Quantify the time saved (e.g., 'saving 10+ hours monthly') for clearer impact.

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Include the original hosting cost to emphasize the financial impact of your re-architecture.

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Provide specific user feedback or metrics that demonstrate the improvement in user experience.

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Quantify the reduction in time spent on reviews or the increase in review efficiency.

Provide specific outcomes from the feedback integration, such as user adoption rates or satisfaction scores.

Add metrics on user engagement or satisfaction improvements post-redesign.

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Reviewer #2 (Public review):

Summary:

In this manuscript, Weiguang Kong et al. investigate the role of immunoglobulin M (IgM) in antiviral defense in the teleost largemouth bass (Micropterus salmoides). The authors employ an in vivo IgM depletion system and viral infection models, complemented by in vitro assays, histology, and gene expression analysis. Assuming the specificity of the MoAb, their findings demonstrate that largemouth bass IgM functions in both systemic and mucosal immunity and exhibits viral neutralization capabilities by acting on viral particles.

Strengths:

The authors utilize multiple complementary methods, including an innovative teleost immunoglobulin depletion approach, to provide strong evidence for the important and conserved role of IgM in anti-viral resistance. The study also highlights the dual role of teleost IgM at both systemic and mucosal levels, challenging the established idea that IgT primarily mediates mucosal protection. Despite variability in IgM depletion levels, the authors demonstrate that fish with depleted IgM+ B cells exhibit significantly higher viral loads, more severe pathological changes, and increased mortality compared to control fish. These results have evolutionary and practical implications, suggesting that IgM's role as an antiviral effector has been conserved across jawed vertebrates for over 500 million years. Insights into IgM's role could inform vaccine strategies targeting mucosal immunity in fish, addressing a key challenge in aquaculture.

Weaknesses:

While the authors validate the specificity of MoAb against IgM and address most of the aspects suggested by the reviewer. Some aspects are missing, mainly concerning the overstatement of the findings' novelty.

Reviewer #1 (Public review):

Wang, Junxiu et al. investigated the underlying molecular mechanisms of the insecticidal activity of betulin against the peach aphid, Myzus persicae. There are two important findings described in this manuscript: (a) betulin inhibits the gene expression of GABA receptor in the aphid, and (b) betulin binds to the GABA receptor protein, acting as an inhibitor. The first finding is supported by RNA-Seq and RNAi, and the second one is convinced with MST and electrophysiological assays. Further investigations on the betulin binding site on the receptor protein provided a fundamental discovery that T228 is the key amino acid residue for its affinity, thereby acting as an inhibitor, backed up by site-directed mutagenesis of the heterologously-expressed receptor in E. coli and by CRISPR-genome editing in Drosophila.

Although the manuscript does have strengths in principle, the weaknesses do exist: the manuscript would benefit from more comprehensive analyses to fully support its key claims in the manuscript. In particular:

(1) The Western blotting results in Figure 5A & B appear to support the claim that betulin inhibits GABR gene expression (L26), as a decrease in target protein levels is often indicative of suppressed gene expression. The result description for Figure 5A & B is found in L312-L316, within Section 3.6 ("Responses of MpGABR to betulin"), where MST and voltage-clamp assays are also presented. It seems the observed decrease in MpGABR protein content is due to gene downregulation, rather than a direct receptor protein-betulin interaction. However, this interpretation lacks discussion or analysis in either the corresponding results section or the Discussion. In contrast, Figures 5C-F are specifically designed to illustrate protein-betulin interactions. Presenting Figure 5A & B alongside these panels might lead to confusion, as they support distinct claims (gene expression vs. protein binding/inhibition). Therefore, I recommend moving Figure 5A & B either to the end of Figure 3 or to a separate figure altogether to improve clarity and logical flow. A minor point in the Western blotting experiment is that although GAPDH was used as a reference protein, there is no explanation in the corresponding M&M section.

(2) The description of the electrophysiological recording experiment is unclear regarding the use of GABA. I didn't realize that GABA, the true ligand of the GABA receptor, was used in this inhibition experiment until I reached the Results section (L321), which states, "In the presence of only GABA, a fast inward current was generated." Crucially, no details are provided on the experiment itself, including how GABA was applied (e.g., concentration, duration, whether GABA was treated, followed by betulin, or vice versa). This information is essential for reproducibility. Please ensure these details are thoroughly described in the corresponding M&M section.

(3) The phylogenetic analysis, particularly concerning Figures 4 and 6B, needs significant attention for clarity and representativeness. First, your claim that MpGABR is only closely related to CAI6365831.1 (L305-L310) is inconsistent with the provided phylogenetic tree, which shows MpGABR as equally close to Metopolophium dirhodum (XP_060864885.1) and Acyrthosiphon pisum (XP_008183008.2). Therefore, singling out only Macrosiphum euphorbiae (CAI6365831.1) is not supported by the data. Second, the representation of various insect orders is insufficient. All 11 sequences in the Hemiptera category (in both Figure 4 and Figure 6B) are exclusively from the Aphididae family. This small subset cannot represent the highly diverse Order Hemiptera. Consequently, statements like "only THR228 was conserved in Hemiptera" (L338), "The results of the sequence alignment revealed that only THR228 was conserved in Hemiptera" (L430), or "THR228... is highly conserved in Hemiptera" (L486) are not adequately supported. Third, similar concerns apply to the Diptera order, which includes 10 Drosophila and 2 mosquito samples (not diverse or representative enough), and likely to other orders as well. Thereby, the Figure 6B alignment should be revised accordingly to reflect a more accurate representation or to clarify the scope of the analysis. Fourth, there's a discrepancy in the phylogenetic method used: the M&M section (L156) states that MEGA7, ClustalW, and the neighbor-joining method were used, while the Figure 4 caption mentions that MEGA X, MUSCLE, and the Maximum likelihood method were employed. This inconsistency needs to be clarified and made consistent throughout the manuscript. Fifth, I have significant concerns about the phylogenetic tree itself (Figure 4). A small glitch was observed at the Danaus plexippus node, which raises suspicion regarding potential manipulation after tree construction. More critically, the tree, especially within Coleoptera, does not appear to be clearly resolved. I am highly concerned about whether all included sequences are true GABR orthologs or if the dataset includes partial or related sequences that could distort the phylogeny. Finally, for Figure 6B, both protein (XP_) and nucleotide (XM_) sequences were mix used. I recommend using the protein sequences instead of nucleotide sequences in this figure panel, as protein sequences are more directly informative.

(4) The Discussion section requires significant revision to provide a more insightful and interpretative analysis of the results. Currently, much of the section primarily restates findings rather than offering deeper discussion. For instance, L409-L419 restate the results, followed by the short sentence "Collectively, these results suggest that betulin may have insecticidal effects on aphids by inhibiting MpGABR expression". It could be further expanded to make it beneficial to elaborate on proposed mechanisms by which gene expression might be suppressed, including any potential transcription factors involved. In contrast, while L422-L442 also initially summarize results, the subsequent paragraph (L445-L472) effectively discusses the potential mechanisms of inhibitory action and how mortality is triggered, which is a good model for other parts of the section. However, all the discussion ends up with a short statement, "implying that betulin acts as a CA of MpGABR" (L472), which appears to be a leap. The inference that betulin acts as a competitive antagonist (CA) is solely based on the location of its extracellular binding site, which does not exactly overlap with the GABA binding site. It needs stronger justification or actually requires further experimental validation. The authors should consider rephrasing this statement to acknowledge the need for additional studies to definitively confirm this mechanism of action.

Reviewer #2 (Public review):

Summary:

This important study shows that betulin from wild peach trees disrupts neural signaling in aphids by targeting a conserved site in the insect GABA receptor. The authors present a nicely integrated set of molecular, physiological, and genetic experiments to establish the compound's species-specific mode of action. While the mechanistic evidence is solid, the manuscript would benefit from a broader discussion of evolutionary conservation and potential off-target ecological effects.

Strengths:

The main strengths of the study lie in its mechanistic clarity and experimental rigor. The identification of a betulin-binding single threonine residue was supported by (1) site-directed mutagenesis and (2) functional assays. These experiments strongly support the specificity of action. Furthermore, the use of comparative analyses between aphids and fruit flies demonstrates an important effort to explore species specificity, and the integration of quantitative data further enhances the robustness of the conclusions.

Weaknesses:

There are several important limitations that need to be addressed. The manuscript does not explore whether the observed sensitivity to betulin reflects a broadly conserved feature of GABA receptors across animal lineages or a more lineage-specific adaptation. This evolutionary context is crucial for understanding the broader significance of the findings.

In addition, while the compound's aphicidal effect is well established, the potential for off-target effects in non-target organisms - especially vertebrates - remains unaddressed, despite prior evidence that betulin interacts with mammalian GABAa receptors. There is little discussion on the ecological or environmental safety of exogenous betulin application, such as persistence, degradation, or exposure risks.

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Reviewer #1 (Public review):

Summary:

Asthenospermia, characterized by reduced sperm motility, is one of the major causes of male infertility. The "9 + 2" arranged MTs and over 200 associated proteins constitute the axoneme, the molecular machine for flagellar and ciliary motility. Understanding the physiological functions of axonemal proteins, particularly their links to male infertility, could help uncover the genetic causes of asthenospermia and improve its clinical diagnosis and management. In this study, the authors generated Ankrd5 null mice and found that ANKRD5-/- males exhibited reduced sperm motility and infertility. Using FLAG-tagged ANKRD5 mice, mass spectrometry, and immunoprecipitation (IP) analyses, they confirmed that ANKRD5 is localized within the N-DRC, a critical protein complex for normal flagellar motility. However, transmission electron microscopy (TEM) and cryo-electron tomography (cryo-ET) of sperm from Ankrd5 null mice did not reveal significant structural abnormalities.

Strengths:

The phenotypes observed in ANKRD5-/- mice, including reduced sperm motility and male infertility, are conversing. The authors demonstrated that ANKRD5 is an N-DRC protein that interacts with TCTE1 and DRC4. Most of the experiments are well designed and executed.

Weaknesses:

The last section of cryo-ET analysis is not convincing. "ANKRD5 depletion may impair buffering effect between adjacent DMTs in the axoneme".

"In WT sperm, DMTs typically appeared circular, whereas ANKRD5-KO DMTs seemed to be extruded as polygonal. (Fig. S9B,D). ANKRD5-KO DMTs seemed partially open at the junction between the A- and B-tubes (Fig. S9B,D)." In the TEM images of 4E, ANKRD5-KO DMTs look the same as WT. The distortion could result from suboptimal sample preparation, imaging or data processing. Thus, the subsequent analyses and conclusions are not reliable.

This paper still requires significant improvements in writing and language refinement. Here is an example: "While N-DRC is critical for sperm motility, but the existence of additional regulators that coordinate its function remains unclear" - ill-formed sentences.

Reviewer #2 (Public review):

Summary:

The manuscript investigates the role of ANKRD5 (ANKEF1) as a component of the N-DRC complex in sperm motility and male fertility. Using Ankrd5 knockout mice, the study demonstrates that ANKRD5 is essential for sperm motility and identifies its interaction with N-DRC components through IP-mass spectrometry and cryo-ET. The results provide insights into ANKRD5's function, highlighting its potential involvement in axoneme stability and sperm energy metabolism.

Strengths:

The authors employ a wide range of techniques, including gene knockout models, proteomics, cryo-ET, and immunoprecipitation, to explore ANKRD5's role in sperm biology.

Weaknesses:

Limited Citations in Introduction: Key references on the role of N-DRC components (e.g.,DRC2, DRC4) in male infertility are missing, which weakens the contextual background.

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Rephrase to 'reducing developer testing setup time by 86% by eliminating test accounts' for conciseness.

Break into two sentences for clarity; consider rephrasing 'increasing overall coverage by 15%' to 'increasing test coverage by 15%'.

Clarify '50+ hr/month' to '50+ hours/month' for grammatical accuracy.

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Use 'May 2025 – Present' for consistency with other dates. Avoid periods after 'May' and 'Present'.

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Reviewer #1 (Public review):

Summary:

The major result in the manuscript is the observation of the higher order structures in a cryoET reconstruction that could be used for understanding the assembly of toroid structures. The cross-linking ability of ZapD dimers result in bending of FtsZ filaments to a constant curvature. Many such short filaments are stitched together to form a toroid like structure. The geometry of assembly of filaments - whether they form straight bundles or toroid like structures - depends on the relative concentrations of FtsZ and ZapD.

Strengths:

In addition to a clear picture of the FtsZ assembly into ring-like structures, the authors have carried out basic biochemistry and biophysical techniques to assay the GTPase activity, the kinetics of assembly, and the ZapD to FtsZ ratio.

Weaknesses:

Future scope of work includes the molecular basis of curvature generation and how molecular features of FtsZ and ZapD affect the membrane binding of the higher order assembly.

Reviewer #3 (Public review):

Summary:

Previous studies have analyzed the binding of ZapD to FtsZ and provided images of negatively stained toroids and straight bundles, where FtsZ filaments are presumably crosslinked by ZapD dimers. Toroids without ZapD have also been previously formed by treating FtsZ with crowding agents. The present study is the first to apply cryoEM tomography, which can resolve the structure of the toroids in 3D. This shows a complex mixture of filaments and sheets irregularly stacked in the Z direction and spaced radially. The most important interpretation would be to distinguish FtsZ filaments from ZapD crosslinks, This is less convincing. The authors seem aware of the ambiguity: "However, we were unable to obtain detailed structural information about the ZapD connectors due to the heterogeneity and density of the toroidal structures, which showed significant variability in the conformations of the connections between the filaments in all directions." Therefore, the reader may assume that the crosslinks identified and colored red are only suggestions, and look for their own structural interpretations.

Strengths:

This is the first cryoEM tomography to image toroids and straight bundles of FtsZ filaments bound to ZapD. A strength is the resolution, which. at least for the straight bundles. is sufficient to resolve the ~4.5 nm spacing of ZapD dimers attached to and projecting subunits of an FtsZ filament. Another strength is the pelleting assay to determine the stoichiometry of ZapD:FtsZ (although this also leads to weaknesses of interpretation).

Reviewer #1 (Public review):

Summary:

In this study, Lamberti et al. investigate how translation initiation and elongation are coordinated at the single-mRNA level in mammalian cells. The authors aim to uncover whether and how cells dynamically adjust initiation rates in response to elongation dynamics, with the overarching goal of understanding how translational homeostasis is maintained. To this end, the study combines single-molecule live-cell imaging using the SunTag system with a kinetic modeling framework grounded in the Totally Asymmetric Simple Exclusion Process (TASEP). By applying this approach to custom reporter constructs with different coding sequences, and under perturbations of the initiation/elongation factor eIF5A, the authors infer initiation and elongation rates from individual mRNAs and examine how these rates covary.

The central finding is that initiation and elongation rates are strongly correlated across a range of coding sequences, resulting in consistently low ribosome density ({less than or equal to}12% of the coding sequence occupied). This coupling is preserved under partial pharmacological inhibition of eIF5A, which slows elongation but is matched by a proportional decrease in initiation, thereby maintaining ribosome density. However, a complete genetic knockout of eIF5A disrupts this coordination, leading to reduced ribosome density, potentially due to changes in ribosome stalling resolution or degradation.

Strengths:

A key strength of this work is its methodological innovation. The authors develop and validate a TASEP-based Hidden Markov Model (HMM) to infer translation kinetics at single-mRNA resolution. This approach provides a substantial advance over previous population-level or averaged models and enables dynamic reconstruction of ribosome behavior from experimental traces. The model is carefully benchmarked against simulated data and appropriately applied. The experimental design is also strong. The authors construct matched SunTag reporters differing only in codon composition in a defined region of the coding sequence, allowing them to isolate the effects of elongation-related features while controlling for other regulatory elements. The use of both pharmacological and genetic perturbations of eIF5A adds robustness and depth to the biological conclusions. The results are compelling: across all constructs and conditions, ribosome density remains low, and initiation and elongation appear tightly coordinated, suggesting an intrinsic feedback mechanism in translational regulation. These findings challenge the classical view of translation initiation as the sole rate-limiting step and provide new insights into how cells may dynamically maintain translation efficiency and avoid ribosome collisions.

Weaknesses:

A limitation of the study is its reliance on exogenous reporter mRNAs in HeLa cells, which may not fully capture the complexity of endogenous translation regulation. While the authors acknowledge this, it remains unclear how generalizable the observed coupling is to native mRNAs or in different cellular contexts.

Additionally, the model assumes homogeneous elongation rates and does not explicitly account for ribosome pausing or collisions, which could affect inference accuracy, particularly in constructs designed to induce stalling. While the model is validated under low-density assumptions, more work may be needed to understand how deviations from these assumptions affect parameter estimates in real data.

Furthermore, although the study observes translation "bursting" behavior, this is not explicitly modeled. Given the growing recognition of translational bursting as a regulatory feature, incorporating or quantifying this behavior more rigorously could strengthen the work's impact.

Assessment of Goals and Conclusions:

The authors successfully achieve their stated aims: they quantify translation initiation and elongation at the single-mRNA level and show that these processes are dynamically coupled to maintain low ribosome density. The modeling framework is well suited to this task, and the conclusions are supported by multiple lines of evidence, including inferred kinetic parameters, independent ribosome counts, and consistent behavior under perturbation.

Impact and Utility:

This work makes a significant conceptual and technical contribution to the field of translation biology. The modeling framework developed here opens the door to more detailed and quantitative studies of ribosome dynamics on single mRNAs and could be adapted to other imaging systems or perturbations. The discovery of initiation-elongation coupling as a general feature of translation in mammalian cells will likely influence how researchers think about translational regulation under homeostatic and stress conditions.

The data, models, and tools developed in this study will be of broad utility to the community, particularly for researchers studying translation dynamics, ribosome behavior, or the effects of codon usage and mRNA structure on protein synthesis.

Context and Interpretation:

This study contributes to a growing body of evidence that translation is not merely controlled at initiation but involves feedback between elongation and initiation. It supports the emerging view that ribosome collisions, stalling, and quality control pathways play active roles in regulating initiation rates in cis. The findings are consistent with recent studies in yeast and metazoans showing translation initiation repression following stalling events. However, the mechanistic details of this feedback remain incompletely understood and merit further investigation, particularly in physiological or stress contexts.

In summary, this is a thoughtfully executed and timely study that provides valuable insights into the dynamic regulation of translation and introduces a modeling framework with broad applicability. It will be of interest to a wide audience in molecular biology, systems biology, and quantitative imaging.

