII across the hippocampus is more convincing in the immunohistochemical preparations than in the western blots. The qualitative data presented in Fig.1p does not convincingly represent the quantitative data presented in Fig.1q. There seem to be multiple bands for COUP-TFII and I wonder exactly how quantifying this was approached?

Behavioural testing is limited to one test of dorsal hippocampus function. other tests for non-spatial memory, e.g. novel object recognition, or ventral hippocampus function, e.g. step through passive avoidance, might have lead to some interesting discriminations between the various knock down animals (see doi: 10.3389/fnagi.2018.00091).

Abnormalities in the trisynaptic circuit. No studies of actual synapses, either physiological or morphological, were carried out. I wonder to what extent these immunohistochemical studies just further reflect the abnormalities in hippocampal morphology presented earlier in the manuscript without specifically telling us about synaptic circuits? Although the immunohistochemical preparations are beautiful, they are inadequate on their own in telling us much about what sort of synaptic circuitry exists in the transgenic animals.

LHX2/LHX5 interaction. The immunohistochemical study, which shows clear differences in LHX5 and LHX2 protein expression at E14.5 in double knockdown mice is more convincing than the qPCR study at E11.5, which show surprisingly small differences in mRNA expression. Could the authors expand upon whether this is due to stage of development, or differences between mRNA and protein expression? Why hasn't both mRNA and protein expression data at both time points been presented?

Response to the re-submission

I am happy that the western blot presentation has been improved, and my minor comments attended to. It is disappointing, although understandable given the timeframe, that the lack of qPCR data at 14.5 ED has not been rectified. The immunohistochemical data alone is qualitative and only indicative of LHX5 expression remaining depressed and LHX2 expression possibly increasing between E11.5 and E14.5. In the absence of qPCR data, a more quantitative immunohistochemical study, such as counting blind the number of LHX5+ Cajal-Retzius cells, or measuring optical density of LHX2 expression under rigorous experimental conditions regarding image collection and processing, would be required to support the hypothesis that COUPTFI/II expression modulates the LHX2/LHX5 axis.

Reviewer #2 (Public Review):

The Author's chose to limit their response to re-doing the Lhx5 immuno using the correct antibody which now displays the expected staining: Lhx5 expression is limited to the hem. They have not however presented a characterization of where the RxCre acts, although this was pointed out by other reviewers as well. It would have been useful to demonstrate the expression domain in particular with respect to the time of its initiation, to explain how it causes a phenotype close to that described for the Lhx5 knockout (Zhao et al., 1999). From the decrease of Lhx5 expression and the CR cells which arise from the hem, it appears that the RxCre does indeed act in the hem. However, the timing and spatial pattern is important to establish, as I had pointed out in my first review, "If [the expression of RxCre] it has a dorso-ventral bias in the early embryo, it could explain the regional difference in the COUPTF phenotypes."

The major interpretive criticisms I made have not been addressed even though these would have only required a re-writing and re-interpretation of the data. The revised manuscript continues to include major errors of interpretation such as the idea that Lhx2 and Lhx5 "inhibit each other", something that is unsupported since the expression domains of these two genes are mutually exclusive as is clear from the authors' own new data and the literature.Lines 355-360: "The expression of Lhx2 was comparable between the control and double-mutant mice at E11.5 (Figure 5Be-h, e'-h'). Interestingly, the expression of the Lhx2 protein was increased in the hippocampal primordium in the COUP-TF double-mutant mice at E13.5 and E14.5 (Figure 5Bm-p, m'-p', u-x, u'-x'). The upregulation of Lhx2 expression is most likely associated with the reduced expression of the Lhx5 gene"There's clearly no Lhx5 in the hippocampal primordium so how is this possible?

The authors have missed the insights from key papers that they cite, e.g. (lines 352-354) " The expression of Lhx2 was expanded ventrally into the choroid plexus in the Lhx5 null mutant mice (Zhao et al., 1999)" - this paper in fact shows there is no choroid plexus. Lhx2 appears to extend to the midline likely because the hem isn't specified. The authors would benefit from reading https://doi.org/10.1101/2022.10.25.513532 in which Lmx1a is shown to be the master regulator of the hem.<br /> A sentence like (lines 77-81) further blurs the literature: "Intriguingly, deficiency of either Lhx5 or Lhx2 results in agenesis of the hippocampus, and more particularly, these genes inhibit each other (Hébert & Fishell, 2008; Mangale et al., 2008; Roy, Gonzalez-Gomez, Pierani, Meyer, & Tole, 2014; Zhao et al., 1999), indicating that the Lhx5 and Lhx2 genes may generate an essential regulatory axis to ensure the appropriate hippocampal development"<br /> First, none of the papers they cite shows that these two factors inhibit each other. Second, the "agenesis of the hippocampus" in the Lhx2 knockout mentioned in Porter et al. (1997) was later shown to be due to a transformation of the hippocampal primordium into an EXPANDED hem (Mangale et al.) In contrast, the "agenesis of the hippocampus" in the Lhx5 mutant appears to be due to the near-complete LOSS of the hem and evidenced by the loss of its derivatives, the choroid plexus and the CR cells (Zhao et al., 1999). The fact that loss of these two factors have opposite effects on the hem (each resulting in loss of the hippocampus, one due to transformation of the hippocampal primordium into hem and the other because of a lack of hipopcampal induction) does not mean that there is an Lhx5-Lhx2 "axis" regulating hippocampal development.

I won't repeat my other comments here, but the majority of them were not addressed in any way.

In conclusion, I find it unfortunate that the authors have chosen not to use the detailed input provided by the reviewers which would have greatly improved their manuscript.

Reviewer #3 (Public Review):

The authors have made significant improvements in addressing my major concerns raised during the previous review. However, I still have some lingering concerns regarding the quantification and statistical analysis presented in the manuscript. Specifically, there is a lack of robust quantification and statistical analysis to support the conclusions drawn, particularly in relation to the numbers of DG, CA1, and CA3 neurons.

To strengthen the validity and reliability of the findings, I would strongly recommend the authors to incorporate a more rigorous quantitative approach in their research. This could involve implementing stereological methods or other appropriate techniques to accurately estimate the numbers of neurons in the DG, CA1, and CA3 regions. By doing so, the authors would enhance the credibility of their conclusions and provide more solid evidence for their claims.

Reviewer #1 (Public Review):

Barlow et al performed a viral insertion screen in larval zebrafish for sleep mutants. They identify a mutant named dreammist (dmist) that displayed defects in sleep, namely, decreased sleep both day and night, accompanied by increased activity. They find that dmist encodes a previously uncharacterized single-pass transmembrane protein that shows structural similarity to Fxyd1, a Na+K+-ATPase regulator. Disruption of fxyd1 or atp1a3a, a Na+,K+-ATPase alpha-3 subunit, decreased night-time sleep. By staining for sodium levels, the authors uncover a global increase of sodium in both dmist and atp1a3a mutants following PTZ treatment, consistent with defects in Na+K+-ATPase function. These genetic data from multiple mutant lines help establish the importance of sodium and/or potassium homeostasis in sleep regulation.

The conclusions of this paper are mostly well supported by data, with the following strengths and weaknesses as described below.

Strengths:<br /> Elegant use of CRISPR knockout methods to disrupt multiple genes that help establish the importance of regulating Na+K+-ATPase function in sleep.<br /> Data are mostly clearly presented.<br /> Double mutant analysis of dmist and atp1a3a help establish an epistatic relationship between these proteins.

Weaknesses:<br /> The authors emphasize the role of increased cellular sodium, but equally plausibly, the phenotypes could be due to decreased cellular potassium. The potassium channel shaker has been previously identified as a critical sleep regulator in Drosophila.<br /> Although the increased sleep rebound after PTZ treatment in the dmist mutant is interesting, I find it difficult to understand, especially in the context of the dmist mutant having decreased sleep.

The similar phenotype between dmist and Fxyd1 in sleep reduction yet very different expression patterns, with dmist being mostly neuronal while fxyd1 being mostly non-neuronal, raise many possible questions: 1) are the sleep phenotypes due to neuronal Na/K imbalance? Or 2) Are the sleep phenotypes due to extracellular Na/K imbalance? Or 3) both? Some feasible experiments may help achieve a better mechanistic understanding of the observed sleep defects.

Reviewer #2 (Public Review):

Barlow and colleagues describe a role for the Na+/K+ pump in sleep/wake regulation. They discovered this role starting with a forward genetic screen in which they tested a biased sample of virus insertion fish lines for sleep phenotypes. They found an insertion in a gene they named dreammist, which is homologous to the gene FXYD1 encoding single membrane-pass modifiers of Na/K pumps. They go on to show that genetic manipulations of either FXYD1 or the Na/K pump also reduce sleep. They use pharmacology and sleep deprivation experiments to provide further evidence that the NA/K pump regulates intracellular sodium and rebound sleep. This study provides additional evidence for the important role of membrane excitability in sleep regulation (prior studies have implicated K+ channel subunits as well as a sodium leak ion channel).

The study is well done and convincing with regard to its major conclusions. I had some minor comments/questions, which they properly addressed in their revision and rebuttal.

Reviewer #1 (Public Review):

In this manuscript, the authors present a valuable new method to represent animal behavior from video data using a variational autoencoder framework that disentangles individual-specific and background variance from variables that can be more reliably compared across individuals. They achieve this aim through the use of a novel Cauchy-Schwatz (C-S) regularization term in their loss function that leads to latents that model continuously varying features in the images. The authors present a variety of validations for the method, including testing across sessions and individuals for a head-fixed task. They also show how the methods could be used for behavioral decoding from neural data, quantifying social behavior in mice, demonstrating the applicability of the method outside of head-fixed environments and for different measurement modalities. While some areas of confusion and questions about the validation exist, this is an overall strong paper and an important contribution to this field.

Strengths:

- The use of the C-S regularizer is novel approach that has potential for wide use across experimental paradigms and model organisms<br /> - The extent of the validations performed was solid, although perhaps not as convincing in a couple of cases as might be ideal<br /> - The GitHub code demo worked well, and the code appears to be accessible and well-written

Weaknesses:

- Some of the validation figures were a bit unclear in their presentation, making it difficult to assess exactly what had been tested<br /> - It is possible that I missed this, but the authors didn't really provide a sense of how to pick a particular distribution to match using the CS term for a specific paradigm/modality and how the choice affects the results<br /> - While the authors' statements about individual training vs. transfer learning accuracy and efficiency in Figure 6 are technically true, the effect size is rather small ( a few percent at most in each case), thus I don't know how much of a big deal I would want to make out of these results<br /> - In general, I would have liked to have seen the Discussion section speak more to the choices and limitations inherent in applying the method. How does the choice of prior/metaparameters/architecture/etc affect the results? In what situations would this method to fail? What are the next advances that are necessary for the field to progress?

Reviewer #2 (Public Review):

This paper presents a valuable contribution to ongoing methods for understanding and modeling structure via latent variable models for neural and behavioral data. Building on the PS-VAE model of Whiteway et al. (2021), which posited a division of latent variables into unsupervised (i.e., useful for reconstruction) and supervised (useful for predicting selected labeled features) variables, the authors propose an additional set of "constrained subspace" latent variables that are regularized toward a prespecified prior via a Cauchy-Schwarz divergence previously proposed.

The authors contend that the added CS latents aid in capturing both patterns of covariance across the data and individual-specific features that are of particular benefit in multi-animal experiments, all without requiring additional labels. They substantiate these claims with a series of computational experiments demonstrating that their CS-VAE outperforms the PS-VAE in several tasks, particularly that of capturing differences between individuals, consistency in behavioral phenotyping, and predicting correlations with neural data.

Strengths of the present work include an extensive and rigorous set of validation experiments that will be of interest to those analyzing behavioral video. Weaknesses include a lack of discussion of key theoretical ideas motivating the design of the model, including the choice of a Cauchy-Schwarz divergence, the specific form of the prior, and arguments for sorts of information the CS latents might capture and why. In addition, the model makes use of a moderate number of key hyperparameters whose effect on training outcomes are not extensively analyzed. As a result, the model may be difficult for less experienced users to apply to their own data. Finally, as with many similar VAE approaches, the lack of a ground truth against which to validate means that much of evidence provided for the model is necessarily subjective, and its appeal lies in the degree to which the discovered latent spaces appear interpretable in particular applications.

In all, this work is a valuable contribution that is likely to have appeal to those interested in applying latent space methods, particularly to multi-animal video data.

Reviewer #3 (Public Review):

As naturalistic neuroscience becomes increasingly popular, the importance of new computational tools that facilitate the study of animals behaving in minimally constrained environments grows. Yi et al convincingly demonstrate the usefulness of their new method on data from neuroethological studies involving multiple animals, including those with social interactions. Briefly, their method improves upon prior semi-supervised machine learning methods in that extracted latent variables can be more cleanly separated into those representing the behavior of individual subjects and those representing social interactions between subjects. Such an improvement is broadly useful for downstream analysis tasks in multi-subject or social neuroethological studies.

Strengths:<br /> The authors tackle an important problem encountered in behavior analyses in an emerging subfield of neuroscience, naturalistic social neuroscience. They make a case for doing so using semi-unsupervised methods, a toolbox which balances competing scientific needs for building models using large neural-behavioral datasets and for model explainability. The paper is well written, with well-designed figures and relevant analyses that make for an enjoyable reading experience.

The authors provide a remarkable variety of examples that make a convincing case for the utility of their method when used by itself or in conjunction with other data analysis techniques commonly used in modern neuroscience (behavioral motif extraction, neural decoding, etc.). The examples show not just that the extracted latents are more disentangled, but also that the improvement in disentangling has positive effects in downstream analysis tasks.

Weaknesses:<br /> While the paper does a great job of applying the method to real world data, the components of the method itself are not as thoroughly investigated. For example, the contribution of the novel Cauchy-Schwarz regularization technique has not been systematically investigated. This could be done either by sharing additional data where hyperparameters control the contribution of the regularizer, or cite relevant papers where such an analysis have been carried out. It would also be valuable to understand what other regularization techniques might potentially have been applicable here.

The authors conclude from their empirical investigations that the specific prior distribution does not matter to the regularization process. This seems reasonable given that the neural network can learn a complex and arbitrary transformation of the data during training. It would be helpful if the authors could cite prior work where this type of prior distribution does matter and how their approach is different from such prior work. If there is a visualization/explainability related motivation for choosing one prior distribution over another, this could be clarified.

Reviewer #1 (Public Review):

In the manuscript by Urban et al., the authors attempt to further delineate the role which non-neuronal CNS cells play in the development of ALS. Toward this goal, the transmembrane signaling molecule ephrinB2 was studied. It was found that there is an increased expression of ephrinB2 in astrocytes within the cervical ventral horn of the spinal cord in a rodent model of ALS. Moreover, the reduction of ephrinB2 reduced motoneuron loss and prevented respiratory dysfunction at the NMJ. Further driving the importance of ephrinB2 is an increased expression in the spinal cords of human ALS individuals. Collectively, these findings present compelling evidence implicating ephrinB2 as a contributing factor towards the development of ALS.

Reviewer #2 (Public Review):

The contribution of glial cells to the pathogenesis of amyotrophic lateral sclerosis (ALS) is of substantial interest and the investigators have contributed significantly to this emerging field via prior publications. In the present study, authors use a SOD1G93A mouse model to elucidate the role of astrocyte ephrinB2 signaling in ALS disease progression. Erythropoietin-producing human hepatocellular receptors (Ephs) and the Eph receptor-interacting proteins (ephrins) signaling is an important mediator of signaling between neurons and non-neuronal cells in the nervous system. Recent evidence suggests that dysregulated Eph-ephrin signaling in the mature CNS is a feature of neurodegenerative diseases. In the ALS model, upregulated Eph4A expression in motor neurons has been linked to disease pathogenesis. In the present study, authors extend previous findings to a new class of ephrinB2 ligands. Urban et al. hypothesize that upregulated ephrinB2 signaling contributes to disease pathogenesis in ALS mice. The authors successfully test this hypothesis and their results generally support their conclusion.

Major strengths of this work include a robust study design, a well-defined translational model, and complementary biochemical and experimental methods such that correlated findings are followed up by interventional studies. Authors show that ephrinB2 ligand expression is progressively upregulated in the ventral horn of the cervical and lumbar spinal cord through pre-symptomatic to end stages of the disease. This novel association was also observed in lumbar spinal cord samples from post-mortem samples of human ALS donors with a SOD1 mutation. Further, they use a lentiviral approach to drive knock-down of ephrinB2 in the central cervical region of SOD1G93A mice at the pre-symptomatic stage. Interestingly, in spite of using a non-specific promoter, authors note that the lentiviral expression was preferentially driven in astrocytes.

Since respiratory compromise is a leading cause of morbidity in the ALS population, the authors proceed to characterize the impact of ephrinB2 knockdown on diaphragm muscle output. In mice approaching the end stage of the disease, electrophysiological recordings from the diaphragm muscle show that animals in the knock-down group exhibited a ~60% larger amplitude. This functional preservation of diaphragm function was also accompanied by the preservation of diaphragm neuromuscular innervation. However, it must be noted that this cervical ephrinB2 knockdown approach had no impact on disease onset, disease duration, or animal survival. Furthermore, there was no impact of ephrinB2 knockdown on forelimb or hindlimb function.

The major limitation of the manuscript as currently written is the conclusion that the preservation of diaphragm output following ephrinB2 knockdown in SOD1 mice is mediated primarily (if not entirely) by astrocytes. The authors present convincing evidence that a reduction in ephrinB2 is observed in local astrocytes (~56% transduction) following the intraspinal injection of the lentivirus. However, the proportion of cell types assessed for transduction with the lentivirus in the spinal cord was limited to neurons, astrocytes, and oligodendrocyte lineage cells. Microglia comprise a large proportion of the glial population in the spinal grey matter and have been shown to associate closely with respiratory motor pools. This cell type, amongst the many others that comprise the ventral gray matter, have not been investigated in this study. Thus, the primary conclusion that astrocytes drive ephrinB2-mediated pathogenesis in ALS mice is largely correlative. Further, it is interesting to note that no other functional outcomes were improved in this study. The C3-C5 region of the spinal cord consists of many motor pools that innervate forelimb muscles. CMAP recordings conducted at the diaphragm are a reflection of intact motor pools. This type of assessment of neuromuscular health is hard to re-capitulate in the kind of forelimb task that is being employed to test motor function (grip strength). Thus, it would be interesting to see if CMAP recordings of forelimb muscles would capture the kind of motor function preservation observed in the diaphragm muscle.

On a similar note, the functional impact of increased CMAP amplitude has not been presented. An increase in CMAP amplitude does not necessarily translate to improved breathing function or overall ventilation. Thus, the impact of this improvement in motor output should be clearly presented to the reader. Further, to the best of my knowledge, expression of Eph (or EphB) receptors has not been explicitly shown at the phrenic motor pool. It is thus speculative at best that the mechanism that the authors suggest in preserving diaphragm function is in fact mediated through Eph-EphrinB2 signaling at the phrenic motor pool. This aspect of the study would warrant a deeper discussion. Lastly, although authors include both male and female animals in this investigation, they do not have sufficient power to evaluate sex differences. Thus, this presents another exciting future of investigation, given that ALS has a slightly higher preponderance in males as compared to females.

In summary, this study by Urban et al. provides a valuable framework for Eph-Ephrin signaling mechanisms imposing pathological changes in an ALS mouse model. The role of glial cells in ALS pathology is a very exciting and upcoming field of investigation. The current study proposes a novel astrocyte-mediated mechanism for the propagation of disease that may eventually help to identify potential therapeutic targets.

Reviewer #1 (Public Review):

Wheeler et al. have discovered a new RNA circuit that regulates T-cell function. They found that the long non-coding RNA Malat1 sponges miR-15/16, which controls many genes related to T cell activation, survival, and memory. This suggests that Malat1 indirectly regulates T-cell function. They used CRISPR to mutate the miR-15/16 binding site in Malat1 and observed that this disrupted the RNA circuit and impaired cytotoxic T-cell responses. While this study presents a novel molecular mechanism of T-cell regulation by Malat1-miR-15/16, the effects of Malat1 are weaker compared to miR-15/16. This could be due to several reasons, including higher levels of miR-15/16 compared to Malat1 or Malat1 expression being mostly restricted to the nucleus. Although the role of miR15/16 in T-cell activation has been previously published, if the authors can demonstrate that miR15/16 and/or Malat1 affect the clearance of Listeria or LCMV, this will significantly add to the current findings and provide physiological context to the study.

Reviewer #2 (Public Review):

This study connects prior findings on MicroRNA15/16 and Malat1 to demonstrate a functional interaction that is consequential for T cell activation and cell fate.

The study uses mice (Malat1scr/scr) with a precise genetic modification of Malat1 to specifically excise the sites of interaction with the microRNA, but sparing all other sequences, and mice with T-cell specific deletion of miR-15/16. The effects of genetic modification on in vivo T-cell responses are detected using specific mutations and shown to be T-cell intrinsic.

It is not known where in the cell the consequential interactions between MicroRNA15/16 and Malat1 take place. The authors depict in the graphical abstract Malat1 to be a nuclear lncRNA. Malat 1 is very abundant, but it is unclear if it can shuttle between the nucleus and cytoplasm. As the authors discuss future work defining where in the cell the relevant interactions take place will be important.

In addition to showing physiological phenotypic effects, the mouse models prove to be very helpful when the effects measured are small and sometimes hard to quantitate in the context of considerable variation between biological replicates (for example the results in Figure 4D).

The impact of the genetic modification on the CD28-IL2- Bcl2 axis is quantitatively small at the level of expression of individual proteins and there are likely to be additional components to this circuitry.

Reviewer #1 (Public Review):

In this manuscript, the authors investigated the role of Elg1 in the regulation of telomere length. The main role of the Elg1/RLC complex is to unload the processivity factor PCNA, mainly after completion of synthesis of the Okazaki fragment in the lagging strand. They found that Elg1 physically interacts with the CST (Cdc13-Stn1-Ten1) and propose that Elg1 negatively regulates telomere length by mediating the interaction between Cdc13 and Stn1 in a pathway involving SUMOylation of both PCNA and Cdc13. Accumulation of SUMOylated PCNA upon deletion of ELG1 or overexpression of RAD30 leads to elongated telomeres. On the other hand, the interaction of Elg1 with Sten1 is SIM-dependent and occurs concurrently with telomere replication in late S phase. In contrast Elg1-Cdc13 interaction is mediated by PCNA-SUMO, is independent on the SIM of Elg1 but still dependent on Cdc13 SUMOylation. The authors present a model containing two main messages 1) PCNA-SUMO acts as a positive signal for telomerase activation 2) Elg1 promotes Cdc13/Stn1 interaction at the expense of Cdc13/Est1 interaction thus terminating telomerase action.

The manuscript contains a large amount of data that make a major inroads on a new type of link between telomere replication and regulation of the telomerase. Nevertheless, the detailed choreography of the events as well as the role of PCNA-SUMO remain elusive and the data do not fully explain the role of the Stn1/Elg1 interaction. The data presented do not convincingly support the claim that SUMO-PCNA is a positive signal for telomerase activation. This was partially addressed in the current version.

Reviewer #2 (Public Review):

This paper purports to unveil a mechanism controlling telomere length through SUMO modifications controlling interactions between PCNA unloader Elg1 and the CST complex that functions at telomeres. This is an extremely interesting mechanism to understand, and this paper indeed reveals some interesting genetic results, leading to a compelling model, with potential impact on the field. Overall, however, the data do not provide sufficient support for the claims. The model may be correct but it is not yet convincingly demonstrated.

The current version addressed some of the issues regarding language describing conclusions and more experimental detail has been provided. However, the authors have not provided new data supporting the model, so the overall evaluation is that the work remains inconclusive.

Reviewer #3 (Public Review):

This paper reveals interesting physical connections between Elg1 and CST proteins that suggest a model where Elg1-mediated PCNA unloading is linked to regulation of telomere length extension via Stn1, Cdc13, and presumably Ten1 proteins. Some of these interactions appear to be modulated by sumolyation and connected with Elg1's PCNA unloading activity. The strength of the paper is in the observations of new interactions between CST, Elg1, and PCNA. These interactions should be of interest to a broad audience interested in telomeres and DNA replication.

What is not well demonstrated from the paper is the functional significance of the interactions described. The model presented by the authors is one interpretation of the data shown, and proposes that the role of sumolyation is temporally regulate the Elg1, PCNA and CST interactions at telomeres. This model makes some assumptions that are not demonstrated by this work (such as Stn1 sumolyation, as noted) and are left for future testing. Alternative models that envision sumolyation as a key in promoting spatial localization could also be proposed based on the data here (as mentioned in the discussion), in addition to or instead of a role for sumolyation in enforcing a series of switches governing a tightly sequenced series of interactions and events at telomeres. Critically, the telomere length data from the paper indicates that the proposed model depicts interactions that are not necessary for telomerase activation or inhibition, as telomeres in pol30-RR strains are normal length and telomeres in elg1∆ strains are not nearly as elongated as in stn1 strains. One possibility mentioned in the paper is the PCNAS and Elg1 interactions are contributing to the negative regulation of telomerase under certain conditions that are not defined in this work. Could it also be possible that the role of these interactions is not primarily directed toward modulating telomerase activity? It will be of interest to learn more about how these interactions and regulation by Sumo function intersect with regulation of telomere extension.

Reviewer #1 (Public Review):

More than ten years ago, it was shown that activity in the primary visual cortex of mice substantially increases when mice are running compared to when they are sitting still. This finding 'revolutionised' our thinking about the visual cortex, turning away from it being a passive image processor and highlighting the influence of non-visual factors. The current study now for the first time repeats this experiment in a primate (the marmoset). The authors find that in contrast to mice, marmoset V1 activity is slightly suppressed during running, and they relate this to differences in gain modulations of V1 activity between the two species.

Strengths:

- Replication in primates of the original finding in mice partly took so long, because of the inherent difficulties with recording from the brain of a running primate. The treadmill for the marmosets in the current study is a very elegant solution to this problem. It allows for true replication of the 'running vs stationary' experiment and undoubtedly opens up many possibilities for other experiments recording from a head-fixed but active marmoset.<br /> - In addition to their own data on the marmoset, the authors run their analyses on a publicly available data set on the mouse. This allows them to directly compare mouse and marmoset findings, which significantly strengthens their conclusions.

Weaknesses:

- The main thing that is missing from the study is a good explanation as to why running has such a different effect on marmoset V1 compared to mouse V1. Differences in neuromodulatory inputs are cited in the discussion as a possible cause for the discrepancy, but an obvious influencing factor that the authors could investigate in their own data set is the retinal input. In Fig1b, the authors even show these data in the form of gaze and pupil size. In these example data, by eye, it looks like the pupil size is positively correlated with the run speed. This would of course have large consequences on the activity in V1, but the authors do not do anything with these data. The study would improve substantially if the authors would correlate their run speed traces with other factors that they have recorded too, such as pupil size and gaze.

- Fig2a shows the 'most correlated mouse session', i.e. the session where the relation between visual cortex activity and running speed was strongest. Looking at the raster plot, however, shows that this strong positive correlation must be due entirely to the lower half of the neurons significantly increasing their firing rate as the mouse starts to run; in fact, the upper 25% or so of the neurons show exactly the opposite (strong suppression of the neurons as the mouse starts running). It would be more balanced if this heterogeneity in the response is at least mentioned somewhere in the text.

Significance:

The paper provides interesting new evidence to the ongoing discussion about the influence of non-visual factors in general, and running in particular, on visual cortex activity. As such, it helps to pull this discussion out of the rodent field mainly and into the field of primate research. The elegant experimental set-up of the marmoset on a treadmill will certainly add new findings to this issue also in the years to come.

Reviewer #2 (Public Review):

This work aims at answering whether activity in the primate visual cortex is modulated by locomotion, as was reported for the mouse visual cortex. The finding that the activity in the mouse visual cortex is modulated by running has changed the concept of primary sensory cortical areas. However, it was an open question whether this modulation generalizes to primates.

To answer this fundamental question the authors established a novel paradigm in which a head-fixed marmoset was able to run on a treadmill while watching a visual stimulus on a display. In addition, eye movements and running speed were monitored continuously and extracellular neuronal activity in the primary visual cortex was recorded using high-channel-count electrode arrays. This paradigm uniquely permitted investigation of whether locomotion modulates sensory-evoked activity in the visual cortex of a marmoset. Moreover, to directly compare the responses in the marmoset visual cortex to responses in the mouse visual cortex the authors made use of a publicly-available mouse dataset from the Allen Institute. In this dataset, the mouse was also running on a treadmill and observing a set of visual stimuli on a display. The authors took extra care to have the marmoset and mouse paradigms as comparable as possible.

To characterize the visually driven activity the authors present a series of moving gratings and estimate receptive fields with sparse noise. To estimate the gain modulation by running the authors split the dataset into epochs of running and non-running which allowed them to estimate the visually evoked firing rates in both behavioral states.

Strengths:<br /> The novel paradigm of head-fixed marmosets running on a treadmill while being presented with a visual stimulus is unique and ideally tailored to answering the question that the authors aimed to answer. Moreover, the authors took extra care to ensure that the paradigm in the marmoset matched as closely as possible to the conditions in the mouse experiments such that the results can be directly compared. To directly compare their data the authors re-analyzed publicly available data from the visual cortex of mice recorded at the Allen Institute. Such a direct comparison, and reuse of existing datasets, is another strong aspect of the work. Finally, the presented new marmoset dataset appears to be of high quality, the comparison between the mouse and marmoset visual cortex is well done and the results and interpretation are straightforward.

Weaknesses:<br /> While the presented results are clear and support the main conclusion of the authors, additional analysis and experimental details could have further strengthened and clarified some aspects of the results. For example, it is known that the locomotion gain modulation varies with layer in the mouse visual cortex, with neurons in the infragranular layers expressing a diversity of modulations (Erisken et al. 2014 Current Biology). However, for the marmoset dataset, it was not reported from which cortical layer the neurons are from, leaving this point unanswered.

Nonetheless, the aim of comparing the locomotion-induced modulation of activity in primate and mouse primary visual cortex was convincingly achieved by the authors. The results shown in the figures support the conclusion that locomotion modulates the activity in primate and mouse visual cortex differently. While mice show a profound gain increase, neurons in the primate visual cortex show little modulation or even a reduction in response strength.

This work will have a strong impact on the field of visual neuroscience but also on neuroscience in general. It revives the debate of whether results obtained in the mouse model system can be simply generalized to other mammalian model systems, such as non-human primates. Based on the presented results, the comparison between the mouse and primate visual cortex is not as straightforward as previously assumed. This will likely trigger more comparative studies between mice and primates in the future, which is important and absolutely needed to advance our understanding of the mammalian brain.

Moreover, the reported finding that neurons in the primary visual cortex of marmosets do not increase their activity during running is intriguing, as it makes you wonder why neurons in the mouse visual cortex do so. The authors discuss a few ideas in the paper which can be addressed in future experiments. In this regard, it is worth noting that the authors report an interesting difference between the foveal and peripheral parts of the visual cortex in marmoset. It will be interesting to investigate these differences in more detail in future studies. Likewise, while running might be an important behavioral state for mice, other behavioral states might be more relevant for marmosets and do modulate the activity of the primate visual cortex more profoundly. Future work could leverage the opportunities that the marmoset model system offers to reveal new insights about behavioral-related modulation in the primate brain.

Reviewer #3 (Public Review):

Prior studies have shown that locomotion (e.g., running) modulates mouse V1 activity to a similar extent as visual stimuli. However, it's unclear if these findings hold in species with more specialized and advanced visual systems such as nonhuman primates. In this work, Liska et al. leverage population and single neuron analyses to investigate potential differences and similarities in how running modulates V1 activity in marmosets and mice. Specifically, they discovered that although a shared gain model could describe well the trial-to-trial variations of population-level neural activity for both species, locomotion more strongly modulated V1 population activity in mice. Furthermore, they found that at the level of individual units, marmoset V1 neurons, unlike mice V1 neurons, experience suppression of their activity during running.

A major strength of this work is the introduction and completion of primate electrophysiology recordings during locomotion. Data of this kind was previously limited, and this work moves the field forward in terms of data collection in a domain previously inaccessible in primates. Another core strength of this work is that it adds to a limited collection of cross-species data collection and analysis of neural activity at the single-unit and population level, attempting to standardize analysis and data collection to be able to make inferences across species.

However, the authors did not take full advantage of the quantity and diversity of the marmoset visual cortex recordings in their analyses. They mention recording and analyzing the activity of peripheral V1 neurons but mainly present results involving foveal V1 neurons. Foveal neurons, with their small receptive fields strongly affected by precise eye position, would seem to be less likely to be comparable to rodent data. If the authors have a reason for not doing so, they should provide an explanation. Given that the marmosets are motivated to run with liquid rewards, the authors should provide more context as to how this may or may not affect marmoset V1 activity. Additionally, the lack of consideration of eye movements or position presents a major absence for the marmoset results, and fails to take advantage of one of the key differences between primate and rodent visual systems - the marmosets have a fovea, and make eye movements that fixate in various locations on the screen during the task. Finally, the model provides a strong basis for comparison at the level of neuronal populations, but some methodological choices are insufficiently described and may have an impact on interpreting the claims.

Overall, the methods and data are supportive of the main claims of the work. The use of single neuron and population level approaches demonstrate that the activity of V1 in mice and marmoset is categorically different. Since primate V1 is so diverse, this limits the interpretation of the cross-species comparison. Still, the work is a great step forward in the field, especially with the novel methodology of collecting neural activity from running primates.

Reviewer #2 (Public Review):

The pear psylla Cacopsylla chinensis has two morphologically different forms, winter- and summer-forms depending on the temperatures. The authors provided solid data showing that the cold sensor CcTRPM is responsible for switching summer- to winter forms, which is in turn regulated by the miRNA miR-252. This finding is interesting and novel.

Reviewer #1 (Public Review):

Here, the authors describe, in detail, the transition between the summer form and the winter form of the pear psyllid, Cacopsylla chinensis. While the authors explore many components of this transition, the central hypotheses they seek to test are (i) that a protein they deem CcTRPM is a cold-sensitive Transient Receptor Potential Melastatin (TRPM) channel, and (ii) that this channel is involved in the summer-to-winter transition, in response to cold.

The authors demonstrate that: both cold and menthol can initiate the summer-to-winter transition; that the protein of interest is required for the summer-to-winter transition (in vivo); that the protein of interest is involved in menthol-dependent Ca2+ transients (in vitro); that miR-252 expression is temperature-dependent, modulates the seasonal transition, and affects the expression of the transcript of interest; and finally, somewhat separately, that the chitin biosynthesis pathway is linked to the summer-to-winter transition.

Although I generally found the evidence to be convincing, I note a few critical weaknesses in the manuscript as it is currently presented. Firstly, there is insufficient methodological detail to understand how the genes/transcripts/proteins in this work were identified. Further, the structural and phylogenetic analyses are incompletely described and the results are inconsistent with our previous understanding of the structure and evolution of TRPMs. It is thus possible (although unlikely) that this protein has been misidentified. Alternatively, this could be a structurally aberrant TRPM from a lineage previously presumed to be lost in insects, but there is not sufficient evidence to conclude this. Perhaps more importantly, the authors conclude that the protein of interest is cold sensitive (i.e., a "temperature receptor") primarily based on menthol sensitivity. Although menthol and cold activate the same receptors in other species, there is no demonstrated reason to think that menthol sensitivity necessitates cold sensitivity, or vice-versa. Thus, the authors' conclusions are, in my opinion, incomplete and overstated. Below are specific comments giving further context to the criticisms summarized above:

1. The method used to identify the various genes/proteins described herein is not described. Relatedly, the alignment in Figure S1 lacks Trpms from non-hemipteran taxa, making it difficult to judge sequence similarity to other more well-characterized Trpms (e.g., from human, mouse, fly, nematode, etc.), and thus difficult to assess homology from the manuscript alone.

2. The authors suggest that the CcTrpm has ankyrin repeats. To my knowledge, this would be the first description of ankyrin repeats in TRPM. It's not stated how the authors identified these putative ankyrin repeats. There's also no description of the absence or presence of previously identified Melastatin Homology Regions (MHRs), a C-terminal coiled-coil that is typically present, other C-terminal domain motifs, or the TRP domain. In the absence of methodological detail, and given the proposed presence of ankyrin repeats, it seems possible that this may not be TRPM.

3. The authors suggest that, because mRNA abundance for CcTRPM is increased in response to cold, it is cold-sensitive. However, this says nothing as to whether cold actually activates the ion channel -- a critical distinction. The authors finally conclude that CcTRPM encodes a cold-sensitive ion channel because menthol elicits Ca2+ activity in vitro. However, this experiment only demonstrates that the protein is likely menthol sensitive. This experiment does not support the authors' conclusion that this is a cold-sensitive receptor (although their later knockdown experiments do, albeit indirectly).

4. The lack of taxonomic representation in the phylogenetic analysis makes it difficult to interpret, especially in the context of methodological detail concerning the initial identification of the gene/transcript/protein of interest. Further, it's not stated if the tree is rooted (if it is, the rooting methodology is not described), the branch lengths are not shown, and the branch support methodology is not described. Many previous phylogenetic analyses have concluded--implicitly or explicitly--that there are at least two ancestral animal TRPM paralogs. From the perspective of vertebrates, one ancestral copy went on to diversify into TRPMs 1, 3, 6, and 7, and the other ancestral copy went on to diversify into TRPMs 2, 8, 4, and 5. The insect Trpms are generally thought to be more closely related to vertebrate TRPMs 1,3,6, and 7. If this phylogeny is rooted, it implies that the hemipteran Trpms are more closely related to vertebrates 2, 8, 4, and 5 (or at least 8, since that is all that is present here), and quite distantly related to other insect Trpms (and presumably, to vertebrates 1,3,6, and 7, which are not present). To my knowledge, this would be the first description of this Trpm subfamily in insects, but there is insufficient evidence or phylogenetic rigor here to conclude that. The most likely explanation is that the tree is unrooted, incorrectly rooted, or that the protein of interest is not TRPM.

Reviewer #1 (Public Review):

The study titled "Distinct states of nucleolar stress induced by anti-cancer drugs" by Potapova and colleagues demonstrates that different chemotherapeutic agents can induce nucleolar stress, which manifests with varying cellular and molecular characteristics. The study also proposes a mechanism for how a novel type of nucleolar stress driven by CDK inhibitors may be regulated. As a reviewer, I appreciate the unbiased screening approach and I am enthusiastic about the novel insights into cell biology and the implications for cancer research and treatment. The study has several significant strengths: i) it highlights the understudied role of nucleolar stress in the on- and off-target effects of chemotherapy; ii) it defines novel molecular and cellular characteristics of the different types of nucleolar stress phenotypes; iii) it proposes novel modes of action for well-known drugs.

However, there are several important points that should be addressed:<br /> • The rationale behind choosing RPE cells for the screen is unclear. It might be more informative to use cancer cells to study the effects of chemotherapeutic agents. Alternatively, were RPE cells selected to evaluate the side effects of these agents on normal cells? Clarifying these points in the introduction and discussion would guide the reader.<br /> • Figure 2F indicates that DLD1 and HCT116 cells are less sensitive to nucleolar changes induced by several inhibitors, including CDK inhibitors. It would be crucial to correlate these differences with cell viability. Are these differences due to cell-type sensitivity or variations in intracellular drug levels? Assessing cell viability and intracellular drug concentration for the same drugs and cells would provide valuable insights.<br /> • Have the authors interpreted nucleolar stress as the primary cause of cell death induced by these drugs? When cells treated with CDK inhibitors exhibit the dissociated nucleoli phenotype, is this effect reversible? Is this phenotype indicative of cell death commitment? Conducting a washout experiment to measure the recovery of nucleolar function and cell viability would address these questions.<br /> • The correlation between the loss of Treacle phosphorylation and nucleolar stress upon CDK inhibition is intriguing. However, it remains unclear how these two events are related. Would Treacle knockdown yield the same nucleolar phenotype as CDK inhibition? Moreover, would point mutations that abolish Treacle phosphorylation prevent its interaction with Pol-I? Experiments addressing these questions would enhance our understanding of the correlation/causation between Treacle phosphorylation and the effects of CDK inhibition on nucleolar stress.

Overall, this study is significant and novel as it sheds light on the importance of nucleolar stress in defining the on-target and off-target effects of chemotherapy in normal and cancer cells.

Reviewer #2 (Public Review):

This is an interesting study with high-quality imaging and quantitative data. The authors devise a robust quantitative parameter that is easily applicable to any experimental system. The drug screen data can potentially be helpful to the wider community studying nucleolar architecture and the effects of chemotherapy drugs. Additionally, the authors find Treacle phosphorylation as a potential link between CDK9 inhibition, rDNA transcription, and nucleolar stress. Therefore I think this would be of broad interest to researchers studying transcription, CDKs, nucleolus, and chemotherapy drug mechanisms. However, the study has several weaknesses in its current form as outlined below.

1. Overall the study seems to suffer from a lack of focus. At first, it feels like a descriptive study aimed at characterizing the effect of chemotherapy drugs on the nucleolar state. But then the authors dive into the mechanism of CDK inhibition and then suddenly switch to studying biophysical properties of nucleolus using NPM1. Figure 6 does not enhance the story in any way; on the contrary, the findings from Fig. 6 are inconclusive and therefore could lead to some confusion.

2. The justification for pursuing CDK inhibitors is not clear. Some of the top hits in the screen were mTOR, PI3K, HSP90, Topoisomerases, but the authors fail to properly justify why they chose CDKi over other inhibitors.

3. In addition to poor justification, it seems like a very superficial attempt at deciphering the mechanism of CDK9i-mediated nucleolar stress. I think the most interesting part of the study is the link between CDK9, Pol I transcription, and nucleolar stress. But the data presented is not entirely convincing. There are several important controls missing as detailed below.

4. The authors did not test if inhibition of CDK7 and/or CDK12 also induces nucleolar stress. CDK7 and CDK12 are also major kinases of RNAPII CTD, just like CDK9. Importantly, there are well-established inhibitors against both these kinases. It is not clear from the text whether these inhibitors were included in the screen library.

5. In Figure 4E, the authors show that Pol I is reduced in nucleolus/on rDNA. The authors should include an orthogonal method like chromatin fractionation and/or ChIP

6. In Fig. 5D, in vitro kinase lacks important controls. The authors should include S to A mutants of Treacle S1299A/S1301A to demonstrate that CDK9 phosphorylates these two residues specifically.

7. To support their model, the authors should test if overexpression of Treacle mutants S1299A/S1301A can partially phenocopy the nucleolar stress seen upon CDK9 inhibition. This would considerably strengthen the author's claim that reduced Treacle phosphorylation leads to Pol I disassociation from rDNA and consequently leads to nucleolar stress.

8. Additionally, it would be interesting if S1299D/S1301D mutants could partially rescue CDK9 inhibition.

Reviewer #1 (Public Review):

In this paper by Lui and colleagues, the authors examine the role of locus coeruleus (LC)-noradrenaline (NA) neurons in the extinction of appetitive instrumental conditioning. They report that optogenetic activation of global LC-NA neurons during the conditioned stimulus (CS) period of extinction enhances long-term extinction memory without affecting within-session extinction. In contrast, LC-NA activation during the intertrial interval doesn't affect extinction and long-term memory. They then show that optogenetic activation of LC-NA neurons doesn't induce conditioned place preference/avoidance. Finally, they assess the necessity of LC-NA neurons in appetitive extinction and find that optogenetic inactivation of LC-NA neurons during the CS period results in the enhancement of within-session extinction. The experiments are well-designed, including offset control in the optogenetic activation study. I think this study adds new insight into the LC-NA system in the context of appetitive extinction.

Strengths:<br /> ・These studies identify that the artificial activation of LC-NA neurons enhances long-term memory of appetitive extinction, while this activation can't induce long-term conditioned place aversion. Thus, optogenetic activation of LC-NA neurons can inhibit spontaneous recovery of appetitive extinction without causing long-term aversive memory.<br /> ・Optoinhibition study demonstrates the reduction of a conditioned response of within-session extinction. Therefore, LC-NA neuronal activity at the CS period of extinction could act as anti-extinction or be important for the expression of the conditioned response.

Weaknesses:<br /> ・It is unclear how LC-NA neurons behave during the CS period of appetitive extinction from this study. This weakens the importance of the optogenetic inactivation result.<br /> ・While authors manipulate global LC-NA neurons, many people find functionally heterogeneous populations in the LC. It remains unsolved if there is a specific LC-NA subpopulation responsible for appetitive extinction.

Reviewer #2 (Public Review):

This study examines the role of the Locus Coeruleus (LC)/noradrenergic (NA) system in extinction in male and female rats. The behavioural task involves three phases i) training on a discriminative procedure in which operant responding was rewarded only during the presentation of a stimulus ii) extinction iii) testing for the expression of extinction at both short (1 day) or long (7 days) delays. Targeting LC/NA cells with optogenetic in TH::Cre rats, the authors found that photoexcitation during extinction led to an increase in the expression of extinguished responding at both short and long delays. By contrast, photo inhibition was found to be without an effect.

1. In such discrimination training, Pavlovian (CS-Food) and instrumental (LeverPress-Food) contingencies are intermixed. It would therefore be very interesting if the authors provided evidence of other behavioural responses (e.g. magazine visits) during extinction training and tests.<br /> 2. In Figure 1, the authors show the behavioural data of the different groups of control animals which were later collapsed in a single control group. It would be very nice if the authors could provide the data for each step of the discrimination training.<br /> 3. Inspection of Figures 2C & 2D shows that responding in control animals is about the same at test 2 as at the end of extinction training. Therefore, could the authors provide evidence for spontaneous recovery in control animals? This is of importance given that the main conclusion of the authors is that LC stimulation during extinction training led to an increased expression of extinction memory as expressed by reduced spontaneous recovery.<br /> 4. Current evidence suggests that there are differences in LC/NA system functioning between males and females. Could the authors provide details about the allocation of male and female animals in each group?<br /> 5. The histology section in both experiments looks a bit unsatisfying. Could the authors provide more details about the number of counted cells and also their distribution along the antero-posterior extent of the LC. Could the authors also take into account the sex in such an analysis?

Reviewer #3 (Public Review):

The introduction/background is excellent. It reviews evidence showing that the extinction of conditioned responding is regulated by noradrenaline and suggests that the locus coeruleus (LC) may be a critical locus of this regulation. This naturally leads to the aim of the study: to determine whether the locus coeruleus is involved in the extinction of an appetitive conditioned response. Overall, the study is well-designed, nicely conducted and the results advance our understanding of the role of the LC in the extinction of conditioned behaviour. As such, I believe that these results will be of interest to readers. I do, however, feel that the paper would benefit from the inclusion of additional data to clarify the impact of the LC manipulations (stimulation and inhibition) on performance in the task; and some comment regarding the likely source of differences between the groups at test.

Reviewer #1 (Public Review):

This is my first review of the article entitled "The canonical stopping network: Revisiting the role of the subcortex in response inhibition" by Isherwood and colleagues. This study is one in a series of excellent papers by the Forstmann group focusing on the ability of fMRI to reliably detect activity in small subcortical nuclei - in this case, specifically those purportedly involved in the hyper- and indirect inhibitory basal ganglia pathways. I have been very fond of this work for a long time, beginning with the demonstration of De Hollander, Forstmann et al. (HBM 2017) of the fact that 3T fMRI imaging (as well as many 7T imaging sequences) do not afford sufficient signal to noise ratio to reliably image these small subcortical nuclei. This work has done a lot to reshape my view of seminal past studies of subcortical activity during inhibitory control, including some that have several thousand citations.

In the current study, the authors compiled five datasets that aimed to investigate neural activity associated with stopping an already initiated action, as operationalized in the classic stop-signal paradigm. Three of these datasets are taken from their own 7T investigations, and two are datasets from the Poldrack group, which used 3T fMRI.

The authors make six chief points:<br /> 1. There does not seem to be a measurable BOLD response in the purportedly critical subcortical areas in contrasts of successful stopping (SS) vs. going (GO), neither across datasets nor within each individual dataset. This includes the STN but also any other areas of the indirect and hyperdirect pathways.<br /> 2. The failed-stop (FS) vs. GO contrast is the only contrast showing substantial differences in those nodes.<br /> 3. The positive findings of STN (and other subcortical) activation during the SS vs. GO contrast could be due to the usage of inappropriate smoothing kernels.<br /> 4. The study demonstrates the utility of aggregating publicly available fMRI data from similar cognitive tasks.<br /> 5. From the abstract: "The findings challenge previous functional magnetic resonance (fMRI) of the stop-signal task"<br /> 6. and further: "suggest the need to ascribe a separate function to these networks."

I strongly and emphatically agree with points 1-5. However, I vehemently disagree with point 6, which appears to be the main thrust of the current paper, based on the discussion, abstract, and - not least - the title.

To me, this paper essentially shows that fMRI is ill-suited to study the subcortex in the specific context of the stop-signal task. That is not just because of the issues of subcortical small-volume SNR (the main topic of this and related works by this outstanding group), but also because of its limited temporal resolution (which is unacknowledged, but especially impactful in the context of the stop-signal task). I'll expand on what I mean in the following.

First, the authors are underrepresenting the non-fMRI evidence in favor of the involvement of the subthalamic nucleus (STN) and the basal ganglia more generally in stopping actions.<br /> - There are many more intracranial local field potential recording studies that show increased STN LFP (or even single-unit) activity in the SS vs. FS and SS vs. GO contrast than listed, which come from at least seven different labs. Here's a (likely non-exhaustive) list of studies that come to mind:<br /> o Ray et al., NeuroImage 2012<br /> o Alegre et al., Experimental Brain Research 2013<br /> o Benis et al., NeuroImage 2014<br /> o Wessel et al., Movement Disorders 2016<br /> o Benis et al., Cortex 2016<br /> o Fischer et al., eLife 2017<br /> o Ghahremani et al., Brain and Language 2018<br /> o Chen et al., Neuron 2020<br /> o Mosher et al., Neuron 2021<br /> o Diesburg et al., eLife 2021<br /> - Similarly, there is much more evidence than cited that causally influencing STN via deep-brain stimulation also influences action-stopping. Again, the following list is probably incomplete:<br /> o Van den Wildenberg et al., JoCN 2006<br /> o Ray et al., Neuropsychologia 2009<br /> o Hershey et al., Brain 2010<br /> o Swann et al., JNeuro 2011<br /> o Mirabella et al., Cerebral Cortex 2012<br /> o Obeso et al., Exp. Brain Res. 2013<br /> o Georgiev et al., Exp Br Res 2016<br /> o Lofredi et al., Brain 2021<br /> o van den Wildenberg et al, Behav Brain Res 2021<br /> o Wessel et al., Current Biology 2022<br /> - Moreover, evidence from non-human animals similarly suggests critical STN involvement in action stopping, e.g.:<br /> o Eagle et al., Cerebral Cortex 2008<br /> o Schmidt et al., Nature Neuroscience 2013<br /> o Fife et al., eLife 2017<br /> o Anderson et al., Brain Res 2020

Together, studies like these provide either causal evidence for STN involvement via direct electrical stimulation of the nucleus or provide direct recordings of its local field potential activity during stopping. This is not to mention the extensive evidence for the involvement of the STN - and the indirect and hyperdirect pathways in general - in motor inhibition more broadly, perhaps best illustrated by their damage leading to (hemi)ballism.

Hence, I cannot agree with the idea that the current set of findings "suggest the need to ascribe a separate function to these networks", as suggested in the abstract and further explicated in the discussion of the current paper. For this to be the case, we would need to disregard more than a decade's worth of direct recording studies of the STN in favor of a remote measurement of the BOLD response using (provably) sub ideal imaging parameters. There are myriads of explanations of why fMRI may not be able to reveal a potential ground-truth difference in STN activity between the SS and FS/GO conditions, beginning with the simple proposition that it may not afford sufficient SNR, or that perhaps subcortical BOLD is not tightly related to the type of neurophysiological activity that distinguishes these conditions (in the purported case of the stop-signal task, specifically the beta band). But essentially, this paper shows that a specific lens into subcortical activity is likely broken, but then also suggests dismissing existing evidence from superior lenses in favor of the findings from the 'broken' lens. That doesn't make much sense to me.

Second, there is actually another substantial reason why fMRI may indeed be unsuitable to study STN activity, specifically in the stop-signal paradigm: its limited time resolution. The sequence of subcortical processes on each specific trial type in the stop-signal task is purportedly as follows: at baseline, the basal ganglia exert inhibition on the motor system. During motor initiation, this inhibition is lifted via direct pathway innervation. This is when the three trial types start diverging. When actions then have to be rapidly cancelled (SS and FS), cortical regions signal to STN via the hyperdirect pathway that inhibition has to be rapidly reinstated (see Chen, Starr et al., Neuron 2020 for direct evidence for such a monosynaptic hyperdirect pathway, the speed of which directly predicts SSRT). Hence, inhibition is reinstated (too late in the case of FS trials, but early enough in SS trials, see recordings from the BG in Schmidt, Berke et al., Nature Neuroscience 2013; and Diesburg, Wessel et al., eLife 2021).<br /> Hence, according to this prevailing model, all three trial types involve a sequence of STN activation (initial inhibition), STN deactivation (disinhibition during GO), and STN reactivation (reinstantiation of inhibition during the response via the hyperdirect pathway on SS/FS trials, reinstantiation of inhibition via the indirect pathway after the response on GO trials). What distinguishes the trial types during this period is chiefly the relative timing of the inhibitory process (earliest on SS trials, slightly later on FS trials, latest on GO trials). However, these temporal differences play out on a level of hundreds of milliseconds, and in all three cases, processing concludes well under a second overall. To fMRI, given its limited time resolution, these activations are bound to look quite similar.

Lastly, further building on this logic, it's not surprising that FS trials yield increased activity compared to SS and GO trials. That's because FS trials are errors, which are known to activate the STN (Cavanagh et al., JoCN 2014; Siegert et al. Cortex 2014) and afford additional inhibition of the motor system after their occurrence (Guan et al., JNeuro 2022). Again, fMRI will likely conflate this activity with the abovementioned sequence, resulting in a summation of activity and the highest level of BOLD for FS trials.

In sum, I believe this study has a lot of merit in demonstrating that fMRI is ill-suited to study the subcortex during the SST, but I cannot agree that it warrants any reappreciation of the subcortex's role in stopping, which are not chiefly based on fMRI evidence.

A few other points:<br /> - As I said before, this team's previous work has done a lot to convince me that 3T fMRI is unsuitable to study the STN. As such, it would have been nice to see a combination of the subsamples of the study that DID use imaging protocols and field strengths suitable to actually study this node. This is especially true since the second 3T sample (and arguably, the Isherwood_7T sample) does not afford a lot of trials per subject, to begin with.<br /> - What was the GLM analysis time-locked to on SS and FS trials? The stop-signal or the GO-signal?<br /> - Why was SSRT calculated using the outdated mean method?<br /> - The authors chose 3.1 as a z-score to "ensure conservatism", but since they are essentially trying to prove the null hypothesis that there is no increased STN activity on SS trials, I would suggest erring on the side of a more lenient threshold to avoid type-2 error.<br /> - The authors state that "The results presented here add to a growing literature exposing inconsistencies in our understanding of the networks underlying successful response inhibition". It would be helpful if the authors cited these studies and what those inconsistencies are.

Reviewer #2 (Public Review):

This work aggregates data across 5 openly available stopping studies (3 at 7 tesla and 2 at 3 tesla) to evaluate activity patterns across the common contrasts of Failed Stop (FS) > Go, FS > stop success (SS), and SS > Go. Previous work has implicated a set of regions that tend to be positively active in one or more of these contrasts, including the bilateral inferior frontal gyrus, preSMA, and multiple basal ganglia structures. However, the authors argue that upon closer examination, many previous papers have not found subcortical structures to be more active on SS than FS trials, bringing into question whether they play an essential role in (successful) inhibition. In order to evaluate this with more data and power, the authors aggregate across five datasets and find many areas that are *more* active for FS than SS, specifically bilateral preSMA, caudate, GPE, thalamus, and VTA, and unilateral M1, GPi, putamen, SN, and STN. They argue that this brings into question the role of these areas in inhibition, based upon the assumption that areas involved in inhibition should be more active on successful stop than failed stop trials, not the opposite as they observed.

As an empirical result, I believe that the results are robust, but this work does not attempt a new theoretical synthesis of the neuro-cognitive mechanisms of stopping. Specifically, if these many areas are more active on failed stop than successful stop trials, and (at least some of) these areas are situated in pathways that are traditionally assumed to instantiate response inhibition like the hyperdirect pathway, then what function are these areas/pathways involved in? I believe that this work would make a larger impact if the author endeavored to synthesize these results into some kind of theoretical framework for how stopping is instantiated in the brain, even if that framework may be preliminary.

I also have one main concern about the analysis. The authors use the mean method for computing SSRT, but this has been shown to be more susceptible to distortion from RT slowing (Verbruggen, Chambers & Logan, 2013 Psych Sci), and goes against the consensus recommendation of using the integration with replacement method (Verbruggen et al., 2019). Therefore, I would strongly recommend replacing all mean SSRT estimates with estimates using the integration with replacement method.

Reviewer #1 (Public Review):

In this manuscript, the authors describe an improved miniscope they name "E-scope", combining in vivo calcium imaging with electrophysiological recording. They use it to examine neural correlates of social interactions with respect to cerebellar and cortical circuits. Through correlations between electrophysiological single units of Purkinje cells and dentate nucleus neurons as well as with calcium signals imaging of neurons from the anterior cingulate cortex, the authors provide correlative data supporting the view that intracerebellar circuits and cerebello-cortical communications take part in the modulation of social behavior. In particular, the electrophysiological dataset reflects the PC-DN connection and strongly suggests its involvement in social interactions. Cross-correlations analyses between PC / DN single units and ACC calcium signals suggest that the recorded cerebellar and cortical structures both take part in the brain networks at play in social behavior.

Strengths:<br /> - This is a timely and important study with solid evidence for correlative conclusions that are not overstated in the manuscript, which is commendable.<br /> - Despite the technical challenge, the experiments presented in this study seem well performed and the quality of the dataset is appropriate.

Weaknesses:<br /> - While the novelty of the device is strongly emphasized, I find that its value is somewhat diminished by the wire-free device developed by the same group as it should thus be possible to perform calcium imaging wire-free and electrophysiological recording via a single conventional cable (or also via wireless headstages).<br /> - The role of the identified network activations in social interactions is not touched upon.

Reviewer #2 (Public Review):

This report by Hur et al. examines simultaneous activity in the cerebellum and anterior cingulate cortex (ACC) to determine how activity in these regions is coordinated during social behavior. To accomplish this, the authors developed a recording device named the E-scope, which combines a head-mounted mini-scope for in vivo Ca2+ imaging with an extracellular recording probe (in the manuscript they use a 32-channel silicon probe). Using the E-scope, the authors find subpopulations of cerebellar neurons with social-interaction-related activity changes. The activity pattern is predominantly decreased firing in PCs and increases in DNs, which is the expected reciprocal relationship between these populations. They also find social-interaction-related activity in the ACC. The authors nicely show the absence of locomotion onset and offset activity in PCs and DNs ruling out that is movement driven. Analysis showed high correlations between cerebellar and ACC populations (namely, Soc+ACC and Soc+DN cells). The finding of correlated activity is interesting because non-motor functions of the cerebellum are relatively little explored. However, the causal relationship is far from established with the methods used, leaving it unclear if these two brain regions are similarly engaged by the behavior or if they form a pathway/loop. Overall, the data are presented clearly, and the manuscript is well written, however, the biological insight gained is rather limited.

Reviewer #3 (Public Review):

Complex behavior requires complex neural control involving multiple brain regions. The currently available tools to measure neural activity in multiple brain regions in small animals are limited and often involve obligatory head-fixation. The latter, obviously, impacts the behaviors under study. Hur and colleagues present a novel recording device, the E-Scope, that combines optical imaging of fluorescent calcium imaging in one brain region with high-density electrodes in another. Importantly, the E-Scope can be implanted and is, therefore, compatible with usage in freely moving mice. The authors used their new E-Scope to study neural activity during social interactions in mice. They demonstrate the presence of neural correlates of social interaction that happen simultaneously in the cerebellum and the anterior cingulate cortex.

The major accomplishment of this study is the development and introduction of the E-Scope. The evaluation of this part can be short: it works, so the authors succeeded.

The authors managed to reduce the weight of the implant to 4.5 g, which is - given all functionality - quite an accomplishment in my view. However, a mouse weighs between 20 and 40 g, so that an implant of 4.5 g is still quite considerable. It can be expected that this has an impact on the behavior and, possibly, the well-being of the animals. Whether this is the case or not, is not really addressed in this study. The authors suffice with the statement that "Recorded animals made more contact with the other mouse than with the object (Figure 2A), suggesting a normal preference for social contact with the E-Scope attached."

Overall, the description of animal behavior is rather sparse. The methods state only that stranger age-matched mice were used, but do not state their gender. The nature of the social interactions was not described? Was their aggressive behavior, sexual approach and/or intercourse? Did the stranger mice attack/damage the E-Scope? Were the interactions comparable (using which parameters?) with and without E-Scope attached? It is not even described what the authors define as an "interaction bout" (Figure 2A). The number of interaction bouts is counted per 7 minutes, I presume? This is not specified explicitly.