Reviewer #2 (Public review):

Summary:

This manuscript uses single-molecule run-off experiments and TASEP/HMM models to estimate biophysical parameters, i.e., ribosomal initiation and elongation rates. Combining inferred initiation and elongation rates, the authors quantify ribosomal density. TASEP modeling was used to simulate the mechanistic dynamics of ribosomal translation, and the HMM is used to link ribosomal dynamics to microscope intensity measurements. The authors' main conclusions and findings are:

(1) Ribosomal elongation rates and initiation rates are strongly coordinated.

(2) Elongation rates were estimated between 1-4.5 aa/sec. Initiation rates were estimated between 0.5-2.5 events/min. These values agree with previously reported values.

(3) Ribosomal density was determined below 12% for all constructs and conditions.

(4) eIF5A-perturbations (KO and GC7 inhibition) resulted in non-significant changes in translational bursting and ribosome density.

(5) eIF5A perturbations resulted in increases in elongation and decreases in initiation rates.

Strengths:

This manuscript presents an interesting scientific hypothesis to study ribosome initiation and elongation concurrently. This topic is highly relevant for the field. The manuscript presents a novel quantitative methodology to estimate ribosomal initiation rates from Harringtonine run-off assays. This is relevant because run-off assays have been used to estimate, exclusively, elongation rates.

Weaknesses:

The conclusion of the strong coordination between initiation and elongation rates is interesting, but some results are unexpected, and further experimental validation is needed to ensure this coordination is valid.

(1) eIF5a perturbations resulted in a non-significant effect on the fraction of translating mRNA, translation duration, and bursting periods. Given the central role of eIF5a, I would have expected a different outcome. I would recommend that the authors expand the discussion and review more literature to justify these findings.

(2) The AAG construct leading to slow elongation is very surprising. It is the opposite of the field consensus, where codon-optimized gene sequences are expected to elongate faster. More information about each construct should be provided. I would recommend more bioinformatic analysis on this, for example, calculating CAI for all constructs, or predicting the structures of the proteins.

(3) The authors should consider using their methodology to study the effects of modifying the 5'UTR, resulting in changes in initiation rate and bursting, such as previously shown in reference Livingston et al., 2023. This may be outside of the scope of this project, but the authors could add this as a future direction and discuss if this may corroborate their conclusions.

(4) The mathematical model and parameter inference routines are central to the conclusions of this manuscript. In order to support reproducibility, the computational code should be made available and well-documented, with a requirements file indicating the dependencies and their versions.

Reviewer #3 (Public review):

Disclaimer:

My expertise is in live single-molecule imaging of RNA and transcription, as well as associated data analysis and modeling. While this aligns well with the technical aspects of the manuscript, my background in translation is more limited, and I am not best positioned to assess the novelty of the biological conclusions.

Summary:

This study combines live-cell imaging of nascent proteins on single mRNAs with time-series analysis to investigate the kinetics of mRNA translation.

The authors (i) used a calibration method for estimating absolute ribosome counts, and (ii) developed a new Bayesian approach to infer ribosome counts over time from run-off experiments, enabling estimation of elongation rates and ribosome density across conditions.

They report (i) translational bursting at the single-mRNA level, (ii) low ribosome density (~10% occupancy {plus minus} a few percents), (iii) that ribosome density is minimally affected by perturbations of elongation (using a drug and/or different coding sequences in the reporter), suggesting a homeostatic mechanism potentially involving a feedback of elongation onto initiation, although (iv) this coupling breaks down upon knockout of elongation factor eIF5A.

Strengths:

(1) The manuscript is well written, and the conclusions are, in general, appropriately cautious (besides the few improvements I suggest below).

(2) The time-series inference method is interesting and promising for broader applications.

(3) Simulations provide convincing support for the modeling (though some improvements are possible).

(4) The reported homeostatic effect on ribosome density is surprising and carefully validated with multiple perturbations.

(5) Imaging quality and corrections (e.g., flat-fielding, laser power measurements) are robust.

(6) Mathematical modeling is clearly described and precise; a few clarifications could improve it further.

Weaknesses:

(1) The absolute quantification of ribosome numbers (via the measurement of $i_{MP}$​) should be improved. This only affects the finding that ribosome density is low, not that it appears to be under homeostatic control. However, if $i_{MP}$​ turns out to be substantially overestimated (hence ribosome density underestimated), then "ribosomes queuing up to the initiation site and physically blocking initiation" could become a relevant hypothesis. In my detailed recommendations to the authors, I list points that need clarification in their quantifications and suggest an independent validation experiment (measuring the intensity of an object with a known number of GFP molecules, e.g., MS2-GFP MS2-GFP-labeled RNAs, or individual GEMs).

(2) The proposed initiation-elongation coupling is plausible, but alternative explanations, such as changes in abortive elongation frequency, should be considered more carefully. The authors mention this possibility, but should test or rule it out quantitatively.

(3) The observation of translational bursting is presented as novel, but similar findings were reported by Livingston et al. (2023) using a similar SunTag-MS2 system. This prior work should be acknowledged, and the added value of the current approach clarified.

(4) It is unclear what the single-mRNA nature of the inference method is bringing since it is only used here to report _average_ ribosome elongation rate and density (averaged across mRNAs and across time during the run-off experiments - although the method, in principle, has the power to resolve these two aspects).

(5) I did not find any statement about data availability. The data should be made available. Their absence limits the ability to fully assess and reproduce the findings.

Reviewer #1 (Public review):

Summary:

In this revised report, Yamanaka and colleagues investigate a proposed mechanism by which testosterone modulates seminal plasma metabolites in mice. The authors have made improvements from the previous version by softening the claim that oleic acid derived from seminal vesicle epithelium strongly affects linear progressive motility in isolated cauda epididymal sperm in vitro. They have also addressed the ambiguous references to the strength of the relationship between fatty acids and sperm motility, making the manuscript more balanced and nuanced.

Strengths:

This study addresses an important gap in our understanding of how testosterone influences seminal plasma metabolites and, in turn, sperm motility. The findings provide valuable insights into the sensitivity of seminal vesicle epithelial cells to testosterone, which could improve in vitro conditions for studying sperm motility. The authors have added methodological details and re-performed experiments with more appropriate control groups, enhancing the robustness of the study. These revisions, along with more carefully modified language reflecting measurement nuances, add significant value to the field. The study's detailed exploration of the physiological role of reproductive tract glandular secretions in modulating sperm behaviors is likely to be of broad interest, providing a strong foundation for future research on the relationship between fatty acid beta-oxidation and sperm motility patterns.

Weaknesses:

While the connection between media fatty acids and sperm motility patterns is still not fully conclusive, the authors have taken substantial steps to clarify and tone down their conclusions. The revised manuscript presents a more balanced view, acknowledging the complexity of the relationship and providing a more solid basis for follow-on studies.

Reviewer #2 (Public review):

Using a combination of in vivo studies with testosterone-inhibited and aged mice with lower testosterone levels, as well as isolated mouse and human seminal vesicle epithelial cells, the authors demonstrate that testosterone induces an increase in glucose uptake. The study reveals that testosterone triggers differential gene expression, particularly focusing on metabolic enzymes. They specifically identify increased expression of enzymes regulating cholesterol and fatty acid synthesis, leading to heightened production of 18:1 oleic acid. The revised version of the manuscript significantly strengthens the role of ACLY as a central regulator of seminal vesicle epithelial cell metabolic programming. The authors suggest that fatty acids secreted by seminal vesicle epithelial cells are taken up by sperm, resulting in a positive impact on sperm function. While the lipid mixture mimicking the lipids secreted by seminal vesicle epithelial cells shows marginal positive effect on sperm motility, the authors have made considerable progress in refining their conclusions. The revised manuscript acknowledges the complexity of pinpointing the specific seminal vesicle fluid component that potentially positively affects sperm function, providing a more measured and credible interpretation of their findings.

Quantify the number of users or sessions tracked to demonstrate the app's usage and impact.

Include user adoption rates or feedback to illustrate the bot's effectiveness and popularity.

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Reviewer #1 (Public review):

Summary:

The authors aim to explore the effects of the electrogenic sodium-potassium pump (Na+/K+-ATPase) on the computational properties of highly active spiking neurons, using the weakly-electric fish electrocyte as a model system. Their work highlights how the pump's electrogenicity, while essential for maintaining ionic gradients, introduces challenges in neuronal firing stability and signal processing, especially in cells that fire at high rates. The study identifies compensatory mechanisms that cells might use to counteract these effects, and speculates on the role of voltage dependence in the pump's behavior, suggesting that Na⁺/K⁺-ATPase could be a factor in neuronal dysfunctions and diseases

Strengths:

(1) The study explores a less-examined aspect of neural dynamics-the effects of Na⁺/K⁺-ATPase electrogenicity. It offers a new perspective by highlighting the pump's role not only in ion homeostasis but also in its potential influence on neural computation.

(2) The mathematical modeling used is a significant strength, providing a clear and controlled framework to explore the effects of the Na⁺/K⁺+-ATPase on spiking cells. This approach allows for the systematic testing of different conditions and behaviors that might be difficult to observe directly in biological experiments.

(3) The study proposes several interesting compensatory mechanisms, such as sodium leak channels and extracellular potassium buffering, which provide useful theoretical frameworks for understanding how neurons maintain firing rate control despite the pump's effects.

Weaknesses:

(1) While the modeling approach provides valuable insights, the lack of experimental data to validate the model's predictions weakens the overall conclusions.

(2) The proposed compensatory mechanisms are discussed primarily in theoretical terms without providing quantitative estimates of their impact on the neuron's metabolic cost or other physiological parameters.

Comments on revisions:

The revised manuscript is notably improved.

Reviewer #2 (Public review):

Summary:

The paper by Weerdmeester, Schleimer, and Schreiber uses computational models to present the biological constraints under which electrocytes - specialized, highly active cells that facilitate electro-sensing in weakly electric fish-may operate. The authors suggest potential solutions that these cells could employ to circumvent these constraints.

Electrocytes are highly active or spiking (greater than 300Hz) for sustained periods (for minutes to hours), and such activity is possible due to an influx of sodium and efflux of potassium ions into these cells after each spike. The resulting ion imbalance must be restored, which in electrocytes, as with many other biological cells, is facilitated by the Na-K pumps at the expense of biological energy, i.e., ATP molecules. For each ATP molecule the pump uses, three positively charged sodium ions from the intracellular space are exchanged for two positively charged potassium ions from the extracellular space. This creates a net efflux of positive ions into the extracellular space, resulting in hyperpolarized potentials for the cell over time. For most cells, this does not pose an issue, as their firing rate is much slower, and other compensatory mechanisms and pumps can effectively restore the ion imbalances. However, in the electrocytes of weakly electric fish, which spike at exceptionally high rates, the net efflux of positive ions presents a challenge. Additionally, these cells are involved in critical communication and survival behaviors, underscoring their essential role in reliable functioning.

In a computational model, the authors test four increasingly complex solutions to the problem of counteracting the hyperpolarized states that occur due to continuous NaK pump action to sustain baseline activity. First, they propose a solution for a well-matched Na leak channel that operates in conjunction with the NaK pump, counteracting the hyperpolarizing states naturally. Their model shows that when such an orchestrated Na leak current is not included, quick changes in the firing rates could have unexpected side effects. Secondly, they study the implications of this cell in the context of chirps-a means of communication between individual fish. Here, an upstream pacemaking neuron entrains the electrocyte to spike, which ceases to produce a so-called chirp - a brief pause in the sustained activity of the electrocytes. In their model, the authors demonstrate that including the extracellular potassium buffer is necessary to obtain a reliable chirp signal. Thirdly, they tested another means of communication in which there was a sudden increase in the firing rate of the electrocyte, followed by a decay to the baseline. For this to occur reliably, the authors emphasize that a strong synaptic connection between the pacemaker neuron and the electrocyte is necessary. Finally, since these cells are energy-intensive, they hypothesize that electrocytes may have energy-efficient action potentials, for which their NaK pumps may be sensitive to the membrane voltages and perform course correction rapidly.

Strengths:

The authors extend an existing electrocyte model (Joos et al., 2018) based on the classical Hodgkin and Huxley conductance-based models of sodium and potassium currents to include the dynamics of the sodium-potassium (NaK) pump. The authors estimate the pump's properties based on reasonable assumptions related to the leak potential. Their proposed solutions are valid and may be employed by weakly electric fish. The authors explore theoretical solutions to electrosensing behavior that compound and suggest that all these solutions must be simultaneously active for the survival and behavior of the fish. This work provides a good starting point for conducting in vivo experiments to determine which of these proposed solutions the fish employ and their relative importance. The authors include testable hypotheses for their computational models.

Weaknesses:

The model for action potential generation simplifies ion dynamics by considering only sodium and potassium currents, excluding other ions like calcium. The ion channels considered are assumed to be static, without any dynamic regulation such as post-translational modifications. For instance, a sodium-dependent potassium pump could modulate potassium leak and spike amplitude (Markham et al., 2013).

This work considers only the sodium-potassium (NaK) pumps to restore ion gradients. However, in many cells, several other ion pumps, exchangers, and symporters are simultaneously present and actively participate in restoring ion gradients. When sodium currents dominate action potentials, and thus when NaK pumps play a critical role, such as the case in Eigenmannia virescens, the present study is valid. However, since other biological processes may find different solutions to address the pump's non-electroneutral nature, the generalizability of the results in this work to other fast-spiking cell types is limited. For example, each spike could include a small calcium ion influx that could be buffered or extracted via a sodium-calcium exchanger.

Reviewer #1 (Public review):

The manuscript by Long et al. focused on SUL1, a gene encoding a sulfate transporter with signaling roles in yeast. The authors claim that the deletion of SUL1, rather than SUL2 (encoding a similar transporter), extended yeast replicative lifespan independent of sulfate transport. They also show that SUL1 loss-of-function mutants display decreased PKA activity, indicated by stress-protective carbohydrate accumulation, relevant transcription factor relocalization (measured during aging in single cells), and changes in gene expression. Finally, they show that loss of SUL1 increases autophagy, which is consistent with the longer lifespan of these cells. Overall, this is an interesting paper, but additional work should strengthen several conclusions, especially for the role of sulfate transport. Specific points include the following:

What prompted the authors to measure the RLS of sul1 mutants? Prior systematic surveys of RLS in the same strain background (which included the same sul1 deletion strain they used) did not report lifespan extension in sul1 cells (PMID: 26456335).

Cells carrying a mutant Sul1 (E427Q), which was reported to be disrupted in sulfate transport, did not have a longer lifespan (Figure 1), leading them to conclude that "lifespan extension by SUL1 deletion is not caused by decreased sulfate uptake". They would need to measure sulfate uptake in the mutants they test to draw that conclusion firmly.

Related to my previous point, another simple experiment would be to repeat the assays in Figure 1 with exogenous sulfur added to see if the lifespan extension is suppressed.

There needs to be more information in the text or the methods about how they did the enrichment analysis in Figure 2B. P-values are typically insufficient, and adjusted FDR values are reported from standard gene ontology platforms (e.g., PANTHER).

It is somewhat puzzling that relocalization of Msn2 was not seen in very old cells (past the 17th generation), but it was evident in younger cells. The authors could consider another possibility, that it was early and midlife experiences that made those cells live longer. Past that window, loss of Sul1 may have no impact on longevity. A conditional shutoff system to regulate SUL1 expression would be needed to test the above, albeit this is probably beyond the scope of this report.

The connections between glucose restriction, autophagy, and sul1 (Figure 4) could be further tested by measuring the RLS of sul1 cells in glucose-restricted cells. If RLS is further extended by glucose restriction, then whatever effects they see should be independent of glucose restriction.

They made and tested the double (sul1, msn2) mutants, but they should also test the sul1, msn4 combination since Msn4 functions similarly to Msn2.

Comments on revisions:

Overall, this is a somewhat improved manuscript, but some prior concerns about the validity of the conclusions remain unresolved.

Reviewer #2 (Public review):

Summary:

In this study, the authors find that deletion of a sulfate transporter in yeast, Sul1, leads to extension of replicative lifespan. They investigate mechanisms underlying this extension, and claim that the effects on longevity can be separated from sulfate transport, and are instead linked to a previously proposed transceptor function of the Sul1 transporter. Through RNA sequencing analysis, the authors find that Sul1 loss triggers activation of several stress response pathways, and conclude that deletion of two pathways, autophagy or Msn2/4, partially prevents lifespan extension in cells lacking Sul1. Overall, while it is well-appreciated that activation of Msn2/4 or autophagy is beneficial for lifespan extension in yeast, the results of this study would add an important new mechanism by which this could achieved, through perceived sulfate starvation. However, as described below, several of the experiments utilized to support the authors conclusion are not experimentally sound, and significant additional experimentation is required to support the authors claims throughout the manuscript.

Strengths:

The major strength of the study is the robust RNA-seq data that identified differentially expressed genes in cells lacking Sul1. This facilitated the authors focus on two of these pathways, autophagy and the Msn2/4 stress response pathway.

Weaknesses:

Several critical experimental flaws need to be addressed by the authors to more rigorously test their hypothesis.

(1) The lifespan assays throughout the manuscript contain inconsistencies in the mean lifespan of the wild type strain, BY4741. For example, in Figure 1A, the lifespan of BY4741 is 24.3, and the extended lifespan of the sul1 mutant is 31. However, although all mutants tested in Figure 1B also have lifespans close to 30 cell divisions, the wild type control is also at 30 divisions in those experiments as well. This is problematic, as it makes it impossible to conclude anything about the lifespan extension of various mutants with the inconsistencies in the wild type lifespan. Additionally, the mutants analyzed in 1B are what the authors use to claim that loss of the transporter does not extend lifespan through sulfate limitation, but instead through a signaling function. Thus, it remains unclear whether loss of sul1 extends lifespan at all, and if it does, whether this is separable from cellular sulfate levels.

(2) While the authors use mutants in Figure 1 that should have differential effects on sulfate levels in cells, the authors need to include experiments to measure sulfate levels in their various mutant cells to draw any conclusions about their data.

(3) Similar to point 2, the authors focused their RNA sequencing analysis on deletion of sul1 and did not include important RNA seq analysis of the specific Sul1 mutation or other mutants in Figure 1B that do not exhibit lifespan extension. The prediction is that they should not see activation of stress response pathways in these mutants as they do not see lifespan extension, but this needs to be tested.

(4) While the RNA-seq data is robust in Figure 2 as well as the follow up quantitative PCR and trehalose/glycogen assays in 2A-B, the follow-up imaging assays for Msn2/4 localization in Figure 2 are not robust and are difficult to interpret. The authors need to include more high-resolution imaging or at least a close up of the cells in Figure 3C.

(5) The autophagy assays utilized in Figure 4 appear to all be done with a C-terminal GFP-tagged Atg8 protein. As C-terminal GFP is removed from Atg8 prior to conjugation to phosphatidylethanolamine, microscopy assays of this reporter cannot be utilized to report on autophagy activity or flux. Instead, the authors need to utilize N-terminally tagged Atg8, which they can monitor for vacuole uptake as an appropriate readout of autophagy levels. As it stands, the authors cannot draw any conclusions about autophagy activity in their studies.

Comments on revisions:

Their autophagy conclusions are weak at best. As was highlighted in the previous review, they need to use an N-terminal Atg8 fusion for these experiments.

Reviewer #3 (Public review):

Summary:

In the revised manuscript, Long et al., showed that sul1∆ mutants have extended replicative lifespan in budding yeast. In comparison, other mutants that have sulfate transport deficiency did not show extended lifespan, suggesting SUL1 deletion extends lifespan independently of sulfate intake. The authors then explored the transcriptome of sul1∆ mutants by RNA-seq, which suggests that SUL1 deletion impacts common longevity pathways. Furthermore, the authors characterized how the PKA pathway is affected in sul1∆ mutants: SUL1 deletion promotes the nuclear localization of Msn2, as well as autophagy, indicating down-regulation of the PKA pathway.

Strengths:

This study raised an interesting point that inorganic transporters may impact cellular stress response pathways and affect lifespan. Some of the characterizations on the sul1∆ mutants, including the RNA-seq and MSN2 localization could provide valuable sources for people in related fields. Compared with the previous version, the writing is significantly improved, making the manuscript clearer.

Weaknesses:

Several critical flaws have not been revised. The claims are still not well supported by the data.

(1) The revised manuscript still uses Atg8-EGFP, in which GFP is likely tagging at the C-terminus of Atg8. No strain information was provided for this strain, so it is unclear whether it is N- or C- terminal tagged. As pointed by reviewers of the previous version, C-terminal tagged Atg8 is not functional. As a result, the conclusions on autophagy (Figure 4) is questionable.

(2) The nuclear localization of Msn2 is much more convincing after the authors updated Figure 3C. However, the rest of the microscopy images (e.g. Figure 3E, 4B, 4E) are still of low resolution. Again, I suggest to separate the DIC and GFP channels. It is really hard to tell where is the GFP signal from these figures.

(3) In the Kankipati et al. 2015 paper, which is cited by the authors, SUL1E427Q is incorporated on a pRS316 (URA3) plasmic and expressed in sul1∆sul2∆ mutants. In this manuscript, the authors used SUL1E427Q mutants but did not give detailed information on how this construct is expressed. Is it endogenously mutated, incorporated into somewhere in the genome, or expressed from an extrachromosomal plasmid?<br /> In Figure 1B, they simply used BY4741 as a control for the SUL1E427Q mutant. This makes me thinking they are using a SUL1E427Q endogenous point mutation mutant. If so, the authors may want to include the information about this strain in their Supplementary table. Or if it is expressed from an extra copy on chromosomes or extrachromosomal plasmids, the authors would need to express this construct in sul1∆ mutant. In this case, the authors may want to use sul1∆ and sul1∆+empty vector as controls, instead of BY4741. As the authors mentioned in their rebuttal letter, lifespan experiments vary between each individual trials and are not comparable between different trials. Thus proper controls are essential to make the results convincing.

(4) As suggested by reviewers of the previous version, the authors tested the sulfate uptake in different mutants within 10 minute of Na2SO4 addition (Figure 1B). The authors concluded from the data that wild type takes up sulfate faster than the mutants but they reach similar concentrations at the end point (as fast as 10 minutes). Are all these cells sulfate-starved before the experiment? If not, the experiment might be affected by the basal level of sulfate in each mutants.

Reviewer #1 (Public review):

The authors aimed to explore the prognostic and therapeutic relevance of immunogenic cell death (ICD)-related genes in bladder cancer, focusing on a risk-scoring model involving CALR, IL1R1, IFNB1, and IFNG. The research indicates that higher expression of certain ICD-related genes is associated with enhanced immune infiltration, prolonged survival, and improved responsiveness to PD1-targeted therapy in bladder cancer patients.

Major strengths:

• The establishment of an ICD-related gene risk model based on publicly available datasets (TCGA and GEO) and further validated through tissue arrays and preliminary single-cell RNA sequencing data provides potential but weak clinical guidance.

• The integration of multi-dimensional data (gene expression, mutation burden, immune infiltration, and treatment responses) strengthens the clinical applicability of the model.

Key limitations and concerns:

(1) Gene Selection and Novelty:

The selection of genes predominantly reflects known regulators of immune responses, somewhat limiting the novelty. Exploring less-characterized ICD markers or extending validation beyond bladder cancer could improve the model's innovative aspect and wider clinical relevance.

(2) Reliance on RNA-Seq for Immune Infiltration:

Immune infiltration analyses based primarily on bulk RNA-Seq data have inherent methodological limitations, such as inability to distinguish cell subsets accurately. Incorporation of robust single-cell sequencing would significantly enhance the reliability of these findings. Although the authors recognize this limitation, future studies should directly address it.

(3) Drug Sensitivity and Immunotherapy Response Data:

While the authors clarify that the drug sensitivity analysis was performed using established databases (TCGA via pRRophetic), the unexpected correlations between ICD-related genes and various targeted therapies need further mechanistic validation. The observed relationships may reflect indirect associations rather than direct biological relevance, which warrants cautious interpretation.

(4) Presentation and Clarity Issues:

Initially noted formatting inconsistencies across figures compromised professional presentation; these have been corrected by the authors. Additionally, the authors have now provided essential methodological details, including clear sample sizes and database versions, enhancing reproducibility.

(5) Immunotherapy Response Evidence:

Conclusions regarding differences in immunotherapy response rates between patient subgroups, although intriguing, remain based on retrospective database analyses with relatively limited demographic and clinical detail. Future prospective studies or more detailed patient characterization would be required to robustly confirm these associations.

(6) Interpretation of ICD Gene Signatures:

The ICD-related gene set includes many genes broadly associated with immune activation rather than specifically ICD. Although this was addressed by the authors, clearly distinguishing ICD-specific versus general immune-response genes in future studies would help clarify biological implications.

Summary and Recommendations for Readers:

Overall, this study presents an interesting and clinically relevant risk-scoring approach to stratify bladder cancer patients based on ICD-related gene expression profiles. It provides useful information about prognosis, immune infiltration, and potential immunotherapy responsiveness. However, readers should interpret the results within the context of its limitations, notably the need for broader validation and careful consideration of the biological significance underlying the observed associations. This work lays a valuable foundation for further investigation into the integration of ICD and immune response signatures in personalized cancer therapy.

Reviewer #1 (Public review):

Summary:

In this study, the authors investigate the role of deubiquitinases (DUBs) in modulating the efficacy of PROTAC-mediated degradation of the cell-cycle kinase AURKA. Using a focused siRNA screen of 97 human DUBs, they identify UCHL5 and OTUD6A as negative regulators of AURKA degradation by PROTACs. They further offer a mechanistic explanation of enhanced AURKA degradation in the nucleus via OTUD6A expression being restricted to the cytosol, thereby protecting the cytoplasmic pool of AURKA. These findings provide important insight into how subcellular localization and DUB activity influence the efficiency of targeted protein degradation strategies, which could have implications for therapy.

Strengths:

(1) The manuscript is well-structured, with clearly defined objectives and well-supported conclusions.

(2) The study employs a broad range of well-validated techniques - including live-cell imaging, proximity ligation assays, HiBiT reporter systems, and ubiquitin pulldowns - to dissect the regulation of PROTAC activity.

(3) The authors use informative experimental controls, including assessment of cell-cycle progression effects, rescue experiments with siRNA-resistant constructs to confirm specificity, and the application of both AURKA-targeting PROTACs with different warheads and orthogonal degrader systems (e.g., dTAG-13 and dTAGv-1) to differentiate between target- and ligase-specific effects.

(4) The identification of OTUD6A as a cytosol-restricted DUB that protects cytoplasmic but not nuclear AURKA is novel and may have therapeutic relevance for selectively targeting oncogenic nuclear AURKA pools.

Weaknesses:

(1) Although UCHL5 and OTUD6A are shown to limit AURKA degradation, direct physical interaction was not assessed.

(2) Although the authors identify a correlation between DUB knockdown-induced cell cycle progression and enhanced PROTAC activity, only one DUB (USP36) is excluded on this basis. In addition, one DUB is shown in the correlation plot (Figure 3B) whose knockdown enhances PROTAC sensitivity without significantly altering cell cycle progression, but it is not identified/discussed.

(3) While the authors suggest that combining PROTACs with DUB inhibition could enhance degradation, this was not experimentally tested.

(4) The study identifies UCHL5 as a general antagonist of CRBN-recruiting PROTACs, yet the ubiquitin pulldown experiments (Figure 5G, H) show no change in AURKA ubiquitination upon UCHL5 knockdown. This raises questions about the precise step or mechanism by which UCHL5 exerts its protective effect.

Reviewer #2 (Public review):

Summary:

In this study, the authors present a screening approach to identify deubiquitylases that may impact PROTAC efficacy/potency, specifically in this case using a previously reported AURKA PROTAC as an initial model. The authors claim that UCHL5 is able to control the level of degradation of both AURKA and dTAG when using CRBN-mediated PROTACs; however, VHL is not impacted by UCHL5 activity. They additionally claim that OTUD6A is able to control the extent of AURKA degradation in a target protein-specific manner and that this effect is specific to cytoplasm-located AURKA.

Overall, whilst the endeavour is of interest and importance, we found that the claims made were overly generalised, the effects observed when knocking down the respective DUBs were very small, the systems used are highly artificial, and the data is not presented in a way that makes understanding absolute changes transparent.

Strengths:

The topic is of high interest and relevance and explores an underappreciated and understudied area of the PROTAC mechanism of action. If findings could be better supported, they would certainly bring value to the field.

Weaknesses:

The overall effects observed are sometimes limited in real terms. Even if statistically significant, the data presented does not fully support that changes in degradation due to UCHL5 activity represent changes of functional relevance. The data provided often omits the absolute changes in protein abundance observed. Data on endogenous/less engineered systems and/or with higher resolution read-outs would greatly strengthen some conclusions.

Reviewer #3 (Public review):

Summary:

Cardno et al. "test the hypothesis that DUBs could oppose PROTAC-mediated degradation of cellular targets, using AURKA as a model target". A screen with a panel of siRNA that depleted 97 DUBs in the presence and absence of AURKA targeted PROTAC-D identified DUBs that regulated AURKA and those that affected the sensitivity of PROTAC-D. Validation studies with DUBs, UCHL5, and OTU6A yielded mixed results. UCHL5 not only affected PROTAC-mediated AURKA degradation but also affected CRBN-associated substrates, OTUD6A, more specifically, affected PROTAC-mediated AURKA degradation, and the effects of OTUD6A were associated with the localisation of AURKA. The findings are interesting; the impact of the findings would be strengthened if the key results are validated in one or more cancer cell lines that have not been modified.

Reviewer #1 (Public review):

Summary:

BK channels are widely distributed and involved in many physiological functions. They have also proven a highly useful tool for studying general allosteric mechanisms for gating and modulation by auxiliary subunits. Tetrameric BK channels are assembled from four separate alpha subunits, which would be identical for homozygous alleles and potentially of five different combinations for heterozygous alleles (Geng et al., 2023, https://doi.org/10.1085/jgp.202213302). Construction of BK channels with concatenated subunits in order to strictly control heteromeric subunit composition had not yet been used because the N-terminus in BK channels is extracellular, whereas the C-terminus is intracellular. In this new work, Chen, Li, and Yan devise clever methods to construct and assemble BK channels of known subunit composition, as well as to fix the number of γ1 axillary subunits per channel. With their novel molecular approaches, Chen, Li and Yan report that a single γ1 axillary subunit is sufficient to fully modulate a BK channel, that the deep conducting pore mutation L312A exhibited a graded effect on gating with each addition mutated subunit replacing a WT subunit in the channel adding an additional incremental left shift in activation, and that the V288A mutation at the selectivity filter must be present on all four alpha subunits in order to induce channel inactivation. Chen, Li, and Yan have been successful in introducing new molecular tools to generate BK channels of known stoichiometry and subunit composition. They validate their methods and provide three examples of their use with useful observations.

Strengths:

Powerful new molecular tools for the study of channel gating have been developed and validated in the study.

Weaknesses:

One example each of auxiliary, deep pore, and selectivity filter allosteric actions is presented, but this is sufficient for the purposes of the paper to establish their methods and present specific examples of applicability.

Reviewer #2 (Public review):

Summary:

This manuscript describes novel BK channel concatemers as a tool to study the stoichiometry of the gamma subunit and mutations in the modulation of the channel. Taking advantage of the modular design of the BK channel alpha subunit, the authors connected S1-S6/1st RCK as two- and four-subunit concatemers and coexpressed with S0-RCK2 to form normal function channels. These concatemers avoided the difficulty that the extracellular N-terminus of S0 was unable to connect with the cytosolic C-terminus of the gamma subunit, allowing a single gamma subunit to be connected to the concatemers. The concatemers also helped reveal the required stoichiometry of mutant BK subunits in modulating channel function. These include L312A in the deep pore region that altered channel function additively with each additional subunit harboring the mutation, and V288A at the selectivity filter that altered channel function cooperatively only when all four subunits were mutated. These results demonstrate that the concatemers are robust and effective in studying BK channel function and molecular mechanisms related to stoichiometry. The different requirement of the gamma subunit and the mutations stoichiometry for altering channel function is interesting, which may relate to the fundamental mechanism of how different motifs of the channel protein control function.

Strengths:

The manuscript presents well-designed experiments with high-quality data, which convincingly demonstrate the BK channel concatemers and their utility. The results are clearly presented.

Weaknesses:

This reviewer did not identify any major concerns with the manuscript.

Reviewer #1 (Public review):

Summary:

This manuscript presents a high-quality, chromosome-level genome assembly of the European cuttlefish (Sepia officinalis), a representative species of the cephalopod lineage. Using state-of-the-art sequencing and scaffolding technologies -including PacBio HiFi long reads and Hi-C chromatin conformation capture - the authors deliver a genome assembly with exceptional contiguity and completeness, as evidenced by high BUSCO scores. This genome resource fills a significant gap in cephalopod genomics and offers a valuable foundation for studies in neurobiology, behavior, and evolutionary biology. However, there are several major aspects that need to be strengthened.

Major Revisions Recommended:

(1) Single-individual genome limitation

The genome assembly is based on a single individual, which appears to be male. While this approach is common in genome projects, it does not capture the full genetic diversity of the species. As S. officinalis exhibits a wide geographical range and possible population structure, future efforts (or discussion in this manuscript) should consider re-sequencing multiple individuals - of both sexes and from diverse geographic origins - to characterize population-level variation, sex-linked features, and structural polymorphisms.

(2) Limited experimental validation of chromosomal inferences

The study reports chromosome-scale scaffolding using Hi-C data and proposes a revised karyotype for S. officinalis. However, these inferences would be significantly strengthened by orthogonal validation methods. In particular, fluorescence in situ hybridization (FISH) or karyotyping from cytogenetic preparations would provide direct confirmation of chromosome number and structural arrangements. The reliance solely on Hi-C contact maps for inferring chromosomal organization should be acknowledged as a limitation or supplemented with such validations.

(3) Shallow discussion of chromosomal evolution

The manuscript briefly mentions chromosomal number differences among cephalopods but does not explore their evolutionary or functional implications. A more thorough comparative analysis - linking chromosomal rearrangements (e.g., fusions, fissions) with ecological adaptation, life history, or neural complexity - would greatly enhance the impact of the findings. Referencing chromosomal dynamics in related taxa and possible links to behavioral innovations would contextualize these results more effectively.

(4) Underdeveloped gene family and pathway analysis

While the authors identify expansions in gene families such as protocadherins and C2H2 zinc finger transcription factors, the functional significance of these expansions remains speculative. The manuscript would benefit from:

a) Functional enrichment analyses (e.g., GO, KEGG) targeting these gene families.

b) Expression profiling across tissues or developmental stages to infer regulatory roles.

c) Comparison with expression or expansion patterns in other cephalopods with known behavioral complexity (e.g., Octopus bimaculoides, Euprymna scolopes).

d) Potential integration of transcriptomic or epigenomic data to support regulatory hypotheses.

Reviewer #2 (Public review):

Summary:

This paper concerns an interesting organism, Sepia officinalis. However, in the opinion of this reviewer, the paper reads somewhat like a genome report. The authors have used 23x PacBio HiFi in conjunction with relatively low coverage (11x) Hi-C to scaffold the genome into a karyotype of 47 chromosomes. They have used a combination of short and long read RNA seq to annotate the genome in what looks like a very good annotation. The paper offers basic analyses of the Busco evaluation, some descriptive analyses of gene family and repeat content, and a bit more focused analysis on synteny among sequenced squids. Generally, the data will be useful.

Strengths:

This is a high-quality annotation, and the data ultimately will be useful to other researchers. I appreciate trying to understand what's happening between assemblies of S. officinalis.

Weaknesses:

I don't believe the data at hand makes a strong case for the argument of 47 chromosomes. This is my biggest sticking point with the paper, and it is for a few reasons:

(1) The authors point to assembly differences between the DToL assembly and the one presented in the manuscript and seem to claim that DToL is incorrect. However, the DToL assembly (xcSepOffi3.1) is based on much deeper HiFi and HiC coverage than the one at hand (51x and 80+x respectively). There are many things to try here, including:

a) Downloading the DToL data and reassembling using a common pipeline.

b) Downsampling the DToL data to similar coverage as what the authors have achieved.

c) Combining your data and that of DToL for even deeper coverage (heterozygosity is low enough that I don't imagine this impeding things too badly).

(2) Looking at Figure 1, there appears to be a misjoin at chromosome 42. Looking carefully at Figure S1, that misjoin does not appear on any of the panels - this is confusing. Given the size of that chromosome and the authors' chromosome numbering, I'm guessing this is a manual merge (as it's larger than most of the chromosomes numerically close (40, 41, 43, etc). Further, staring closely at Figure 1, there appear to be cross-scaffold contacts between 42 and 43 and 42 and 44. Secondarily there are contacts between 43 and 44. This bit of the assembly seems potentially problematic.

Reviewer #3 (Public review):

Summary:

In this study, authors Simone Rencken and co-authors present and investigate the genome of the common cuttlefish Sepia officinalis.