In Figure 1 D-G, the authors present raw data from the neurophysiological recordings. In panel D, we see events with vastly different amplitudes. It would be very insightful if the authors would describe which events they considered to be action potentials, and which not. Similarly, the raw traces of Figure 1E are declared to be single-unit recordings of Purkinje cells. Partially due to the small size of the traces (invisible in print and pixelated in the digital version), I have a hard time recognizing complex spikes and simple spikes in these traces. This is a bit worrisome, as the authors declare the typical duration of the pause in simple spike firing after a complex spike to be 20-100 ms. In my experience, such long pauses are rare in this region, and definitely not typical. In the right panel of Figure 1A, an example of a complex spike-induced pause is shown. This pause is around 15 ms, so not typical according to the text, and starts only around 4 ms after the complex spike, which should not be the case and suggests either a misalignment of the figure or the detection of complex spike spikelets as simple spikes, while the abnormally long pause suggests that the authors fail to detect a lot of simple spikes. The authors could provide more confidence in their data by including more raw data, making explicit how they analyzed the signals, and by reporting basic statistics of firing properties (like rate, cv or cv2, pause duration). In this respect, Figure 2 - figure supplement 3 shows quite a large percentage of cells to have either a very low or a very high firing rate.

The number of Purkinje cells recorded during social interactions is quite low: only 11 cells showed a modulation in their spiking activity (unclear whether in complex spikes, simple spikes or both. During object interaction, only 4 cells showed a significant modulation. Unclear is whether the latter 4 are a subset of the former 11, or whether "social cells" and "object cells" are different categories. Having so few cells, and with these having different types of modulation, the group of cells for each type of modulation is really small, going down to 2 cells/group. It is doubtful whether meaningful interpretation is possible here.

This brings us to the next point: neural correlates of social interaction are notoriously difficult to interpret. Social behavior is complex, and involves the processing of sensory cues (olfaction, touch (whiskers), visual and auditory), the production of ultrasonic vocalizations (in specific contexts), movements, and emotional behavior (fear, pleasure, sexual interest). In other words, neural activity patterns observed during social interaction do not necessarily relate specifically to social interaction, but can also occur in a non-social context. The authors control this by comparing social interactions with object interactions, but I miss a direct comparison between the two conditions, both in terms of behavior (now only the number of interactions is counted, not their duration or intensity), and in terms of neural activity. There is some analysis done on the interaction between movement and cerebellar activity (Figure 2 - figure supplement 4), but it is unclear to what extent social interactions and movements are separated here. It would already help to indicate in the plots with trajectories (e.g., Fig. 2H) indicate the social interactions (e.g., social interaction-related movements in red, the rest of the trajectories in black).

The neuron count in the anterior cingulate cortex is much higher than for the cerebellum, but also here it is not so clear what is "social" and what is "non-social". In Figure 3G-H, the authors indicate a near-perfect separation between cells active during social encounters and those active during object encounters. This could indicate that there is here indeed a social aspect, but as we do not know to what extent the sensory and motor aspects differ between social and non-social interactions, this is still hard to interpret.

Finally, the authors show that there are correlations between the modulation in neurons of the anterior cingulate cortex and cerebellar neurons related to bouts of social activity. Here, it could be interesting to see whether there are differences in latency between the two brain areas.

In conclusion, the authors present a novel method to record neural activity with single cell-resolution in two brain regions in freely moving mice. Given the challenges associated with understanding of complex behaviors, this approach can be useful for many neuroscientists. The authors demonstrate the potential of their approach by studying social interactions in mice. Clearly, there are correlations in the activity of neurons in the anterior cingulate cortex and the cerebellum related to social interactions. To bring our understanding of these patterns to a higher level, more detailed analyses (and probably also larger group sizes of cerebellar neurons) are required, though.

Reviewer #1 (Public Review):

This study conducted a series of experiments to comprehensively support the allocentric rather than egocentric visual spatial reference updating for the path-integration mechanism in the control of target-oriented locomotion. Authors firstly manipulated the waiting time before walking to tease apart the influence from spatial working memory in guiding locomotion. They demonstrated that the intrinsic bias in perceiving distance remained constant during walking and that the establishment of a new spatial layout in the brain took a relatively longer time beyond the visual-spatial working memory. In the following experiments, the authors then uncovered that the strength of the intrinsic bias in distance perception along the horizontal direction is reduced when participants' attention is distracted, implying that world-centered path integration requires attentional effort. This study also revealed horizontal-vertical asymmetry in a spatial coding scheme that bears a resemblance to the locomotion control in other animal species such as desert ants.

The overall design of the behavioral experiments is elegant and statistics are well performed to support the authors' viewpoint in the allocentric rather than egocentric visual spatial coding scheme for distance perception along the horizontal line.

It is however worth noting the statement from Gibson in 1979 that for egocentric distances, tangible information arises from the effort required to walk a distance, thus, effort becomes associated through experience with visual distance cues. Accordingly, visual information alone is insufficient to support the awareness of distance. Perceived distance is rather specified by an invariant relationship between distal extent and a persons' potential to perform gross motion actions such as walking. This view is supported later by Proffitt et al. (2003) in which participants wore backpacks and their perceived distance increased compared with the baseline condition. Authors need to acknowledge the physical effort in addition to visual information for the spatial coding and may consider the manipulation of physical efforts in the future to support the robustness of constant intrinsic bias in ground-based spatial coding during walking.

Furthermore, it would be more comprehensive and fit into the Neuroscience Section if the authors can add in current understandings of the spatial reference frames in neuroscience in the introduction and discussion, and provide explanations on how the findings of this study supplement the physiological evidence that supports our spatial perception as well. For instance, world-centered representations of the environment, or cognitive maps, are associated with hippocampal formation while self-centered spatial relationships, or image spaces, are associated with the parietal cortex (see Bottini, R., & Doeller, C. F. (2020). Knowledge Across Reference Frames: Cognitive Maps and Image Spaces. Trends in Cognitive Sciences, 24(8), 606-619. https://doi.org/10.1016/j.tics.2020.05.008 for details)

Reviewer #2 (Public Review):

The study provides a valuable contribution by demonstrating the use of an allocentric spatial reference frame in the perception of the location of a dimly lit target in the dark. While the evidence presented in support of the authors' claims is solid and convincing, it would be beneficial for the study to address potential limitations, such as its ecological validity.

Strengths:<br /> Unlike previous research where observers were stationary during a visual-spatial perception task, this recent study expanded upon prior findings by incorporating bodily movements for the observers. This study is a valuable addition to the literature as it not only discovered that the intrinsic bias is grounded on the home base, but also identified several key characteristics through a series of follow-up experiments. The findings suggest that this "allocentric" spatial coding decays over time, requires attentional resources, can be based solely on vestibular signals, and is most effective in the horizontal direction. In general, this study is interesting, clearly presented, well-thought-out and executed. The results confirmed the conclusions and the study's comprehensive approach offers valuable insights into the nature of intrinsic bias in spatial perception.

The counter-intuitive results presented in the manuscript are intriguing and add to the study's overall appeal. Moreover, the manuscript draws an interesting parallel between human spatial navigation and that of desert ants. This comparison helps to underscore the importance of understanding spatial coding mechanisms across different species and highlights potential avenues for future research.

One aspect I particularly valued about this study was the authors' thorough description of the experimental methods. This level of detail not only highlights the rigor of the research but also enhances the reproducibility of the study, making it more accessible for future researchers.

Weaknesses:<br /> While the current study provides valuable insights into the nature of intrinsic bias in spatial perception, there is a concern regarding its ecological validity. The experimental design involved stringent precautions, such as a very dark room and a small target, to minimize the presence of depth cues. This is in contrast to the real world, where depth information is readily available from the ground and surrounding objects, aiding in our perception of space and depth. As a result, it is unclear to what extent this "allocentric" intrinsic bias is involved in our everyday spatial perception. To provide more context for the general audience, it would be beneficial for the authors to address this issue in their discussion.

The current findings on the "allocentric" coding scheme raise some intriguing questions as to why such a mechanism would be developed and how it could be beneficial. The finding that the "allocentric" coding scheme results in less accurate object localization and requires attentional resources seems counterintuitive and raises questions about its usefulness. However, this observation presents an opportunity for the manuscript to discuss the potential evolutionary advantages or trade-offs associated with this coding mechanism.

The manuscript lacks a thorough description of the data analysis process, particularly regarding the fitting of the intrinsic bias curve (e.g., the blue and gray dashed curve in Figure 3c) and the calculation of the horizontal separation between the curves. It would be beneficial for the authors to provide more detailed information on the specific function and parameters used in the fitting process and the formula used for the separation calculation to ensure the transparency and reproducibility of the study's results.

Reviewer #3 (Public Review):

This study investigated what kind of reference (allocentric or egocentric) frame we used for perception in darkness. This question is essential and was not addressed much before. The authors compared the perception in the walking condition with that in the stationary condition, which successfully separated the contribution of self-movement to the spatial representation. In addition, the authors also carefully manipulated the contribution of the waiting period, attentional load, vestibular input, testing task, and walking direction (forward or backward) to examine the nature of the reference frame in darkness systematically.

I am a bit confused by Figure 2b. Allocentric coordinate refers to the representation of the distance and direction of an object relative to other objects but not relative to the observer. In Figure 2, however, the authors assumed that the perceived target was located on the interception between the intrinsic bias curve and the viewing line from the NEW eye position to the target. This suggests that the perceived object depends on the observer's new location, which seems odd with the allocentric coordinate hypothesis.

According to Fig 2b, the perceived size should be left-shifted and lifted up in the walking condition compared to that in the stationary condition. However, in Figure 3C and Fig 4, the perceived size was the same height as that in the baseline condition.

Is the left-shifted perceived distance possibly reflecting a kind of compensation mechanism? Participants could not see the target's location but knew they had moved forward. Therefore, their brain automatically compensates for this self-movement when judging the location of a target. This would perfectly predict the left-shifted but not upward-shifted data in Fig 3C. A similar compensation mechanism exists for size constancy in which we tend to compensate for distance in computing object size.

According to Fig 2a, the target, perceived target, and eye should be aligned in one straight line. This means that connecting the physical targets and the corresponding perceived target results in straight lines that converge at the eye position. This seems, however, unlikely in Figure 3c.

Reviewer #1 (Public Review):

The manuscript by Lin et al describes a wide biophysical survey of the molecular mechanisms underlying full-length BTK regulation. This is a continuation of this lab's excellent work on deciphering the myriad levels of regulation of BTKs downstream of their activation by plasma membrane localised receptors.

The manuscript uses a synergy of cryo EM, HDX-MS and mutational analysis to delve into the role of how the accessory domains modify the activity of the kinase domain. The manuscript essentially has three main novel insights into BTK regulation.

1. Cryo EM and SAXS show that the PHTH region is dynamic compared to the conserved Src module.<br /> 2. A 2nd generation tethered PH-kinase construct crystal of BTK reveals a unique orientation of the PH domain relative to the kinase domain, that is different from previous structures.<br /> 3. A new structure of the kinase domain dimer shows how trans-phosphorylation can be achieved.

Excitingly these structural works allow for the generation of a model of how BTK can act as a strict coincidence sensor for both activated BCR complex as well as PIP3 before it obtains full activity. To my eye the most exciting result of this work is describing how the PH domain can inhibit activity once the SH3/SH2 domain is disengaged, allowing for an additional level of regulatory control.

I have very few experimental concerns as the methods and figures are well-described and clear. As the authors are potentially saying that the previously solved PH domain-kinase interface is artefactual, additional evidence strengthening their model would be helpful to resolve any possible controversies.

Reviewer #2 (Public Review):

In this study, multiple biophysical techniques were employed to investigate the activation mechanism of BTK, a multi-domain non-receptor protein kinase. Previous studies have elucidated the inhibitory effects of the SH3 and SH2 domains on the kinase and the potential activation mechanism involving the membrane-bound PIP3 inducing transient dimerization of the PH-TH domain, which binds to lipids.

The primary focus of the present study was on three new constructs: a full-length BTK construct, a construct where the PH-TH domain is connected to the kinase domain, and a construct featuring a kinase domain with a phosphomimetic at the autophosphorylation site Y551. The authors aimed to provide new insights into the autoinhibition and allosteric control of BTK.

The study reports that SAXS analysis of the full-length BTK protein construct, along with cryoEM visualization of the PH-TH domain, supports a model in which the N-terminal PH-TH domain exists in a conformational ensemble surrounding a compact/autoinhibited SH3-SH2-kinase core. This finding is interesting because it contradicts previous models proposing that each globular domain is tightly packed within the core.

Furthermore, the authors present a model for an inhibitory interaction between the N-lobe of the kinase and the PH-TH domain. This model is based on a study using a tethered complex with a longer tether than a previously reported construct where the PH-TH domain was tightly attached to the kinase domain (ref 5). The authors argue that the new structure is relevant. However, this assertion requires further explanation and discussion, particularly considering that the functional assays used to assess the impact of mutating residues within the PH-TH/kinase domain contradict the results of the previous study (ref 5).

Additionally, the study presents the structure of the kinase domain with swapped activation loops in a dimeric form, representing a previously unseen structure along the trans-phosphorylation pathway. This structure holds potential relevance. To better understand its significance, employing a structure/function approach like the one described for the PH-TH/kinase domain interface would be beneficial.

Overall, this study contributes to our understanding of the activation mechanism of BTK and sheds light on the autoinhibition and allosteric control of this protein kinase. It presents new structural insights and proposes novel models that challenge previous understandings. However, further investigation and discussion would significantly strengthen the study.

Reviewer #3 (Public Review):

Yin-wei Lin et al set out to visualize the inactive conformation of full-length Bruton's Tyrosine Kinase (BTK), a molecule that has evaded high-resolution structural studies in its full-length form to this date. An open question in the field is how the Pleckstrin Homology-Tec Homology (PHTH) domain inhibits BTK activity, with multiple competing models in the field. The authors used a complimentary set of biophysical techniques combined with well-thought-out stabilizing mutations to obtain structural insights into BTK regulation in its full-length form. They were able to crystallize the full-length construct of BTK but unfortunately, the PHTH was not resolved yielding a structure similar to that previously obtained in the field. The investigation of the same construct by SAXS yielded an elongated structural model, consistent with previous SAXS studies. Using cryo-EM the authors obtained a low-resolution model for the FL BTK with a loosely connected density assigned to the dynamic PHTH around the compact SH2-SH3-Kinase Domain (KD) core. To gain further molecular insights into PHTH-KD interactions the authors followed a previously reported strategy and generated a fusion of PHTH-KD with a longer linker, yielding a crystal structure with a novel PHTH-KD interface which they tested in biochemical assays. Lastly, Yin-wei Lin et al crystallized the BTK KD in a novel partially active state in a "face-to-face" dimer with kinases exchanging the activation loops, although partially disordered, being theoretically perfectly positioned for transphosphorylation. Overall this presents a valiant effort to gain molecular insights into what clearly is a dynamic regulatory motif on BTK and is a valuable addition to the field.

However, this work can be improved by considering these points:

1) The cryo-EM reconstructions are potentially over-interpreted. The reported resolution for all of the analyzed reconstructions is better than 8Å, at which point helices should be recognized as well-resolved structural elements. In the current view/depiction of the cryo-EM maps/models it is hard to see such structural features and it would be great if the authors could include a panel showing maps at higher thresholds to show correspondence between the helices in the kinase C lobe and the cryo-EM maps. Otherwise, the overall positioning of the models within the cryo-EM maps is hard to evaluate and may very well be wrong. (Fig 4, S2).

2) With the above in mind, if the maps are not at the point where helices are well resolved, it may be beneficial to low-pass filter the maps to a more conservative resolution for fitting, analysis, and representation. (Fig 4, S2).

3) It would be valuable to get a quantitative metric on the model/map fitting for the cryo-EM work. One good package for this is Situs which provides cross-correlation values for the top orthogonal fits, without user input for initial fitting. This would again increase the confidence in the correctness of model positioning on the map. (Fig 4, S2).

4) It would be great to see 2D class averages from the particles contributing to each of the 3D classes. Theoretically, a clear bright "blob" (hypothesized to be the PHTH domain) should be observable in the 2D class averages. In the current 2D class averages that region is unconvincingly weak. (Fig 4, S2).

5) It seems like there was quite a large circular mask applied during 2D classification. Are authors confident that the weak density attributed to the PHTH domain is not neighboring particles making their way into the extraction box? It would be great if the authors would trim their particle stack with a very stringent inter-particle distance cutoff (or report the cutoff in the manuscript if already done so) to minimize this possibility.

6) The cryo-EM processing may benefit from more stringent particle picking. The authors picked over 2M particles from 750 micrographs which likely represents very heavy overpicking. I would encourage the authors to re-pick the micrographs with 2D class averages and use more stringent metrics to reduce the overpicking. This may result in higher-resolution reconstructions. (Fig 4, S2).

7) The Dmax from SAXS for the Full Length BTK is at 190Å. It would be great if the authors could make a cartoon of what domain arrangement may satisfy this distance, as it is quite extended for such a small particle. Can the authors rule out dimerization at SAXS concentrations? (Fig 1).

8) In Figure S1 (C) it seems that the curves are just scattering curves with Guinier plots in the inserts, but are labeled as Guinier plots in the legend. The Guinier plots for some samples (FL 4P1F) show signs of aggregation, which may complicate the analysis, it could be beneficial to redo.

9) Have the authors verified that the activation loop mutations that they introduce do not disrupt the PHTH binding as they previously reported an activation loop on BTK to interact with PHTH, an interaction they do not see here? If so, a citation would be helpful in the text. If not, testing this would strengthen the paper.

10) Can the authors comment on the surfaces which are accessible and inaccessible to the PHTH in the crystal (Fig 3E)? The fact that PHTH doesn't adopt a stable conformation in the solvent channel to some degree indicates that the accessible interaction surfaces are not suitable for PHTH interactions, as the "effective concentration" of the PHTH would be quite high. Are these surfaces consistent with the cryo-EM analysis?

11) For the novel active state dimer of the Kinase Domain it would be great to see some functional validation of the dimerization interface. It is structurally certainly quite suggestive, but without such experiments the functional significance is unclear. If appropriate mutations have been published previously a citation would be helpful.

Reviewer #1 (Public Review):

First, I agree with the authors of this manuscript that conformational changes in the XFEL structures with 2.8 A resolution are not reliable enough for demonstrating the subtle changes in the electron transfer events in this bacterial photosynthesis system. Actually, the data statistics in the paper by Dods et al. showed that the high-resolution range of some of the XFEL datasets may include pretty high noise (low CC1/2 and high Rsplit) so the comparison of the subtle conformational changes of the structures is problematic.

The manuscript by Gai Nishikawa investigated time-dependent changes in the energetics of the electron transfer pathway based on the structures by Dods et al. by calculating redox potential of the active and inactive branches in the structures and found no clear link between the time-dependent structural changes and the electron transfer events in the XFEL structures published by Dods, R.et al. (2021). This study provided validation for the interpretation of the structures of those electron-transferring proteins.

The paper was well prepared.

Reviewer #2 (Public Review):

The manuscript by Nishikawa et al. addresses time-dependent changes in the electron transfer energetics in the photosynthetic reaction center from Blastochloris viridis, whose time-dependent structural changes upon light illumination were recently demonstrated by time-resolved serial femtosecond crystallography (SFX) using X-ray free-electron laser (XFEL) (Dods et al., Nature, 2021). Based on the redox potential Em values of bacteriopheophytin in the electron transfer active branch (BL) by solving the linear Poisson-Boltzmann equation, the authors found that Em(HL) values in the charge-separated 5-ps structure obtained by XFEL are not clearly changed, suggesting that the P+HL- state is not stabilized owing to protein reorganization. Furthermore, chlorin ring deformation upon HL- formation, which was expected from their QM/MM calculation, is not recognized in the 5-ps XFEL structure. Then the authors concluded that the structural changes in the XFEL structures are not related to the actual time course of charge separation. They argued that their calculated changes in Em and chlorin ring deformations using the XEFL structures may reflect the experimental errors rather than the real structural changes; they mentioned this problem is due to the fact that the XFEL structures were obtained at not high resolutions (mostly at 2.8 Å). I consider that their systematic calculations may suggest a useful theoretical interpretation of the XFEL study. However, the present manuscript insists as a whole negatively that the experimental errors may hamper to provide the actual structural changes relevant to the electron transfer events. My concerns are the following two points:<br /> Is the premise of the authors for the electron transfer energetics obviously valid?<br /> Could the authors find any positive aspect(s) in the XFEL study?

The authors' argument is certainly due to their premise "Em(HL) is expected to be exclusively higher in the 5-ps and 20-ps structures than in the other XFEL structures due to the stabilization of the [PLPM]•+HL•- state by protein reorganization" as noted in the Results and Discussion (p. 12, lines 180-182); however, it is unknown whether this premise can be applied to the ps-timescale electron transfer events. The above premise is surely based on the Marcus theory, as the authors also noted in the Introduction "The anionic state formation induces not only reorganization of the protein environment (ref. 5: Marcus and Sutin, 1985) but also out-of-plane distortion of the chlorin ring (ref. 6: two of the authors, Saito and Ishikita, co-authored, 2012)"; however, it is unknown whether protein reorganization can follow the ps-timescale electron transfer events. Indeed, Dods et al. mentioned in the Nature paper (2021) "The primary electron-transfer step from SP (special pair PLPM) to BPhL (HL) occurs in 2.8 {plus minus} 0.2 ps across a distance of 10 Å by means of a two-step hopping mechanism via the monomeric BChL molecule and is more rapid than conventional Marcus theory". It was also mentioned, "By contrast, the 9 Å electron-transfer step from BPhL to QA has a single exponential decay time of 230 {plus minus} 30 ps, which is consistent with conventional Marcus theory". As for the primary electron-transfer step from PLPM to HL, Wang et al. (2007, Science 316, 747; cited as ref. 8 in the Nature paper 2021) reported, by monitoring tryptophan absorbance changes in various reaction centers in which the driving forces (namely, the Em gaps between PLPM and HL) are different, that the protein relaxation kinetics is independent of the charge separation kinetics on the picosecond timescale. On the other hand, in the EPR study cited by the authors as ref. 7 (Muh et al. (1998) Biochemistry 37, 13066), although the authors described "two distinct conformations of HL- were reported in spectroscopic studies" (p. 3, lines 44-45), it should be noted that conformation of HL- was formed by 1 or 45 s illumination prior to freezing, and hence the second-order reorganized conformations may differ from picosecond-order conformations observed by the XFEL study (Nature, 2021) and/or the transient absorption spectroscopy (Science, 2007).

Therefore, I consider there is a possibility that the authors' findings may reflect not experimental errors but the actual ps-timescale phenomena presented by the first-time XFEL study on the timescale of the primary charge-separation reactions of photosynthesis. Thus I would like to suggest that the authors reconsider the premise for the electron transfer energetics on the picosecond timescale.

In any case, to discuss the experimental errors in the XFEL study, it is better to calculate the Em(QA) changes in the 300-ps and 8-us XFEL structures, which showed distinctive structural changes even at the 2.8 Å resolution as discussed by Dods et al. Then, if the Em(QA) values are changed as expected from theoretical calculations, such calculated results may suggest a useful theoretical interpretation of the XFEL study as a positive aspect. If the Em(QA) values are not higher in the 300-ps and 8-us structures than in the other structures, it may be argued that the experimental errors would be so large that the XFEL structures are irrelevant to the electron transfer events expected from theoretical calculations.

Reviewer #1 (Public Review):

The authors aimed to establish a cell culture system to investigate muscle tissue development and homeostasis. They successfully developed a complex 3D cell model and conducted a comprehensive molecular and functional characterization. This approach represents a critical initial step towards using human cells, rather than animals, to study muscular disorders in vitro. Although the current protocol is time-consuming and the fetal cell model may not be mature enough to study adult-onset diseases, it nonetheless provides a valuable foundation for future disease modelling studies using isogenic iPSC lines or patient-derived cells with specific mutations. The manuscript does not explore whether or how this stem cell model can advance our understanding of muscular diseases, which would be an exciting avenue for future research. Overall, the detailed protocol presented in this paper will be useful for informing future studies and provides an important resource to the stem cells community. The inclusion of data on disease modelling using isogenic iPSC lines or patient-derived cells would further enhance the manuscript's impact.

Reviewer #2 (Public Review):

This paper illustrates that PSCs can model myogenesis in vitro by mimicking the in vivo development of the somite and dermomyotome. The advantages of this 3D system include (1) better structural distinctions, (2) the persistence of progenitors, and (3) the spatial distribution (e.g. migration, confinement) of progenitors. The finding is important with the implication in disease modeling. Indeed the authors tried DMD model although it suffered the lack of deeper characterization.

The differentiation protocol is based on a current understanding of myogenesis and compelling. They characterized the organoids in depth (e.g. many time points and immunofluorescence). The evidence is solid, and can be improved more by rigorous analyses and descriptions as described below.

Major comments:

1. Consistency between different cell lines.<br /> I see the authors used a few different PSC lines. Since organoid efficiency differ between lines, it is important to note the consistency between lines.

2. Heterogeneity among each organoid<br /> Let's say authors get 10 organoids in one well. Are they similar to each other? Does each organoid possess similar composition of cells? To determine the heterogeneity, the authors could try either FACS or multiple sectioning of each organoid.

3. Consistency of Ach current between organoids.<br /> Related to comment 2, are the currents consistent between each organoid? How many organoids were recorded in the figures? Also, please comment if the current differ between young and aged organoids.

4. Communication between neural cells and muscle?<br /> The authors did scRNAseq, but have not gone deep analysis. I would recommend doing Receptor-ligand mapping and address if neural cells and muscle are interacting.

5. More characterization of DMD organoids.<br /> One of the key applications of muscle organoids is disease model. They have generated DMD muscle organoids, but rarely characterized except for currents. I recommend conducting immunofluorescence of DMA organoids to confirm structure change. Very intriguing to see scRNAseq of DMD organoids and align with disease etiology.

6. More characterization of engraft.<br /> Authors could measure the size of myotube between mice and human. Does PAX7+ Sattelite cell exist in engraft? To exclude cell fusion events make up the observation, I recommend to engraft in GFP+ immunodeficient mice. Could the authors comment how long engraft survive.

Joint Public Review:

Throughout the study, there is insufficient information about how experiments were performed and how often (imaging, pull-downs etc), how data was acquired, modified and analysed (especially imaging data, see below), how statistical analyses were done and what is presented in the figures (single planes or maximum intensity projections etc). This makes it difficult to evaluate the data and results.

There is insufficient information about tools and reporters used. This is misleading and impacts the conclusions that can be made from the results presented. To give an example, in Figure 1D-F, the authors present data that HDA-1::GFP and LIN-53::mNeonGreen (both components of the nucleosome remodeling and deacetylation complex) but not the histone acetyltransferase MYS-1::GFP are 'asymmetrically segregated' during QR.a division. However, the authors do not mention that HDA-1::GFP and LIN-53::mNeonGreen are expressed at endogenous levels (they are CRISPR alleles) whereas MYS-1::GFP is overexpressed (integration of a multi-copy extrachromosomal array). The difference in 'segregation' could therefore be a consequence of different levels of expression rather than different modes of segregation ('asymmetric' versus 'symmetric').

There is insufficient information about the phenotypes of the animals used (RNAi knock-downs of hda-1, lin-53 RNAi, pig-1 etc). Again this is misleading and impacts the conclusions that can be made. To give some examples, (1) in Figure 3A-G, control RNAi embryos are compared to hda-1 RNAi and lin-53 RNAi embryos. What the authors do not mention is that hda-1 RNAi and lin-53 RNAi embryos have severe developmental defects and essentially cannot be compared to control RNAi embryos. The differences between the embryos can be seen in Figure S7B where bright-field images of control RNAi, hda-1 RNAi and lin-53 RNAi embryos are shown. At the 350 min time point, a normal embryo is visible for the control, a 'ball of cells' embryo for hda-1 RNAi and an embryo that seems to have arrested at an earlier developmental stage (and therefore have much larger cells) for lin-53 RNAi. Because of these pleiotropic phenotypes, it is unclear whether differences seen for example in sAnxV::GFP positive cells (Figure 3A) are the result of a direct effect of hda-1(RNAi) on cell death or whether they are the result of global changes in development and cell fate induced by hda-1(RNAi). hda-1(RNAi) and lin-53(RNAi) embryos are also used for the data shown in Figures S6 and S7, raising the same concerns; (2) the authors do not mention what the impact of Baf A1 treatment is on animals; however, the images provided in Figure 5E indicate that Baf A1 treatment causes pleiotropic effects in L1 larvae.

There is a lack of adequate controls. Because of this, some of the data presented must be considered as preliminary. To give some examples: (1) controls are lacking for the data shown in Figure 3D-G (i.e. genes other than egl-1). Since hda-1 RNAi has a pleiotropic effect and most likely affects H3K27 acetylation genome-wide, this is critical. Based on what is shown, it is unclear whether the results presented are specific to egl-1 or not; (2) the co-IP and mass spec data shown in Figure 4A, C and Figure S8 also lack a critical control, which is GFP only. Because of this, it is unclear whether subunits of the V-ATPase bind to HDA-1 or GFP. The co-IP and mass spec data forms the basis of Figures 5 and 6 as well as Figure S9. Data presented in these figures therefore has to be considered preliminary as well.

Inappropriate methods are used. For this reason, some of the data again must be considered preliminary. To give some examples: (1) in Figure 5A, B, the authors used super-ecliptic pHluorin to look at changes in pH in the daughter cells. However, the authors used quenching of super-ecliptic pHluorin fluorescence rather than a ratio-metric method to 'measure' changes in pH. Because of this, it is unclear whether the changes in fluorescence observed are due to changes in pH or changes in the amount of pHluorin protein. Figure 5A, B forms the basis for the experiments presented in the remaining parts of Figure 5 as well as in Figure 6 and Figure S9; (2) the authors' description of how some images were modified before quantitative analysis raises concerns. The figures of concern are particularly Figure 1 and Figure S4, where background subtraction with denoising and deconvolution was used. Background subtraction, with denoising and deconvolution is an image manipulation that enhances the contrast between background and what looks like foreground. Therefore, background subtraction should be applied primarily in experiments involving image segmentation not fluorescence intensity measurement. Not being provided any information by the authors about the kind of subtraction that was made, this processing could lead to an uneven subtraction across the image, which can easily lead to artefacts. Since the fluorescence intensity in the smaller daughter cell is lower, and thus closer to background, the algorithm the authors used may have misinterpreted the grey value information in the smaller daughter cell pixels. This could have led to an asymmetric subtraction of background in the two daughter cells, leading to a stronger subtraction in the smaller daughter cell. Ultimately, their processing could have artificially increased the intensity asymmetry between the two daughter cells in all their results.

The imaging data is of low quality (for example Figures 1, 2, 5, 6; Figures S2, S3, S5, S6, S9). Since much of the study and the findings are based on imaging, this is a major concern. Critical parameters are not mentioned (number of sections in z-stack, size of the field-of-view, laser power used etc), which makes it difficult to understand what was done and what one is looking at. To give some specific examples, (1) the images shown in Figure 2B are of very low quality with severe background from neighbouring cells. In addition, the outline of the cells (plasma membrane) or the nuclei of the daughter cells is unknown. Based on this it is not clear how the authors could have measured 'Fluorescence intensity ratio between sister nuclei' in an accurate and unbiased way (what is clear from these images is that there is an increase in HDA-1::GFP signal in ALL surviving daughters (asymmetric and symmetric divisions) post cytokinesis but not in the daughter cell that is about to die (asymmetric and unequal division); (2) the images in Figure 6A and Figure S9A on VHA-17 segregation and its colocalization to ER and lysosome segregation during QR.a division are of very low quality and it is unclear to the reviewer how such images were used to obtain the quantitative data shown.