Strengths:

The authors explain in a detailed yet concise manner the main steps for a genome assembly, with very robust methods for validation, and according to current best practices. In addition to the chromosomal assembly, the authors confirmed the presence of 47 chromosomes using Hi-C data and multiple species synteny. They also generated a comprehensive gene annotation, with assessments of gene completeness, providing a useful resource for the community of researchers interested in cuttlefish biology and comparative genomics.

Weaknesses:

While the study touches upon the subjects of gene content, TE activity, or species-level comparisons, the study does not provide in-depth investigations of these.

Reviewer #1 (Public review):

Summary:

This research investigates how the cellular protein quality control machinery influences the effectiveness of cystic fibrosis (CF) treatments across different genetic variants. CF is caused by mutations in the CFTR gene, with over 1,700 known disease-causing variants that primarily work through protein misfolding mechanisms. While corrector drugs like those in Trikafta therapy can stabilize some misfolded CFTR proteins, the reasons why certain variants respond to treatment while others don't remain unclear. The authors hypothesized that the cellular proteostasis network-the machinery that manages protein folding and quality control-plays a crucial role in determining drug responsiveness across different CFTR variants. The researchers focused on calnexin (CANX), a key chaperone protein that recognizes misfolded glycosylated proteins. Using CRISPR-Cas9 gene editing combined with deep mutational scanning, they systematically analyzed how CANX affects the expression and corrector drug response of 234 clinically relevant CF variants in HEK293 cells.

In terms of findings, this study revealed that CANX is generally required for robust plasma membrane expression of CFTR proteins, and CANX disproportionately affects variants with mutations in the C-terminal domains of CFTR and modulates later stages of protein assembly. Without CANX, many variants that would normally respond to corrector drugs lose their therapeutic responsiveness. Furthermore, loss of CANX caused broad changes in how CF variants interact with other cellular proteins, though these effects were largely separate from changes in CFTR channel activity.

This study has some limitations: the research was conducted in HEK293 cells rather than lung epithelial cells, which may not fully reflect the physiological context of CF. Additionally, the study only examined known disease-causing variants and used methodological approaches that could potentially introduce bias in the data analysis.

How cellular quality control mechanisms influence the therapeutic landscape of genetic diseases is an emerging field. Overall, this work provides important cellular context for understanding CF mutation severity and suggests that the proteostasis network significantly shapes how different CFTR variants respond to corrector therapies. The findings could pave the way for more personalized CF treatments tailored to patients' specific genetic variants and cellular contexts.

Strengths:

(1) This work makes an important contribution to the field of variant effect prediction by advancing our understanding of how genetic variants impact protein function.

(2) The study provides valuable cellular context for CFTR mutation severity, which may pave the way for improved CFTR therapies that are customized to patient-specific cellular contexts.

(3) The research provides further insight into the biological mechanisms underlying approved CFTR therapies, enhancing our understanding of how these treatments work.

(4) The authors conducted a comprehensive and quantitative analysis, and they made their raw and processed data as well as analysis scripts publicly available, enabling closer examination and validation by the broader scientific community.

Weaknesses:

(1) The study only considers known disease-causing variants, which limits the scope of findings and may miss important insights from variants of uncertain significance.

(2) The cellular context of HEK293 cells is quite removed from lung epithelia, the primary tissue affected in cystic fibrosis, potentially limiting the clinical relevance of the findings.

(3) Methodological choices, such as the expansion of sorted cell populations before genetic analysis, may introduce possible skew or bias in the data that could affect interpretation.

(4) While the impact on surface trafficking is convincingly demonstrated, how cellular proteostasis affects CFTR function requires further study, likely within a lung-specific cellular context to be more clinically relevant.

Reviewer #2 (Public review):

In this work, the authors use deep mutational scanning (DMS) to examine the effect of the endogenous chaperone calnexin (CANX) on the plasma membrane expression (PME) and potential pharmacological stabilization cystic fibrosis disease variants. This is important because there are over 1,700 loss-of-function mutations that can lead to the disease Cystic Fibrosis (CF), and some of these variants can be pharmacologically rescued by small-molecule "correctors," which stabilize the CFTR protein and prevent its degradation. This study expands on previous work to specifically identify which mutations affect sensitivity to CFTR modulators, and further develops the work by examining the effect of a known CFTR interactor-CANX-on PME and corrector response.

Overall, this approach provides a useful atlas of CF variants and their downstream effects, both at a basal level as well as in the context of a perturbed proteostasis. Knockout of CANX leads to an overall reduced plasma membrane expression of CFTR with CF variants located at the C-terminal domains of CFTR, which seem to be more affected than the others. This study then repeats their DMS approach, using PME as a readout, to probe the effect of either VX-445 or VX-455 + VX-661-which are two clinically relevant CFTR pharmacological modulators. I found this section particularly interesting for the community because the exact molecular features that confer drug resistance/sensitivity are not clear. When CANX is knocked out, cells that normally respond to VX-445 are no longer able to be rescued, and the DMS data show that these non-responders are CF variants that lie in the VX-445 binding site. Based on computational data, the authors speculate that NBD2 assembly is compromised, but that remains to be experimentally examined. Cells lacking CANX were also resistant to combinatorial treatment of VX-445 + VX-661, showing that these two correctors were unable to compensate for the lack of this critical chaperone.

One major strength of this manuscript is the mass spectrometry data, in which 4 CF variants were profiled in parental and CANX KO cells. This analysis provides some explanatory power to the observation that the delF508 variant is resistant to correctors in CANX KO cells, which is because correctors were found not to affect protein degradation interactions in this context. Findings such as this provide potential insights into intriguing new hypothesis, such as whether addition of an additional proteostasis regulators, such as a proteosome inhibitor, would facilitate a successful rescue. Taken together, the data provided can be generative to researchers in the field and may be useful in rationalizing some of the observed phenotypes conferred by the various CF variants, as well as the impact of CANX on those effects.

To complete their analysis of CF variants in CANX KO cells, the research also attempted to relate their data, primarily based on PME, to functional relevance. They observed that, although CANX KO results in a large reduction in PME (~30% reduction), changes in the actual activation of CFTR (and resultant quenching of their hYFP sensor) were "quite modest." This is an important experiment and caveat to the PME data presented above since changes in CFTR activity does not strictly require changes in PME. In addition, small molecule correctors also do not drastically alter CFTR function in the context of CANX KO. The authors reason that this difference is due to a sort of compensatory mechanism in which the functionally active CFTR molecules that are successfully assembled in an unbalanced proteostasis system (CANX KO) are more active than those that are assembled with the assistance of CANX. While I generally agree with this statement, it is not directly tested and would be challenging to actually test.

The selected model for all the above experiments was HEK293T cells. The authors then demonstrate some of their major findings in Fischer rat thyroid cell monolayers. Specifically, cells lacking CANX are less sensitive to rescue by CFTR modulators than the WT. This highlights the importance of CANX in supporting the maturation of CFTR and the dependence of chemical correctors on the chaperone. Although this is demonstrated specifically for CANX in this manuscript, I imagine a more general claim can be made that chemical correctors depend on a functional/balanced proteostasis system, which is supported by the manuscript data. I am surprised by the discordance between HEK293T PME levels compared to the CTFR activity. The authors offer a reasonable explanation about the increase in specific activity of the mature CFTR protein following CANX loss.

For the conclusions and claims relevant to CANX and CF variant surveying of PME/function, I find the manuscript to provide solid evidence to achieve this aim. The manuscript generates a rich portrait of the influence of CF mutations both in WT and CANX KO cells. While the focus of this study is a specific chaperone, CANX, this manuscript has the potential to impact many researchers in the broad field of proteostasis.

Reviewer #1 (Public review):

This was a clearly written manuscript that did an excellent job summarizing complex data. In this manuscript, Cuevas-Zuviría et al. use protein modeling to generate over 5,000 predicted structures of nitrogenase components, encompassing both extant and ancestral forms across different clades. The study highlights that key insertions define the various Nif groups. The authors also examined the structures of three ancestral nitrogenase variants that had been previously identified and experimentally tested. These ancestral forms were shown in earlier studies to exhibit reduced activity in Azotobacter vinelandii, a model diazotroph.

This work provides a useful resource for studying nitrogenase evolution. However, its impact is somewhat limited due to a lack of evidence linking the observed structural differences to functional changes. For example, in the ancestral nitrogenase structures, only a small set of residues (lines 421-431) were identified as potentially affecting interactions between nitrogenase components. Why didn't the authors test whether reverting these residues to their extant counterparts could improve nitrogenase activity of the ancestral variants?

Additionally, the paper feels somewhat disconnected. The predicted nitrogenase structures discussed in the first half of the manuscript were not well integrated with the findings from the ancestral structures. For instance, do the ancestral nitrogenase structures align with the predicted models? This comparison was never explicitly made and could have strengthened the study's conclusions.

Comments on revisions:

I appreciate the authors responding to my comments. I think Fig. S10 helps put the structural data into more context. It would be helpful to make clearer in the legend what proteins are being compared, especially in 10C.

Although I can see why the authors focus on the NifK extension and its potential connection to oxygen protection, I would point out that Vnf and Anf do not have this extension in their K subunit, and you find both Vnf and Anf in aerobic and facultative anaerobic diazotrophs. This is a minor point, but I think it is important to mention in the discussion.

Reviewer #2 (Public review):

Summary:

This work aims to study the evolution of nitrogenanses, understanding how their structure and function adapted to changes in environment, including oxygen levels and changes in metal availability.

The study predicts > 3000 structures of nitrogenases, corresponding to extant, ancestral and alternative ancestral sequences. It is observed that structural variations in the nitrogenases correlate with phylogenetic relationships. The amount of data generated in this study represents a massive and admirable undertaking. The study also provides strong insight into how structural evolution correlates with environmental and biological phenotypes

Reviewer #1 (Public review):

Summary:

In this study, the authors utilized in situ cryo-electron tomography (cryo-ET) to uncover the native thylakoid architecture of spinach chloroplasts and mapped the molecular organization of these thylakoids with single-molecule resolution. The obtained images show the detailed ultrastructural features of grana membranes and highlight interactions between thylakoids and plastoglobules. Interestingly, despite the distinct three-dimensional architecture of vascular plant thylakoids, their molecular organization closely resembles that of green algae. The pronounced lateral segregation of PSII and PSI was observed at the interface between appressed and non-appressed thylakoid regions, without evidence of a specialized grana margin zone where these complexes might intermix. Furthermore, unlike isolated thylakoid membranes, photosystem II (PSII) did not form a semi-crystalline array and was distributed uniformly within the membrane plane and across stacked grana membranes in intact chloroplasts. Based on the above observations, the authors propose a simplified two-domain model for the molecular organization of thylakoid membranes, which can be applied to both green algae and vascular plants. This study suggests that the general understanding of the functional separation of thylakoid membranes in vascular plants requires reconsideration.

Strengths:

By employing and refining AI-driven computational tools for the automated segmentation of membranes and identification of membrane proteins, this study successfully quantifies the spatial organization of photosynthetic complexes both within individual thylakoid membranes and across neighboring stacked membranes.

Weaknesses:

This study's weakness is that it requires the use of chloroplasts isolated from leaves and the need to freeze them on a grid for observation. However, the authors have correctly identified the limitations of this approach and have made some innovations, such as rapid sample preparation. The reliability of the interpretation of the results in light of previous results can be evaluated as high.

Comments on revised version:

The author has responded appropriately to the peer review comments and revised the paper.

Reviewer #2 (Public review):

Summary:

For decades, the macromolecular organization of photosynthetic complexes within the thylakoids of higher plant chloroplasts has been a topic of significant debate. Using focused ion beam milling, cryo-electron tomography, and advanced AI-based image analysis, the authors compellingly demonstrate that the macromolecular organization in spinach thylakoids closely mirrors the patterns observed in their earlier research on Chlamydomonas reinhardtii. Their findings provide strong evidence challenging long-standing assumptions about the existence of a 'grana margin'-a region at the interface between grana and stroma lamellae domains that was thought to contain intermixed particles from both areas. Instead, the study establishes that this mixed zone is absent and reveals a distinct, well-defined boundary between the grana and stroma lamellae.

Strengths:

By situating high-resolution structural data within the broader cellular context, this work contributes valuable insights into the molecular mechanisms governing the spatial organization of photosynthetic complexes within thylakoid membranes.

Comments on revised version:

All reviewer comments have been fully addressed, and I have no further comments.

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Reviewer #1 (Public review):

Munday, Rosello, and colleagues compared predictions from a group of experts in epidemiology with predictions from two mathematical models on the question of how many Ebola cases would be reported in different geographical zones over the next month. Their study ran from November 2019 to March 2020 during the Ebola virus outbreak in Democratic Republic of the Congo. Their key result concerned predicted numbers of cases in a defined set of zones. They found that neither the ensemble of models nor the group of experts produced consistently better predictions. Similarly, neither model performed consistently better than the other, and no expert's predictions were consistently better than the others'. Experts were also able to specify other zones in which they expected to see cases in the next month. For this part of the analysis, experts consistently outperformed the models. In March, the final month of the analysis, the models' accuracy was lower than in other months, and consistently poorer than the experts' predictions.

A strength of the analysis is use of consistent methodology to elicit predictions from experts during an outbreak that can be compared to observations, and that are comparable to predictions from the models. Results were elicited for a specified group of zones, and experts were also able to suggest other zones that were expected to have diagnosed cases. This likely replicates the type of advice being sought by policymakers during an outbreak.

A potential weakness is that the authors included only two models in their ensemble. Ensembles of greater numbers of models might tend to produce better predictions. The authors do not address whether a greater number of models could outperform the experts.

The elicitation was performed in four months near the end of the outbreak. The authors address some of the implications of this. A potential challenge for the transferability of this result is that the experts' understanding of local idiosyncrasies in transmission may have improved over the course of the outbreak. The model did not have this improvement over time. The comparison of models to experts may therefore not be applicable to early stages of an outbreak when expert opinions may be less well-tuned.

This research has important implications for both researchers and policy-makers. Mathematical models produce clearly-described predictions that will later be compared to observed outcomes. When model predictions differ greatly from observations, this harms trust in the models, but alternative forms of prediction are seldom so clearly articulated or accurately assessed. If models are discredited without proper assessment of alternatives then we risk losing a valuable source of information that can help guide public health responses. From an academic perspective, this research can help to guide methods for combining expert opinion with model outputs, such as considering how experts can inform models' prior distributions and how model outputs can inform experts' opinions.

Comments on revisions:

I am grateful to the authors for their responses to my previous comments. I think their updates have made the paper much clearer. I do not think the updates change the opinions already given in the public review so I have not modified it.

Reviewer #2 (Public review):

The manuscript by Munday et al. presents real-time predictions of geographic spread during an Ebola epidemic in north-eastern DRC. Predictions were elicited from individual experts engaged in outbreak response and from two mathematical models. The authors found comparable performance between experts and models overall, although the models outperformed experts in a few dimensions.

Both individual experts and mathematical models are commonly used to support outbreak response, but the relative strengths of each information source are rarely quantified. The manuscript presents an in-depth analysis of the accuracy and decision-relevance of the information provided by each source individually and in combination for a real-time outbreak response effort.

While this paper presents an important and unique comparison, forecast performance is known to be inconsistent and unpredictable across many dimensions such as pathogen, location, forecasting target, and phase of the outbreak. Thus, as the authors note, continuing to replicate such studies will be important for verifying the robustness of their conclusions in other contexts.

Comments on revisions:

I have no further comments. I commend the authors for an interesting and important contribution.

Reviewer #1 (Public review):

Summary:

The authors sought to identify the relationships between gut microbiota, lipid metabolites and the host in type 2 diabetes (T2DM) by using spontaneously developed T2DM in macaques, considered among the best human models.

Strengths:

The authors compared comprehensively the gut microbiota, plasma fatty acids between spontaneous T2DM and the control macaques, verifying the results with macaques in a high-fat diet-fed mice model.

Comments on revisions:

The authors responded to the comments raised, and the manuscript has been improved.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors explore the role of the conserved transcription factor POU4-2 in planarian maintenance and regeneration of mechanosensory neurons. The authors explore the role of this transcription factor and identify potential targets of this transcription factor. Importantly, many genes discovered in this work are deeply conserved, with roles in mechanosensation and hearing, indicating that planarians may be a useful model with which to study the roles of these key molecules. This work is important within the field of regenerative neurobiology, but also impactful for those studying the evolution of the machinery that is important for human hearing.

Strengths:

The paper is rigorous and thorough, with convincing support for the conclusions of the work.

Weaknesses:

Weaknesses are relatively minor and could be addressed with additional experiments or changes in writing.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigate the role of the transcription factor Smed-pou4-2 in the maintenance, regeneration, and function of mechanosensory neurons in the freshwater planarian Schmidtea mediterranea. First, they characterize the expression of pou4-2 in mechanosensory neurons during both homeostasis and regeneration, and examine how its expression is affected by the knockdown of soxB1, 2, a previously identified transcription factor essential for the maintenance and regeneration of these neurons. Second, the authors assess whether pou4-2 is functionally required for the maintenance and regeneration of mechanosensory neurons.

Strengths:

The study provides some new insights into the regulatory role of pou4-2 in the differentiation, maintenance, and regeneration of ciliated mechanosensory neurons in planarians.

Weaknesses:

The overall scope is relatively limited. The manuscript lacks clear organization, and many of the conclusions would benefit from additional experiments and more rigorous quantification to enhance their strength and impact.

Reviewer #1 (Public review):

Summary:

While previous studies by this group and others have demonstrated the anti-inflammatory properties of osteoactivin, its specific role in cartilage homeostasis and disease pathogenesis remains unknown. Building on current knowledge, Asaad and colleagues investigated the functional role of this protein using both in vitro systems and an in vivo post-traumatic osteoarthritis model. In line with existing literature, the authors report that osteoactivin exerts inhibitory effects in these experimental settings. This study thus offers novel evidence supporting the cartilage-protective effects of osteoactivin in various experimental models.

Strengths:

Strengths of the study include its clinical relevance, given the lack of curative treatments for osteoarthritis, as well as the clarity of the narrative and the quality of most results.

Weaknesses:

A limitation of the study is the reliance on standard techniques; however, this is a minor concern that does not diminish the overall impact or significance of the work.

Reviewer #2 (Public review):

Summary:

This manuscript presents compelling evidence for a novel anti-inflammatory function of glycoprotein non-metastatic melanoma protein B (GPNMB) in chondrocyte biology and osteoarthritis (OA) pathology. Through a combination of in vitro, ex vivo, and in vivo models, including the destabilization of the medial meniscus (DMM) surgery in mice, the authors demonstrate that GPNMB expression is upregulated in OA-affected cartilage and that recombinant GPNMB treatment reduces the expression of key catabolic markers (MMPs, Adamts-4, and IL-6) without impairing anabolic gene expression. Notably, DBA/2J mice lacking functional GPNMB exhibit exacerbated cartilage degradation post-injury. Mechanistically, GPNMB appears to mitigate inflammation via the MAPK/ERK pathway. Overall, the work is thorough, methodologically sound, and significantly advances our understanding of GPNMB as a protective modulator in osteoarthritic joint disease. The findings could open pathways for therapeutic development.

Strengths:

(1) Clear hypothesis addressing a well-defined knowledge gap.

(2) Robust and multi-modal experimental design: includes human, mouse, cell-line, explant, and surgical OA models.

(3) Elegant use of DBA/2J GPNMB-deficient mice to mimic endogenous loss-of-function.

(4) Mechanistic insight provided through MAPK signaling analysis.

(5) Statistical analysis appears rigorous, and figures are informative.

Weaknesses:

(1) Clarify the strain background of the DBA/2J GPNMB+ mice: While DBA/2J GPNMB+ is described as a control, it would help to explicitly state whether these are transgenically rescued mice or another background strain. Are they littermates, congenic, or a separate colony?

(2) Provide exact sample sizes and variance in all figure legends: Some figures (e.g., Figure 2 panels) do not consistently mention how many replicates were used (biological vs. technical) for each experimental group. Standardizing this across all panels would improve reproducibility.

(3) Expand on potential sex differences: The DMM model is applied only in male mice, which is noted in the methods. It would be helpful if the authors added 1-2 lines in the discussion acknowledging potential sex-based differences in OA progression and GPNMB function.

(4) Visual clarity in schematic (Figure 7): The proposed mechanism is helpful, but the text within the schematic is somewhat dense and could be made more readable with spacing or enlarged font. Also, label the MAPK/ERK pathway explicitly in panel B.

Reviewer #1 (Public review):

Summary:

The manuscript by Gamen et al. analyzed the functional role of HIF signaling in the epicardium, providing evidence that stabilization of the hypoxia signaling pathway might contribute to neonatal heart regeneration. By generating different conditionally mouse mutants and performing pharmacological interventions, the authors demonstrate that stabilizing HIF signaling enhances cardiac regeneration after MI in P7 neonatal hearts.

Strengths:

The study presents convincing genetic and pharmacological approaches to the role of hypoxia signaling in enhancing the regenerative potential of the epicardium.

Weaknesses:

The major weakness is the lack of convincing evidence demonstrating the role of hypoxia signaling in EMT modulation in epicardial cells. Additionally, novel experimental approaches should be performed to allow for the translation of these findings to the clinical arena.

Reviewer #2 (Public review):

Summary:

In this study, Gamen et al. investigated the roles of hypoxia and HIF1a signaling in regulating epicardial function during cardiac development and neonatal heart regeneration. They found that WT1⁺ epicardial cells become hypoxic and begin expressing HIF1a from mid-gestation onward. During development, epicardial HIF1a signaling regulates WT1 expression and promotes coronary vasculature formation. In the postnatal heart, genetic and pharmacological upregulation of HIF1a sustained epicardial activation and improved regenerative outcomes.

Strengths:

HIF1a signaling was manipulated in an epicardium-specific manner using appropriate genetic tools.

Weaknesses:

There appears to be a discrepancy between some of the conclusions and the provided histological data. Additionally, the study does not offer mechanistic insight into the functional recovery observed.

Reviewer #3 (Public review):

Summary:

The authors' research here was to understand the role of hypoxia and hypoxia-induced transcription factor Hif-1a in the epicardium. The authors noted that hypoxia was prevalent in the embryonic heart, and this persisted into neonatal stages until postnatal day 7 (P7). Hypoxic regions in the heart were noted in the outer layer of the heart, and expression of Hif-1a coincided with the epicardial gene WT1. It has been documented that at P7, the mouse heart cannot regenerate after myocardial infarction, and the authors speculated that the change in epicardial hypoxic conditions could play a role in regeneration. The authors then used genetic and pharmacological tools to increase the activity of Hif genes in the heart and noted that there was a significant improvement in cardiac function when Hif-1a was active in the epicardium. The authors speculated that the presence of Hif-1a improved cell survival.

Strengths:

A focus on hypoxia and its effects on the epicardium in development and after myocardial infarction. This study outlines the potential to extend the regenerative time window in neonatal mammalian hearts.

Weaknesses:

While the observations of improved cardiac function are clear, the exact mechanism of how increased Hif-1a activity causes these effects is not completely revealed. The authors mention improved myocardium survival, but do not include studies to demonstrate this.

There is an indication that fibrosis is decreased in hearts where Hif activity is prolonged, but there are no studies to link hypoxia and fibrosis.

Reviewer #1 (Public review):

Summary:

This study presents a new Bayesian approach to estimate importation probabilities of malaria, combining epidemiological data, travel history, and genetic data through pairwise IBD estimates. Importation is an important factor challenging malaria elimination, especially in low-transmission settings. This paper focuses on Magude and Matutuine, two districts in southern Mozambique with very low malaria transmission. The results show isolation-by-distance in Mozambique, with genetic relatedness decreasing with distances larger than 100 km, and no spatial correlation for distances between 10 and 100 km. But again, strong spatial correlation in distances smaller than 10 km. They report high genetic relatedness between Matutuine and Inhambane, higher than between Matutuine and Magude. Inhambane is the main source of importation in Matutuine, accounting for 63.5% of imported cases. Magude, on the other hand, shows smaller importation and travel rates than Matutuine, as it is a rural area with less mobility. Additionally, they report higher levels of importation and travel in the dry season, when transmission is lower. Also, no association with importation was found for occupation, sex, and other factors. These data have practical implications for public health strategies aiming for malaria elimination, for example, testing and treating travelers from Matutuine in the dry season.

Strengths:

The strength of this study lies in the combination of different sources of data - epidemiological, travel, and genetic data - to estimate importation probabilities, and the statistical analyses.

Weaknesses:

The authors recognize the limitations related to sample size and the biases of travel reports.

Reviewer #2 (Public review):

Summary:

Based on a detailed dataset, the authors present a novel Bayesian approach to classify malaria cases as either imported or locally acquired.

Strengths:

The proposed Bayesian approach for case classification is simple, well justified, and allows the integration of parasite genomics, travel history, and epidemiological data. The work is well-written, very organized, and brings important contributions both to malaria control efforts in Mozambique and to the scientific community. Understanding the origin of cases is essential for designing more effective control measures and elimination strategies.

Weakness:

While the authors aim to classify cases as imported or locally acquired, the work lacks a quantification of the contribution of each case type to overall transmission.

The Bayesian rationale is sound and well justified; however, the formulation appears to present an inconsistency that is replicated in both the main text and the Supplementary Material.

Reviewer #3 (Public review):

The authors present an important approach to identify imported P. falciparum malaria cases, combining genetic and epidemiological/travel data. This tool has the potential to be expanded to other contexts. The data was analyzed using convincing methods, including a novel statistical model; although some recognized limitations can be improved. This study will be of interest to researchers in public health and infectious diseases.

Strengths:

The study has several strengths, mainly the development of a novel Bayesian model that integrates genomic, epidemiological, and travel data to estimate importation probabilities. The results showed insights into malaria transmission dynamics, particularly identifying importation sources and differences in importation rates in Mozambique. Finally, the relevance of the findings is to suggest interventions focusing on the traveler population to help efforts for malaria elimination.

Weaknesses:

The study also has some limitations. The sample collection was not representative of some provinces, and not all samples had sufficient metadata for risk factor analysis, which can also be affected by travel recall bias. Additionally, the authors used a proxy for transmission intensity and assumed some conditions for the genetic variable when calculating the importation probability for specific scenarios. The weaknesses were assessed by the authors.

Reviewer #1 (Public review):

This study investigates how ant group demographics influence nest structures and group behaviors of Camponotus fellah ants, a ground-dwelling carpenter ant species (found locally in Israel) that build subterranean nest structures. Using a quasi-2D cell filled with artificial sand, the authors perform two complementary sets of experiments to try to link group behavior and nest structure: first, the authors place a mated queen and several pupae into their cell and observe the structures that emerge both before and after the pupae eclose (i.e., "colony maturation" experiments); second, the authors create small groups (of 5,10, or 15 ants, each including a queen) within a narrow age range (i.e., "fixed demographic" experiments) to explore the dependence of age on construction. Some of the fixed demographic instantiations included a manually induced catastrophic collapse event; the authors then compared emergency repair behavior to natural nest creation. Finally, the authors introduce a modified logistic growth model to describe the time-dependent nest area. The modification introduced parameters that allow for age-dependent behavior, and the authors use their fixed demographic experiments to set these parameters, and then apply the model to interpret the behavior of the colony maturation experiments. The main results of this paper are that for natural nest construction, nest areas, and morphologies depend on the age demographics of ants in the experiments: younger ants create larger nests and angled tunnels, while older ants tend to dig less and build predominantly vertical tunnels; in contrast, emergency response seems to elicit digging in ants of all ages to repair the nest.

The experimental results are solid, providing new information and important insights into nest and colony growth in a social insect species. As presented, I still have some reservations about the model's contribution to a deeper understanding of the system. Additional context and explanation of the model, implications, and limitations would be helpful for readers.

Reviewer #2 (Public review):

I enjoyed this paper and its examination of the relationship between overall density and age polyethism to reduce the computational complexity required to match nest size with population. I had some questions about the requirement that growth is infinite in such a solution, but these have been addressed by the authors in the responses and the updated manuscript. I also enjoyed the discussion of whether collective behaviour is an appropriate framework in systems in which agents (or individuals) differ in the behavioural rules they employ, according to age, location, or information state. This is especially important in a system like social insects, typically held as a classic example of individual-as-subservient to whole, and therefore most likely to employ universal rules of behaviour. The current paper demonstrates a potentially continuous age-related change in target behaviour (excavation), and suggests an elegant and minimal solution to the requirement for building according to need in ants, avoiding the invocation of potentially complex cognitive mechanisms, or information states that all individuals must have access to in order to have an adaptive excavation output.

The authors have addressed questions I had in the review process and the manuscript is now clear in its communication and conclusions.

The modelling approach is compelling, also allowing extrapolation to other group sizes and even other species. This to me is the main strength of the paper, as the answer to the question of whether it is younger or older ants that primarily excavate nests could have been answered by an individual tracking approach (albeit there are practical limitations to this, especially in the observation nest setup, as the authors point out). The analysis of the tunnel structure is also an important piece of the puzzle, and I really like the overall study.

Reviewer #1 (Public review):

The medicinal leech preparation is an amenable system in which to understand how the underlying cellular networks for locomotion function. A previously identified non-spiking neuron (NS) was studied and found to alter the mean firing frequency of a crawl-related motoneuron (DE-3), which fires during the contraction phase of crawling. The data are mostly solid. Identifying upstream neurons responsible for crawl motor patterning is essential for understanding how rhythmic behavior is controlled.

Review of Revision:

Reviewer: On a positive note, the rationale for the study is clearer to me now after reading the authors' responses to both reviewers, but that information, as described in the authors' responses, is minimally incorporated into the current revised paper. Incorporating a discussion of previous work on the NS cell has, indeed, improved the paper.

I suggested earlier that the paper be edited for clarity but not much text has been changed since the first draft. I will provide an example of the types of sentences that are confusing. The title of the paper is: "Phase-specific premotor inhibition modulates leech rhythmic motor output". Are the authors referring to the inhibition created by premotor neurons (e.g., on to the motoneurons) or the inhibition that the premotor neurons receive?

I also find the paper still confusing with regard to the suggested "functional homology" with the vertebrate Renshaw cells. When the authors set up this expectation of homology (should be analogy) in the introduction and other sections of the paper, one would assume that the NS cell would be directly receiving excitation from a motoneuron (like DE-3) and, in turn, the motoneuron would then receive some sort of inhibitory input to regulate its firing frequency. Essentially, I have always viewed the Renshaw cells as nature's clever way to monitor the ongoing activity of a motoneuron while also providing recurrent feedback or "recurrent inhibition" to modify that cell's excitatory state. The authors present their initial idea below on line 62. Authors write: "These neurons are present as bilateral pairs in each segmental ganglion and are functional homologs of the mammalian Renshaw cells (Szczupak, 2014). These spinal cord cells receive excitatory inputs from motoneurons and, in turn, transmit inhibitory signals to the motoneurons (Alvarez and Fyffe, 2007)."

[Reviewer (minor note): I suggest re-writing this last sentence as "these" is confusing. Change to: 'In the spinal cord, Renshaw interneurons receive excitatory inputs from motoneurons and, in turn, transmit inhibitory signals to them (Alvarez and Fyffe, 2007).']

Reviewer: Furthermore, the authors note that (line 69 on): "In the context of this circuit the activity of excitatory motoneurons evokes chemically mediated inhibitory synaptic potentials in NS. Additionally, the NS neurons are electrically coupled......In physiological conditions this coupling favors the transmission of inhibitory signals from NS to motoneurons." Based on what is being conveyed here, I see a disconnect with the "functional homology" being presented earlier. I may be missing something, but the Renshaw analogy seems to be quite different compared to what looks like reciprocal inhibition in the leech. If the authors want to make the analogy to Renshaw cells clearer, then they should make a simple ball and stick diagram of the leech system and visually compare it to the Renshaw/motoneuron circuit with regard to functionality. This simple addition would help many readers.

Reviewer: The Abstract, Authors write (line 19), "Specifically, we analyzed how electrophysiological manipulation of a premotor nonspiking (NS) neuron, that forms a recurrent inhibitory circuit (homologous to vertebrate Renshaw cells)...."<br /> First, a circuit would not be homologous to a cell, and the term homology implies a strict developmental/evolutionary commonality. At best, I would use the term functionally analogous but even then I am still not sure that they are functionally that similar (see comments above). Line 22: "The study included a quantitative analysis of motor units active throughout the fictive crawling cycle that shows that the rhythmic motor output in isolated ganglia mirrors the phase relationships observed in vivo." This sentence must be revised to indicate that not all of the extracellular units were demonstrated to be motor units. Revise to: "The study included a quantitative analysis of identified and putative motor units active throughout the fictive crawling cycle that shows.....'

Line 187 regarding identifying units as motoneurons: Authors write, "While multiple extracellular recordings have been performed previously (Eisenhart et al., 2000), these results (Figure 4) present the first quantitative analysis of motor units activated throughout the crawling cycle in this type of recordings." The authors cannot assume that the units in the recorded nerves belong only to motoneurons. Based on their first rebuttal, the authors seem to be reluctant to accept the idea that the extracellularly recorded units might represent a different class of neurons. They admit that some sensory neurons (with somata located centrally) do, indeed, travel out the same nerves recorded, but go on to explain why they would not be active.

The leech has a variety of sensory organs that are located in the periphery, and some of these sensory neurons do show rhythmic activity correlated with locomotor activity (see Blackshaw's early work). The numerous stretch receptors, in fact, have very large axons that pass through all the nerves recorded in the current paper. In Fig. 4, it is interesting that the waveforms of all the units recorded in the PP nerve exhibit a reversal in waveform as compared to those in the DP nerve, which might indicate (based on bipolar differential recording) that the units in the PP nerve are being propagated in the opposite direction (i.e., are perhaps afferent). Rhythmic presynaptic inhibition and excitation is commonly seen for stretch receptors within the CNS (see the work of Burrows) and many such cells are under modulatory control.

Most likely, the majority of the units are from motoneurons, but we do not really know at this point. The authors should reframe their statements throughout the paper as: 'While multiple extracellular recordings have been performed previously (Eisenhart et al., 2000), these results (Figure 4) present the first quantitative analysis of multiple extracellular units, using spike sorting methods, which are activated throughout the crawling cycle.' In cases where the identity of the unit is known, then it is fine to state that, but when the identity of the unit is not known, then there should be some qualification and stated as 'putative motor units'

Reviewer, the Methods section: needs to include the full parameters that were used to assess whether bursting activity was qualified in ways to be considered crawling activity or not. Typically, crawl-like burst periods of no more than 25 seconds have been the limit for their qualification as crawling activity. In Fig 2F, for example, the inter-burst period is over 35 seconds; that coupled with an average 5 second burst duration would bring the burst period to 40 seconds, which is substantially out of range for there to be bursting relevant to crawl activity. Simply put, long DE-3 burst periods are often observed but may not be indicative of a crawl state as the CV motoneurons are no longer out of phase with DE-3. A number of papers have adopted this criterion.

Reviewer #1 (Public review):

This work addresses an important question in the field of Drosophila aggression and mating. Prior social isolation is known to increase aggression in males, manifesting as increased lunging, which is suppressed by group housing (GH). However, it is also known that single housed (SH) males, despite their higher attempts to court females, are less successful. Here, Gao et al., develop a modified aggression assay to address this issue by recording aggression in Drosophila males for 2 hours, with a virgin female immobilized by burying its head in the food. They found that while SH males frequently lunge in this assay, GH males switch to higher intensity but very low frequency tussling. Constitutive neuronal silencing and activation experiments implicate cVA sensing Or67d neurons in promoting high frequency lunging, similar to earlier studies, whereas Or47b neurons promote low frequency but higher intensity tussling. Optogenetic activation revealed that three pairs of pC1SS2 neurons increase tussling. Cell-type-specific DsxM manipulations combined with morphological analysis of pC1SS2 neurons and side-by-side tussling quantification link the developmental role of DsxM to the functional output of these aggression-promoting cells. In contrast, although optogenetic activation of P1a neurons in the dark did not increase tussling, thermogenetic activation under visible light drove aggressive tussling. Using a further modified aggression assay, GH males exhibit increased tussling and maintain territorial control, which could contribute to a mating advantage over SH males, although direct measures of reproductive success are still needed

Strengths:

Through a series of clever neurogenetic and behavioral approaches, the authors implicate specific subsets of ORNs and pC1 neurons in promoting distinct forms of aggressive behavior, particularly tussling. They have devised a refined territorial control paradigm, which appears more robust than earlier assays using a food cup (Chen et al., 2002). This new setup is relatively clutter-free and could be amenable to future automation using computer vision approaches. The updated Figure 5, which combines cell-type-specific developmental manipulation of pC1SS2 neurons with behavioral output, provides a link between developmental mechanisms and functional aggression circuits. The manuscript is generally well written, and the claims are largely supported by the data.