In some cases, there is a discrepancy between what is shown in figures and what the authors state in the text. To give some examples: (1) on page 7, the authors state "..., we found that nuclear HDA-1 or LIN-53 asymmetry gradually increased from 1.1-fold at the onset of anaphase to 1.5 or 1.8-fold at cytokinesis, respectively (Figure 1D-E)." Looking at the images for HDA-1 and LIN-53 in Figure 1D, the increase in the ratio mainly occurs between 4 min and 6 min, which is post cytokinesis and NOT prior to cytokinesis; (2) these images (Figure 1D) also show that there is an increase in the HDA-1 and LIN-53 signals in the larger daughter cells (QR.ap), which suggests that the increase in ratios (Figure 1E) is the result of increased HDA-1 and LIN-53 synthesis post cytokinesis. However, on top of page 8, the authors state "The total fluorescence of HDA-1, LIN-53 and MYS-1 remained constant during ACDs, suggesting that protein redistribution may establish NuRD asymmetry (Figure S4C)." In Figure S4C, the authors present straight lines for 'relative total fluorescence' for imaging (probably z-stacks) that was done every min over the course of 7 min. If there was no increase in material as the authors claim, they should have seen significant photobleaching over the course of the 7 min and therefore reduced level of 'relative total fluorescence' over time. How the data presented in Figure S4C was generated is therefore unclear. (Despite the fact that the authors claim that the asymmetry seen is not due to new synthesis in the larger daughter cell post cytokinesis, it would be more consistent with the first experiment presented in this study (Figure S1) that shows that there is more hda-1 mRNA in egl-1(-) cells compared to egl-1(+) cells); (3) On page 12, the authors state "..., in Baf A1-treated animals, QRaa inherited similar levels of HDA-1::GFP as its sister cell,...". However, looking at the image provided in Figure 5E (0 min), there seems to be a similar ratio of HDA-1::GFP between the daughter cells in DMSO and Baf A1-treated animals.

Reviewer #1 (Public Review):

The study as a concept is well designed, although there are two issues I see in the methodology (these may be just needing further explanation or if I am correct in my interpretation of what was done, may need reanalysis to take into account). Both issues relate to the data that was extracted from the published literature on zoonotic malaria prevalence in the study area.

1. No limit was set on the temporal range<br /> With no temporal limit on the range of studies, the landscape in many cases will have changes between the study being conducted and the spatial data. This will be particularly marked in areas where there has been clearing since the zoonotic malaria prevalence study. Also, population changes (either through population growth, decline or movement) will have occurred. All research is limited in what it can do with the available data, so I realise that there may not be much the authors can do to correct this. One possible solution would be to look at the land use change at each site between the prevalence study and the remote sensing data. I'm not sure if this is feasible, but if it is I would recommend the authors attempt this as it will make their results stronger.

2. Most studies only gave a geographic area or descriptive location.<br /> The spatial analysis was based on a 5km and 20km radius of the 'study site' location, but for many of the studies the exact site is not known. Therefore the 'study site' was artificially generated using a polygon centroid. Considering that the polygon could be an administrative boundary (ie district/state/country), this is an extremely large area for which a 5km radius circle in the middle of the polygon is being taken as representative of the 'study site'. This doesn't make sense as it assumes that the landscape is uniform across the district, which in most cases it will not be (in rural areas it is going to be a mixture of villages, forest, plantation, crops etc which will vary across the landscape). This might just be a case of misunderstanding what was done (in which case the text needs rewording to make it clearer) or if I have interpreted it correctly the selection of the centroid to represent the study area does not make sense. I am not sure how to overcome this as it probably not possible to get exact locations for the study sites. One possibility could be to make the remote sensing data the same scale as the prevalence data ie if the study site is only identifiable at the polygon level, then the remote sensing data (fragmentation, cover and population) is used at the polygon level.

Both these issues could have an impact on the study's findings. I would think that in both cases it might make the relationship between the environmental variables and prevalence even clearer.

Reviewer #2 (Public Review):

This is the first comprehensive study aimed at assessing the impact of landscape modification on the prevalence of P. knowlesi malaria in non-human primates in Southeast Asia. This is a very important and timely topic both in terms of developing a better understanding of zoonotic disease spillover and the impact of human modification of landscape on disease prevalence.

This study uses the meta-analysis approach to incorporate the existing data sources into a new and completely independent study that answers novel research questions linked to geospatial data analysis. The challenge, however, is that neither the sampling design of previous studies nor their geospatial accuracy are intended for spatially-explicit assessments of landscape impact. On the one hand, the data collection scheme in existing studies was intentionally opportunistic and does not represent a full range of landscape conditions that would allow for inferring the linkages between landscape parameters and P. knowlesi prevalence in NHP across the region as a whole. On the other hand, the absolute majority of existing studies did not have locational precision in reporting results and thus sweeping assumptions about the landscape representation had to be made for the modeling experiment. Finally, the landscape characterization was oversimplified in this study, making it difficult to extract meaningful relationships between the NHP/human intersection on the landscape and the consequences for P. knowlesi malaria transmission and prevalence.

Despite many study limitations, the authors point to the critical importance of understanding vector dynamics in fragmented forested landscapes as the likely primary driver in enhanced malaria transmission. This is an important conclusion particularly when taken together with the emerging evidence of substantially different mosquito biting behaviors than previously reported across various geographic regions.

Another important component of this study is its recognition and focus on the value of geospatial analysis and the availability of geospatial data for understanding complex human/environment interactions to enable monitoring and forecasting potential for zoonotic disease spillover into human populations. More multi-disciplinary focus on disease modeling is of crucial importance for current and future goals of eliminating existing and preventing novel disease outbreaks.

Reviewer #1 (Public Review):

The authors investigated the metabolic effects of ∆9-THC, the main psychoactive component of cannabis, on early mouse embryonic cell types. They found that ∆9-THC increases proliferation in female mouse embryonic stem cells (mESCs) and upregulates glycolysis. Additionally, primordial germ cell-like cells (PGCLCs) differentiated from ∆9-THC-exposed cells also show alterations to their metabolism. The study is valuable because it shows that physiologically relevant ∆9-THC concentrations have metabolic effects on cell types from the early embryo, which may cause developmental effects. However, the claim of "metabolic memory" is not justified by the current data, since the effects on PGCLCs could potentially be due to ∆9-THC persisting in the cultured cells over the course of the experiment, even after the growth medium without ∆9-THC was added.

The study shows that ∆9-THC increases the proliferation rate of mESCs but not mEpiLCs, without substantially affecting cell viability, except at the highest dose of 100 µM which shows toxicity (Figure 1). Treatment of mESCs with rimonabant (a CB1 receptor antagonist) blocks the effect of 100 nM ∆9-THC on cell proliferation, showing that the proliferative effect is mediated by CB1 receptor signaling. Similarly, treatment with 2-deoxyglucose, a glycolysis inhibitor, also blocks this proliferative effect (Figure 4G-H). Therefore, the effect of ∆9-THC depends on both CB1 signaling and glycolysis. This set of experiments strengthens the conclusions of the study by helping to elucidate the mechanism of the effects of ∆9-THC.

Although several experiments independently showed a metabolic effect of ∆9-THC treatment, this effect was not dose-dependent over the range of concentrations tested (10 nM and above). Given that metabolic effects were observed even at 10 nM ∆9-THC (see for example Figure 1C and 3B), the authors should test lower concentrations to determine the dose-dependence and EC50 of this effect. The authors should also compare their observed EC50 with the binding affinity of ∆9-THC to cellular receptors such as CB1, CB2, and GPR55 (reported by other studies).

The study also profiles the transcriptome and metabolome of cells exposed to 100 nM ∆9-THC. Although the transcriptomic changes are modest overall, there is upregulation of anabolic genes, consistent with the increased proliferation rate in mESCs. Metabolomic profiling revealed a broad upregulation of metabolites in mESCs treated with 100 nM ∆9-THC.

Additionally, the study shows that ∆9-THC can influence germ cell specification. mESCs were differentiated to mEpiLCs in the presence or absence of ∆9-THC, and the mEpiLCs were subsequently differentiated to mPGCLCs. mPGCLC induction efficiency was tracked using a BV:SC dual fluorescent reporter. ∆9-THC treated cells had a moderate increase in the double positive mPGCLC population and a decrease in the double negative population. A cell tracking dye showed that mPGCLCs differentiated from ∆9-THC treated cells had undergone more divisions on average. As with the mESCs, these mPGCLCs also had altered gene expression and metabolism, consistent with an increased proliferation rate.

My main criticism is that the current experimental setup does not distinguish between "metabolic memory" vs. carryover of THC (or its metabolites) causing metabolic effects. The authors assume that their PGCLC induction was performed "in the absence of continuous exposure" but this assumption may not be justified. ∆9-THC might persist in the cells since it is highly hydrophobic. In order to rule out the persistence of ∆9-THC as an explanation of the effects seen in PGCLCs, the authors should measure concentrations of ∆9-THC and THC metabolites over time during the course of their PGCLC induction experiment. This could be done by mass spectrometry. This is particularly important because 10 nM of ∆9-THC was shown to have metabolic effects (Figure 1C, 3B, etc.). Since the EpiLCs were treated with 100 nM, if even 10% of the ∆9-THC remained, this could account for the metabolic effects. If the authors want to prove "metabolic memory", they need to show that the concentration of ∆9-THC is below the minimum dose required for metabolic effects.

Overall, this study is promising but needs some additional work in order to justify its conclusions. The developmental effects of ∆9-THC exposure are important for society to understand, and the results of this study are significant for public health.

Reviewer #2 (Public Review):

In the study conducted by Verdikt et al, the authors employed mouse Embryonic Stem Cells (ESCs) and in vitro differentiation techniques to demonstrate that exposure to cannabis, specifically Δ9-tetrahydrocannabinol (Δ9-THC), could potentially influence early embryonic development. Δ9-THC was found to augment the proliferation of naïve mouse ESCs, but not formative Epiblast-like Cells (EpiLCs). This enhanced proliferation relies on binding to the CB1 receptor. Moreover, Δ9-THC exposure was noted to boost glycolytic rates and anabolic capabilities in mESCs. The metabolic adaptations brought on by Δ9-THC exposure persisted during differentiation into Primordial Germ Cell-Like Cells (PGCLCs), even when direct exposure ceased, and correlated with a shift in their transcriptional profile. This study provides the first comprehensive molecular assessment of the effects of Δ9-THC exposure on mouse ESCs and their early derivatives. The manuscript underscores the potential ramifications of cannabis exposure on early embryonic development and pluripotent stem cells. However, it is important to note the limitations of this study: firstly, all experiments were conducted in vitro, and secondly, the study lacks analogous experiments in human models.

Reviewer #3 (Public Review):

Verdikt et al. focused on the influence of Δ9-THC, the most abundant phytocannabinoid, on early embryonic processes. The authors chose an in vitro differentiation system as a model and compared the proliferation rate, metabolic status, and transcriptional level in ESCs, exposure to Δ9-THC. They also evaluated the change of metabolism and transcriptome in PGCLCs derived from Δ9-THC-exposed cells. All the methods in this paper do not involve the differentiation of ESCs to lineage-specific cells. So the results cannot demonstrate the impact of Δ9-THC on preimplantation developmental stages. In brief, the authors want to explore the impact of Δ9-THC on preimplantation developmental stages, but they only detected the change in ESCs and PGCLCs derived from ESCs, exposure to Δ9-THC, which showed the molecular characterization of the impact of Δ9-THC exposure on ESCs and PGCLCs.

Reviewer #1 (Public Review):

Trebino et al. investigated the BRAF activation process by analysing the interactions of BRAF N-terminal regulatory regions (CRD, RBD, and BSR) with the C-terminal kinase domain and with the upstream regulators HRAS and KRAS. To this end, they generated four constructs comprising different combinations of N-terminal domains of BRAF and analysed their interaction with HRAS as well as conformational changes that occur. By HDX-MS they confirmed that the RBD is indeed the main mediator of interaction with HRAS. Moreover, they observed that HRAS binding leads to conformational changes exposing the BSR to the environment. Next, the authors used OpenSPR to determine the binding affinities of HRAS to the different BRAF constructs. While BSR+RBD, RBD+CRD, and RBD bound HRAS with nanomolar affinity, no binding was observed with the construct comprising all three domains. Based on these experiments, the authors concluded that BSR and CRD negatively regulate binding to HRAS and hypothesised that BSR may confer some RAS isoform specificity. They corroborated this notion by showing that KRAS bound to BRAF-NT1 (BSR+RBD+CRD) while HRAS did not. Next, the authors analysed the autoinhibitory interaction occurring between the N-terminal regions and the kinase domain. Through pulldown and OpenSPR experiments, they confirm that it is mainly the CRD that makes the necessary contacts with the kinase domain. In addition, they show that the BSR stabilizes these interactions and that the addition of HRAS abolishes them. Finally, the D594G mutation within the KD of BRAF is shown to destabilise these autoinhibitory interactions, which could explain its oncogenic potential.

Overall, the in vitro study provides new insights into the regulation of BRAF and its interactions with HRAS and KRAS through a comprehensive in vitro analysis of the BRAF N-terminal region. Also, the authors report the first KD values for the N- and C-terminal interactions of BRAF and show that the BSR might provide isoform specificity towards KRAS. While these findings could be useful for the development of a new generation of inhibitors, the overall impact of the manuscript could probably be enhanced if the authors were to investigate in more detail how the BSR-mediated specificity of BRAF towards certain RAS isoforms is achieved. Moreover, though the very "clean" in vitro approach is appreciated, it also seems useful to examine whether the observed interactions and conformational changes occur in the full-length BRAF molecule and in more physiological contexts. Some of the results could be compared with studies including full-length constructs.

Reviewer #2 (Public Review):

In the manuscript entitled 'Unveiling the Domain-Specific and RAS Isoform-Specific Details of BRAF Regulation', the authors conduct a series of in vitro experiments using N-terminal and C-terminal BRAF fragments (SPR, HDX-MS, pull-down assays) to interrogate BRAF domain-specific autoinhibitory interactions and engagement by H- and KRAS GTPases. Of the three RAF isoforms, BRAF contains an extended N-terminal domain that has yet to be detected in X-ray and cryoEM reconstructions but has been proposed to interact with the KRAS hypervariable region. The investigators probe binding interactions between 4 N-terminal (NT) BRAF fragments (containing one more NT domain (BRS, RBD, and CRD)), with full-length bacterial expressed HRAS, KRAS as well as two BRAF C-terminal kinase fragments to tease out the underlying contribution of domain-specific binding events. They find, consistent with previous studies, that the BRAF BSR domain may negatively regulate RAS binding and propose that the presence of the BSR domain in BRAF provides an additional layer of autoinhibitory constraints that mediate BRAF activity in a RAS-isoform-specific manner. One of the fragments studied contains an oncogenic mutation in the kinase domain (BRAF-KDD594G). The investigators find that this mutant shows reduced interactions with an N-terminal regulatory fragment and postulate that this oncogenic BRAF mutant may promote BRAF activation by weakening autoinhibitory interactions between the N- and C-terminus.

While this manuscript sheds light on B-RAF specific autoinhibitory interactions and the identification and partial characterization of an oncogenic kinase domain (KD) mutant, several concerns exist with the vitro binding studies as they are performed using tagged-isolated bacterial expressed fragments, 'dimerized' RAS constructs, lack of relevant citations, controls, comparisons and data/error analysis. Detailed concerns are listed below.

1. Bacterial-expressed truncated BRAF constructs are used to dissect the role of individual domains in BRAF autoinhibition. Concerns exist regarding the possibility that bacterial expression of isolated domains or regions of BRAF could miss important posttranslational modifications, intra-molecular interactions, or conformational changes that may occur in the context of the full-length protein in mammalian cells. This concern is not addressed in the manuscript.

2. The experiments employ BRAF NT constructs that retain an MBP tag and RAS proteins with a GST tag. Have the investigators conducted control experiments to verify that the tags do not induce or perturb native interactions?

3. The investigators state that the GST tag on the RAS constructs was used to promote RAS dimerization, as RAS dimerization is proposed to be key for RAF activation. However, recent findings argue against the role of RAS dimers in RAF dimerization and activation (Simanshu et al, Mol. Cell 2023). Moreover, while GST can dimerize, it is unclear whether this promotes RAS dimerization as suggested. In methods for the OpenSPR experiments probing NT BRAF:RAS interactions, it is stated that "monomeric KRAS was flowed...". This terminology is a bit confusing. How was the monomeric state of KRAS determined and what was the rationale behind the experiment? Is there a difference in binding interactions between "monomeric vs dimeric KRAS"?

4. The investigators determine binding affinities between GST-HRAS and NT BRAF domains (NT2 7.5 {plus minus} 3.5; NT3 22 {plus minus} 11 nM) by SPR, and propose that the BRS domain has an inhibitory role HRAS interactions with the RAF NT. However, it is unclear whether these differences are statistically meaningful given the error.

5. It is unclear why NT1 (BSR+RBD+CRD) was not included in the HDX experiments, which makes it challenging to directly compare and determine specific contributions of each domain in the presence of HRAS. Including NT1 in the experimental design could provide a more comprehensive understanding of the interplay between the domains and their respective roles in the HRAS-BRAF interaction. Further, excluding certain domains from the constructs, such as the BSR or CRD, may overlook potential domain-domain interactions and their influence on the conformational changes induced by HRAS binding.

6. The authors perform pulldown experiments with BRAF constructs (NT1: BSR+RBD+CRD, NT2: BSR+RBD, NT3: RBD+CRD, NT4: RBD alone), in which biotinylated BRAF-KD was captured on streptavidin beads and probed for bound His/MBP-tagged BRAF NTs. Western blot results suggest that only NT1 and NT3 bind to the KD (Figure 5). However, performing a pulldown experiment with an additional construct, CRD alone, it would help to determine whether the CRD alone is sufficient for the interaction or if the presence of the RBD is required for higher affinity binding. This additional experiment would strengthen the authors' arguments and provide further insights into the mechanism of BRAF autoinhibition.

7. While the investigators state that their findings indicate that H- and KRAS differentially interact with BRAF, most of the experiments are focused on HRAS, with only a subset on KRAS. As SPR & pull-down experiments are only conducted on NT1 and NT2, evidence for RAS isoform-specific interactions is weak. It is unclear why parallel experiments were not conducted with KRAS using BRAF NT3 & NT4 constructs.

8. The investigators do not cite the AlphaFold prediction of full-length BRAF (AF-P15056-F1) or the known X-ray structure of the BRAF BRS domain. Hence, it is unclear how Alpha-Fold is used to gain new structural information, and whether it was used to predict the structure of the N-terminal regulatory or the full-length protein.

9. In HDX-MS experiments, it is unclear how the authors determine whether small differences in deuterium uptake observed for some of the peptide fragments are statistically significant, and why for some of the labeling reaction times the investigators state " {plus minus} HRAS only" for only 3 time points?

10. The investigators find that KRAS binds NT1 in SPR experiments, whereas HRAS does not. However, the pull-down assays show NT1 binding to both KRAS and HRAS. SI Fig 5 attributes this to slow association, yet both SPR (on/off rates) and equilibrium binding measurements are conducted. This data should be able to 'tease' out differences in association.

11. The model in Figure 7B highlights BSR interactions with KRAS, however, BSR interactions with the KRAS HVR (proximal to the membrane) are not shown, as supported by Terrell et al. (2019).

12. The investigators state that 'These findings demonstrate that HRAS binding to BRAF directly relieves BRAF autoinhibition by disrupting the NT1-KD interaction, providing the first in vitro evidence of RAS-mediated relief of RAF autoinhibition, the central dogma of RAS-RAF regulation. However, in Tran et al (2005) JBC, they report pull-down experiments using N-and C-terminal fragments of BRAF and state that 'BRAF also contains an N-terminal autoinhibitory domain and that the interaction of this domain with the catalytic domain was inhibited by binding to active HRAS'. This reference is not cited.

13. In Fig 2, panels A and C, it is unclear what the grey dotted line in is each plot.

14. In Fig 3, error analysis is not provided for panel E.

15. How was RAS GMPPNP loading verified?

Reviewer #1 (Public Review):

As this experience as a reviewer has been unusual, it may be helpful to outline some relevant parameters of the task at the outset. While I was invited to review the Fuentes et al. study only, two additional papers concerning the claimed engravings and burials associated with Homo naledi by Berger and colleagues were also provided as components of the reviewer package. The two manuscripts presenting the archaeological evidence are accessible as preprints in bioRxiv, by Lee Berger and colleagues ('2023a, 2023b').

Unfortunately, the arguments in the Fuentes et al manuscript hinge entirely on the strength of archaeological evidence for engravings and intentional burial by Homo naledi (presented in the abovementioned two preprints). All inferences regarding hominin behaviour and biology of Homo naledi, discussed by Fuentes and colleagues, are wholly dependent on the evidence presented in the archaeology preprints being true.

Yet both of the archaeological manuscripts are unfortunately weak. In short, the claims for engravings depend on the demonstration of several elements of association that are rather standard for linking material traces found in the archaeological record with particular hominin behaviours. For the particular arguments by Berger and colleagues to be demonstrated, the traces on the rock surface need to be linked causally with hominin agency, in other words, their anthropogenic nature need to be established. The author of the engravings needs to be demonstrated as a particular hominin species (Homo naledi in this case), and the activity of engraving needs to have taken place ~241-335 kya. After reading the manuscript on the engravings, however, what is clear is that the scratches could as easily have been made by a modern-day farmer 50 years ago, as Homo naledi ~335 kya. Berger and colleagues do not present any evidence to the contrary, they simply describe their narrative as the most parsimonious scenario. A particularly curious piece of information presented as evidence is a list of individuals known to have entered the Dinaledi system in recent times (and known not to have scratched the walls, one presumes, though this is not stated).

The question of intentional burial is more complex. What we know from other widely accepted early burials is that documenting the geoarchaeological context of the hominin remains is critical to assess the likelihood of an intentional burial - this needs to be established at the outset through high quality fieldwork. Yet even the boundaries of the excavation presented in the burial manuscript appear so angled or skew relative to one another (Fig. 2a) that the individual squares look to be aligned with different XY grids, which does not instill confidence in the quality of field documentation. One can make out very little from the sediment section images - which are key to identifying intrusive features associated with burials - and the multivariate geochemical analysis of sediments is unconvincing: a scatterplot (not a biplot) should have been provided showing the geochemistry of the burial sediment samples relative to the immediately surrounding sediment characteristics. While one remains excited about the potential for a spectacular archaeological discovery within the Dinaledi cave system, unfortunately, the three manuscripts provided do not present convincing evidence to that effect.

Reviewer #2 (Public Review):

Fuentes et al. provide a detailed and thoughtful commentary on the evolutionary and behavioral implications of complex behaviors associated with a small-brained hominin, Homo naledi. Within the Rising Star Cave of South Africa, Berger et al. 2023a,b proposed evidence that Homo naledi intentionally buried their dead through complex mortuary practices and engaged in symbolic expression by engraving the cave walls in cross-hatching motifs. Two burials were identified in the Rising Star cave subsystems: Feature 1 in the Dinaledi Chamber and a feature in the Hill Antechamber. The engravings are located in the Hill Antechamber near the passageway leading into the Dinaledi chamber. The authors aimed to provide evidence for burials by (1) testing sediment samples for mineral composition from within and outside the burial feature; (2) demonstrating an interruption in the stratigraphy indicative of a "bowl-shaped" feature; (3) evaluating the anatomical coherence of the skeletal remains; (4) demonstrate matrix-supported positioning of skeletal elements; and (5) determine the compatibility of non-articulated material with decomposition and subsequent collapse. Berger et al. 2023b evaluated the engravings through high resolution photography, cross-polarization, and 3D photogrammetry. Neither article involved radiometric dating of materials. While the review by Fuentes et al. highlights important assumptions about the relationship between hominin brain size, cognition, and complex behaviors, the evidence presented by Berger et al. 2023a,b does not support the claim that Homo naledi engaged in burial practices or symbolic expression through wall engravings.

The major weaknesses for Berger et al. 2023a are as follows:

1) The mineral composition from sediment sampled from within Dinaledi Feature 1 is not different compared to the surrounding sediment, which is one rationale proposed by the authors that would lead to the conclusion of a burial pit. An effort to replicate the multivariate statistical analysis using the data provided in SI Table 1 by this reviewer failed, and thus, the results are not replicable.

2) The authors failed to provide clear visualizations or analysis that showed an unambiguous interruption in the stratigraphy surrounding the Dinaledi Feature 1.

3) Attempts 1 and 2 were applied solely to Dinaledi Feature 1, not the Hill Antechamber Feature.

4) Skeletal cohesion does suggest that the bodies were likely covered or protected by external environment. However, given the geological context, there is minimal opportunity for scavengers or other agents to scatter the skeletal remains within such an isolated location. Thus, this alone cannot solely support intentional burials as this line of evidence is subject to equifinality.

5) Similar to the preceding statement, evidence for matrix-supported elements was inconclusive at best. There was no mention of sedimentary rate or expectations for how quickly sediments would naturally bury the remains of whole bodies in the chamber compared with the rate of decomposition of buried remains.

The major weaknesses for Berger et al. 2023b are as follows:

6) While this is incredibly difficult to accomplish, dating rock art or other cave wall engravings is the only method to ensure that the etchings were created during the time of Homo naledi. Unfortunately, this was not attempted. Instead, the authors state that "This description is intended to document the discovery and provide spatial and contextual information prior to any further analyses that may require invasive sampling." Yet, the authors assign a date to the engravings in the title of the paper. Here, the authors are generating interpretations before analyses are attempted.

7) The engravings are indeed very interesting and are likely anthropogenic in origin. However, the argument that these engravings were created by Homo naledi is based on the bold assumption that "No physical or cultural evidence of any other hominin population occurs within this part of the cave system, and there is no evidence that recent humans or earlier hominins ever entered any adjacent area of the cave until surveys by human cave explorers during the last 40 years." (page 6). To assume that no other individual entered the cave system from the time of Homo naledi until 40 years ago is an unrealistic and faulty assumption. This reviewer does not discount that the engravings could have been made by Homo naledi, but the evidence must be sufficient to support this statement or provide other alternatives as working hypotheses.

As a discipline, paleoanthropology aims to understand the evolutionary history of the hominin clade through fossil remains, material culture, and, most recently, ancient DNA. The methods and approaches that we as paleoanthropologists use to understand the past often bridge both the humanities and the hard sciences to create a unique understanding of our shared history. We are only limited by the conditions in which time and attrition has erased pieces of our collective story from the earth. Thus, it is our responsibility to ensure that our interpretations of the past are supported by measurable and testable means, to the best of our ability, and that hypotheses are not presented as conclusions.

Unfortunately, this is not the case for Berger et al. 2023a,b. The work presented by the authors is imprudent and incomplete and does not meet the requirements set forth by our discipline. While it is important that scholars publish their work in a dutiful timeline, it is arguably more critical for scholars to take the necessary time to ensure the integrity and resolution of the work. The consequences for rushing publications with such a significant unsubstantiated find will likely result in perilous ramifications, as it is more difficult to correct an idea than to introduce one.

Reviewer #3 (Public Review):

This paper presents the cognitive implications of claims made in two accompanying papers (Berger et al. 2023a, 2023b) about the creation of rock engravings, the intentional disposal of the dead, and fire use by Homo naledi. The importance of the paper, therefore, relies on the validity of the claims for the presence of socio-culturally complex and cognitively demanding behaviors that are presented in the associated papers. Given the archaeological, hominin, and taphonomic analyses in the associated papers are not adequate to enable the exceptional claims for naledi-associated complex behaviors, the inferences made in this paper are currently inadequate and incomplete.

The claimed behaviors are widely recognized as complex and even quintessential to Homo sapiens. The implications of their unequivocal association with such a small-brained Middle Pleistocene hominin are thus far reaching. Accordingly, the main thrust of the paper is to highlight that greater cognition and complex socio-cultural behaviors were not necessarily associated with a positively encephalized brain. This argument begs the obvious question of whether absolute brain size and/or encephalization quotient (i.e., the actual brain volume of a given species relative the expected brain size for a species of the same average body size) can measure cognitive capacity and the complexity of socio-cultural behaviors among late Middle Pleistocene hominins.

Claims for a positive correlation between absolute and/or relative brain size and cognitive ability are not common in discussions surrounding the evolution of Middle- and Late Pleistocene hominin behavior. Currently, the bulk of the evidence for early complex technological and social behaviors derives from multiple sites across South Africa and postdates the emergence of H. sapiens by more than 100,000 years. Such lag in the expression of complex technologies and behaviors within our species renders the brain size-implies-cognitive capacity argument moot. Instead, a rich body of research over the past several decades has focused on aspects related to socio-cultural, environmental, and even the wiring of the brain in order to understand factors underlying the expression of the capacity for greater behavioral variability. In this regard, even if the claimed evidence for complex behaviors among the small-brained naledi populations proves valid, the exploration of the specific/potential socio-cultural, neuro-structural, ecological and other factors will be more informative than the emphasis on absolute/relative brain size.

The paper presents as supporting evidence previous claims for the appearance of similar complex behaviors predating the emergence of our species, H. sapiens, although it does acknowledge their controversial nature. It then uses the current claims for the association of such behaviors with H. naledi as decisive. Given the inadequate analyses in the accompanying papers and the lack of evidence for stone tools in the naledi sites, the present claims for the expression of culturally and symbolically mediated behaviors by this small-brained hominin must be adequately established. The importance of the paper thus rests on the validity of the claimed evidence--including contextual aspects--for rock engraving, mortuary practices, and the use of fire presented in the associated two papers. The claims in both associated papers are inadequate, incomplete, and largely assumption- (rather than evidence) based. As responsible and ethical researchers, the team must return to the sites, conduct the required standard chronomoetric and taphonomic studies and weigh the strength of the evidence before proceeding with the current claims.

Reviewer #1 (Public Review):

I think it is important to note up front that I recognize that the goal of this paper was to announce the discovery of what appear to be intentionally-made marks in Rising Star cave in South Africa. This was not meant to be an in-depth analysis or a declaration of definitive results. With this in mind, I appreciate that the authors did not try to overstate this new discovery, but instead simply reported what had been observed, provided a little bit of background on the current state of the field in regards to the evolution of hominin visual mark-making, made a few tentative identifications, but then clearly acknowledged that a lot more documentation, sampling, and study would be needed before we could understand the full scope and potential importance of this find.

This is a big claim. If it proves to be true, it has the potential to be paradigm-shifting as the identification of intentional engraved marks, made by a small-brained distant human cousin 200,000+ years ago in South Africa, would completely change our understanding of where, when and who made the first graphic marks. Twenty years ago, this claim would probably have been dismissed out of hand as being too far-fetched to be taken seriously, but there have been some major shifts in the field in recent years, in regard to the age of the art and the identity of the artists, that means this is a claim that should be approached with a scientifically cautious, but open mind. There is now mounting evidence for Neanderthals, and potentially other closely related species as well, to have been engaging in similar art-making practices to our own Homo sapiens ancestors. What makes this particular claim so extraordinary is that these marks are some of the oldest in the world and that Homo naledi is a more distant relation with a smaller brain. This is also what makes the further study of this discovery such a fascinating exercise in scientific inquiry.