Weakness:

Although most concerns have been addressed, the manuscript still lacks a rigorous, objective method for quantifying lunging and tussling. Because scoring appears to have been done manually and a single lunge in a 30 fps video spans only 2-3 frames, the 0.2 s cutoff seems arbitrary, and there are no objective criteria distinguishing reciprocal lunging from tussling. Despite this, the study offers valuable insights into the neural and behavioral mechanisms of Drosophila aggression.

Reviewer #2 (Public review):

Summary:

Gao et al. investigated the change of aggression strategies by the social experience and its biological significance by using Drosophila. Two modes of inter-male aggression in Drosophila are known: lunging, high-frequency but weak mode, and tussling, low-frequency but more vigorous mode. Previous studies have mainly focused on the lunging. In this paper, the authors developed a new behavioral experiment system for observing tussling behavior and found that tussling is enhanced by group rearing, while lunging is suppressed. They then searched for neurons involved in the generation of tussling. Although olfactory receptors named Or67d and Or65a have previously been reported to function in the control of lunging, the authors found that these neurons do not function in the execution of tussling and another olfactory receptor, Or47b, is required for tussling, as shown by the inhibition of neuronal activity and the gene knockdown experiments. Further optogenetic experiments identified a small number of central neurons pC1[SS2] that induce the tussling specifically. These neurons express doublesex (dsx), a sex-determination factor, and knockdown of dsx strongly suppresses the induction of tussling. In order to further explore the ecological significance of the aggression mode change in group-rearing, a new behavioral experiment was performed to examine the territorial control and the mating competition. And finally, the authors found that differences in the social experience (group vs. solitary rearing) and the associated change in aggression strategy are important in these biologically significant competitions. These results add a new perspective to the study of aggression behavior in Drosophila. Furthermore, this study proposes an interesting general model in which the social experience modified behavioral changes play a role in reproductive success.

Strengths:

A behavioral experiment system that allows stable observation of tussling, which could not be easily analyzed due to its low-frequency, would be very useful. The experimental setup itself is relatively simple, just the addition of a female to the platform, so it should be applicable to future research. The finding about the relationship between the social experience and the aggression mode change is quite novel. Although the intensity of aggression changes with the social experience was already reported in several papers (Liu et al., 2011 etc), the fact that the behavioral mode itself changes significantly has rarely been addressed, and is extremely interesting. The identification of sensory and central neurons required for the tussling makes appropriate use of the genetic tools and the results are clear. A major strength of this study in neurobiology is the finding that another group of neurons (Or47b-expressing olfactory neurons and pC1[SS2] neurons), distinct from the group of neurons previously thought to be involved in low-intensity aggression (i.e. lunging), function in the tussling behavior. Furthermore, the results showing that the regulation of aggression by pC1[SS2] neurons is based on the function of the dsx gene will bring a new perspective to the field. Further investigation of the detailed circuit analysis is expected to elucidate the neural substrate of the conflict between the two aggression modes. The experimental systems examining the territory control and the reproductive competition in Fig. 6 are novel and have advantages in exploring their biological significance. It is important to note that in addition to showing the effects of age and social experience on territorial and mating behaviors, the authors experimentally demonstrated that altered fighting strategy has effects with respect to these behaviors.

Reviewer #3 (Public review):

In this revised manuscript, Gao et al. presented a series of well-controlled behavioral data showing that tussling, a form of high-intensity fighting among male fruit flies (Drosophila melanogaster) is enhanced specifically among socially experienced and relatively old males. Moreover, results of behavioral assays led authors to suggest that increased tussling among socially experienced males may increase mating success. They also concluded that tussling is controlled by a class of olfactory sensory neurons and sexually dimorphic central neurons that are distinct from pathways known to control lunges, a common male-type attack behavior.

A major strength of this work is that it is the first attempt to characterize behavioral function and neural circuit associated with Drosophila tussling. Many animal species use both low-intensity and high-intensity tactics to resolve conflicts. High-intensity tactics are mostly reserved for escalated fights, which are relatively rare. Because of this, tussling in the flies, like high-intensity fights in other animal species, have not been systematically investigated. Previous studies on fly aggressive behavior have often used socially isolated, relatively young flies within a short observation duration. Their discovery that 1) older (14-days old) flies tend to tussle more often than younger (2 to 7-days-old) flies, 2) group-reared flies tend to tussle more often than socially isolated flies, and 3) flies tend to tussle at later stage (mostly ~15 minutes after the onset of fighting), are the result of their creativity to look outside of conventional experimental settings. These new findings are key for quantitatively characterizing this interesting yet under-studied behavior.

Newly presented data have made several conclusions convincing. Detailed descriptions of methods to quantify behaviors help understand the basis of their claims by improving transparency. However, I remain concerned about authors' persistent attempt to link the high intensity aggression to reproductive success. The authors' effort to "tone down" the link between the two phenomena remains insufficient. There are purely correlational. I reiterate this issue because the overall value of the manuscript would not change with or without this claim.

Reviewer #1 (Public review):

The manuscript by Feng et al. reported that Endothelin B receptor (ETBR) expressed by the satellite glial cells (SGCs) in the dorsal root ganglions (DRG) acted to inhibit sensory axon regeneration in both adult and aged mice. Thus, pharmacological inhibition of ETBR with specific inhibitors resulted in enhanced sensory axon regeneration in vitro and in vivo. In addition, sensory axon regeneration significantly reduces in aged mice and inhibition of ETBR could restore such defect in aged mice. Moreover, the study provided some evidence that the reduced level of gap junction protein connexin 43 might act downstream of ETBR to suppress axon regeneration in aged mice. Overall, the study revealed an interesting SGC-derived signal in the DRG microenvironment to regulate sensory axon regeneration. It provided additional evidence that non-neuronal cell types in the microenvironment function to regulate axon regeneration via cell-cell interaction.

However, the molecular mechanisms by which ETBR regulates axon regeneration are unclear, and the structure of the manuscript is relatively not well organized, especially the last section. Some discussion and explanation about the data interpretation are needed to improve the manuscript.

(1) The result showed that the level of ETBR was not changed after the peripheral nerve injury. Does it mean that its endogenous function is to limit the spontaneous sensory axon regeneration? In other words, the results suggest that SGCs expressing ETBR or vascular endothelial cells expressing its ligand ET-1 act to suppress sensory axon regeneration. Some explanation or discussion about this are necessary. Moreover, does the protein level of ETBR or its ligand change during aging?

(2) In ex vivo experiments, NGF was added in the culture medium. Previous studies have shown that adult sensory neurons could initiate fast axon growth in response to NGF within 24 hours. In addition, dissociated sensory neurons could also initiate spontaneous regenerative axon growth without NGF after 48 hours. Some discussion or rationale is needed to explain the difference between NGF-induced or spontaneous axon growth of culture adult sensory neurons and the roles of ETBR and SGCs.

(3) In cultured dissociated sensory neurons, inhibiting ETBR also enhanced axon growth, which meant the presence of SGCs surrounding the sensory neurons. Some direct evidence is needed to show the cellular relationship between them in culture.

(4) In Figure 3, the in vivo regeneration experiments first showed enhanced axon regeneration either at 1 day or 3 days after the nerve injury. The study then showed that inhibiting ETBR could enhance sensory axon growth in vitro from uninjured naïve neurons or conditioning lesioned neurons. To my knowledge, in vivo sensory axon regeneration is relatively slow during the first 2 days after the nerve injury and then enter the fast regeneration mode in the 3rd day, representing the conditioning lesion effect in vivo. Some discussion is needed to compare the in vitro and the in vivo model of axon regeneration.

(5) In Figure 5, the study showed that the level of connexin 43 increased after ETBR inhibition in either adult or aged mice, proposing an important role of connexin 43 in mediating the enhancing effect of ETBR inhibition on axon regeneration. However, in the study there was no direct evidence supporting that ETBR directly regulate connexin 43 expression in SGCs. Moreover, there was no functional evidence that connexin 43 acted downstream of ETBR to regulate axon regeneration.

In the revised manuscript, most comments have been addressed with some new experiments or text revisions in the results or discussion. For representative images showing in vitro cultured DRG neurons, it would be much more convincing if several neurons in the same imaging field are shown, rather than a single neuron (Figure 2A, 3J).

Reviewer #2 (Public review):

Summary:

Feng and colleagues set out to investigate the effect of manipulating endothelin signaling on nerve regeneration, focusing on the crosstalk between endothelial cells (ECs) in dorsal root ganglia (DRG), which secrete ET-1, and satellite glial cells (SGCs), which express the ETBR receptor. ETBR signaling limits axon growth. Using in vitro explant assays coupled with pharmacological inhibition in mouse models of nerve injury, the authors demonstrate that the ETAR/ETBR antagonist Bosentan promotes axon regeneration, and that this effect is maintained in aged mice. Although Bosentan inhibits both endothelin receptors A and B, comparison with an ETAR-specific antagonist suggests primary involvement of the ET-1/ETBR pathway. In the DRG, ETBR is mostly expressed by SGCs, a cell type implicated in nerve regeneration. SGCs ensheath and couple with DRG neurons through gap junctions formed by Cx43. The pro-regenerative effects of ETBR inhibition are attributed in part to an increase in Cx43 levels, which are expected to enhance neuron-SGC coupling. snRNA sequencing and TEM analysis reveal a decline in SGC numbers, morphological changes, and transcriptional reprogramming that may impair their pro-regenerative capacity.

Strengths:

The study is well-executed, and the main conclusion (that ETBR signaling inhibits axon regeneration after nerve injury and contributes to the age-related decline in regenerative capacity) is well supported by the data. In addition, the study highlights the importance of vascular signals in nerve regeneration, a topic that has gained traction in recent years. Importantly, these results further emphasize the contribution of long-neglected SGCs to nerve tissue homeostasis and repair. Although the study does not provide a complete mechanistic understanding, the findings are robust and are likely to attract the interest of a broad readership.

Weaknesses:

While certain aspects could have been further addressed experimentally, these points were either technically challenging or considered beyond the scope of the current study, and are appropriately addressed in the Discussion.

(1) It remains to be determined whether the accelerated axon regrowth observed after nerve injury depends on cellular crosstalk mediated by ET-1 at the lesion site. Are ECs along the nerve secreting ET-1? What cells are present in the nerve stroma that could respond and participate in the repair process? Would these interactions be sensitive to Bosentan? Dissecting these contributions would require cell-specific manipulations. The potential roles of ECs, fibroblast and SCs in the nerve are discussed.

(2) It is suggested that the permeability of DRG vessels may facilitate the release of vascular-derived signals. The possibility that the ET-1/ETBR pathway modulates vascular permeability, and that this in turn contributes to the observed effects on regeneration, is discussed.

(3) It cannot be excluded that ET-3 in fibroblasts is relevant for controlling SGC responses. The possibility that both ET-1 and ET-3 participate in ETBR- dependent effect on axon regeneration is discussed.

(4) The discovery that ET-1/ETBR signaling in SGC curtails the growth capacity of axons at baseline raises questions about the physiological role of this pathway. This remains to be elucidated with cell type-specific knockout approaches.

(5) The modulation of Cx43 expression by ET-1/ETBR is examined by immunostaining, but a complementary analysis by quantitative RT-PCR on sorted SGCs would have been a valuable addition. However, quantifying Cx43 on purified SGCs was not attainable due to technical complications.

(6) The conclusion "that ETBR inhibition in SGCs contributes to axonal regeneration by increasing Cx43 levels, gap junction coupling or hemichannels and facilitating SGC-neuron communication" are consistent with previous studies (Procacci et al., 2008) but in apparent discrepancy with increased gap junctions and dye coupling in SGCs of aged mice (Huang et al., 2006). More experiments are required to clarify what distinguishes a beneficial increase in coupling after ETBR inhibition, from what is observed in aging.

(7) The effect of Bosentan likely extends beyond the modulation of Cx43 levels. Cell type-specific knockout of Cx43 and ETBR, studies of SGCs-neuron coupling, and biochemical analysis of Cx43 functions would clarify the link between ETBR, Cx43 regulation, and axon regeneration. A discussion of alternative mechanisms is provided.

Reviewer #1 (Public review):

Summary:

The authors study the steady-state solutions of ODE models for molecular signaling involving ligand binding coupled to multi-site phosphorylation at saturating ligand concentrations. Although the results are in principle general, the work highlights the receptor tyrosine kinases (RTK) as model systems. After presenting previous ODE model solutions, the authors present their own "kinetic sorting" model, which is distinguished by ligand-induced phosphorylation-dependent receptor degradation and the property that every phosphorylation state is signaling competent. The authors show that this model recovers the two types of non-monotonicity experimentally reported for RTKs: maximum activity for intermediate ligand affinity and maximum activity for intermediate kinase activity.

The main contribution of the work is in demonstrating that their model can capture both types of non-monotonicity, whereas previous models could at most capture non-monotonicity of ligand binding.

Strengths:

The question of how energy-dissipating, and thus non-equilibrium, molecular systems can achieve steady-state solutions not accessible to equilibrium systems is of fundamental importance in biomolecular information processing and self-organization. Although the authors do not address the energy requirements of their non-equilibrium model, their comparative analysis of different alternative non-equilibrium models provides insight into the design choices necessary to achieve non-monotonic control, a property that is inaccessible at equilibrium.

The paper is succinctly written and easy to follow, and the authors achieve their aims by providing convincing numerical solutions demonstrating non-monotonicity over the range of parameter values encompassing the biologically relevant regime.

Weaknesses:

(1) A key motivating framework for this work is the argument that the ability to tune to recognize intermediate ligand affinities provides a control knob for signal selection that is available to non-equilibrium systems. As such, this seems like a compelling type of ligand selectivity, which is a question of broad interest. However, as the authors note in the results, the previously published "limited signaling model" already achieves such non-monotonicity in ligand binding affinity. The introduction and abstract do not clearly delineate the new contributions of the model.

The novel benefit of the model introduced by the authors is that it also achieves a non-monotonic response to kinase activity. Because such non-monotonicity is observed for RTK, this would make the authors' model a better fit for capturing RTK behavior. However, the broad significance of achieving non-monotonicity to kinase activity is not motivated or supported by empirical evidence in the paper. As such, the conceptual significance of the modified model presented by the authors is not clear.

(2) Whereas previous models used in the literature are schematized in Figure 1, the model proposed by the authors is missing (see line 97 of page 3). Without the schematic, the text description of the model is incomplete.

(3) The authors use the activity of the first phosphorylation site as the default measure of activity. This choice needs to be justified. Why not use the sum of the activities at all sites?

Reviewer #2 (Public review):

Summary:

In classical models of signaling networks, the signaling activity increases monotonically with the ligand affinity. However, certain receptors prefer ligands of intermediate affinity. In the paper, the authors present a new minimal model to derive generic conditions for ligand specificity. In brief, this requires multi-site phosphorylation and that high-affinity complexes be more prone to degrade. This particular type of kinetic discrimination allows for overcoming equilibrium constraints.

Strengths:

The model is simple, and it adds only a few parameters to classical generic models. Moreover, the authors vary these additional parameters in ranges based on experimental observations. They explain how the introduction of these new parameters is essential to ligand specificity. Their model quantitatively reproduces the ligand specificity of a certain receptor. Finally, they provide a testable prediction.

Weaknesses:

The naming of certain variables may be confusing to readers.

Reviewer #1 (Public review):

Summary:

The study by Teplenin and coworkers assesses the combined effects of localized depolarization and excitatory electrical stimulation in myocardial monolayers. They study the electrophysiological behaviour of cultured neonatal rat ventricular cardiomyocytes expressing the light-gated cation channel Cheriff, allowing them to induce local depolarization of varying area and amplitude, the latter titrated by the applied light intensity. In addition, they used computational modeling to screen for critical parameters determining state transitions and to dissect the underlying mechanisms. Two stable states, thus bistability, could be induced upon local depolarization and electrical stimulation, one state characterized by a constant membrane voltage and a second, spontaneously firing, thus oscillatory state. The resulting 'state' of the monolayer was dependent on the duration and frequency of electrical stimuli, as well as the size of the illuminated area and the applied light intensity, determining the degree of depolarization as well as the steepness of the local voltage gradient. In addition to the induction of oscillatory behaviour, they also tested frequency-dependent termination of induced oscillations.

Strengths:

The data from optogenetic experiments and computational modelling provide quantitative insights into the parameter space determining the induction of spontaneous excitation in the monolayer. The most important findings can also be reproduced using a strongly reduced computational model, suggesting that the observed phenomena might be more generally applicable.

Weaknesses:

While the study is thoroughly performed and provides interesting mechanistic insights into scenarios of ventricular arrhythmogenesis in the presence of localized depolarized tissue areas, the translational perspective of the study remains relatively vague. In addition, the chosen theoretical approach and the way the data are presented might make it difficult for the wider community of cardiac researchers to understand the significance of the study.

Reviewer #2 (Public review):

In the presented manuscript, Teplenin and colleagues use both electrical pacing and optogenetic stimulation to create a reproducible, controllable source of ectopy in cardiomyocyte monolayers. To accomplish this, they use a careful calibration of electrical pacing characteristics (i.e., frequency, number of pulses) and illumination characteristics (i.e., light intensity, surface area) to show that there exists a "sweet spot" where oscillatory excitations can emerge proximal to the optogenetically depolarized region following electrical pacing cessation, akin to pacemaker cells. Furthermore, the authors demonstrate that a high-frequency electrical wave-train can be used to terminate these oscillatory excitations. The authors observed this oscillatory phenomenon both in vitro (using neonatal rat ventricular cardiomyocyte monolayers) and in silico (using a computational action potential model of the same cell type). These are surprising findings and provide a novel approach for studying triggered activity in cardiac tissue.

The study is extremely thorough and one of the more memorable and grounded applications of cardiac optogenetics in the past decade. One of the benefits of the authors' "two-prong" approach of experimental preps and computational models is that they could probe the number of potential variable combinations much deeper than through in vitro experiments alone. The strong similarities between the real-life and computational findings suggest that these oscillatory excitations are consistent, reproducible, and controllable.

Triggered activity, which can lead to ventricular arrhythmias and cardiac sudden death, has been largely attributed to sub-cellular phenomena, such as early or delayed afterdepolarizations, and thus to date has largely been studied in isolated single cardiomyocytes. However, these findings have been difficult to translate to tissue and organ-scale experiments, as well-coupled cardiac tissue has notably different electrical properties. This underscores the significance of the study's methodological advances: the use of a constant depolarizing current in a subset of (illuminated) cells to reliably result in triggered activity could facilitate the more consistent evaluation of triggered activity at various scales. An experimental prep that is both repeatable and controllable (i.e., both initiated and terminated through the same means).

The authors also substantially explored phase space and single-cell analyses to document how this "hidden" bi-stable phenomenon can be uncovered during emergent collective tissue behavior. Calibration and testing of different aspects (e.g., light intensity, illuminated surface area, electrical pulse frequency, electrical pulse count) and other deeper analyses, as illustrated in Appendix 2, Figures 3-8, are significant and commendable.

Given that the study is computational, it is surprising that the authors did not replicate their findings using well-validated adult ventricular cardiomyocyte action potential models, such as ten Tusscher 2006 or O'Hara 2011. This may have felt out of scope, given the nice alignment of rat cardiomyocyte data between in vitro and in silico experiments. However, it would have been helpful peace-of-mind validation, given the significant ionic current differences between neonatal rat and adult ventricular tissue. It is not fully clear whether the pulse trains could have resulted in the same bi-stable oscillatory behavior, given the longer APD of humans relative to rats. The observed phenomenon certainly would be frequency-dependent and would have required tedious calibration for a new cell type, albeit partially mitigated by the relative ease of in silico experiments.

For all its strengths, there are likely significant mechanistic differences between this optogenetically tied oscillatory behavior and triggered activity observed in other studies. This is because the constant light-elicited depolarizing current is disrupting the typical resting cardiomyocyte state, thereby altering the balance between depolarizing ionic currents (such as Na+ and Ca2+) and repolarizing ionic currents (such as K+ and Ca2+). The oscillatory excitations appear to later emerge at the border of the illuminated region and non-stimulated surrounding tissue, which is likely an area of high source-sink mismatch. The authors appear to acknowledge differences in this oscillatory behavior and previous sub-cellular triggered activity research in their discussion of ectopic pacemaker activity, which is canonically expected more so from genetic or pathological conditions. Regardless, it is exciting to see new ground being broken in this difficult-to-characterize experimental space, even if the method illustrated here may not necessarily be broadly applicable.

Reviewer #1 (Public review):

MPRAs are a high-throughput and powerful tool for assaying the regulatory potential of genomic sequences. However, linking MPRA-nominated regulatory sequences to their endogenous target genes and identifying the more specific functional regions within these sequences can be challenging. MPRAs that tile a genomic region, and saturation mutagenesis-based MPRAs, can help to address these challenges. In this work, Tulloch et al. describe a streamlined MPRA system for the identification and investigation of the regulatory elements surrounding a gene of interest with high resolution. The use of BACs covering a locus of interest to generate MPRA libraries allows for an unbiased and high-coverage assessment of a particular region. Follow-up degenerate MPRAs, where each nucleotide in the nominated sequences is systematically mutated, can then point to key motifs driving their regulatory activity. The authors present this MPRA platform as straightforward, easily customizable, and less time- and resource-intensive than traditional MPRA designs. They demonstrate the utility of their design in the context of the developing mouse retina, where they first use the LS-MPRA to identify active regulatory elements for select retinal genes, followed by d-MPRA, which allowed them to dissect the functional regions within those elements and nominate important regulatory motifs. These assays were able to recapitulate some previously known cis-regulatory modules (CRMs), as well as identify some new potential regulatory regions. Follow-up experiments assessing co-localization of the gene of interest with the CRM-linked GFP reporter in the target cells, and CUT&RUN assays to confirm transcription factor binding to nominated motifs, provided support linking these CRMs to the genes of interest. Overall, this method appears flexible and could be an easy-to-implement tool for other investigators aiming to study their locus of interest with high resolution.

Strengths:

(1) The method of fragmenting BACs allows for high, overlapping coverage of the region of interest.

(2) The d-MPRA method was an efficient way to identify key functional transcription factor motifs and nominate specific transcription factor-driven regulatory pathways that could be studied further.

(3) Additional assays like co-expression analyses using the endogenous gene promoter, and use of the Notch inhibitor in the case of Olig2, helped correlate the activity of the CRMs to the expression of the gene of interest, and distinguish false positives from the initial MPRA.

(4) The use of these assays across different time points, tissues, and even species demonstrated that they can be used across many contexts to identify both common and divergent regulatory mechanisms for the same gene.

Weaknesses:

The LS-MPRA assay most strongly identified promoters, which are not usually novel regulatory elements you would try to discover, and the signal-to-noise ratio for more TSS-distal, non-promoter regulatory elements was usually high, making it difficult to discriminate lower activity CRMs, like enhancers, from the background. For example, NR2 and NR3 in Figure 3 have very minimal activity peaks (NR3 seems non-existent). The ex vivo data in Figure 2 are similarly noisy. Is there a particular metric or calculation that was or could be used to quantitatively or statistically call a peak above the background? The authors mention in the discussion some adjustments that could reduce the noise, such as increased sequencing depth, which I think is needed to make these initial LS-MPRA results and the benchmarking of this assay more convincing and impactful.

Reviewer #2 (Public review):

Summary:

In this study, Tulloch et al. developed two modified massively parallel reporter assays (MPRAs) and applied them to identify cis-regulatory modules (CRMs) - genomic regions that activate gene expression, controlling retinal gene expression. These CRMs usually function at specific developmental stages and in distinct cell types to orchestrate retinal development. Studying them provides insights into how retinal progenitor cells give rise to various retinal cell types.

The first assay, named locus-specific MPRA (LS-MPRA), tests all genomic regions within 150-300 kb of the gene of interest, rather than relying on previously predicted candidate regulatory elements. This approach reduces potential bias introduced during candidate selection, lowers the cost of synthesizing a library of candidate sequences, and simplifies library preparation. The LS-MPRA libraries were electroporated into mouse retinas in vivo or ex vivo. To benchmark the method, the authors first applied LS-MPRA near stably expressed retinal genes (e.g., Rho, Cabp5, Grm6, and Vsx2), and successfully identified both known and novel CRMs. They then used LS-MPRA to identify CRMs in embryonic mouse retinas, near Olig2 and Ngn2, genes expressed in subsets of retinal progenitor cells. Similar experiments were conducted in chick retinas and postnatal mouse retinas, revealing some CRMs with conserved activity across species and developmental stages.

Although the study identified CRMs with robust reporter activity in Olig2+ or Ngn2+ cells, the data do not provide sufficient evidence to support the claims that these CRMs regulate Olig2 or Ngn2, rather than other nearby genes, in a cell-type-specific manner. For example, the authors propose that three regions (NR1/2/3) regulate Olig2 specifically in retinal progenitor cells based on: (1) the three regions are close to Olig2, (2) increased Olig2 expression and NR1/2/3 activity upon Notch inhibition, and (3) reporter activity observed in Olig2+ cells (though also present in many Olig2- cells). While these are promising findings, they do not directly support the claims.

The second assay, called degenerate MPRA (d-MPRA), introduces random point mutations into CRMs via error-prone PCR to assess the impact of sequence variations on regulatory activity. This approach was used on NR1/2/3 to identify mutations that alter CRM activity, potentially by influencing transcription factor binding. The authors inferred candidate transcription factors, such as Mybl1 and Otx2, through motif analysis, co-expression with Olig2 (based on single-cell RNA-seq), and CUR&RUN profiling. While some transcription factors identified in this way overlapped with the d-MPRA results, others did not. This raises questions about how well d-MPRA complements other methods for identifying transcriptional regulators.

Strengths:

(1) The study introduces two technically robust MPRA protocols that offer advantages over standard methods, such as avoiding reliance on predefined candidate regions, reducing cost and labor, and minimizing selection bias.

(2) The identified regulatory elements and transcription factors contribute to our understanding of gene regulation in retinal development and may have translational potential for cell-type-specific gene delivery into developing retinas.

Weaknesses:

(1) The claims for gene-specific and cell type-specific CRMs would benefit from further validation using complementary approaches, such as CRISPR interference or Prime editing.

Reviewer #3 (Public review):

Summary:

Use of reporter assays to understand the regulatory mechanisms controlling gene expression moves beyond simple correlations of cis-regulatory sequence accessibility, evolutionary sequence conservation, and epigenetic status with gene expression, instead quantifying regulatory sequence activity for individual elements. Tulloch et al., provide a systematic characterization of two new reporter assay techniques (LS-MPRA and d-MPRA) to comprehensively identify cis-regulatory sequences contained within genomic loci of interest during retinal development. The authors then apply LS-MPRA and d-MPRA to identify putative cis-regulatory sequences controlling Olig2 and Ngn2 expression, including potential regulatory motifs that known retinal transcription factors may bind. Transcription factor binding to regulatory sequences is then assessed via CUT&RUN. The broader utility of the techniques is then highlighted by performing the assays across development, across species, and across tissues.

Strengths:

(1) The authors validate the reporter assays on retinal loci for which the regulatory sequences are known (Rho, Vsx2, Grm6, Cabp5) mostly confirming known regulatory sequence activity but highlighting either limitations of the current technology or discrepancies of previous reporter assays and known biology. The techniques are then applied to loci of interest (Olig2 and Ngn2) to better understand the regulatory sequences driving expression of these transcription factors across retinal development within subsets of retinal progenitor cells, identifying novel regulatory sequences through comprehensive profiling of the region.

(2) LS-MPRA provides broad coverage of loci of interest.

(3) d-MPRA identifies sequence features that are important for cis-regulatory sequence activity.

(4) The authors take into account transcript and protein stability when determining the correlation of putative enhancer sequence activity with target gene expression.

Weaknesses:

(1) In its current form, the many important controls that are standard for other MPRA experiments are not shown or not performed, limiting the interpretations of the utility of the techniques. This includes limited controls for basal-promoter activity, limited information about sequence saturation and reproducibility of individual fragments across different barcode sequences, limitations in cloning and assay delivery, and sequencing requirements. Additional quantitative metrics, including locus coverage and number of barcodes/fragments, would be beneficial throughout the manuscript.

(2) There are no statistical metrics for calling a region/sequence 'active'. This is especially important given that NR3 for Olig2 seems to have a small 'peak' and has non-significant activity in Figure 4.

(3) The authors present correlational data for identified cis-regulatory sequences with target gene expression. Additionally, the significance of transcription factor binding to the putative regulatory sequences is not currently tested, only correlated based on previous single-cell RNA-sequencing data. While putative regulatory sequences with potential mechanisms of regulation are identified/proposed, the lack of validation (and discrepancies with previous literature) makes it hard to decipher the utility of the techniques.

(4) While the interpretations that Olig2 mRNA/protein expression is dynamically regulated improved the proportions of cells that co-expressed CRM-regulated GFP and Olig2, alternate explanations (some noted) are just as likely. First, the electroporation isn't specific to Olig2+ progenitors. Also, the tested, short CRM fragments may have activating signals outside of Olig2 neurogenic cells because chromatin conformation, histone modifications, and DNA methylation are not present on plasmids to precisely control plasmid activity. Alternatively, repressive elements that control Olig2 expression are not contained in the reporter vectors.

(5) It is unclear as to why the d-MPRA uses a different barcoding strategy, placing a second copy of the cis-regulatory sequence in the 3' UTR. As acknowledged by the author, this will change the transcript stability by changing the 3' UTR sequence. Because of this, comparisons of sequence activity between the LS-MPRA and d-MPRA should not be performed as the experiments are not equivalent.

(6) Furthermore, details of the mutational burden in d-MPRA experiments are not provided, limiting the interpretations of these results.

(7) Many figures are IGV screenshots that suffer from low resolution. Many figures could be consolidated.

Reviewer #1 (Public review):

(1) Presentation of Figures in the Response Letter

I would like to note that the figures included in the response letter would benefit from improved organization. For example, Author response image 1 lacks clarity for experimental conditions. From the response letter, my understanding is that a "Labeling rate index", Rg−Rn, was calculated to represent the difference in the rate of increase in labeling between neurons and glial across two time intervals based on experiments shown in Figure 2-figure supplement 1C and G. It seems that a mean convergence index was calculated for each experimental condition at each time point for glial and neurons, and then the differences in mean convergence index increase between time intervals were calculated for glial and neurons. The legend needs more detail to enhance clarity.

Furthermore, the manuscript should clearly distinguish between figures generated from re-analysis of existing data and those based on newly conducted experiments. This distinction should be explicitly stated in the figure legends and/or main text.<br /> I recommend that all response figures containing data integral to the authors' rebuttal be properly integrated into the manuscript's existing supplementary figure set, rather than remaining isolated in the response document. This would enhance clarity and ensure that key supporting data are fully accessible to readers. For instance, Author response image 1 can be integrated with Figure 2-figure supplement.

(2) Glial Cell Labeling and Specificity of Trans-Synaptic Spread

The authors provided a comprehensive and well-reasoned response to the concern regarding the labeling of radial glial cells. The inclusion of a dedicated section in the revised Discussion and response figures (possibly to be integrated with supplementary figures), strengthens the manuscript.

The authors have made an interesting observation in Author response image 2 that glial labeling was frequently observed near the soma and dendrites of starter cells, suggesting that transneuronal labeled glial cells may be synaptically associated with the starter neurons. Also astroglia starter cells lead to infection of nearby TVA-negative astroglia, suggesting astroglia-to- astroglia transmission.

I find the response scientifically satisfactory and appreciate the authors' transparency in addressing the limitations of their approach.

(3) Temperature Effects and Larval Viability

The authors' justification for raising larvae at 36C to improve labeling efficiency is reasonable. The supporting data indicating minimal impact on larval viability within the experimental timeframe are convincing. Referencing prior behavioral studies and including survival data under controlled conditions adds credibility to their claims. I find this issue satisfactorily addressed.

(4) Viral Toxicity and Dosage Considerations, Secondary Starter Cells

The authors present a well-reasoned explanation that viral cytotoxicity is primarily driven by replication and not by viral titer or injection volume. However, the inclusion of experimental data directly testing the effects of higher titer or volume on starter cell viability would have strengthened this point, particularly since such tests are relatively straightforward to perform.

Regarding the potential contribution of secondary starter cells, the authors provide a convincing rationale for why such effects are unlikely under their sparse labeling conditions. However, in cases where TVA and G are broadly expressed-such as under the vglut2a promoter, as shown in Author response image 2-it would be valuable to directly evaluate this possibility experimentally. While the authors' interpretation is reasonable, empirical validation would further strengthen their conclusions.

Reviewer #2 (Public review):

The study by Chen, Deng et al. aims to develop an efficient viral transneuronal tracing method that allows efficient retrograde tracing in the larval zebrafish. The authors utilize pseudotyped-rabies virus that can be targeted to specific cell types using the EnvA-TvA systems. Pseudotyped rabies virus has been used extensively in rodent models and, in recent years, has begun to be developed for use in adult zebrafish. However, compared to rodents, the efficiency of spread in adult zebrafish is very low (~one upstream neuron labeled per starter cell). Additionally, there is limited evidence of retrograde tracing with pseudotyped rabies in the larval stage, which is the stage when most functional neural imaging studies are done in the field. In this study, the authors systematically optimized several parameters of rabies tracing, including different rabies virus strains, glycoprotein types, temperatures, expression construct designs, and elimination of glial labeling. The optimal configurations developed by the authors are up to 5-10 fold higher than more typically used configurations.

The results are convincing and support the conclusions. There are some additional changes that are recommended:

(1) The new data included in the response to reviewer's letter are important to support the main conclusions and should be included in the manuscript.

(2) Line 357-362: This section should include all of the Author response image and associated details. Additionally, the Author response image 3 is at odds with Fig 2-supplement 1G. In Author response image 3, ~75% of glial cells labeled at 4 dpi loses their fluorescence by 10 dpi. However, Figure 2-supplement 1G shows that glial overall labeling increases ~2 fold from 4 dpi to 10 dpi. This would suggest that the de novo labeling rate for glia is much higher than the net labeling rate calculated from the convergence index. The authors should clarify these findings.

Reviewer #1 (Public review):

The authors conducted a comprehensive investigation into sleep and circadian rhythm disturbances in Fmr1 knockout (KO) mice, a model for Fragile X Syndrome (FXS). They began by monitoring daily home cage behaviors to identify disruptions in sleep and circadian patterns, then assessed the mice's adaptability to altered light conditions through photic suppression and skeleton photoperiod experiments. To uncover potential mechanisms, they examined the connectivity between the retina and the suprachiasmatic nucleus. The study also included an analysis of social behavior deficits in the mutant mice and tested whether scheduled feeding could alleviate these issues. Notably, scheduled feeding not only improved sleep, circadian, and social behaviors but also normalized plasma cytokine levels. The manuscript is strengthened by its focus on a significant and underexplored area-sleep deficits in an FXS model-and by its robust experimental design, which integrates a variety of methodological approaches to provide a thorough understanding of the observed phenomena and potential therapeutic avenues.

Reviewer #2 (Public review):

Summary:

In the present study, the authors, using a mouse model of Fragile X syndrome, explore the intriguing hypothesis that restricting food access over the daily schedule will improve sleep patterns and subsequently enhanced behavioral capacities. By restricting food access from 12h to 6h over the nocturnal period (the active period for mice), they show, in these KO mice, an improvement in the sleep pattern accompanied by reduced systemic levels of inflammatory markers and improved behavior. These data, using a classical mouse model of neurodevelopmental disorder (NDD), suggest that modifying eating patterns might improve sleep quality, leading to reduced inflammation and enhanced cognitive/behavioral capacities in children with NDD.

Overall, the paper is well-written and easy to follow. The rationale of the study is generally well introduced. Data are globally sound. The interpretation is overall supported by the provided data.

Reviewer #1 (Public review):

Summary:

The manuscript titled "Introduction of cytosine-5 DNA methylation sensitizes cells to oxidative damage" proposes that 5mC modifications to DNA, despite being ancient and wide-spread throughout life, represent a vulnerability, making cells more susceptible to both chemical alkylation and, of more general importance, reactive oxygen species. Sarkies et al take the innovative approach of introducing enzymatic genome-wide cytosine methylation system (DNA methyltransferases, DNMTs) into E. coli, which normally lacks such a system. They provide compelling evidence that the introduction of DNMTs increases the sensitivity of E. coli to chemical alkylation damage. Surprisingly they also show DNMTs increase the sensitivity to reactive oxygen species and propose that the DNMT generated 5mC presents a target for the reactive oxygen species that is especially damaging to cells. Evidence is presented that DNMT activity directly or indirectly produces reactive oxygen species in vivo, which is an important discovery if correct, though the mechanism for this remains obscure.