From a technical and methodological perspective, there is an excellent range of tools and technologies that can be used to study these engravings, so I have no doubt that further studies will help answer some of the "nuts and bolts" questions. Then there is also the opportunity created by this discovery to really open a broader dialogue in the field about who were the first artists and at what point does the hominin brain become "primed" for making visual marks. I look forward to all sorts of lively debates in the future and to seeing the results of further in-depth studies.

Reviewer #2 (Public Review):

Patterns scored into or painted on durable media have long been considered important markers of the cognitive capabilities of hominins. More specifically, the association of such markers with Homo sapiens has been used to argue that our evolutionary success was in part shaped by our unique ability to code, store and convey information through abstract conventions.

That singularity of association has been cast into doubt in the last decade with finds of designs apparently painted or carved by Neanderthals, and potentially by even earlier hominins. Even allowing for these developments, however, extending the capability to generate putatively abstract designs to a relatively small-brained hominin like Homo naledi is contentious. The evidential bar for such claims is necessarily high, and I don't believe that it has been cleared here.

The central issue is that the engravings themselves are not dated. As the authors themselves note, the minimum age constraint provided by U/Th on flowstone does not necessarily relate to the last occupation of the Dinaledi cave system, as the earlier ESR age on teeth does not necessarily document first use of the cave. The authors state that "At present we have no evidence limiting the time period across which H. naledi was active in the cave system". On those grounds though, assigning the age range of presently dated material within the cave system to the engravings - as the current title unambiguously does - is not justifiable.

Because we don't know when they were made, the association between the engravings and Homo naledi rests on the assertion that no humans entered and made alterations to the cave system between its last occupation by Homo naledi, and its recent scientific recording. This is argued on page 6 with the statement that "No physical or cultural evidence of any other hominin population occurs within this part of the cave system".

There is an important contrast between the quotes I have referred to in the last two paragraphs. In the earlier quote, the absence of evidence for Homo naledi in the cave system >335 ka and <241 ka is not considered evidence for their absence before or after these ages. Just because we have no evidence that Homo naledi was in the cave at 200 ka doesn't mean they weren't there, which is an argument I think most archaeologists would accept. When it comes to other kinds of humans, though - per the latter quote - the opposite approach is taken. Specifically, the present lack of physical evidence of more recent humans in the cave is considered evidence that no such humans visited the cave until its exploration by cavers 40 years ago. I don't think many archaeologists would consider that argument compelling. I can see why the authors would be drawn to make that assertion, but an absence of evidence cannot be used to argue in one way for use of the cave by Homo naledi and in another way for use of the cave by all other humans.

A second problem is with what Homo naledi might have made engravings. The authors state that "The lines appear to have been made by repeatedly and carefully passing a pointed or sharp lithic fragment or tool into the grooves". The authors then describe one rock with superficial similarities to a flake from the more recent site of Blombos to suggest that sharp-edge stones with which to make the engravings were available to Homo naledi. Blombos is considered relevant here presumably because it has evidence for Middle Stone Age engravings. The authors do not, however, demonstrate any usewear on that stone object such as might be expected if it was used to carve dolomite. Given that it is presented as the only such find in the cave system so far, this seems important.

My greater concern is that the authors did not compare the profile morphology of the Dinaledi engravings with the extensive literature on the morphology of scored lines caused by sharp-edge stone implements (e.g., Braun et al. 2016, Pante et al. 2017). I appreciate that the research group is reticent to undertake any invasive work until necessary, but non-destructive techniques could have been used to produce profiles with which to test the proposition that the engravings were made with a sharp edge stone.

One thing I noticed in this respect is that the engravings seem very wide, both in absolute terms and relative to their depth. The data I collected from the Middle Stone Age engraved ochre from Klein Kliphuis suggested average line widths typically around 0.1-0.2 mm (Mackay and Welz 2008). The engraved lines at Dinaledi appear to be much wider, perhaps 2-5 mm. This doesn't discount the possibility that the engravings in the Dinaledi system were carved with a sharp edge stone - the range of outcomes for such engravings in soft rock can be quite variable (Hodgskiss 2010) - only that detailed analysis should precede rather than follow any assertion about their mode of formation.

None of this is to say that the arguments mounted here are wrong. It should be considered possible that Homo naledi made the engravings in the Dinaledi cave system. The problem is that other explanations are not precluded.

As an example, the western end of the Dinaledi subsystem has a particular geometry to the intersection of its passages, with three dominant orientations, one vertical (which is to say, north-south), and two diagonal (Figure 1). The major lines on Panel A have one repeated vertical orientation and two repeated diagonal orientations (Figure 16), particularly in the upper area not impacted by stromatolites. The lines in both the cave system and engravings in Panel A appear to intersect at similar angles. Several of the cave features appear, superficially at least, to be replicated. In fact, scaled, rotated, and super-imposed, Figure 16 is a plausible 'mud map' of the western end of the Dinaledi system carved incrementally by people exploring the caves. A figure showing this is included here:

Of course, there are problems with this suggestion. The choice of the upper part of Panel A is selective, the similarity is superficial, and the scales are not necessarily comparable. (Note, btw, that all of those caveats hold equally well for the comparison the authors make between the unmodified rock from Dinaledi and the flake from Blombos in Figure 19). However, the point is that such a 'mud map hypothesis' is, as with the arguments mounted in this paper, both plausible and hard to prove.

Having read this paper a few times, I am intrigued by the engravings in the Dinaledi system and look forward to learning more about them as this research unfolds. Based on the evidence presently available, however, I feel that we have no robust grounds for asserting when these engravings were made, by whom they were made, or for what reason they were made.

References:

• Braun, D. R., et al. (2016). "Cut marks on bone surfaces: influences on variation in the form of traces of ancient behaviour." Interface Focus 6: 20160006.

• Hodgskiss, T. (2010). "Identifying grinding, scoring and rubbing use-wear on experimental ochre pieces." Journal of Archaeological Science 37: 3344-3358.

• Mackay, A. & A. Welz (2008). "Engraved ochre from a Middle Stone Age context at Klein Kliphuis in the Western Cape of South Africa." Journal of Archaeological Science 35: 1521-1532.

• Pante, M. C., et al. (2017). "A new high-resolution 3-D quantitative method for identifying bone surface modifications with implications for the Early Stone Age archaeological record." J Hum Evol 102: 1-11.

Reviewer #3 (Public Review):

Lee Berger and colleagues argue here that markings they have found in a dark isolated space in the Rising Star Cave system are likely over a quarter of a million years old and were made intentionally by Homo naledi, whose remains nearby they have previously reported. As in a European and much later case they reference ('Neanderthal engraved 'art' from the Pyrenees'), the entangled issues of demonstrable intentionality, persuasive age and likely authorship will generate much debate among the academic community of rock art specialists. The title of the paper and the reference to 'intentional designs', however, leave no room for doubt as to where the authors stand, despite avoidance of the word art, entering a very disputed terrain. Iain Davidson's (2020) 'Marks, pictures and art: their contributions to revolutions in communication', also referenced here, forms a useful and clearly articulated evolutionary framework for this debate. The key questions are: 'are the markings artefactual or natural?', 'how old are they?' and 'who made them?, questions often intertwined and here, as in the Pyrenees, completely inseparable. I do not think that these questions are definitively answered in this paper and I guess from the language used by the authors (may, might, seem etc) that they do not think so either.

First, a few referencing issues: the key reference quoted for distinguishing natural from artefactual markings (Fernandez-Jalvo et al. 2014), whilst mentioned in the text, is not included in the references. In the acknowledgements, the claim that "permits to conduct research in the Rising Star Cave system are provided by the South African National Research Foundation" should perhaps refer rather to SAHRA? In the primary description of their own markings from Rising Star and their presumed significance, there are, oddly, several unacknowledged quotes from the abstract of one of the most significant European references (Rodriguez-Vidal et al. 2014). These need attention.

Before considering the specific arguments of the authors to justify the claims of the title, we should recognise the shift in the academic climate of those concerned with 'ancient markings' that has taken place over the past two or three decades. Before those changes, most specialists would probably have expected all early intentional markings to have been made by Homo sapiens after the African diaspora as part of the explosion of innovative behaviours thought to characterise the 'origins of modern humans'. Now, claims for earlier manifestations of such innovations from a wider geographic range are more favourably received, albeit often fiercely challenged as the case for Pyrenean Neanderthal 'art' shows (White et al. 2020). This change in intellectual thinking does not, however, alter the strict requirements for a successful assertion of earlier intentionality by non-sapiens species. We should also note that stone, despite its ubiquity in early human evolutionary contexts, is a recalcitrant material not easily directly dated whether in the form of walling, artefact manufacture or potentially meaningful markings. The stakes are high but the demands are no less so.

Why are the markings not natural? Berger and co-authors seem to find support for the artefactual nature of the markings in their location along a passage connecting chambers in the underground Rising Star Cave system. The presumption is that the hominins passed by the marked panel frequently. I recognise the thinking but the argument is weak. More confidently they note that "In previous work researchers have noted the limited depth of artificial lines, their manufacture from multiple parallel striations, and their association into clear arrangement or pattern as evidence of hominin manufacture (Fernandez-Jalvo et al. 2014)". The markings in the Rising Star Cave are said to be shallow, made by repeated grooving with a pointed stone tool that has left striations within the grooves and to form designs that are "geometric expressions" including crosshatching and cruciform shapes. "Composition and ordering" are said to be detectable in the set of grooved markings. Readers of this and their texts will no doubt have various opinions about these matters, mostly related to rather poorly defined or quantified terminology. I reserve judgement, but would draw little comfort from the similarities among equally unconvincing examples of early, especially very early, 'designs'. Two or even three half-convincing arguments do not add up to one convincing one.

The authors draw our attention to one very interesting issue: given the extensive grooving into the dolomite bedrock by sharp stone objects, where are these objects? Only one potential 'lithic artefact' is reported, a "tool-shaped rock [that] does resemble tools from other contexts of more recent age in southern Africa, such as a silcrete tool with abstract ochre designs on it that was recovered from Blombos Cave (Henshilwood et al. 2018)", also figured by Berger and colleagues. A number of problems derive from this comparison. First, 'tool-shaped rock' is surely a meaningless term: in a modern toolshed 'tool-shaped' would surely need to be refined into 'saw-shaped', 'hammer-shaped' or 'chisel-shaped' to convey meaning? The authors here seem to mean that the Rising Star Cave object is shaped like the Blombos painted stone fragment. But the latter is a painted fragment, not a tool and so any formal similarity is surely superficial and offers no support to the 'tool-ness' of the Rising Star Cave object. Does this mean that Homo naledi took (several?) pointed stone tools down the dark passageways, used them extensively and, whether worn out or still usable, took them all out again when they left? Not impossible, of course. And the lighting?

The authors rightly note that the circumstance of the markings "makes it challenging to assess whether the engravings are contemporary with the Homo naledi burial evidence from only a few metres away" and more pertinently, whether the hominins did the markings. Despite this honest admission, they are prepared to hypothesise that the hominin marked, without, it seems, any convincing evidence. If archaeologists took juxtaposition to demonstrate authorship, there would be any number of unlikely claims for the authorship of rock paintings or even stone tools. The idea that there were no entries into this Cave system between the Homo naledi individuals and the last two decades is an assertion, not an observation, and the relationship between hominins and designs no less so. In fact, the only 'evidence' for the age of the markings is given by the age of the Homo naledi remains, as no attempt at the, admittedly very difficult, perhaps impossible, task of geochronological assessment, has been made.

The claims relating to artificiality, age and authorship made here seem entangled, premature and speculative. Whilst there is no evidence to refute them, there isn't convincing evidence to confirm them.

References:

• Davidson, I. 2020. Marks, pictures and art: their contribution to revolutions in communication. Journal of Archaeological Method and Theory 27: 3 745-770.

• Henshilwood, C.S. et al. 2018. An abstract drawing from the 73,000-year-old levels at Blombos Cave, South Africa. Nature 562: 115-118.

• Rodriguez-Vidal, J. et al. 2014. A rock engraving made by Neanderthals in Gibralter. Proceedings of the National Academy of Sciences.

• White, Randall et al. 2020. Still no archaeological evidence that Neanderthals created Iberian cave art.

Reviewer #4 (Public Review):

This is potentially a landmark study with far-reaching consequences for archaeology, palaeoanthropology, and more widely. The antiquity of intentional human mark marking is a hot topic but this study – understood as initial – has as yet incomplete sources of evidence and methods; and it will be interesting to follow how the study develops in subsequent studies.

Strengths and points to build on:

* Heuristic potential: As knowledge advances it poses a risk to accepted knowledge – and we should accept that one such risk is moving on from long-held disciplinary tenets. In this case, there has been a growing quantum of evidence – all hotly debated – for the deep antiquity of mark-making and even symbolism by species other than ourselves. Most researchers now accept Neanderthal symbolic capacity actualised in burials, intentional mark-making and the like. The evidence here presented is not unequivocal but is very suggestive and an ideal test case for applying multi-disciplinary techniques of analysis and interpretation beyond the expertise of the listed authors *see comments in 'weaknesses'). This work by itself may be equivocal but when taken together with other such work, points to a 'human' sensu lato past that is as complex as it is long. This work then helps all researchers to at least be alive to the possibility of things like anthropic marks and residues in a context not normally thought to have it.

* Decentering speciesism: As per the above comment, I appreciate empirical studies that erode speciesism – in particular studies that open up our minds to the possibility that multiple members of the Genus Homo were capable of intentional mark-making and even 'symbolic' behaviour, though this latter term is not well understood or uniformly used. This is probably because of continuous unconscious bias on our part as currently the only exemplar of our genus living - in contrast to most of the past in which different species and genera co-existed - if not on the same landscape and/or at exactly the same time, then with enough overlap that people would have realised 'others' were about either by sight and/or by encountering their physical remains and artefacts.

* Problematising 'firsts' and deep time: A strength – but which needs to be developed in this manuscript – is our understanding of time and change. We have a plethora of dating techniques but relatively few substantive monographs, articles, and think tanks on time – and especially on how change comes about and what causes it. This leads us to privilege 'firsts' and the 'oldest' finds in 'deep' time above those that are more recent and in 'shallow' time. I would suggest in addition to the claims for the oldest of the reported marks, the authors develop nascent remarks on the possibility the suite of marks may have been made over time. This will help counter criticism that these marks – if established to be anthropic – were not just a singularity, but part of patterned behaviour, which would move it towards the realm of 'symbolic' cognitive behaviour. And indeed, it would be good to hear more about why in this place, these marks were made to establish a replicable model for identifying early anthropic marks.

Ultimately, this manuscript presents evidence that those who are pro the deep antiquity of intentional mark-making by Homo (and possibly even other genera) will find enough evidence to support; while those sceptical of such claims will find enough methodological flaws and evidential limits to refute those claims. The next decade of work will likely be definitive and this article makes a key contribution to the debate.

Weaknesses and points to attend to:

* Definitions: The term 'rock engraving' is used rather uncritically and also the term 'etching' – and it would be useful to have a short definition of how the authors understand the term. Rock art scholars regularly debate these terms and whether they are or are not 'rock art' with its overwhelmingly visual bias; which this discovery may usefully help overthrow and advance.

* Dating: There is no evidence provided for dating the marks found in the cave system. They could, for example, have been made more recently than the dates claimed – and by another species (if we accept their anthropogenic authorship). This is a perennial problem of much rock art research – especially when it comes to understanding the wider archaeological/palaeoanthropological context. More crucially, accurate dating allows a more reliable understanding of authorship and who/what was responsible for a particular artefact or feature. This has not been demonstrated in this case, though we do have fossil evidence of Homo naledi in the cave system. The article title is this incorrect / and unsupported claim as the marks, if they are anthropic, have not been dated and are of unknown age. The authors allow that there may have been multiple episodes, but not that the marks can belong to a time other than they posit – either earlier, later, or distributed over a long period as the authors allow for in their concluding remarks.

* Authorship: The study does not utilise either a geoscientist as one of the authorial team, or a rock art specialist. These are key oversights as the former would help better contextualise the dating of the marks reported on, as well as explore alternative non-anthropogenic agents that may have created the marks reported on. For example, the marks and 'pitting' etc may be the result of water bringing abrasive agents during times of flooding, hitting prominent rock features in the cave system. Some explanation is given from lines 114-124, but are uncited. The overlying 'sediment' may be similar to the mondmilch found in cave systems and which is of natural origin. It may be that these non-anthropogenic causes are easy to discount; but the arguments do need to be made. Or, that the polishing was made by Homo naledi brushing against the surfaces as they moved in the cave system, independent of any mark-making. A Table showing the pros and cons of intentional anthropic versus natural authorship would be very effective - as well as showing some of the natural linear marks in the cave system to avoid any confirmation or similar bias. FTIR analysis of the panel A-C would be more than useful to determine whether an additional layer of material has been added. This is mentioned for future work, but this seems a rather post-hoc research programme.

* Use-wear analysis: If the marks are anthropic in origin; they are likely to have been made by a stone tool, which would leave characteristic marks, directionality and sequencing, distinct from natural causes. It is vital this work – such as was done on the Blombos engraved ochre – is done here – for example, linking to the chert and other tools described on lines 152-158. Note Figure 19, of such a tool, is very hard to make out. The Blombos – and Klasies River Mouth engraved ochres (curiously not referenced) – have very similar geometric markings and there is a real opportunity to compare these in securely dated contexts of 70-120 kya –which could support the argument made here for Homo naledi's cognitive capacity. On figure 16 it would be good to know on what basis some marks were selected as anthropic – and why others were not; this would help demonstrate the methodology and ability to distinguish between the two kinds of marks.

* Viewshed: The rock art specialist would have added essential expertise on how to study anthropic marks. For example, the images of the marks shown are all of individual or small collections of motifs rather than showing each panel as well as all panels together, to help understand the iconographic context as an ensemble – a 'feature' rather than isolated 'artefacts' or 'motifs'. Line 60 mentions being able to see these as a 'triptych' but the reader is not able to have this view in this manuscript. From the cave map, it is not clear whether all three 'panels' (an unfortunate art historical term that suggests a framed entity - better to use a term like 'cluster') can be viewed simultaneously or in sequence. The view shed in relation to the area where the bodies were recovered is vaguely stated as 'only a few metres away' and is worth developing. I understand 3D scans have been made so it would be useful to have a version showing the marks in relation to where the bodies were recovered and as a 3-cluster ensemble.

* Image enhancements: Also, in addition to polarised images, have colour enhancement tools like DStretch been tried to see if, for example, attempts at colouring with different coloured sands were made? Similarly, a 3D scan of the motif and panel – (Metashape is mentioned but not shown) – might assist in understanding how the marks and the rock they are on might relate to each other- as research in European upper Palaeolithic contexts has shown. Here, experimenting with different kinds of lighting - or in the absence of lighting, of tactility and how these marks and their rock support may have been experienced by those who may have made and interacted with them? As a note, it would be useful to have a scale in each image of the 'engravings' and it is a pity the one in situ photograph with the scale is not a standard rock art colour-corrected scale as is commonly used in rock art research.

Reviewer #1 (Public Review):

The discovery of Homo naledi fossils and the rising star cave system is unquestionably important for paleoanthropology. The fossils themselves hold a wealth of information about the diversity and complexity of morphological and evolutionary change on the hominin family tree. It is a truly amazing find and important site and it is important that information about this site continues to be produced so that more can be known. It is equally important that the papers produced from the site be fully reviewed for scientific rigor. I hope to assist with this to the best of my ability.

In its current form the paper, "Evidence for the deliberate burial of the dead by Homo naledi," does not meet the standards of our field. The paper is hard to follow. It lacks key citations, contextual background information to inform the reader about the geological and depositional structure of the caves, and concise understandable descriptions of the methods and the significance of the results.

The main point of the paper is to describe three possible burial features. The working hypothesis is that the features are intentional burials, and the authors seek to support this hypothesis throughout rather than test it. The authors do this by noting mineralogical differences in sediment and possible bowl-shaped sedimentological distinctions where fossil bones occur. As stated above, this evidence needs to be elaborated on the in text, contextualized, and edited for clarity. In addition, throughout the paper, the authors only consider two depositional scenarios for burial and body decomposition: 1) a body was intentionally buried in a pit that was dug into the cave sediments, and then buried in sediment (without detailing in the main text what sediment was used to backfill the pit); and 2) the body was left in a natural pit and decayed in the open. A major problem with only considering these two scenarios for body decomposition is that previous reports about cave geology and sedimentology show that it is a dynamic system involving erosion, sediment slumping and drainage, and contraction of clay, which is a major component of the sediment, etc. The authors are very clear that flooding is not a viable option for the movement of skeletal elements in the cave. However, they do not mention other processes such as erosion or sediment slumping, that are known to occur and could be responsible for moving sediment and fossils in each chamber of the cave. They also do not consider carnivore involvement which has been suggested by Val (2016) and Egeland et al. (2018). Such processes could naturally transport bodies, shift them around, and sediment erosion could bury them. The articulation of some skeletal elements is a major argument for intentional burial, yet within the cave substructure, articulated bones are often commingled with disarticulated elements from the same or different individuals. This same situation exists in the features included in this paper. It does appear that some skeletal material was covered in sediment before decomposition and remains in articulation, but bodies decompose at different rates, and can decompose slowly, especially in environments that lack insects (see Simmons et al. 2010 Journal of Forensic Sciences https://doi-org.aurarialibrary.idm.oclc.org/10.1111/j.1556-4029.2009.01206.x). Wiersma et al., 2019 describe the cave system as very humid, but dry due to little standing water, mildly acidic, with an average temperature today of 18{degree sign}C and a minimum of 12{degree sign}C over the last million years. The starting null hypothesis should be that the bodies were naturally covered in sediment. Intentional burial requires extraordinary circumstances and requires multiple lines of solid evidence to support the hypothesis. In testing for natural burial processes, the rate of body decomposition should be reconstructed given the environmental parameters of the cave.

In keeping with supporting their starting hypothesis that Homo naledi intentionally buried individuals in the cave, the authors conclude that "A parsimonious explanation for this configuration of skeletal remains is that these remains may be a palimpsest of burials that have sequentially disrupted each other. In this hypothesis, early burials were disturbed when pits were dug for subsequent burials. Other occurrences of remains outside of the Dinaledi Chamber and Hill Antechamber (Hawks et al., 2017; Brophy et al., 2021) are discussed as possible evidence of mortuary practices in SI 4.2. Instances where parts of individuals occur in remote narrow passages cannot be explained as a result of carnivore or water transport (Elliott et al., 2021; Brophy et al., 2021), making it necessary to consider that H. naledi may have placed these partial remains in these locations, possibly representing a form of funerary caching." After reviewing the evidence presented in the current manuscript, it is not clear why this is a parsimonious explanation. The authors have repeatedly described how incredibly challenging it is to get into and out of this cave system and all of its chambers. How could any species, even small bodied species, drag/pull/shove dead bodies through small crevasses, shove or drop them down a narrow shoot, continue to move through the hill antechamber to the Dineledi chamber and bury bodies? It is not impossible but given the previously published descriptions of the dynamic process of sedimentation movement in the cave it is certainly not a parsimonious explanation. To support this will take many more lines of evidence than presented here such as micromorphological analysis of the overall cave system and each feature (discussed in the supplementary information but briefly), full detailed reconstruction of sediment, water, fossil, and debris movement throughout the cave system coupled with reconstructions of body decomposition rates. Scientifically precise computer-generated reconstructions of all of this are possible working with specialists affiliated with National Geographic. An analysis also needs to start by testing a null hypothesis, not deciding on the conclusion and setting out to "prove" it.

Reviewer #2 (Public Review):

In this study (Berger et al.), geological and fossil data from the Rising Star Cave System in South Africa are presented to provide evidence for intentional burials of Homo naledi individuals. The authors focus on describing and interpreting what they refer to as "delimited burial features." These features include two located on the floor of the Dinaledi Chamber (referred to as 'Dinaledi Features' 1 and 2) and one from the floor of the Hill Antechamber.

'Dinaledi Feature 1' consists of a collection of 108 skeletal elements recovered from sub-unit 3b deposits. These remains are believed to primarily represent the remains of a single adult individual, along with at least one additional juvenile individual. Although additional anatomical elements associated with 'Dinaledi Feature 1' are mentioned, they are not described as they remain unexcavated. The study states that the spatial arrangement of the skeletal remains is indicative of the primary burial of a fleshed body. On the other hand, 'Dinaledi Feature 2' is not extensively discussed, and its complete extent was not thoroughly investigated.

Regarding the Hill Antechamber feature, it was divided into three separate plaster jackets for removal from the excavation. Through micro-CT and medical CT scans of these plaster jackets, a total of 90 skeletal elements and 51 dental elements were identified. From these data, three individuals were identified, along with a fourth individual described as significantly younger. Individuals 1 and 2 are classified as juveniles.

I feel that there is a significant amount of missing information in the study presented here, which fails to convince me that the human remains described represent primary burials, i.e. singular events where the bodies are placed in their final resting places. Insufficient evidence is provided to differentiate between natural processes and intentional funerary practices. In my opinion, the study should include a section that distinguishes between taphonomic changes and deliberate human modifications of the remains and their context, as well as reconstruct the sequence and timeline of events surrounding death and deposition. A deliberate burial involves a complex series of changes, including decomposition of soft tissues, disruption of articulations between bones, and the sequence of skeletonization. While the geological information is detailed, the archaeothanatological reasoning (see below) is largely absent and, when presented, it lacks clarity and unambiguousness.

My main concern is that the study does not apply or cite the basic principles of archaeothanatology, which combines taphonomy, anatomy, and knowledge of human decomposition to interpret the arrangement of human bones within the Dinaledi Chamber and the Hill Antechamber. Archaeothanatology has been developed since the 1970s (see Duday et al., 1990; Boulestin and Duday, 2005; Duday and Guillon, 2006) and has been widely used by archaeologists and osteologists to reconstruct various aspects such as the original treatment of the body, associated mortuary practices, the sequence of body decomposition, and the factors influencing changes in the skeleton within the burial.

Specifically, the study lacks a description of the relative sequence of joint disarticulation during decomposition and the spatial displacement of bones. A detailed assessment of the anatomical relationships of bones, both articulated and disarticulated, as well as the direction and extent of bone displacement, is missing. For instance, while it is mentioned that "many elements are in articulation or sequential anatomical position," a comprehensive list of these articulated elements and their classification (as labile or not) is not provided.

Furthermore, the patterns described are not illustrated in sufficient detail. If Homo naledi was deliberately buried, it would be crucial to present illustrations depicting the individuals in their burial positions, as well as the representation and proportions of the larger and smaller anatomical elements for each individual. While Figure 2B provides an overall view of 'Dinaledi Feature 1,' it is challenging to determine the relationships of bones, whether articulated or disarticulated, in Figures 2C or 2D. Such information is essential to determine whether the bones are in a primary or secondary position, differentiate between collective and multiple burials, ascertain the body's stage of decomposition at the time of burial, identify postmortem and post-depositional manipulation of the body and grave (e.g., intentional removal of bodies/body parts), and establish whether burial occurred immediately after death or was delayed.

Moreover, the study does not address bone displacements within secondary voids created after the decomposition of soft tissues, nor does it provide assessments of the position of bones within or outside of the original body volume. Factors such as variations in soft tissue volume between individuals of different sizes/corpulence, and the progressive filling (i.e., sediment continually fills newly formed voids) or delayed filling (causing the 'flattening' of the ribcage and 'hyper-flexed' burials, for instance) of secondary open spaces with sediment over time should also be discussed.

In conclusion, while I acknowledge the importance of investigating potential deliberate burials in Homo naledi, I do not think that in its present form, the evidence presented in this study is as robust as it should be.

Reviewer #3 (Public Review):

This paper provides new information on the Dinaledi Chamber at the Rising Star Cave System. In short, a previously excavated area was expanded and resulted in the discovery of a cluster of bones appearing to be of one individual, a second similar cluster, and a third cluster with articulated elements (though with several individuals). Two of these clusters are argued to be intentionally buried individuals (the third one has not been investigated) and thus Homo naledi not only placed conspecifics in deep and hard to reach parts of caves but also buried them (apparently in shallow graves). This would be the oldest evidence of intentional burial. The main issue with the paper is that the purported burials were not fully excavated. Two are still in the ground, and one was removed in blocks but left unexcavated. As burials are mostly about sediments, it means the authors are lacking important lines of evidence. Instead, they bring other lines of argument as outlined below. While their preferred scenario is possible, there are important issues with the evidence as presented and they are severely hampered by the lack of detailed archaeological and geoarchaeological information both from the specific skeletal contexts and more generally from the chamber (because in fact the amount of excavation conducted here is still quite limited in scope). I also found that while the presentations of the various specialists in the team was quite good, the integration of these contributions into the main text was not. In particular, the geology of the cave system and the chamber need (especially what is known of the depositional and post-depositional processes) need to be better integrated into the presentation of the archaeology and the interpretation of the finds.

Often times the presence of articulated or mostly articulated skeletons is used to argue for intentional burial. This argument, however, is based on the premise that if not buried, these skeletons would have otherwise become disarticulated. Normally disarticulation would happen as a result of subsequent use of the site by hominins (e.g. purported burials in Neandertal cave sites) or by carnivores scavenging the body. Indeed this latter point is why bodies are buried so deeply in many Western societies (i.e. beyond the reach and smell of carnivores). Bodies can also be disarticulated by natural processes of deposit and erosion.

However, here in the case of the Dinaledi Chamber, we apparently don't have any of these other processes. The chamber was not used by carnivores and it was not a living area where H. naledi would have frequently returned and cleared out the space. As for depositional processes, it is more complex, but it is clear from Wiersma et al. that there is a steady, constant movement of these sediments towards drains. They also think that this process can account for the mix of articulated and non-articulated elements in the cave. Importantly, that same paper makes the argument that the formation of these sediments is not the result of water movement and that the cave has been dry since the formation of this deposit. So bodies lying on the surface and slowly covered by the formation of the deposit and slowly moving towards the drains could perhaps account for the pattern observed, meaning burial is not needed to account for articulations (note that more information on fabrics would be good in this context - orientation analysis of surface finds or of excavated finds is either completely lacking or minimal - see figure 13b and c report orientations on 79 bones of unknown context that appear to show perhaps elevated plunge angles and some slightly patterning in bearing but there is no associated statistics or text explaining the significance).

So, unless the team can provide some process that would have otherwise disarticulated these skeletons after the bodies arrived here and decomposed, their articulated state is not evidence of burial (no more than finding an articulated or mostly articulated bear skeleton deep in a European cave would suggest that it was buried).

As for the elemental analysis, what I understood from the paper is that the sediment associated with bones is different from the sediment not associated with bones. It is therefore unsurprising that the sediment associated with the reported skeletons clusters with sediments with bones. The linking argument for why this makes this sediment pit fill is unclear to me. Perhaps it is there, but as written I didn't follow it.

What the elemental analysis could suggest, I think, is that there has not been substantial reworking of the sediments (as opposed to the creep suggested by Wiersma et al.) since the bones leached these minerals into the sediment. What I don't know, and what is not reported, is how long after deposition we can expect the soil chemistry to change. If this elemental analysis were extended in a systematic way across the chamber (both vertically and horizontally) after more extensive excavations, I could see it perhaps being useful for better understanding the site formation processes and depositional context. As it is now, I did not see the argument in support of a burial pit.

The other line of evidence here is that some bones are sediment supported. The argument here is that when a body decomposes, bones that were previously held in place by soft tissues will be free to move and will shift their position. How the bones shift will differ depending on whether the body is surrounded by matrix (as they argue here in an excavated burial pit) or whether it is in the open (say, for instance, in a coffin) (and there are other possibilities as well - for instance wrapped in a shroud). Experiments have also shown the order in which the tendons, for instance, decompose and therefore which bones are likely to be free to move first or last.

I will note that this literature is poorly cited. I think the only two papers cited for how bodies decompose are Roksandic 2002 and Mickleburgh and Wescott 2019. The former is a review paper that summarizes a great many contexts that are clearly not appropriate here, and it generally makes the point that it is difficult to sort out, and it notes that progressively filled is an additional alternative to not buried/buried. The other looks at experimental data of bodies decomposing without being buried. In the paper here, this citation is used to argue that the body must have been buried. I don't see the linking argument at all. And the cited paper is mostly about how complicated it is to figure this all out and how many variables are still unaccounted for (including the initial positioning of the body and the consumption of the body by insects - something that is attested to at Naledi - plus snails - see not just Val but also Wiersma et al. and I think the initial Dirk et al. paper).

So the team here instead simply speaks of how the body decomposes in burials as if it is known. For the Feature 1 skeleton, the authors note that the ribs are "apparently" sediment supported and that a portion of the partial cranium is vertical or subvertical and sediment supported. For both of these, the figures show it very poorly. We really have to take their word for it. Second, I would have liked to have seen some reference and comparison to the literature for how the ribs should be in sediment burial cases. For the cranium, seems like a broken cranium resting on a surface will have vertical aspects regardless of sediment support. To the contrary, the orientation of the cranium will change depending on whether there is sediment holding it in place or not. But that argument is not made here. It is very hard from the figures to have a detailed idea of how these skeletons are oriented in the sediments, to know which elements are in articulation, which are missing, etc.

In the case of the Hill Antechamber Feature, an additional argument is made about the orientation of the finds in relation to the natural stratigraphy in this location. The team argues that the skeleton is lying more horizontally than the sediments and that in fact the foot is lying against the slope. First, there is no documentation of the slope of the layers here (e.g. a stratigraphic profile with the layers marked or a fabric analysis). There is a photo in the SI that says it shows sloping, but it needs some work. Second, this skeleton was removed in three blocks and then scanned. So the position of the skeleton is being worked out separate from its context. This is doable, but I would have liked to have seen some mention of how the blocks were georeferenced in the field and then subsequently in the lab and of how the items inside the block (i.e. the data coming from the CT scanner) were then georeferenced. I can think of ways I would try to do this, but without some discussion of this critical issue, the argument presented in Figure 10c is difficult to evaluate. Further, even if we accept this work, it is hard for me to see how the alignment of the foot is 15 degrees opposite the slope (the figure in the SI is better). It is also hard to understand the argument that the sediment separating the lower limb from the torso means burial. The team gives the explanation that if the body was in an open pit it would have been flat with no separation. Maybe. I mean I guess if the pit was flat. But there is no evidence here of a pit (at all). And what if the body was stuffed down the chute and was resting on a slope and covered with additional sediments from the chute (or additional bodies) as it decomposed? It seems that this should be the starting point here rather than imagining a pit.

One of the key pieces of evidence for demonstrating deliberate burial is the recognition of a pit. Pits can be identified because of the rupture they create in the stratigraphy when older sediments are brought to the surface, mixed, and then refilled into the pit with a different color, texture, compaction, etc. In some homogenous sediments a pit can be hard to detect and in some instances post-depositional processes (e.g. burrowing) can blur the distinction between the pit and the surrounding sediments. But the starting point of any discussion of deliberate burial has to be the demonstration of a pit. And I don't see it here. It might just be that the figures need to be improved. But I am skeptical because the team has taken the view that these finds can't be excavated. While I appreciate the scanning work done on the Antechamber find, it is not the same as excavating. Same comment for Features 1 and 2.

In short, my view is that they have an extremely interesting dataset. That H. naledi buried their dead here can't be excluded based on the data, but neither is it supported here. My view is that this paper is premature and that more excavation and the use of geoarchaeological techniques (especially micromorphology) are required to sort this out (or go a long way towards sorting it out).

Reviewer #4 (Public Review):

Berger et al. 2023a argues that Homo naledi intentionally buried their dead within the Rising Star cave system by digging pits and covering the bodies with infilled sediment. The authors identified two burials: Dinaledi Feature 1 from the Dinaledi Chamber, and the Hill Antechamber Feature from the Hill Antechamber. The evolutionary and behavioral implications for such behavior are highly significant and would be the first instance of a relatively small-brained hominin engaging is complex behavior that is often found in association with Homo sapiens and Homo neanderthalensis. Thus, the scientific rigor to validate these findings should be of the highest quality, and thus, provide clear documentation of intentional burial. In an attempt to meet these standards, the authors stated a series of tests that would support their hypothesis of intentional burials in the Rising Star Cave system:

"The key observations are (1) the difference in sediment composition within the feature compared to surrounding sediment; (2) the disruption of stratigraphy; (3) the anatomical coherence of the skeletal remains; (4) the matrix-supported position of some skeletal elements; and (5) the compatibility of non-articulated material with decomposition and subsequent collapse." (page 5)

To find support for the first (1) test, the authors collected sediment samples from various locations within the Rising Star Cave system, including sediment from within and outside Dinaledi Feature 1. However:

• The authors did not select sediment samples from within the Hill Antechamber Feature, so this test was only used to assess Dinaledi Feature 1.

• The sediment samples were analyzed using x-ray diffraction (XRD) and x-ray fluorescence (XRF) to test the mineralogy and chemistry of the samples from within and outside the feature. The XRF results were presented as weighted percentages (not intensities) with no control source reported. The weighted percentages were analyzed using a principal components analysis (PCA) while the particle-size distribution was analyzed using GRADISTAT statistics package and the Folk and Ward Method to summarize "mean grain size, sorting, skewness and kurtosis in addition to the percentages of clay, silt and sand in each sample." (page 28).

• The PCA results were reported solely as a biplot without showing the PC scores projected into the loading space, which is unusual and does not present the data accurately. Instead, the authors present the scores of a single component (PC2, figure 3) because the authors interpreted this component as "distinctly delineates fossil-bearing sediments from sterile sediments based on the positive loadings of P and S" (Page 6). However, the supplementary table that reports XRF bulk chemistry results as a weighted percentage of minerals within each sample (SI Table 1) shows mostly an absence of data for both Na and S. Since Na is at the lower end of detection limits for the method, and S seems to just be absent from the list, the intentions of the authors for showing the inclusion of these elements in their PCA results is unclear. Given that this is the author's primary method for demonstrating a burial, this issue is particularly concerning and requires additional attention.

• Regardless of the missing data, this reviewer attempted to replicate the XRF PCA results using the data provided in SI Table 1 and was unsuccessful. The samples that were collected from within the feature (SB) cluster with samples collected from sterile sediments and other locations around the cave system. Thus, these results are not replicable as currently reported.

• Visual comparisons of sediment grain size, shape, and composition were qualitatively summarized. Grain size was plotted as a line graph and is buried as supplemental Figure S13 showing sample by color and area, but these results do not distinguish samples from WITHIN the burial compared to OUTSIDE the burial as the authors state in the methods as a primary goal.

To test the second (2) aim, the "stratigraphy" was primarily described in text.

• For Dinaledi Feature 1, the authors state that the layer around Feature 1 "is continuous in the profile immediately to the east of the feature; it is disrupted in the sediment profile at the southern extent of the feature (fig. 3b)." Upon examination of figure 3b, the image shows an incredibly small depiction of the south (?) profile view with an extremely large black box overlaying a large portion of the photograph containing a small 5 cm scale. Visually, there is no difference in the profile that would suggest a disruption in the form of a pit. The LORM (orange-red mud layer) does seem to become fragmentary, but no micromorphological analysis was conducted on this section to provide an evaluation of stratigraphic composition. Also, by only excavating a portion of the feature, the authors were unable to adequately demonstrate the full extent of this feature.

• The authors attempt to describe "a bowl-shaped concave layer of clasts and sediment-free voids make up the bottom of the feature" (page 13) and refer to figures and supplementary information that do not depict any stratigraphic profile. Moreover, the authors state that "the leg, foot, and adjacent [skeletal?] material cut across stratigraphy" indicating that the skeleton is orientated on a flat plane against the surrounding stratigraphy that is "30{degree sign} slope of floor and underlying strata" (page 51, fig. 10c captions). There is no mention of infilled sediment from a pit and how this relates to the skeleton or the slope of the floor. It is therefore extremely unclear what the authors are meaning to describe without any visual or micromorphological supplementation to demonstrate a "bowl-shaped concave layer".

The third (3) test was to evaluate the anatomical coherence of the skeletal remains using macro- and micro-CT (computed tomography) of the Hill Antechamber Feature that was removed during excavation. To visually assess the anatomy of the Dinaledi Feature 1 burial, the authors describe the spatial relationship of skeletal elements as they were being excavated but halted partway through the excavation.

• The authors do not provide any documentation (piece-plotting, 3D rendering of stages of excavation, etc.) of the elements that were removed from the Dinaledi Feature. Figure 4 and SI Fig. S22 show the spatial relationship between identifiable skeletal elements that remain in the Feature. However, in Fig. 4, it is unclear why the authors chose to plot 2023-2014 excavated material along with material reported here, and it's even more difficult to understand the anatomical positioning of the elements given their color and point size choices. Although, the authors do provide a 3D rendering of the unexcavated remains showing some skeletal cohesion, apart from the mandible and teeth being re-located near the pelvis (Fig. 9). That said, it is very difficult to visually confirm the elements from this model or understand the original placement of the skeleton.

• 3D renderings of the Hill Antechamber feature skeletal material is clearly shown in SI Fig. S26. Contrary to what the authors state in text, there is a rather wide dispersal and rearrangement of elements for a "burial" that is theoretically protected from scavengers and other agents that would aid in dispersing bone from the surface. The authors do not offer any alternatives to explain disturbance, such as human activity, which clearly took place.

• Moreover, there does not appear to be any intentional arrangement of limbs that may suggest symbolic orientation of the dead (another line of evidence often used to support intentional burial but omitted by the authors). Thus, skeletal cohesion is not enough evidence to support the hypothesis of an intentional burial.

The fourth (4) test was attempted by evaluating whether some elements were vertically aligned from 3D reconstructed models of Hill Antechamber Feature and a photogrammetric model of the Dinaledi Feature 1. The authors state that "the spatial arrangement of the skeletal remains is consistent with primary burial of the fleshed body" (page 8 in reference to Dinaledi Feature 1) without providing any evidence, qualitative or quantitative, that this is the case for either burial.

Since this reviewer was unable to understand the fifth (5) test as it was written by the authors, I am unable to comment on the evidence to support this test and will default to the other reviewers for evaluation of this claim.

In addition to a lack of evidence to support the claims of intentional burial, this paper was also written extremely poorly. For example, the authors often overused 'persuasive communication devices' (see eLife article, https://elifesciences.org/articles/88654) to mislead readers:

"During this excavation, we recognized that the developing evidence was suggestive of a burial, due to the spatial configuration of the feature and the evidence that the excavated material seemed to come from a single body." (page 5)

As an opening statement to introduce Dinaledi Feature 1, the authors state the interpretation and working hypothesis as fact before the authors present any evidence. This is known as "HARKing" and "gives the impression that a hypothesis was formulated before data were collected" (Corneille et al. 2023). This type of writing is pervasive throughout the manuscript and requires extensive editing. I recommend that the authors review the article provided by eLife (https://elifesciences.org/articles/88654) and carefully review the manuscript. Moreover, as this text demonstrates, the authors’ word choice is indicative of storytelling for a popular news article instead of a scientific paper. I highly suggest that the authors review the manuscript carefully and present the data prior to giving conclusions in a clear and concise manner.

Moreover, the writing structure is inconsistent. Information that should be included in results is included in the methods, text in the results should be in discussions, and so forth. This inconsistency is pervasive throughout the entire manuscript, making it incredibly difficult to adequately understand what the authors had done and how the results were interpreted.

Finally, the "artifact" that was described and visualized using CT models is just that - a digitally colored model. The object in question has not been analyzed. Until this object is removed from the dirt and physically analyzed, this information needs to be removed from the manuscript as there is nothing to report before the object is physically examined.

Overall, there is not enough evidence to support the claim that Homo naledi intentionally buried their dead inside the Rising Star Cave system. Unfortunately, the manuscript in its current condition is deemed incomplete and inadequate, and should not be viewed as finalized scholarship.

Public Review:

In this article, a novel technique allowing the linking of viral transcription levels and progeny virion production is presented. Barcoded libraries of an H1N1 influenza virus (two genes were barcoded near the 3'end) were used to infect cells using an experimental approach ensuring that, in the low multiplicity of infection condition, each cell is infected by one virion and that nearly every virion has a unique barcode. This allows then, upon single-cell RNA sequencing and sequencing of the supernatants, to infer back the cells that were producing certain barcoded viruses. Assessing detection frequencies of barcodes in the single-cell sequencing and in the sequencing of the supernatants allows us to compare the levels of viral transcription and progeny virion production.

Observations that viral transcription levels are very heterogenous at the single-cell level are not novel, but reinforce those from previous studies. The major findings of this study are (i) progeny virion production is also very heterogenous, i.e., a few cells produce most of the progeny virions and (ii) there is a poor correlation between viral transcription levels and progeny virion production at the single-cell level.

Strengths:<br /> The article is very well written, the experimental choices are very well justified and the methods are very detailed, allowing the possibility of reproducing the work performed in this study. The conclusions are very well supported by the data and the limitations of the study and how those might influence the conclusions are also clearly explained. In addition, several experimental caveats, such as PCR cross-overs in next-generation sequencing and cell multiplets in single-cell sequencing, were well accounted for, which is not always the case in studies using these techniques.

Weaknesses:<br /> It seems that the results presented here are from one single experiment. How reproducible are the results?

As explained in the article, it is important that nearly every virion has a unique barcode. This was assessed by sequencing the barcodes in the virus libraries. Between 92% to 96% of the barcodes were unique. With this information, it should be possible to assess whether non-unique barcodes were detected in infected cells, and if yes, remove these from the downstream analysis.

It seems like all the information available in this very rich dataset was not fully exploited. For instance, Figure 5C suggests that cells missing the expression of one viral gene might still be able to produce progeny viruses. It would be interesting to have the information regarding which gene was not expressed in these cells.

The introduction and discussion are rather short and the article could benefit from expanding them. Additional speculations about viral or cellular factors (e.g. differences in innate immune responses, differences in cell division status) that might explain the observed heterogeneity, both at the viral transcription and viral progeny virus production levels, would be interesting.

Reviewer #3 (Public Review):

This study explores how condensin and telomere proteins cooperate to facilitate sister chromatid disjunction at chromosome ends during anaphase. Building upon previous results published by the same group (Reyes et al. 2015, Berthezene et al. 2020), the authors demonstrate that condensin is essential for sister telomere disjunction in anaphase in fission yeast. The primary role of condensin appears to be counteracting cohesin, which holds sister telomeres together. Furthermore, condensin is found to be enriched at telomeres, and this enrichment partially relies on Taz1, the principal telomere factor in S. pombe. The loss of Taz1 does not cause an obvious defect in sister telomere disjunction, which prevents drawing strong conclusions about its role in this process.

Reviewer #1 (Public Review):

Colin et al demonstrated that condensin is a key factor for the disjunction of sister-telomeres during mitosis and proposed that it is due to that condensin restrains the telomere association of cohesin. The authors first showed that condensin binds telomeres in mitosis evidenced by ChIP-qPCR and calibrated ChIP-seq. They further demonstrated that compromising condensin's activity leads to a failure in the disjunction of telomeres, with convincing cytological and HI-seq evidence. Two telomeric proteins Taz1 and Mit1 were identified to specifically regulate the telomere association of condensin. Deletion of these genes decreased/increased condensin's telomere association and exacerbated/remedied the defected telomere disjunction in a condensin mutant, echoing the role of condensin in telomere disjunction. They proposed that the underlying mechanism is that condensin inhibits cohesin's accumulation at telomeres. However, the evidence for this claim might need to be further strengthened. Nevertheless, this study uncovered a novel role of condensin in the separation of telomeres of sister chromosomes and open a question of how condensin regulates the structure of chromosomal ends.

Reviewer #2 (Public Review):

This manuscript presents a comprehensive investigation into the role of condensin complexes in telomere segregation in fission yeast. The authors employ chromatin immunoprecipitation analysis to demonstrate the enrichment of condensin at telomeres during anaphase. They then use condensin conditional mutants to confirm that this complex plays a crucial role in sister telomere disjunction as well as the unclustering of telomeric regions from the preceding Rabl configuration. Interestingly, they show that condensin's role in telomere disjunction is unlikely related to catenation removal but rather related to the organization of telomeres in cis and/or the elimination of structural constraints or proteins that hinder separation.

The authors also investigate the regulation of condensin localization to telomeres and reveal the involvement of the shelterin subunit Taz1 in promoting condensin's association with telomeres while demonstrating that the chromatin remodeler Mit1 prevents excessive loading of condensin onto telomeres. Finally, they show that cohesin acts as a negative regulator of telomere separation, counteracting the positive effects of condensin.

Overall, the manuscript is well-executed, and the authors provide sufficient supporting evidence for their claims. There are a couple of aspects that arise from this study that when fully elucidated will lead to a mechanistic understanding of important biological processes. For instance, the exact mechanism by which Taz1 affects condensin loading or the mechanistic link between cohesin and condensin, especially in the context of their opposing roles, are exciting prospects for the future and it is possible that future work within the context of telomeres might provide valuable insights into this question.

Another crucial point emphasized by the manuscript is that the role of condensin in telomere segregation extends beyond facilitating catenation removal.

Reviewer #2 (Public Review):

The authors have addressed most of the concerns. Yet, I still think the authors should at least mention in the article the residues involved in the intra-pore lipid binding pockets for further experimental validation (not only for those residues involve in disease). This is important because the lipid-like density information usually does not come integrated into the PDB structures, so it is not easily accessible for non-structural biologists. The structural data seems solid, and the MD data supports the notion that the GJC is in a putative close state.

Reviewer #1 (Public Review):

Gap junctions, formed from connexins, are important in cell communication, allowing ions and small molecules to move directly between cells. While structures of connexins have previously been reported, the structure of Connexin 43, which is the most widely expressed connexin and is important in many physiological processes was not known. Qi et al used cryo-EM to solve the structure of Connexin 43. They then compared this structure to structures of other connexins. Connexin gap junctions are built from two "hemichannels" consisting of hexamers of connexins. Hemichannels from two opposing cells dock together to form a complete channel that allows the movement of molecules between cells. N-terminal helices from each of the 6 subunits of each hemichannel allow control of whether the channels are open or closed. Previously solved structures of Cx26 and Cx46/50 have the N-termini pointing down into the pore of the protein leaving a central pore and so these channels have been considered to be open. The structure that Qi et al observed has the N-termini in a more raised position with a narrower pore through the centre. This led them to speculate whether this was the "closed" form of the protein. They also noted that, if only the protein was considered, there were gaps between the N-terminal helices, but these gaps were filled with lipid-like molecules. They, therefore, speculated that lipids were important in the closure mechanism. To address whether their structure was open or closed with respect to ions they carried out molecular dynamics studies, and demonstrated that under the conditions of the molecular dynamics ions did not traverse the channel when the lipids were present.

Strengths:

The high resolution cryo-EM density maps clearly show the structure of the protein with the N-termini in a lateral position and lipid density blocking the gaps between the neighbouring helices. The conformation that they observe when they have solved the structure from protein in detergent is also seen when they reconstitute the protein into nanodiscs, which is ostensibly a more membrane-like environment. They, therefore, would appear to have trapped the protein in a stable conformational state.<br /> The molecular dynamics simulations are consistent with the channel being closed when the lipid is present and raises the possibility of lipids being involved in regulation.<br /> A comparison of this structure with other structures of connexin channels and hemichannels gives another representation of how the N-terminal helix of connexins can variously be involved in the regulation of channel opening.

Weaknesses:

While the authors have trapped a relatively stable state of the protein and shown that, under the conditions of their molecular dynamics simulations, ions do not pass through, it is harder to understand whether this is physiologically relevant. Determining this would be beyond the scope of the article. To my knowledge there is no direct evidence that lipids are involved in regulation of connexins in this way, but this is also an interesting area for future exploration. It is also possible that lipids were trapped in the pore during the solubilisation process making it non-physiological. The authors acknowledge this and they describe the structure as a "putative" closed state.

The positions of the mutations in disease shown in Figure 4 is interesting. However, the authors don't discuss/speculate how any of these mutations could affect the binding of the lipids or the conformational state of the protein.

It should also be noted that a structure of the same protein has recently been published. This shows a very similar conformation of the N-termini with lipids bound in the same way, despite solubilising in a different detergent.

Reviewer #1 (Public Review):

The authors report a study, where they have sequenced whole genomes of four individuals of an extinct species of butterfly from western North America (Glaucopsyche xerces), along with seven genomes of a closely related species (Glaucopsyche lygdamus), mainly from museum specimens, several to many decades old. They then compare these fragmented genomes to a high-quality, chromosome-level assembly of a genome of a European species in the same genus (Glaucopsyche alexis). They find that the extinct species shows clear signs of declining population sizes since the last glacial period and an increase in inbreeding, perhaps exacerbating the low viability of the populations and contributing to the extinction of the species.

The study really highlights how museum specimens can be used to understand the genetic variability of populations and species in the past, up to a century or more ago. This is an incredibly valuable tool, and can potentially help us to quickly identify whether current populations of rare and declining species are in danger due to inbreeding, or whether at least their genetic integrity is in good condition and other factors need to be prioritised in their conservation. In the case of extinct species, sequencing museum specimens is really our only window into the dynamics of genomic variability prior to extinction, and such information can help us understand how genetic variation is related to extinction.

I think the authors have achieved their goal admirably, they have used a careful approach to mapping their genomic reads to a related species with a high-quality genome assembly. They might miss out on some interesting genetic information in the unmapped reads, but by and large, they have captured the essential information on genetic variability within their mapped reads. Their conclusions on the lower genetic variability in the extinct species are sound, and they convincingly show that Glaucopyche xerces is a separate species to Glaucopsyche lygdamus (this has been debated in the past).

Reviewer #2 (Public Review):

The Xerces Blue is an iconic species, now extinct, that is a symbol for invertebrate conservation. Using genomic sequencing of century-old specimens of the Xerces Blue and its closest living relatives, the authors hypothesize about possible genetic indicators of the species' demise. Although the limited range and habitat destruction are the most likely culprits, it is possible that some natural reasons have been brewing to bring this species closer to extinction.

The importance of this study is in its generality and applicability to any other invertebrate species. The authors find that low effective population size, high inbreeding (for tens of thousands of years), and higher fraction of deleterious alleles characterize the Xerces colonies prior to extinction. These signatures can be captured from comparative genomic analysis of any target species to evaluate its population health.

It should be noted that it remains unclear if these genomic signatures are indeed predictive of extinction, or populations can bounce back given certain conditions and increase their genetic diversity somehow.

Methods are detailed and explained well, and the study could be replicated. I think this is a solid piece of work. Interested researchers can apply these methods to their chosen species and eventually, we will assemble datasets to study extinction process in many species to learn some general rules.

Several small questions/suggestions:

1) The authors reference a study concluding that Shijimiaeoides is Glaucopsyche. Their tree shows the same, confirming previous publications. And yet they still use Shijimiaeoides, which is confusing. Why not use Glaucopsyche for all these blues?

2) Plebejus argus is a species much more distant from P. melissa than Plebejus anna (anna and melissa are really very close to each other), and yet their tree shows the opposite. What is the problem? Misidentification? Errors in phylogenetic analyses?

3) Wouldn't it be nicer to show the underside of butterfly pictures that reveals the differences between xerces and others? Now, they all look blue and like one species, no real difference.

4) The authors stated that one of five xerces specimens failed to sequence, and yet they show 5 specimens in the tree. Was the extra specimen taken from GenBank?

Reviewer #1 (Public Review):

Multiple sclerosis (MS) is a debilitating autoimmune disease that causes loss of myelin in neurons of the central nervous system. MS is characterized by the presence of inflammatory immune cells in several brain regions as well as the brain barriers (meninges). This study aims to understand the local immune hallmarks in regions of the brain parenchyma that are adjacent to the leptomeninges in a mouse model of MS. The leptomeninges are known to be a foci of inflammation in MS and perhaps "bleed" inflammatory cells and molecules to adjacent brain parenchyma regions. To do so, they use novel technology called spatial transcriptomics so that the spatial relationships between the two regions remain intact. The study identifies canonical inflammatory genes and gene sets such as complement and B cells enriched in the parenchyma in close proximity to the leptomeninges in the mouse model of MS but not control. The manuscript is very well written and easy to follow. The results will become a useful resource to others working in the field and can be followed by time series experiments where the same technology can be applied to the different stages of the disease.

Reviewer #2 (Public Review):

Accumulating data suggests that the presence of immune cell infiltrates in the meninges of the multiple sclerosis brain contributes to the tissue damage in the underlying cortical grey matter by the release of inflammatory and cytotoxic factors that diffuse into the brain parenchyma. However, little is known about the identity and direct and indirect effects of these mediators at a molecular level. This study addresses the vital link between an adaptive immune response in the CSF space and the molecular mechanisms of tissue damage that drive clinical progression. In this short report the authors use a spatial transcriptomics approach using Visium Gene Expression technology from 10x Genomics, to identify gene expression signatures in the meninges and the underlying brain parenchyma, and their interrelationship, in the PLP-induced EAE model of MS in the SJL mouse. MRI imaging using a high field strength (11.7T) scanner was used to identify areas of meningeal infiltration for further study. They report, as might be expected, the upregulation of genes associated with the complement cascade, immune cell infiltration, antigen presentation, and astrocyte activation. Pathway analysis revealed the presence of TNF, JAK-STAT and NFkB signaling, amongst others, close to sites of meningeal inflammation in the EAE animals, although the spatial resolution is insufficient to indicate whether this is in the meninges, grey matter, or both.

UMAP clustering illuminated a major distinct cluster of upregulated genes in the meninges and smaller clusters associated with the grey matter parenchyma underlying the infiltrates. The meningeal cluster contained genes associated with immune cell functions and interactions, cytokine production, and action. The parenchymal clusters included genes and pathways related to glial activation, but also adaptive/B-cell mediated immunity and antigen presentation. This again suggests a technical inability to resolve fully between the compartments as immune cells do not penetrate the pial surface in this model or in MS. Finally, a trajectory analysis based on distance from the meningeal gene cluster successfully demonstrated descending and ascending gradients of gene expression, in particular a decline in pathway enrichment for immune processes with distance from the meninges.

Although these results confirm what we already know about processes involved in the meninges in MS and its models and gradients of pathology in sub-pial regions, this is the first to use spatial transcriptomics to demonstrate such gradients at a molecular level in an animal model that demonstrates lymphoid like tissue development in the meninges and associated grey matter pathology. The mouse EAE model being used here does reproduce many, although not all, of the pathological features of MS and the ability to look at longer time points has been exploited well. However, this particular spatial transcriptomics technique cannot resolve at a cellular level and therefore there is a lot of overlap between gene expression signatures in the meninges and the underlying grey matter parenchyma.

The short nature of this report means that the results are presented and discussed in a vague way, without enough molecular detail to reveal much information about molecular pathogenetic mechanisms.

The trajectory analysis is a good way to explore gradients within the tissues and the authors are to be applauded for using this approach. However, the trajectory analysis does not tell us much if you only choose 2 genes that you think might be involved in the pathogenetic processes going on in the grey matter. It might be more useful to choose some genes involved in pathogenetic processes that we already know are involved in the tissue damage in the underlying grey matter in MS, for which there is already a lot of literature, or genes that respond to molecules we know are increased in MS CSF, although the animal models may be very different. Why were C3 and B2m chosen here?

Strengths:<br /> - The mouse model does exhibit many of the features of the compartmentalized immune response seen in MS, including the presence of meningeal immune cell infiltrates in the central sulcus and over the surface of the cortex, with the presence of FDC's HEVs PNAd+ vessels and CXCL13 expression, indicating the formation of lymphoid like cell aggregates. In addition, disruption of the glia limitans is seen, as in MS. Increased microglial reactivity is also present at the pial surface.<br /> - Spatial transcriptomics is the best approach to studying gradients in gene expression in both white matter and grey matter and their relationship between compartments.<br /> - It would be useful to have more discussion of how the upregulated pathways in the two compartments fit with what we know about the cellular changes occurring in both, for which presumably there is prior information from the group's previous publications.

Limitations:<br /> - EAE in the mouse is not MS and may be far removed when one considers molecular mechanisms, especially as MS is not a simple anti-myelin protein autoimmune condition. Therefore, this study could be following gene trajectories that do not exist in MS. This needs a significant amount of discussion in the manuscript if the authors suggest that it is mimicking MS.<br /> - The model does not have the cortical subpial demyelination typical of MS and it is unknown whether neuronal loss occurs in this model, which is the main feature of cytokine-mediated neurodegeneration in MS. If it does not then a whole set of genes will be missing that are involved in the neuronal response to inflammatory stimuli that may be cytotoxic.<br /> - Visium technology does not get down to single cell level and does not appear to allow resolution of the border between the meninges and the underlying grey matter.<br /> - Neuronal loss in the MS cortex is independent of demyelination and therefore not related to remyelination failure. There does not appear to be any cortical grey matter demyelination in these animals, so it is difficult to relate any of the gene changes seen here to demyelination.<br /> - No mention of how the ascending and descending patterns of gene expression may be due to the gradient of microglial activation that underlies meningeal inflammation, which is a big omission.

Reviewer #3 (Public Review):

In this study, Gadani et al. induced EAE in SJL/J mice and performed a comprehensive spatial transcriptomic analysis in areas of meningeal inflammation during the relapse phase of the disease. The authors found specific enrichment in spatial gene signatures (cluster 11) in the regions of increased contrast-enhancement by MRI (where meningeal extravasation of activated immune cells is observed) that overlap with signatures in the adjacent brain parenchyma, namely the thalamus. Several pathways were similarly upregulated in the meningeal-associated cluster 11 and adjacent parenchymal clusters (like adaptive mediated immunity, and antigen processing and presentation), suggestive of a "leakage" of inflammatory mediators from the meninges into the brain during the re-activation of disease. The tested hypothesis, as well as the data presented in this study, is quite interesting and novel.

Reviewer #1 (Public Review):

In this paper, the authors developed an image analysis pipeline to automatically identify individual ‎neurons within a population of fluorescently tagged neurons. This application is optimized to deal with ‎multi-cell analysis and builds on a previous software version, developed by the same team, to resolve ‎individual neurons from whole-brain imaging stacks. Using advanced statistical approaches and ‎several heuristics tailored for C. elegans anatomy, the method successfully identifies individual ‎neurons with a fairly high accuracy. Thus, while specific to C. elegans, this method can become ‎instrumental for a variety of research directions such as in-vivo single-cell gene expression analysis ‎and calcium-based neural activity studies.‎

The analysis procedure depends on the availability of an accurate atlas that serves as a reference map ‎for neural positions. Thus, when imaging a new reporter line without fair prior knowledge of the ‎tagged cells, such an atlas may be very difficult to construct. Moreover, usage of available reference ‎atlases, constructed based on other databases, is not very helpful (as shown by the authors in Fig 3), ‎so for each new reporter line a de-novo atlas needs to be constructed.‎

I have a few comments that may help to better understand the potential of the tool to become handy:

‎1) I wonder the degree by which strain mosaicism affects the analysis (Figs 1-4) as it was performed on ‎a non-integrated reporter strain. As stated, for constructing the reference atlas, the authors used ‎worms in which they could identify the complete set of tagged neurons. But how sensitive is the ‎analysis when assaying worms with different levels of mosaicism? Are the results shown in the paper ‎stem from animals with a full neural set expression? Could the authors add results for which the ‎assayed worms show partial expression where only 80%, 70%, 50% of the cells population are ‎observed, and how this will affect identification accuracy? This may be important as many non-‎integrated reporter lines show high mosaic patterns and may therefore not be suitable for using this ‎analytic method. In that sense, could the authors describe the mosaic degree of their line used for ‎validating the method.‎<br /> ‎<br /> 2) For the gene expression analysis (Fig 5), where was the intensity of the GFP extracted from? As it has ‎no nuclear tag, the protein should be cytoplasmic (as seen in Fig 5a), but in Fig 5c it is shown as if the ‎region of interest to extract fluorescence was nuclear. If fluorescence was indeed extracted from the ‎cytoplasm, then it will be helpful to include in the software and in the results description how this was ‎done, as a huge hurdle in dissecting such multi-cell images is avoiding crossreads between ‎adjacent/intersecting neurons.‎<br /> ‎<br /> 3) In the same matter: In the methods, it is specified that the strain expressing GCAMP was also used ‎in the gene expression analysis shown in Figure 5. But the calcium indicator may show transient ‎intensities depending on spontaneous neural activity during the imaging. This will introduce a ‎significant variability that may affect the expression correlation analysis as depicted in Figure 5.‎

Reviewer #2 (Public Review):

The authors succeed in generalizing the pre-alignment procedure for their cell identification method to allow it to work effectively on data with only small subsets of cells labeled. They convincingly show that their extension accurately identifies head angle, based on finding auto fluorescent tissue and looking for a symmetric l/r axis. They demonstrate that the method works to identify known subsets of neurons with varying accuracy depending on the nature of underlying atlas data. Their approach should be a useful one for researchers wishing to identify subsets of head neurons in C. elegans, for example in whole brain recording, and the ideas might be useful elsewhere.

The authors also strive to give some general insights on what makes a good atlas. It is interesting and valuable to see (at least for this specific set of neurons) that 5-10 ideal examples are sufficient. However, some critical details would help in understanding how far their insights generalize. I believe the set of neurons in each atlas version are matched to the known set of cells in the sparse neuronal marker, however this critical detail isn't explicitly stated anywhere I can see. In addition, it is stated that some neuron positions are missing in the neuropal data and replaced with the (single) position available from the open worm atlas. It should be stated how many neurons are missing and replaced in this way (providing weaker information). It also is not explicitly stated that the putative identities for the uncertain cells (designated with Greek letters) are used to sample the neuropal data. Large numbers of openworm single positions or if uncertain cells are misidentified forcing alignment against the positions of nearby but different cells would both handicap the neuropal atlas relative to the matched florescence atlas. This is an important question since sufficient performance from an ideal neuropal atlas (subsampled) would avoid the need for building custom atlases per strain.

Reviewer #1 (Public Review):

Summary of the major findings -

1. The authors used saturation mutagenesis and directed evolution to mutate the highly conserved fusion loop (98 DRGWGNGCGLFGK 110) of the Envelope (E) glycoprotein of Dengue virus (DENV). They created 2 libraries with parallel mutations at amino acids 101, 103, 105-107, and 101-105 respectively. The in vitro transcribed RNA from the two plasmid libraries was electroporated separately into Vero and C6/36 cells and passaged thrice in each of these cells. They successfully recovered a variant N103S/G106L from Library 1 in C6/36 cells, which represented 95% of the sequence population and contained another mutation in E outside the fusion loop (T171A). Library 2 was unsuccessful in either cell type.

2. The fusion loop mutant virus called D2-FL (N103S/G106L) was created through reverse genetics. Another variant called D2-FLM was also created, which in addition to the fusion loop mutations, also contains a previously published, evolved, and optimized prM-furin cleavage sequence that results in a mature version of the virus (with lower prM content). Both D2-FL and D2-FLM viruses grew comparably to wild type virus in mosquito (C6/36) cells but their infectious titers were 2-2.5 log lower than wild<br /> type virus when grown in mammalian (Vero) cells. These viruses were not compromised in thermostability, and the mechanism for attenuation in Vero cells remains unknown.

4. Next, the authors probed the neutralization of these viruses using a panel of monoclonal antibodies (mAbs) against fusion loop and domain I, II and III of E protein, and against prM protein. As intended, neutralization by fusion loop mAbs was reduced or impaired for both D2-FL and D2-FLM, compared to wild type DENV2. D2-FLM virus was equivalent to wild type with respect to neutralization by domain I, II, and III antibodies tested (except domain II-C10 mAb) suggesting an intact global antigenic landscape of the mutant virion. As expected, D2-FLM was also resistant to neutralization by prM mAbs (D2-FL was not tested in this batch of experiments).

5. Finally, the authors evaluated neutralization in the context of polyclonal serum from convalescent humans (n=6) and experimentally infected non-human primates (n=9) at different time points (27 total samples). Homotypic sera (DENV2) neutralized D2-FL, D2-FLM, and wild type DENV similarly, suggesting that the contribution of fusion loop and prM epitopes is insignificant in a serotype-specific neutralization response. However, heterotypic sera (DENV4) neutralized D2-FL and D2-FLM less potently than wild type DENV2, especially at later time points, demonstrating the contribution of fusion loop- and prM-specific antibodies to heterotypic neutralization.

Impact of the study-

1. The engineered D2-FL and D2-FLM viruses are valuable reagents to probe antibodies targeting the fusion loop and prM in the overall polyclonal response to DENV.

2. Though more work is needed, these viruses can facilitate the design of a new generation of DENV vaccine that does not elicit fusion loop- and prM-specific antibodies, which are often poorly neutralizing and lead to antibody-dependent enhancement effect (ADE).

3. This work can be extended to other members of the flavivirus family.

4. A broader impact of their work is a reminder that conserved amino acids may not always be critical for function and therefore should not be immediately dismissed in substitution/mutagenesis/protein design efforts.

Evaluating this study in the context of prior literature -

The authors write "Although the extreme conservation and critical role in entry have led to it being traditionally considered impossible to change the fusion loop, we successfully tested the hypothesis that massively parallel directed evolution could produce viable DENV fusion-loop mutants that were still capable of fusion and entry, while altering the antigenic footprint."<br /> ".....Previously, a single study on WNV successfully generated a viable virus with a single mutation at the fusion loop, although it severely attenuated neurovirulence. Otherwise, it has not been generated in DENV or other mosquito-borne flaviviruses"

The above claims are a bit overstated. In the context of other flaviviruses:

- A previous study applied a similar saturation mutagenesis approach to the *full length* E protein of Zika virus and found that while the conserved fusion loop was mutationally constrained, some mutations, including at amino acid residue 106 were tolerated (PMID 31511387).<br /> - The Japanese encephalitis virus (JEV) SA14-14-2 live vaccine strain contains a L107F mutation in the fusion loop (in addition to other changes elsewhere in the genome) relative to the parental JEV SA14 strain (PMID: 25855730).<br /> - For tickborne encephalitis virus (TBEV-DENV4 chimera), H104G/L107F double mutant has been described (PMID: 8331735)

There have also been previous examples of functionally tolerated mutations within the DENV fusion loop:

- Goncalvez et al., isolated an escape variant of DENV 2 using chimpanzee Fab 1A5, with a mutation in the fusion loop G106V (PMID: 15542644). G106 is also mutated in D2-FL clone (N103S/G106L) described in the current study.<br /> - In the context of single-round infectious DENV, mutation at site 102 within the fusion loop has been shown to retain infectivity (PMID 31820734).

Appraisal of the results -

The data largely support the conclusions, but some improvements and extensions can benefit the work.

1. Line 92-93: "This major variant comprised ~95% of the population, while the next most populous variant comprised only 0.25% (Figure 1C)".<br /> What is the sequence of the next most abundant variant?

2. Lines 94-95: "Residues W101, C105, and L107 were preserved in our final sequence, supporting the structural importance of these residues."<br /> L107F is viable in other flaviviruses.

3. Figure 2c: The FLM sample in the western blot shows hardly any E protein, making E/prM quantitation unreliable.

4. Lines 149 -151: "Importantly, D2-FL and D2-FLM were resistant to antibodies targeting the fusion loop. While neutralization by 1M7 is reduced by ~2-logs, no neutralization was observed for 1N5, 1L6, and 4G2 for either variant (Figure 3 A)".

a) Partial neutralization was observed for 1N5, for D2-FL.<br /> b) Do these mAbs cover the full spectrum of fusion loop antibodies identified thus far in the field?<br /> c) Are the epitopes known for these mAbs? It would be useful to discuss how the epitope of 1M7 differs from the other mAbs? What are the critical residues?<br /> d) Maybe the D2-FL mutant can be further evolved with selection pressure with fusion loop mAbs 1M7 +/-1N5 and/or other fusion loop mAbs.

5. It would have been useful to include D2-M for comparison (with evolved furin cleavage sequence but no fusion loop mutations).

6. Data for polyclonal serum can be better discussed. Table 1 is not discussed much in the text. For the R1160-90dpi-DENV4 sample, D2-FL and D2-FLM are neutralized better than wild type DENV2? The authors' interpretation in lines 181-182 is inconsistent with the data presented in Figure 3C, which suggests that over time, there is INCREASED (not waning) dependence on FL- and prM-specific antibodies for heterotypic neutralization.

Suggestions for further experiments-

1. It would be interesting to see the phenotype of single mutants N103S and G106L, relative to double mutant N103S/G106L (D2-FL).<br /> 2. The fusion capability of these viruses can be gauged using liposome fusion assay under different pH conditions and different lipids.<br /> 3. Correlative antibody binding vs neutralization data would be useful.

Reviewer #2 (Public Review):

Antibody-dependent enhancement (ADE) of Dengue is largely driven by cross-reactive antibodies that target the DENV fusion loop or pre-membrane protein. Screening polyclonal sera for antibodies that bind to these cross-reactive epitopes could increase the successful implementation of a safe DENV vaccine that does not lead to ADE. However, there are few reliable tools to rapidly assess the polyclonal sera for epitope targets and ADE potential. Here the authors develop a live viral tool to rapidly screen polyclonal sera for binding to fusion loop and pre-membrane epitopes. The authors performed a deep mutational scan for viable viruses with mutations in the fusion loop (FL). The authors identified two mutations functionally tolerable in insect C6/36 cells, but lead to defective replication in mammalian Vero cells. These mutant viruses, D2-FL and D2-FLM, were tested for epitope presentation with a panel of monoclonal antibodies and polyclonal sera. The D2-FL and D2-FLM viruses were not neutralized by FL-specific monoclonal antibodies demonstrating that the FL epitope has been ablated. However, neutralization data with polyclonal sera is contradictory to the claim that cross-reactive antibody responses targeting the pre-membrane and the FL epitopes wane over time.

Overall the central conclusion that the engineered viruses can predict epitopes targeted by antibodies is supported by the data and the D2-FL and D2-FLM viruses represent a valuable tool to the DENV research community.

Reviewer #1 (Public Review):

Masson et al. leveraged the natural genetic diversity presented in a large cohort of the Diversity Outbred in Australia (DOz) mice (n=215) to determine skeletal muscle proteins that were associated with insulin sensitivity. The hits were further filtered by pQTL analysis to construct a proteome fingerprint for insulin resistance. These proteins were then searched against Connectivity Map (CMAP) to identify compounds that could modulate insulin sensitivity. In parallel, many of these compounds were screened experimentally alongside other compounds in the Prestwick library to independently validate some of the compound hits. These two analyses were combined to score for compounds that would potentially reverse insulin resistance. Thiostrepton was identified as the top candidate, and its ability to reverse insulin resistance was validated using assays in L6 myotubes.

Below are several comments made on the original version of this study, addressed by the authors in the current version:

(1) Please describe the rationale of trypsinizing the tissue prior to mitochondrial isolation.

(2) The authors mentioned that the proteomics data were Log2 transformed and median-normalized. Please provide a bit more details on this, including whether the subjects were randomized.

(3) In Figure 1D, please give the numbers of mice the authors used for the CV comparisons in each group, whether they were of similar age and sex, and whether the differences in CV values were statistically significant

(4) The authors stated in lines 155-157 that proteins negatively associated with Matsuda index were further filtered by presence of their cis-pQTLs. Please provide more explanations to justify this filtering criterion.

(5) Please explain why the first half of the paper focused extensively on the authors' discoveries in the mitochondrial proteome, and how proteins involved in mitochondrial processes (such as complex I) were associated with Matsuda Index, but the final fingerprint list of insulin resistance, which contained 76 proteins, only had 7 mitochondrial proteins.

(6) The authors found that thiostrepton-induced insulin resistance reversal effects were not through insulin signalling. Please list the proteins in the fingerprint list that led to identification of thiostrepton on CMAP, and discuss whether you think that thiostrepton directly or indirectly acts on these protein targets.

Reviewer #2 (Public Review):

In the present study, Masson et al. provide an elegant and profound demonstration of utilization of systems genetics data to fuel discovery of actionable therapeutics. The strengths of the study are many: generation of a novel skeletal muscle genetics proteomic dataset which is paired with measures of glucose metabolism in mice, systematic utilization of these data to yield potential therapeutic molecules which target insulin resistance, cross-referencing library screens from connectivity map with an independent validation platform for muscle glucose uptake and preclinical data supporting a new mechanism for thiostrepton in alleviating muscle insulin resistance. Future studies evaluating similar integrations of omics data from genetic diversity with compound screens, as well as detailed characterization of mechanisms such as thiostrepton on muscle fibers will further inform some remaining questions. In general, the thorough nature of this study not only provides strong support for the conclusions made but additionally offers a new framework for analysis of systems-based data. I had made several comments on the prior submission, all of which have been fully addressed and incorporated.

Reviewer #1 (Public Review):

The authors investigated the molecular correlates in potential neural centers in the Japanese quail brain associated with photoperiod-induced life-history states. The authors simulated photoperiod to attain winter and summer-like physiology and samples of neural tissues at spring, and autumn life-history states, daily rhythms in transcripts in solstices and equinox, and lastly studies FSHb transcripts in the pituitary. The experiments are based on a series of changes in photoperiod and gave some interesting results. The experiment did not have a control for no change in photoperiod so it seems possible that endogenous rhythms could be another aspect of seasonal rhythms that lack in this study. The short-day group does not explain the endogenous seasonal response.

The manuscript would benefit from further clarity in synthesizing different sections. Additionally, there are some instances of unclear language and numerous typos throughout the manuscript. A thorough revision is recommended, including addressing sentence structure for improved clarity, reframing sentences where necessary, correcting typos, conducting a grammar check, and enhancing overall writing clarity.

Data analysis needs more clarity particularly how transcriptome data explains different physiological measures across seasonal life-history states. It seems the discussion is built around a few genes that have been studied in other published literature on quail seasonal response. Extending results on the promotor of DEGs and building discussion is an extrapolating discussion on limited evidence and seems redundant.

Last, I wondered if it would be possible to add an ecological context for the frequent change in the photoperiod schedule and not take account of the endogenous annual response. Adding discussion on ecological relevance would make more sense.

Reviewer #2 (Public Review):

This study is carefully designed and well executed, including a comprehensive suite of endpoint measures and large sample sizes that give confidence in the results. I have a few general comments and suggestions that the authors might find helpful.

1) I found it difficult to fully grasp the experimental design, including the length of light treatment in the three different experiments (which appears to extend from 2 weeks up to 8 weeks). A graphical description of the experimental design along a timeline would be very helpful to the reader. I suggest adding the respective sample sizes to such a graphic, because this information is currently also difficult to keep track of.

2) The authors use a lot of terminology that is second nature to a chronobiologist but may be difficult for the general reader to keep track of. For example, what is the difference between "photoinducibility" and "photosensitivity"? Similarly, "vernal" and "autumnal" should be briefly explained at the outset, or maybe simply say "spring equinox" and "fall equinox."

3) What was the rationale for using only male birds in this study? The authors may want to include a brief discussion on whether the expected results for females might be similar to or different from what they found in males, and why.

4) The authors used the Bonferroni correction method to account for multiple hypothesis testing of measures of testes mass, body mass, fat score, vimentin immunoreactivity and qPCR analyses in Study 1. I don't think Bonferroni is ever appropriate for biological data: these methods assume that all variables are independent of each other, an assumption that is almost never warranted in biology. In fact, the data show clear relationships between these endpoint measures. Alternatively, one might use Benjamini-Hochberg's FDR correction or various methods for calculating the corrected alpha level.

5) The graphical interpretations of the results shown in Figure 1n and Figure 3e, along with the hypothesized working model shown in Figure S5, might best be combined into a single figure that becomes part of the Discussion. As is, I do not think these interpretative graphics (which are well done and super helpful!) are appropriate for the Results section.

Reviewer #3 (Public Review):

It is well known that as seasonal day length increases, molecular cascades in the brain are triggered to ready an individual for reproduction. Some of these changes, however, can begin to occur before the day length threshold is reached, suggesting that short days similarly have the capacity to alter aspects of phenotype. This study seeks to understand the mechanisms by which short days can accomplish this task, which is an interesting and important question in the field of organismal biology and endocrinology.

The set of studies that this manuscript presents is comprehensive and well-controlled. Many of the effects are also strong and thus offer tantalizing hints about the endo-molecular basis by which short days might stimulate major changes in body condition. Another strength is that the authors put together a compelling model for how different facets of an animal's reproductive state come "on line" as day length increases and spring approaches. In this way, I think the authors broadly fulfill their aims.

I do, however, also think that there are a few weaknesses that the authors should consider, or that readers should consider when evaluating this manuscript. First, some of the molecular genetic analyses should be interpreted with greater caution. By bioinformatically showing that certain DNA motifs exist within a gene promoter (e.g., FSHbeta), one is not generating robust evidence that corresponding transcription factors actually regulate the expression of the gene in question. In fact, some may argue that this line of evidence only offers weak support for such a conclusion. I appreciate that actually running the laboratory experiments necessary to generate strong support for these types of conclusions is not trivial, and doing so may even be impossible. I would therefore suggest a clear admission of these limitations in the paper.

Second, I have another issue with the interpretation of data presented in Figure 3. The data show that FSHbeta increases in expression in the 8Lext group, suggesting that endogenous drivers likely act to increase the expression of this gene despite no change in day length. However, more robust effects are reported for FSHbeta expression in the 10v and 12v groups, even compared to the 8Lext group. Doesn't this suggest that both endogenous mechanisms and changes in day length work together to ramp up FSHbeta? The rest of the paper seemed to emphasize endogenous mechanisms and gloss over the fact that such mechanisms likely work additively with other factors. I felt like there was more nuance to these findings than the authors were getting into.

Third, studies 1 - 3 are well controlled; however, I'm left wondering how much of an effect the transitions in day length might have on the underlying molecular processes that mediate changes in body condition. While the changes in day length are themselves ecologically relevant, the transitions between day length states are not. How do we know, for example, that more gradual changes in day length that occur over long timespans do not produce different effects at the levels of the brain and body? This seemed especially relevant for study 3, where animals experience a rather sudden change in day length. I recognize that these experimental methods are well described in the literature, and they have been used by endocrinologists for a long time; nonetheless, I think questions remain.

Reviewer #1 (Public Review):

In this study, the authors set out to investigate spatial RNA processing events, specifically alternative splicing and 3' UTR usage, in mouse brain and kidney tissues using ReadZS and SpliZ methodologies on spatial transcriptomics data. The research contributes to understanding tissue-specific gene expression regulation from a spatial perspective. The study introduces a novel approach for analyzing spatial transcriptomics data, allowing for the identification of RNA processing and regulation patterns directly from 10X Visium data. The authors present convincing evidence supporting the identification of novel RNA processing patterns using their methodology, which holds significant implications for researchers in the field of spatial transcriptomics and the study of alternative splicing and 3' UTR usage

The conclusions of the study are mostly well-supported by the data; however, certain aspects could be improved to strengthen the findings.<br /> 1) The conclusions of this study would be strengthened by conducting a more extensive tissue sample analysis and including biological replicates. Additionally, appropriate batch effect corrections should be applied when dealing with biological replicates.<br /> 2) The 3' UTR usage and alternative splicing should be compared among clearly labeled clusters for a more comprehensive analysis.<br /> 3) The authors should clarify their rationale for choosing ReadZS and SpliZ approaches and provide comparisons with other methods to demonstrate the advantages and potential limitations of their chosen methodologies.

Reviewer #2 (Public Review):

The authors applied existing ReadZS and the SpliZ methods, previously developed to analyze RNA process in scRNA-seq data, to Visium data to study spatial splicing and RNA processing events in tissues by Moran's I. The authors showed several example genes in mouse brain and kidney, whose processing are spatially regulated, such as Rps24, Myl6, Gng13.

The paper touches on an important question in RNA biology about how RNA processing is regulated spatially. Both experimental and computational challenges remain to address it. Despite some potentially interesting findings, most claims remain to be validated by orthogonal methods such as RNA FISH and simulations. In addition, the percentage of spatial processing events (splicing in 0.8-2.2% of detected genes, i.e. 8-17 genes and RNA processing in 1.1-5.5% of detected genomic windows, i.e. 57-161 windows) discovered is low. Does it suggest that most of RNA processing events were not spatially regulated across the tissue? Or does it question the assumption of treating spatial transcriptomics data similar to scRNA-seq data? The unique features for ST data, such as mixture of neighboring cells, different capture biases and much smaller number of spots (pseudo cells here), may have significant effects on the power of scRNA-seq based methods, but it is not discussed in the manuscript. The lack of careful evaluation and low discovery rates could limit application of the approach to other tissues and subcellular data.

Reviewer #1 (Public Review):

Trypanosoma brucei undergoes antigenic variation to evade the mammalian host's immune response. To achieve this, T. brucei regularly expresses different VSGs as its major surface antigen. VSG expression sites are exclusively subtelomeric, and VSG transcription by RNA polymerase I is strictly monoallelic. It has been shown that T. brucei RAP1, a telomeric protein, and the phosphoinositol pathway are essential for VSG monoallelic expression. In previous studies, Cestari et al. (ref. 24) have shown that PIP5Pase interacts with RAP1 and that RAP1 binds PI(3,4,5)P3. RNAseq and ChIPseq analyses have been performed previously in PIP5Pase conditional knockout cells, too (ref. 24). In the current study, Touray et al. did similar analyses except that catalytic dead PIP5Pase mutant was used and the DNA and PI(3,4,5)P3 binding activities of RAP1 fragments were examined. Specifically, the authors examined the transcriptome profile and did RAP1 ChIPseq in PIP5Pase catalytic dead mutant. The authors also expressed several C-terminal His6-tagged RAP1 recombinant proteins (full-length, aa1-300, aa301-560, and aa 561-855). These fragments' DNA binding activities were examined by EMSA analysis and their phosphoinositides binding activities were examined by affinity pulldown of biotin-conjugated phosphoinositides. As a result, the authors confirmed that VSG silencing (both BES-linked and MES-linked VSGs) depends on PIP5Pase catalytic activity, but the overall knowledge improvement is incremental. The most convincing data come from the phosphoinositide binding assay as it clearly shows that N-terminus of RAP1 binds PI(3,4,5)P3 but not PI(4,5)P2, although this is only assayed in vitro, while the in vivo binding of full-length RAP1 to PI(3,4,5)P3 has been previously published by Cestari et al (ref. 24) already. Considering that many phosphoinositides exert their regulatory role by modulating the subcellular localization of their bound proteins, it is reasonable to hypothesize that binding to PI(3,4,5)P3 can remove RAP1 from the chromatin. However, no convincing data have been shown to support the author's hypothesis that this regulation is through an "allosteric switch". Therefore, the title should be revised.

There are serious concerns about many conclusions made by Touray et al., according to their experimental approaches:<br /> 1. The authors have been studying RAP1's chromatin association pattern by ChIPseq in cells expressing a C-terminal HA tagged RAP1. According to data from tryptag.org, RAP1 with an N-terminal or a C-terminal tag does not seem to have identical subcellular localization patterns, suggesting that adding tags at different positions of RAP1 may affect its function. It is therefore essential to validate that the C-terminally HA-tagged RAP1 still has its essential functions. However, this data is not available in the current study. RAP1 is essential. If RAP1-HA still retains its essential functions, cells carrying one RAP1-HA allele and one deleted allele are expected to grow the same as WT cells. In addition, these cells should have the WT VSG expression pattern, and RAP1-HA should still interact with TRF. Without these validations, it is impossible to judge whether the ChIPseq data obtained on RAP1-HA reflect the true chromatin association profile of RAP1.

2. Touray et al. expressed and purified His6-tagged recombinant RAP1 fragments from E. coli and used these recombinant proteins for EMSA analysis: The His6 tag has been used for purifying various recombinant proteins. It is most likely that the His6 tag itself does not convey any DNA binding activities. However, using His6-tagged RAP1 fragments for EMSA analysis has a serious concern. It has been shown that His6-tagged human RAP1 protein can bind dsDNA, but hRAP1 without the His6 tag does not. It is possible that RAP1 proteins in combination with the His6 tag can exhibit certain unnatural DNA binding activities. To be rigorous, the authors need to remove the His6 tag from their recombinant proteins before the in vitro DNA binding analyses are performed. This is a standard procedure for many in vitro assays using recombinant proteins.

3. It is unclear why Nanopore sequencing was used for RNAseq and ChIPseq experiments. The greatest benefit of Nanopore sequencing is that it can sequence long reads, which usually helps with mapping, particularly at genome loci with repetitive sequences. This seems beneficial for RAP1 ChIPseq analysis as RAP1 is expected to bind telomere repeats. However, for ChIPseq, the chromatin needs to be fragmented. Larger DNA fragments from ChIPseq experiments will decrease the accuracy of the final calculated binding sites. Therefore, ChIPseq experiments are not supposed to have long reads to start with, so Nanopore sequencing does not seem to bring any advantage. In addition, compared to Illumina sequencing, Nanopore sequencing usually yields smaller numbers of reads, and the sequencing accuracy rate is lower. The Nanopore sequencing accuracy may be a serious concern in the current study. All telomeres have the perfect TTAGGG repeats, all VSG genes have a very similar 3' UTR, and all 70 bp repeats have very similar sequences. In fact, the active and silent ESs have 90% sequence identity. Are sequence reads accurately mapped to different ESs? How is the sequencing and mapping quality controlled? Furthermore, it is unclear whether the read depth for RNAseq is deep enough.

4. Many statements in the discussion section are speculations without any solid evidence. For example, lines 218 - 219 "likely due to RAP1 conformational changes", no data have been shown to support this at all. In lines 224-226, the authors acknowledged that more experiments are necessary to validate their observations, so it is important for the authors to first validate their findings before they draw any solid conclusions. Importantly, RAP1 has been shown to help compact telomeric and subtelomeric chromatin a long time ago by Pandya et al. (2013. NAR 41:7673), who actually examined the chromatin structure by MNase digestion and FAIRE. The authors should acknowledge previous findings. In addition, the authors need to revise the discussion to clearly indicate what they "speculate" rather than make statements as if it is a solid conclusion.

There are also minor concerns:

1. In the PIP5Pase conditional knockout system, the WT or mutant PIP5Pase with a V5 tag is constitutively expressed from the tubulin array. What's the relative expression level of this allele and the endogenous PIP5Pase? Without a clear knowledge of the mutant expression level, it is hard to conclude whether the mutant has any dominant negative effects or whether the mutant phenotype is simply due to a lower than WT PIP5pase expression level.

2. In EMSA analysis, what are the concentrations of the protein and the probe used in each reaction? The amount of protein used in the binding assay appears to be very high, and this can contribute to the observation that many complexes are stuck in the well. Better quality EMSA data need to be shown to support the authors' claims.

Reviewer #2 (Public Review):

This manuscript by Touray, et al. provides a significant new twist to our understanding of how antigenic variation may be regulated in T. brucei. Key aspects of antigenic variation are the mutually exclusive expression of a single antigen per cell and the periodic switching from expression of one antigen isoform to another. In this manuscript, the authors show, as they have previously shown, that depletion of the nuclear phosphatidylinositol 5-phosphatase (PIP5Pase) results in a loss of mutually exclusive VSG expression. Furthermore, using ChIP-seq, the authors show that the repressor/activator protein 1 (RAP1) binds to regions upstream and downstream of VSG genes located in transcriptionally repressed expression sites and that this binding is lost in the absence of a functional PIP5Pase. Importantly, the authors decided to further investigate this link between PIP5Pase and RAP1, a protein that has previously been implicated in antigenic variation in T. brucei, and found that inactivation of PIP5Pase results in the accumulation of PI(3,4,5)P3 bound to the RAP1 N-terminus and that this binding impairs the ability of RAP1 to bind DNA. Based on these observations, the authors suggest that the levels of PI(3,4,5)P3 may determine the cellular function of RAP1, either by binding upstream of VSG genes and repressing their function, or by not binding DNA and allowing the simultaneous expression of multiple VSG genes in a single parasite.

While I find most of the data presented in this manuscript compelling, there are aspects of Figure 1 that are not clear to me. Based on Figure 1F, the authors claim that transient inactivation of PIP5Pase results in a switch from the expression of one VSG isoform to another. However, I am not exactly sure what the authors are showing in this panel, nor do the data in Figure 1F seem to be consistent with those shown in Figure 1C. Based on Figure 1F, a transient inactivation of PIP5Pase appears to result in an almost exclusive switch to a VSG located in BES12. However, based on Figure 1E, the VSG transcripts most commonly found after a transient inactivation of PIP5Pase are those from the previously active VSG (BES1) and VSGs located on chr 1 and 6 (I believe). The small font and the low resolution make it impossible to infer the location of the expressed VSG genes, nor to confirm that ALL VSG genes located in expression sites are activated, as the authors claim. Also, I was not able to access the raw ChIP-seq and RNA-seq reads. Thus, could not evaluate the quality of the sequencing data.

Reviewer #3 (Public Review):

In this manuscript, Touray et al investigate the mechanisms by which PIP5Pase and RAP1 control VSG expression in T. brucei and demonstrate an important role for this enzyme in a signalling pathway that likely plays a role in antigenic variation in T. brucei.

The methods used in the study are rigorous and well-controlled. The authors convincingly demonstrate that RAP1 binds to PI(3,4,5)P3 through its N-terminus and that this binding regulates RAP1 binding to VSG expression sites, which in turn regulates VSG silencing. Overall their results support the conclusions made in the manuscript.

There are a few small caveats that are worth noting. First, the analysis of VSG derepression and switching in Figure 1 relies on a genome that does not contain minichromosomal (MC) VSG sequences. This means that MC VSGs could theoretically be misassigned as coming from another genomic location in the absence of an MC reference. As the origin of the VSGs in these clones isn't a major point in the paper, I do not think this is a major concern, but I would not over-interpret the particular details of switching outcomes in these experiments.

The authors state that "our data imply that antigenic variation is not exclusively stochastic." I am not sure this is true. While I also favor the idea that switching is not exclusively stochastic, evidence for a signaling pathway does not necessarily imply that antigenic variation is not stochastic. This pathway could be important solely for lifecycle-related control of VSG expression, rather than antigenic variation during infection. Nevertheless, these data are critical for establishing a potential pathway that could control antigenic variation and thus represent a fundamental discovery.

Another aspect of this work that is perhaps important, but not discussed much by the authors, is the fact that signalling is extremely poorly understood in T. brucei. In Figure 1B, the RNA-seq data show many genes upregulated after expression of the Mut PIP5Pase (not just VSGs). The authors rightly avoid claiming that this pathway is exclusive to VSGs, but I wonder if these data could provide insight into the other biological processes that might be controlled by this signaling pathway in T. brucei.

Overall, this is an excellent study that represents an important step forward in understanding how antigenic variation is controlled in T. brucei. The possibility that this process could be controlled via a signalling pathway has been speculated for a long time, and this study provides the first mechanistic evidence for that possibility.

Reviewer #1 (Public Review):

Bierman et al. have developed a set of metrics for measuring the spatial patterning of mRNAs in high-throughput fluorescence in situ hybridisation experiments and applied these to identify a subset of mRNAs whose spatial patterning correlates with 3'UTR length. A strength of the study is the clarity and honesty with which the authors have outlined the strengths and weaknesses of their own approach and reported negative results. A key benefit of the tool is that the methodological choices allow wide applicability to existing datasets. However, these choices also feed into a limitation of the method, which is the difficulty in interpreting the biology underpinning the metrics - raising the question of how users will understand the output of the tool.

Reviewer #2 (Public Review):

The authors develop SPRAWL (Subcellular Patterning Ranked Analysis With Labels), a statistical framework to identify cell-type specific subcellular RNA localization from multiplexed imaging datasets. The tool is able to assign to each gene and in each annotated cell type, a score (with a p-value) that measures:<br /> - Peripheral/central localization of RNAs within the cell, based on a previous segmentation step defining cell boundaries and the centroid coordinate.<br /> - Radial/punctuate localization of RNAs within the cell

The method is applied to three multiplexed imaging datasets, identifying defined and cell-type specific patterns for several transcripts.

In the second part of the manuscript, the authors couple SPRAWL with ReadZS, a computational tool developed by the same group and recently published (Meyer et al, 2022). Starting from single-cell datasets, ReadZS is able to quantify 3'UTR length in each cell type. The authors find a subset of genes showing a positive, or negative correlation between the predicted localization and the predicted 3'UTR length across cell types.

Strengths:<br /> As the authors state in the introduction, the study of subcellular RNA localization, with the characterization of organizational principles and of molecular regulation mechanisms, is extremely relevant. The authors develop a strategy to detect statistically significant and non-random patterns of RNA sub-cellular localization in MERFISH and SeqFISH+ datasets, i.e. emerging platforms producing spatially resolved maps of hundreds of transcripts with cellular resolution.

Weaknesses:<br /> Although the method and the presented results have strengths in principle, the main weakness of the paper is that these strengths are not directly demonstrated. That is, insufficient validations are performed to show the biological significance of the results and to fully support the key claims in the manuscript by the data presented.

In particular, the authors imply that their tool is unique and not comparable to any other method. Therefore there is no comparison of SPRAWL with any other method. For example, a comparison could be made with Baysor (Petukhov, V et al. Nat Biotechnol. https://doi.org/10.1038/s41587-021-01044-w). According to the authors, this method is able to identify "small molecular neighbourhoods with stereotypical transcriptional composition" and provides a "General approach for statistical labeling of spatial data".

The authors claim that SPRAWL is able to identify spatial patterns of localization and generated relevant hypotheses to be tested, yet the manuscript contains little proof that the results have biological significance (for example association of RNAs with specific subcellular compartments) and there is no experimental validation for the results obtained applying this method.

The correlation between localization scores and 3'UTR length across cell types for certain genes is also not experimentally validated: results are based on inference from single-cell or imaging data, with no complementary experimental validation.

It is therefore very difficult to assess the biological relevance of the results produced by SPRAWL.

Reviewer #3 (Public Review):

Bierman et al. present a novel statistical framework for examining the subcellular localisation of RNA molecules. Subcellular Patterning Ranked Analysis With Labels, SPRAWL, uses the data available in multiplexed single-cell imaging datasets to assign four metrics of localisation patterns to RNA at a gene per cell level. These easy-to-understand scores, ranging from -1 to 1, can be averaged to detect cell-type specific spatial patterns or used in tandem with tools for RNA 3' UTR length or splicing state to determine the correlation between subcellular localisation and RNA isoforms. Such quantitive association between RNA isoforms and localisation provides a useful tool to determine candidate genes for future studies.

The peripheral and central scores indicate the proximity of RNA molecules to the cell boundary and centre of the cell respectively in relation to other RNA present in the cell. Whilst understanding whether a gene tends to be localised to the cellular membrane is important, it is unclear what biological benefits the central metric gives compared to high "anti-peripheral" scores considering that no single organelle (eg. the nucleus) is located specifically at the centre of the cell in all cell-types.

The punctate and radial patterning scores provide information on the spatial aggregation of RNA molecules of a given gene within a cell. Whilst the punctate score is easy to understand as simply the distance between RNA, the radial score, the angle between RNA, is harder to understand from the main text and would benefit from a schematic showing how this is in respect to the cell-boundary centroid.

Despite endeavouring to create a robust statistical measure of RNA subcellular localisation, this paper is full of inconsistencies. Values (eg. Pearson correlation coefficient values, number of significant genes, number of total genes) and names (eg. cell types, gene names) stated throughout the main text and figures/table do not match repeatedly and without fixing these disparities, the conclusions from this paper are hard to believe.

Reviewer #1 (Public Review):

This study focuses on molecular and cellular mechanisms underlying the sorting of miRNAs into exosomes originating from multivesicular bodies (MVBs). Following up on their previous work, the authors analysed the biochemical basis of miRNA selection by the RNA-binding protein YBX1 which is known to participate in this sorting. Using electrophoretic mobility shift assays (EMSA) involving a series of YBX1 constructs, they pinpointed the key role of the cold shock domain of YBX1 (supported by the C-terminal domain) in miRNA binding. By comparing a secreted model miRNA (miR223), a control cytoplasmic miRNA that is not enriched in exosomes (miR190), and a series of their swap mutants, the authors identified what could be a sequence motif enabling YBX1 to discriminate - through direct binding - between miRNAs to be secreted or to be retained.

The authors then wondered from which subcellular pool miR223 could be mobilised for secretion. They turned their attention to the mitochondria and found evidence of miR223 association with these organelles. Interestingly, when mitochondria were depleted by Parkin overexpression and CCCP treatment, the cellular level of miR223, but not of miR190, increased, whereas its enrichment in extracellular vesicles dropped. This observation permitted to forward a hypothesis whereby mitochondria could be involved in miR223 mobilisation into exosomes. This process would be mediated by YBX1 which shuttles between mitochondria and endosomes, as was elegantly shown in live imaging experiments.

Finally, the authors provide initial data implicating in this process the mitochondrial matrix protein YBAP1, broadly known as C1QBP, or p32. YBAP1 was found to interact with YBX1 and miR223 in pull-down assays. Moreover, direct and moderately strong miR223 binding by YBAP1 was confirmed by EMSA. Interestingly, just like YBX1, YBAP1 seems to prefer this substrate over miR190, indicating certain binding specificity. The observation that YBAP1 knockout resulted in the decreased association of miR223 with mitochondria, paralleled by its correspondingly better mobilisation into exosomes, enabled the authors to propose that YBAP1 could negatively control miR223 secretion at the level of mitochondria.

Strengths

This is a very interesting study proposing an elegant hypothesis and featuring a creative panel of methods, many of which will certainly be of interest to biochemists and cell biologists working with extracellular RNA and mitochondria (e.g. the Parkin/CCCP-mediated mitochondria depletion and the time-lapse imaging of RNA-binding proteins against cellular organelles).

The authors did a good job of dissecting the YBX1 interaction with miR223 versus miR190. These experiments are performed at a high technical level, and their interpretation is straightforward and convincing. The nearly two orders of magnitude difference in affinity provides a plausible means by which YBX1 could recognise and funnel one, but not the other, miRNA into the secretion pathway.<br /> Another valuable piece of data is related to YBAP1. This important, deeply conserved protein, strongly implicated in severe mitochondrial diseases and cancer, remains poorly understood at the level of basic molecular mechanisms, and even its subcellular localisation is debated. The data presented by the authors reinforce the idea of its primarily mitochondrial localisation, in agreement with earlier studies. They also provided new information about the RNA-binding activity of YBAP1. First proposed to interact with RNA by Yagi et al., Nucleic Acids Res 2012 (doi:10.1093/nar/gks774), YBAP1 is confirmed in the present study as a reasonably affine RNA-binding protein, based on direct EMSA experiments involving a highly purified protein and natural RNAs. These data should encourage the community to explore the full RNA-binding potential of YBAP1/C1QBP/p32 in a wider variety of models, especially in the context of mitochondrial gene expression.

Weaknesses

While the authors might be right about the existence of a sequence motif that specifies miRNAs for exosome sorting by YBX1, it is at present difficult to disentangle the sequence and structure contributions to YBX1 binding within the variants described in the paper. RNA structure predictions, however imperfect, suggest that miR223-3p is a fully single-stranded transcript (ensemble ΔG = -0.33 kcal/mol, RNAfold), while miR190-5p is a tightly base-paired one (ensemble ΔG = -2.85 kcal/mol). This likely explains the differential affinity to YBX1, known to strongly prefer single-stranded RNAs. When mutating the putative sorting motif in miR223 (UCAGU>AGACA), the authors introduced some amount of secondary structure (ΔG = -1.04 kcal/mol), which could have impeded YBX1 binding. By contrast, the mutation of miR190 (AUAUG>UCAGU) significantly weakened the structure (ensemble ΔG = -2.21 kcal/mol), which might explain the improvement in YBX1 interaction.

Mitochondria appear to be a plausible location for mobilisable RNAs, given their multiple associations with ribosomes, RNA-containing condensates, and other organelles. However, the presented evidence of the mitochondrial localisation of miR223 is limited. The colocalisation pattern of the ATTO 647-labelled miR223 with the well-behaved mitochondrial marker Tom22 is remarkable; such a neat overlap has so far only been observed for some abundant mtDNA-encoded transcripts, but not for an extraneous transcript. The interpretation of this result will depend a great deal on experimental details which, unfortunately, are missing for this section. ATTO 647N is known to be quantitatively recruited to mitochondria, producing just the same kind of complete colocalisation, making it a perfect tool to visualise mitochondria in the cell (Han et al., Nat Commun 2017, doi:10.1038/s41467-017-01503-6). There is a worry that the colocalisation observed here might have been driven by the dye alone.

Furthermore, the definition of the topology of RNA localisation with respect to the mitochondrial membranes remains challenging, and a number of more robust methods have been recently proposed to address this contentious issue. At minima, one would expect that the authors would use RNase treatment, with or without Triton X-100 (like they did in the in vitro packaging assay), to see whether miR223 is indeed protected by the mitochondrial membranes and, therefore, resides in the interior of the organelles. As for now, based on the presented data, one can safely conclude that miR223 is associated with the mitochondria, without claiming that it is necessary inside them.

The Parkin/CCCP method is very powerful, which is its strength and weakness at the same time. miR223 secretion does decrease when the mitochondria are depleted. However, it is unclear how direct and specific this effect is. The destruction of mitochondria likely crashed the cellular ATP levels, which could have generally affected vesicular transport, not only miR223 sorting. A more detailed analysis of the overall abundance of extracellular vesicles and their cargo under these conditions could reveal the true scope of the mitochondrial contribution to RNA secretion.

YBAP1 is a difficult, indeed "treacherous", protein to work with. Its strong negative charge (pI = 4) makes it easily stick to positively charged proteins, such as YBX1 (pI = 9.9). Such interactions are routinely observed in pulldown assays from cell lysates, where all components are intermixed (but often cannot be corroborated by in situ or in vivo approaches). The authors carefully showed that YBX1 and YBAP1 do not significantly colocalise in the cell, which makes the interplay between the two proteins in miR223 sorting difficult to stage. They also studied the miR223 distribution between mitochondria and extracellular vesicles using YBAP1 knockout cells. However, such cells are known to be very sick and have an extremely pleiotropic mitochondrial and metabolic phenotype. Therefore, the apparent implication of YBAP1 in miR223 sorting might be less direct than currently envisaged.

Reviewer #2 (Public Review):

The manuscript by Ma et al, "Two RNA-binding proteins mediate the sorting of miR223 from mitochondria into exosomes" examines the contribution of two RNA-binding proteins on the exosomal loading of miR223. The authors conclude that YBX1 and YBAP1 work in tandem to traffic and load miR223 into the exosome. The manuscript is interesting and potentially impactful. It proposes the following scenario regarding the exosomal loading of miR223: (1) YBAP1 sequesters miR223 in the mitochondria, (2) YBAP1 then transfers miR223 to YBX1, and (3) YBX1 then delivers miR223 into the early endosome for eventual secretion within an exosome. While the authors propose plausible explanations for this phenomenon, they do not specifically test them and no mechanism by which miR223 is shuttled between YBAP1 and YBX1, and the exosome is shown. Thus, the paper is missing critical mechanistic experiments that could have readily tested the speculative conclusions that it makes.

Comments:<br /> 1. The major limitation of this paper is that it fails to explore the mechanism of any of the major changes it describes. For example, the authors propose that miR223 shuttles from mitochondrially localized YBAP1 to P-body-associated YBX1 to the exosome. This needs to be tested directly and could be easily addressed by showing a transfer of miR223 from YBAP1 to YBX1 to the exosome.<br /> 2. If YBAP1 retains miR223 in mitochondria, what is the trigger for YBAP1 to release it and pass it off to YBX1? The authors speculate in their discussion that sequestration of mito-miR223 plays a "role in some structural or regulatory process, perhaps essential for mitochondrial homeostasis, controlled by the selective extraction of unwanted miRNA into RNA granules and further by secretion in exosomes...". This is readily testable by altering mitochondria dynamics and/or integrity.<br /> 3. Much of the miRNA RT-PCR analysis is presented as a ratio of exosomal/cellular. This particular analysis assumes that cellular miRNA is unaffected by treatments. For example, Figure 1a shows that the presence of exosomal miR223 is significantly reduced when YBX1 is knocked out. This analysis does not consider the possibility that YBX1-KO alters (up or down-regulates) intracellular miR223 levels. Should that be the case, the ratiometric analysis is greatly skewed by intracellular miRNA changes. It would be better to not only show the intracellular levels of the miRs but also normalize the miRNA levels to the total amount of RNA isolated or an irrelevant/unchanged miRNA.<br /> 4. In figure 1, the authors show that in YBX1-KO cells, miR223 levels are decreased in the exosome. They further suggest this is because YBX1 binds with high affinity to miR223. This binding is compared to miR190 which the authors state is not enriched in the exosome. However, no data showing that miR190 is not present in the exosome is shown. A figure showing the amount of cellular and exosomal miR223 and 190 should be shown together on the same graph.<br /> 5. Figure 2 Supplement 1 - As to determine the nucleotides responsible for interacting with YBX1, the authors made several mutations within the miR223 sequence. However, no explanation is given regarding the mutant sequences used or what the ratios mean. Mutant sequences need to be included. How do the authors conclude that UCAGU is important when the locations of the mutations are unclear? Also, the interpretation of this data would benefit from a binding affinity curve as shown in Fig 2C.<br /> 6. While the binding of miR223mut to YBX1 is reduced, there is still significant binding. Does this mean that the 5nt binding motif is not exact? Do the authors know if there are multiple nucleotide possibilities at these positions that could facilitate binding? Perhaps confirming binding "in vivo" via RIP assay would further solidify the UCAGU motif as critical for binding to YBX1.<br /> 7. Figures 2g, h - It would be nice to show that miR190mut also packages in the cell-free system. This would confirm that the sequence is responsible. Also, to confirm that the sorting of miR223 is YBX1-dependent, a cell-free reaction using cytosol and membranes from YBX1 KO cells is needed.<br /> 8. In Figure 3a, the authors show that miR223 is mitochondrially localized. Does the sequence of miR223 (WT or Mut) matter for localization? Does it matter for shuttling between YBAP1 and YBX1?<br /> 9. Supplement 3c - Is it strange that miR190 is not localized to any particular compartment? Is miR190 present ubiquitously and equally among all intracellular compartments?<br /> 10. Figure 3h - Why would the miR223 levels increase if you remove mitochondria? Does CCCP also cause miR223 upregulation? I would have thought miR223 would just be mis-localized to the cytosol.<br /> 11. Figure 3i - What is the meaning of "Urd" in the figure label? This isn't mentioned anywhere.<br /> 12. Figure 3j - The data is presented as a ratio of EV/cell. Again, this inaccurately represents the amount of miR223 in the EV. This issue is apparent when looking at Figures 3h and 3j. In 3h, CCCP causes an upregulation of intracellular miR223. As such, the presumed decrease in EV miR233 after CCCP (3j) could be an artifact due to increased levels of intracellular miR223. Both intracellular and EV levels of miRs need to be shown.<br /> 13. In Figure 4, the authors show that when overexpressed, YBX1 will pulldown YBAP1. Can the authors comment as to why none of the earlier purifications show this finding (Figure 1 for example)? Even more curious is that when YBAP1 is purified, YBX1 does not co-purify (Figure 4 supplement 1a, b).<br /> 14. Figure 4f, g - The text associated with these figures is very confusing, as is the labeling for the input. Also, what is "miR223 Fold change" in this regard? Seeing as your IgG should not have IP'd anything, normalizing to IgG can amplify noise. As such, RIP assays are typically presented as % input or fold enrichment.<br /> 15. Figure 4h - The authors show binding between miR223 and YBAP1 however it is not clear how significant this binding is. There is more than a 30-fold difference in binding affinity between miR223 and YBX1 than between miR223 and YBAP1. Even more, when comparing the EMSAs and fraction bound from figures 1 and 2 to those of Figure 4h, the binding between miR223 and YBAP1 more closely resembles that of miR190 and YBX1, which the authors state is a non-binder of YBX1. The authors will need to reconcile these discrepancies.<br /> 16. Can the authors present the Kd values for EMSA data?<br /> 17. Figure 5 - Does YBAP1-KO affect mitochondrial protein integrity or numbers?<br /> 18. Figure 6a - Are the authors using YBAP1 as their mitochondrial marker? Please include TOM20 and/or 22.<br /> 19. Figure 6b - Rab5 is an early endosome marker and may not fully represent the organelles that become MVBs. Co-localization at this point does not suggest that associating proteins will be present in the exosome, and it is possible that the authors are looking at the precursor of a recycling endosome. Even more, exosome loading does not occur at the early endosome, but instead at the MVB. Perhaps looking at markers of the late endosome such as Rab7 or ideally markers of the MVB such as M6P or CD63 would help draw an association between YBX1, YBAP1, and the exosome. Also, If the authors want to make the claim that interactions at the early endosome leads to secretion as an exosome, the authors should show that isolated EVs from Rab5Q79L-expressing cells contain miR223.<br /> 20. The mentioning of P-bodies is interesting but at no time is an association addressed. This is therefore an overly speculative conclusion. Either show an association or leave this out of the manuscript.<br /> 21. In lines 55-58, the authors make the comment "However, many of these studies used sedimentation at ~100,000 g to collect EVs, which may also collect RNP particles not enclosed within membranes which complicates the interpretation of these data." Do RNPs not dissolve when secreted? Can the authors give a reference for this statement?

Reviewer #3 (Public Review):

The article by Ma et al pursues the previous work of the Schekman group, exploring the mechanisms of targeting of miRNAs into extracellular vesicles (EVs), or possibly exosomes, in HEK293 and U2OS cells. The authors had identified YBX1 as an RNA-binding protein required for the sorting of miR223 into CD63-expressing small EVs, probably mainly exosomes. Here they further observed that YBX1 directly binds miR223, which also binds to another protein, YBAP1, localized in mitochondria, where it sequesters miR223, thus preventing its targeting to MVBs' intraluminal vesicles. They observe the association of YBX1-containing P-bodies in the cytoplasm with mitochondria and with enlarged Rab5-endosomes and propose that this step is required for the exchange of miR223 for its loading into MVBs intraluminal vesicles and future exosomes.

The biochemical parts of the article, with quantitative experiments to decipher the molecular interactions of YBX1 and YBAP1 with miR223, are nicely performed and convincing. By contrast, the parts on the involvement of YBX1 and of YBAP1 in the release of miR223 in EVs or exosomes are more correlative than demonstrative and lack some controls. In particular, it is far-fetched to conclude from the observed movement (which may be serendipitous) of 2 P-bodies between mitochondria and enlarged endosomes (without any visualization of the miR) that this movement may be instrumental in the transfer of miR223 between mitochondria and putative exosomes (figures 6 and model in figure 7).

The experiments designed to evidence the mechanisms of miR223 release in EVs are also not sufficiently controlled and analysed to really support the interpretations. And the EV isolation steps are not performed in a way that supports the actual exosomal nature (i.e. exclusive origin from multivesicular endosome) of the EV analysed.

Another experimental weakness is that the authors make strong conclusions on MVBs and exosomes when they only analyse artificially-enlarged endosomes induced by overexpression of mutant Rab5. Although this approach has been used previously and shown CD63 in these induced enlarged compartments, it is an artificial blocking of normal endosomal trafficking, and may not reflect the situation of intracellular trafficking of miR223 in normal cells.

Reviewer #1 (Public Review):

This study is based on the hypothesis that tumor treating fields, a form of cancer therapy that exposes tumors to alternating electrical fields, has an effect on tunneling microtubes, fine actin-rich protrusions that connect cancer cells and allow intercellular communication, contributing to the tumor microenvironment and therapeutic resistance. This is an interesting hypothesis and may be of importance. To prove their hypothesis better data presentation and mechanistic studies are needed, as it is not clear based on this study how the proposed effect is working.

Reviewer #2 (Public Review):

The authors tested TTFields' effect on TNT formation in two mesothelioma cell lines, MSTO-211H and VMAT. The MSTO-211H is a biphasic cell line with epithelioid and sarcomatoid features while VMAT only has sarcomatoid morphology. They treated their cell lines at 150 or 200 kHz either unidirectionally or bidirectionally. The experiments took place within 72 hours of plating, after which the cells will become confluent on coverslips and their TNT formation drops.

Under these experimental conditions, they found: (i) Unidirectional is more effective than bidirectional TTFields in reducing TNT formation, (ii) TNT formation was markedly reduced after 48 hours of treatment in MSTO-211H but not VMAT cells, (iii) no difference in actin polymerization or actin filament bundling after one hour of TTFields treatment, (iv) reduced TNT formation when TTFields were combined with cisplatin but not with both cisplatin and pemetrexed, (v) analysis TNT cargo transport using markers of gondolas and mitochondria did not show changes in transport velocity, and (vi) in vivo spatial transcriptomic analysis revealed EMT markers and immunogenic markers.

Reviewer #3 (Public Review):

Sarkari et al. describe the effects of TTFields on inter-cellular communication structures called tunneling nanotubes in malignant pleural mesothelioma cells. Recent studies have implicated these F-actin-based nanotubes in promoting malignant transformation and biology by allowing long-range communications between malignant cells. The authors suggest that TTFields disrupt these structures by impacting the expression of genes involved in nanotube formation and cell proliferation. Although TTFields are thought to affect tubulin-based structures, recent studies suggest that TTFields also impact actin-based structures. Therefore, the authors' findings are in keeping with this new understanding. They also found that TTFields upregulated marker genes in immunity. This is one of the first studies that implicate TTFields in these tunneling nanotube structures. Overall, the study adds to our understanding of TTFields on various cellular structures. However, conclusions are only partially supported by the data presented. The study is largely descriptive and there are many areas that need to be addressed to substantively improve the premise and rigors and strengthen the conclusions.

Reviewer #1 (Public Review):

In this study, Shin and colleagues investigate the role of the posttranslational modification of the DNA methyltransferase by covalent linkage of the N-Acetylglucosamine (O-GlcNAc).

The authors present compelling evidence showing that a prolonged high fat/sucrose diet causes global protein O-GlcNAcylation in the liver and DNMT1 is among the proteins that increase their O-GlcNAc level. This result is significant because of the paucity of in vivo data addressing the interplay between metabolism and protein O-GlcNAcylation. The paper also shows that DNMT1's O-GlcNAcylation level correlated to the extracellular glucose levels in other cell types.

Using mass spectrometry, the authors identify S878 as the main site for O-GlcNAcylation. It is noteworthy that the mapping was performed with hyper-O-GlcNAcylated cells and may be different in a physiological situation. To investigate how O-GlcNAcylation of S878 of DNMT1 impacts its activity and ultimately DNA methylation patterns, Shin and colleagues mostly use a cellular model of hyper O-GlcNAcylation induced by the combination of high glucose and a chemical inhibitor of OGA (the only enzyme responsible for O-GlcNAc removal). The data shows that increased O-GlcNAcylation resulting from the combination of high glucose and OGA inhibition causes a reduction of DNMT1 activity and local loss of DNA methylation specifically at partially methylated domains.

This study brings completely new knowledge on the regulatory function of glycosylation of DNMT1 and its impact on its methyl-transferase activity and downstream genomic methylation. Furthermore, the manuscript introduces new data on the interplay between cellular metabolism and O-GlcNAcylation on DNMT1 and other proteins. The experiments are well-controlled, and their interpretation is sound. This study should be of special interest to the fields of fundamental and environmental epigenetics, as well as metabolism.

The main limitation of the study is the convolution of the functional experiments where the perturbation is a combination of high glucose and chemical inhibition of OGA. The relative contribution of the two variables is partially addressed in Figure 3-figure supplement 1B which shows that high glucose increases DNMT1 activity (Hep3B cells) while Figure 3D shows that high glucose when combined with OGA inhibitor decreases DNMT1 activity (Hep3B cells). As discussed, the data suggest that high-glucose and OGA inhibition may have an antagonistic effect on DNMT1 activity. An experiment of treatment of the cells with the OGA inhibitor in physiological glucose conditions would address this gap of knowledge.

To understand the impact of the environment (in this study: extracellular glucose level) on the epigenome, one should keep in mind the variation of cytosine methylation patterns between individuals and over time. A recent large-scale profiling of DNA methylation of 137 individuals shows a near absence of individual variation between replicates of the same cell type, suggesting that genomic methylation patterns are largely insensitive to the environment (https://doi.org/10.1038/s41586-022-05580-6).

Comparative methylomes of healthy and diabetic individuals are needed to examine the medical significance of the findings presented here. It is possible that the modulation of DNMT1 activity by O-GlcNAc modification is relevant for a specific cell type or developmental stage that remains to be discovered.

Reviewer #2 (Public Review):

I've read the manuscript by Shin et al with great interest. The authors describe the identification of O-GlcNAcylation of DNMT1 and the impact this modification has on the maintenance activity of DNMT1 genome-wide and that modification of S878 leads to enzyme inhibition.<br /> The manuscript is written in a clear and understandable way making it easy for the reader to understand the logic as well as the steps of the experimental approach.

The authors identify O-GlcNAcylation of DNMT1 in a number of different cell lines by combining inhibition studies and WB and further on they identify the modification sites with LC/MS, predictions, and mutational studies. I really like the experimental approach, which while being straightforward (albeit technically challenging), is powerful and well-controlled in this case to unequivocally prove the modification of DNMT1 and identify the site. However, mutation of the two identified modification sites does not remove all the O-GlcNAcylation signal associated with DNMT1, thus possibly not all the possible sites were identified. While this is not a criticism of this manuscript, it would be interesting to know what other sites are modified and the enzymatic/biological effects associated.

Also, the authors isolate the modified DNMT1 from cells using immunoprecipitation, which is indeed useful to study the changes in catalytic activity but does not provide any information if the cellular localisation of modified DNMT1 changes. Subsequently, the authors checked the impact of high glucose diet on the genome-wide DNA methylation patterns. The observed effects (Fig 4A) are very strong, almost as strong as observed with Aza treatment and therefore I wonder if LINE/IAP or other elements are getting activated (as observed with genome-wide demethylation with Aza). Do the authors see any changes in cell phenotype, slower/faster proliferation, or increased apoptosis due to the activation of mobile elements (not only ROS)? Another point is that the S878A mutant seems not to be able to fully maintain the DNA methylation (Fig 4A). Does O-GlcNAcylation recruit any additional interactors? Given that the authors immunoprecipitated DNMT1 and use it for activity assay, it is possible, that the modification attracts an additional protein factor that could in turn inhibit DNMT1 activity (as observed). Therefore, the observed kinetic effect could be indirect, while still interesting and important, the mechanism of inhibition would be different.

DNA methylation clock can be used to estimate the biological age of a tissue/cells. While not directly in the line of the manuscript, I was wondering if the DNA methylation changes in the high glucose diet would affect the methylation sites used for the DNAme clock. Meaning, would the cells/tissue epigenetically age faster when in high glucose media, and if the Ala mutant could provide resistance to that?

In discussion, the authors write that this is the first investigation of O-GlcNAcylation in relation to DNA methylation, while this is true for DNMTs, TET enzymes, that oxidise 5mC and trigger active DNA demethylation have been shown before to also be modified.

A nice and rigorous study, with important observations and connections to biological effects. It would be nice to prove that the effects are direct and not associated with other factors that could be recruited by the modification and impact the activity of DNMT1. I find it a bit surprising that phosphorylation of the target serine does not impact DNMT1 activity as well.

Reviewer #3 (Public Review):