I am satisfied that the points #2, #3 and #4 relating to non-addativity, transcriptional changes and ROS generation have been appropriately addressed in this revised manuscript. The most important point (previously #1) has not been addressed beyond the acknowledgement in the results section that: "Alternatively, 3mC induction by DNMT may lead to increased levels of ssDNA, particularly in alkB mutants, which could increase the risk of further DNA damage by MMS exposure and heighten sensitivity." This slightly miss-represents the original point that 5mC the main enzymatic product of DNMTs rather or in addition to 3mC is likely to lead to transient damage susceptible ssDNA, especially in an alkB deficient background. And more centrally to the main claims of this manuscript, the authors have not resolved whether methylated cytosine introduced into bacteria is deleterious in the context of genotoxic stress because of the oxidative modification to 5mC and 3mC, or because of oxidative/chemical attack to ssDNA that is transiently exposed in the repair processing of 5mC and 3mC, especially in an alkB deficient background. This is a crucial distinction because chemical vulnerability of 5mC would likely be a universal property of cytosine methylation across life, but the wide-spread exposure of ssDNA is expected to be peculiarity of introducing cytosine methylation into a system not evolved with that modification as a standard component of its genome.

These two models make different predictions about the predominant mutation types generated, in the authors system using M.SssI that targets C in a CG context - if oxidative damage to 5mC dominates then mutations are expected to be predominantly in a CG context, if ssDNA exposure effects dominate then the mutations are expected to be more widely distributed - sequencing post exposure clones could resolve this.

Strengths:

This work is based on an interesting initial premise, it is well motivated in the introduction and the manuscript is clearly written. The results themselves are compelling.

Weaknesses:

I am not currently convinced by the principal interpretations and think that other explanations based on known phenomena could account for key results. Specifically the authors have not resolved whether oxidative modification to 5mC and 3mC, or chemical attack to ssDNA that is transiently exposed in the repair processing of 5mC and 3mC is the principal source of the observed genotoxicity.

(1) Original query which still stands: As noted in the manuscript, AlkB repairs alkylation damage by direct reversal (DNA strands are not cut). In the absence of AlkB, repair of alklylation damage/modification is likely through BER or other processes involving strand excision and resulting in single stranded DNA. It has previously been shown that 3mC modification from MMS exposure is highly specific to single stranded DNA (PMID:20663718) occurring at ~20,000 times the rate as double stranded DNA. Consequently the introduction of DNMTs is expected to introduce many methylation adducts genome-wide that will generate single stranded DNA tracts when repaired in an AlkB deficient background (but not in an AlkB WT background), which are then hyper-susceptible to attack by MMS. Such ssDNA tracts are also vulnerable to generating double strand breaks, especially when they contain DNA polymerase stalling adducts such as 3mC. The generation of ssDNA during repair is similarly expected follow the H2O2 or TET based conversion of 5mC to 5hmC or 5fC neither of which can be directly repaired and depend on single strand excision for their removal. The potential importance of ssDNA generation in the experiments has not been [adequately] considered.

Reviewer #2 (Public review):

5-methylcytosine (5mC) is a key epigenetic mark in DNA and plays a crucial role in regulating gene expression in many eukaryotes including humans. The DNA methyltransferases (DNMTs) that establish and maintain 5mC, are conserved in many species across eukaryotes, including animals, plants, and fungi, mainly in a CpG context. Interestingly, 5mC levels and distributions are quite variable across phylogenies with some species even appearing to have no such DNA methylation.

This interesting and well-written paper discusses continuation of some of the authors' work published several years ago. In that previous paper, the laboratory demonstrated that DNA methylation pathways coevolved with DNA repair mechanisms, specifically with the alkylation repair system. Specifically, they discovered that DNMTs can introduce alkylation damage into DNA, specifically in the form of 3-methylcytosine (3mC). (This appears to be an error in the DNMT enzymatic mechanism where the generation 3mC as opposed to its preferred product 5-methylcytosine (5mC), is caused by the flipped target cytosine binding to the active site pocket of the DNMT in an inverted orientation.) The presence of 3mC is potentially toxic and can cause replication stress, which this paper suggests may explain the loss of DNA methylation in different species. They further showed that the ALKB2 enzyme plays a crucial role in repairing this alkylation damage, further emphasizing the link between DNA methylation and DNA repair.

The co-evolution of DNMTs with DNA repair mechanisms suggest there can be distinct advantages and disadvantages of DNA methylation to different species which might depend on their environmental niche. In environments that expose species to high levels of DNA damage, high levels of 5mC in their genome may be disadvantageous. This present paper sets out to examine the sensitivity of an organism to genotoxic stresses such as alkylation and oxidation agents as the consequence of DNMT activity. Since such a study in eukaryotes would be complicated by DNA methylation controlling gene regulation, these authors cleverly utilize Escherichia coli (E.coli) and incorporate into it the DNMTs from other bacteria that methylate the cytosines of DNA in a CpG context like that observed in eukaryotes; the active sites of these enzymes are very similar to eukaryotic DNMTs and basically utilize the same catalytic mechanism (also this strain of E.coli does not specifically degrade this methylated DNA) .

The experiments in this paper more than adequately show that E. coli expression of these DNMTs (comparing to the same strain without the DNMTS) do indeed show increased sensitivity to alkylating agents and this sensitivity was even greater than expected when a DNA repair mechanism was inactivated. Moreover, they show that this E. coli expressing this DNMT is more sensitive to oxidizing agents such as H2O2 and has exacerbated sensitivity when a DNA repair glycosylase is inactivated. Both propensities suggest that DNMT activity itself may generate additional genotoxic stress. Intrigued that DNMT expression itself might induce sensitivity to oxidative stress, the experimenters used a fluorescent sensor to show that H2O2 induced reactive oxygen species (ROS) are markedly enhanced with DNMT expression. Importantly, they show that DNMT expression alone gave rise to increased ROS amounts and both H2O2 addition and DNMT expression has greater effect that the linear combination of the two separately. They also carefully checked that the increased sensitivity to H2O2 was not potentially caused by some effect on gene expression of detoxification genes by DNMT expression and activity. Finally, by using mass spectroscopy, they show that DNMT expression led to production of the 5mC oxidation derivatives 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) in DNA. 5fC is a substrate for base excision repair while 5hmC is not; more 5fC was observed. Introduction of non-bacterial enzymes that produce 5hmC and 5fC into the DNMT expressing bacteria again showed a greater sensitivity than expected. Remarkedly, in their assay with addition of H2O2, bacteria showed no growth with this dual expression of DNMT and these enzymes.

Overall, the authors conduct well thought-out and simple experiments to show that a disadvantageous consequence of DNMT expression leading to 5mC in DNA is increased sensitivity to oxidative stress as well as alkylating agents.

Again, the paper is well-written and organized. The hypotheses are well-examined by simple experiments. The results are interesting and can impact many scientific areas such as our understanding of evolutionary pressures on an organism by environment to impacting our understanding about how environment of a malignant cell in the human body may lead to cancer.

In a new revised version of the paper, the authors have adequately addressed issues put forth by other reviewers. The result is even a better manuscript. Additions to the Results and Discussion sections and a new Supplemental Figure 2 give further credence to their conclusions.

Reviewer #3 (Public review):

Summary:

Krwawicz et al., present evidence that expression of DNMTs in E. coli results in (1) introduction of alkylation damage that is repaired by AlkB; (2) confers hypersensitivity to alkylating agents such as MMS (and exacerbated by loss of AlkB); (3) confers hypersensitivity to oxidative stress (H2O2 exposure); (4) results in a modest increase in ROS in the absence of exogenous H2O2 exposure; and (5) results in the production of oxidation products of 5mC, namely 5hmC and 5fC, leading to cellular toxicity. The findings reported here have interesting implications for the concept that such genotoxic and potentially mutagenic consequences of DNMT expression (resulting in 5mC) could be selectively disadvantageous for certain organisms. The other aspect of this work which is important for understanding the biological endpoints of genotoxic stress is the notion that DNA damage per se somehow induces elevated levels of ROS.

Strengths:

The manuscript is well-written, and the experiments have been carefully executed providing data that support the authors' proposed model presented in Fig. 7 (Discussion, sources of DNA damage due to DNMT expression).

Weaknesses:

(1) The authors have established an informative system relying on expression of DNMTs to gauge the effects of such expression and subsequent induction of 3mC and 5mC on cell survival and sensitivity to an alkylating agent (MMS) and exogenous oxidative stress (H2O2 exposure). The authors state (p4) that Fig. 2 shows that "Cells expressing either M.SssI or M.MpeI showed increased sensitivity to MMS treatment compared to WT C2523, supporting the conclusion that the expression of DNMTs increased the levels of alkylation damage." This is a confusing statement and requires revision as Fig. 2 does ALL cells shown in Fig. 2 are expressing DNMTs and have been treated with MMS. It is the absence of AlkB and the expression of DNMTs that that causes the MMS sensitivity.

(2) It would be important to know whether the increased sensitivity (toxicity) to DNMT expression and MMS is also accompanied by substantial increases in mutagenicity. The authors should explain in the text why mutation frequencies were not also measured in these experiments.

(3) Materials and Methods. ROS production monitoring. The "Total Reactive Oxygen Species (ROS) Assay Kit" has not been adequately described. Who is the Vendor? What is the nature of the ROS probes employed in this assay? Which specific ROS correspond to "total ROS"?

(4) The demonstration (Fig. 4) that DNMT expression results in elevated ROS and its further synergistic increase when cells are also exposed to H2O2 is the basis for the authors' discussion of DNA damage-induced increases in cellular ROS. S. cerevisiae does not possess DNMTs/5mC, yet exposure to MMS also results in substantial increases in intracellular ROS (Rowe et al, (2008) Free Rad. Biol. Med. 45:1167-1177. PMC2643028). The authors should be aware of previous studies that have linked DNA damage to intracellular increases in ROS in other organisms and should comment on this in the text.

Reviewer #1 (Public review):

In the current article, Octavia Soegyono and colleagues study "The influence of nucleus accumbens shell D1 and D2 neurons on outcome-specific Pavlovian instrumental transfer", building on extensive findings from the same lab. While there is a consensus about the specific involvement of the Shell part of the Nucleus Accumbens (NAc) in specific stimulus-based actions in choice settings (and not in General Pavlovian instrumental transfer - gPIT, as opposed to the Core part of the NAc), mechanisms at the cellular and circuitry levels remain to be explored. In the present work, using sophisticated methods (rat Cre-transgenic lines from both sexes, optogenetics, and the well-established behavioral paradigm outcome-specific PIT-sPIT), Octavia Soegyono and colleagues decipher the differential contribution of dopamine receptors D1 and D2 expressing spiny projection neurons (SPNs).

After validating the viral strategy and the specificity of the targeting (immunochemistry and electrophysiology), the authors demonstrate that while both NAc Shell D1- and D2-SPNs participate in mediating sPIT, NAc Shell D1-SPNs projections to the Ventral Pallidum (VP, previously demonstrated as crucial for sPIT), but not D2-SPNs, mediates sPIT. They also show that these effects were specific to stimulus-based actions, as value-based choices were left intact in all manipulations.

This is a well-designed study, and the results are well supported by the experimental evidence. The paper is extremely pleasant to read and adds to the current literature.

Reviewer #2 (Public review):

Summary:

This manuscript by Soegyono et al. describes a series of experiments designed to probe the involvement of dopamine D1 and D2 neurons within the nucleus accumbens shell in outcome-specific Pavlovian-instrumental transfer (osPIT), a well-controlled assay of cue-guided action selection based on congruent outcome associations. They used an optogenetic approach to phasically silence NAc shell D1 (D1-Cre mice) or D2 (A2a-Cre mice) neurons during a subset of osPIT trials. Both manipulations disrupted cue-guided action selection but had no effects on negative control measures/tasks (concomitant approach behavior, separate valued guided choice task), nor were any osPIT impairments found in reporter-only control groups. Separate experiments revealed that selective inhibition of NAc shell D1 but not D2 inputs to ventral pallidum was required for osPIT expression, thereby advancing understanding of the basal ganglia circuitry underpinning this important aspect of decision making.

Strengths:

The combinatorial viral and optogenetic approaches used here were convincingly validated through anatomical tract-tracing and ex vivo electrophysiology. The behavioral assays are sophisticated and well-controlled to parse cue and value-guided action selection. The inclusion of reporter-only control groups is rigorous and rules out nonspecific effects of the light manipulation. The findings are novel and address a critical question in the literature. Prior work using less decisive methods had implicated NAc shell D1 neurons in osPIT but suggested that D2 neurons may not be involved. The optogenetic manipulations used in the current study provide a more direct test of their involvement and convincingly demonstrate that both populations play an important role. Prior work had also implicated NAc shell connections to ventral pallidum in osPIT, but the current study reveals the selective involvement of D1 but not D2 neurons in this circuit. The authors do a good job of discussing their findings, including their nuanced interpretation that NAc shell D2 neurons may contribute to osPIT through their local regulation of NAc shell microcircuitry.

Weaknesses:

The current study exclusively used an optogenetic approach to probe the function of D1 and D2 NAc shell neurons. Providing a complementary assessment with chemogenetics or other appropriate methods would strengthen conclusions, particularly the novel demonstration of D2 NAc shell involvement. Likewise, the null result of optically inhibiting D2 inputs to the ventral pallidum leaves open the possibility that a more complete or sustained disruption of this pathway may have impaired osPIT.

Reviewer #3 (Public review):

Summary:

The authors present data demonstrating that optogenetic inhibition of either D1- or D2-MSNs in the NAc Shell attenuates expression of sensory-specific PIT while largely sparing value-based decision on an instrumental task. They also provide evidence that SS-PIT depends on D1-MSN projections from the NAc-Shell to the VP, whereas projections from D2-MSNs to the VP do not contribute to SS-PIT.

Strengths:

This is clearly written. The evidence largely supports the authors' interpretations, and these effects are somewhat novel, so they help advance our understanding of PIT and NAc-Shell function.

Weaknesses:

I think the interpretation of some of the effects (specifically the claim that D1-MSNs do not contribute to value-based decision making) is not fully supported by the data presented.

Reviewer #1 (Public review):

Wojcik et al. conducted a working memory (WM) experiment in which participants had to press the right or left button after being presented with a square (upright) or diamond stimulus. The response mapping ('context') depended on a colour cue presented at the start of each trial. This results in an XOR task, requiring participants to integrate colour and shape information. Importantly, multiple colours could map onto the same context, allowing the authors to disentangle the (neural) representations of context from those of colour.

The authors report that participants learn the appropriate context mappings quickly over the course of the experiment. Neural context representation is evident in the WM delay and emerges later in the experiment, unlike colour representation, which is present only during colour presentation and does not evolve over experimental time. There are furthermore results on neural geometry (averaged cross-generalized decoding) and neural dimensionality (averaged decoding after shattering all task dimensions), which are somewhat harder to interpret.

Overall, the findings are likely Important, as they highlight the flexible and future-oriented nature of WM. The strength of support at the moment is incomplete: there are some loose ends on the context/colour generalization, and the evidence for the XOR neural representation is not (yet) well-established.

I have one (major) concern and several suggestions for improvement.

(1a) As the authors also acknowledge in several places, the XOR dimension is strongly correlated with motor responses, in any case toward the end of the task (and by definition for all correct trials). This should be dealt with properly. Right now, e.g. Figures 2g/i, 2h/j, 3e/g, 3f/h are highly similar, respectively, because of this strong collinearity. I would remove the semi-duplicate graphs and/or deal with this explicitly through some partial regression, trial selection, or similar (and report these correlations).

(1b) Most worrisome in this respect is that one of the key results presented is that XOR decoding increases with learning. But also task accuracy increases, meaning that the proportion of correct trials increases with learning, meaning that the XOR and motor regressors become more similar over experimental time. This means that any classifier picking up on motor signals will be better able to do so later on in the task than earlier on. (In other words, the XOR regressor may be a noisy version of the motor regressor early on, and a more precise version of the motor regressor later on.) Therefore, the increase in XOR decoding over experimental time may be (entirely) due to an increase in similarity between the XOR and motor dimensions. The authors should either rule out this explanation, and/or remove/tone down the conclusions regarding the XOR coding increase. (Note that the takeaway regarding colour/context generalization does not depend on this analysis, fortunately.) The absence of a change in motor decoding with learning (as reported on page 11) does not affect this potential confound; in fact it is made more likely with it.

(2) Bayes factors would be valuable in several places, especially with null results (p. 5) or cases with borderline-significant p-values.

(3) The authors' interpretation of the key results implies that the abstract coding learned over the task should be relevant for behaviour. The current results do not show a particularly strong behavioural relevance of coding, to put it mildly. It might be worth exploring whether neural coding expresses itself in reaction times, rather than (in)correct responses, and reflecting on the (lack of) behavioural relevance in the Discussion.

(4) All data and experiment/analysis code should be made available, in public repositories (i.e., not "upon request").

Reviewer #2 (Public review):

This manuscript describes an experiment in which subjects learned to apply an XOR rule in a task in which an initial color cue conditioned the instruction ("press left" or "press right") conveyed by a subsequent shape.

This manuscript gives the impression of being written to address a sophisticated computational framework, but the experiment was not designed to test this framework. Stated differently, the memory-as-resource-for-computations framework may not be needed to account for the results presented here. Variants of this task have been used for decades, often in the context of prospective processing, and although the authors emphasize a dimensionality reduction operation, the task may actually only require the recoding of retrospectively relevant sensory information into the prospectively relevant rule that is needed to guide the response on that trial. Consequently, many of the claims are only partially supported.

The framework invoked by the authors is summarized in the second paragraph of the manuscript:

"Insights from machine learning and computational neuroscience further highlight the idea that memory processes can be viewed as a resource for computations rather than a passive mechanism for storage (Dasgupta & Gershman, 2021; Ehrlich & Murray, 2022). In this light, working memory adapts computations to the current task demands (Dasgupta & Gershman, 2021); pre-computed information can be stored in working memory, and thus reduce the computation time at the moment of the decision (Braver, 2012; Hunt et al., 2021). This perspective is further supported by computational modelling of neural circuits that contends that working memory will change neural geometry in a way that supports the temporal decomposition of computations (Ehrlich & Murray, 2022). This work suggests that the computational load at the moment of action can be thus alleviated by decomposing complex operations into several simple problems solved sequentially in time."

However, the relevance, certainly the necessity, of this framework leads to mischaracterizations of some elements of the task (including about a hypothesis), the emphasis of constructs that don't actually exist in the task, some logical inconsistencies, and the repeated invocation of operations like "dimensionality reduction" despite the fact that the authors find no evidence for them.

Beginning with the final point, the task presented here is a variant of a Badre-style hierarchical control task, one requiring solution at the second order of abstraction (i.e., the color conditions the interpretation of the shape [2nd order], which then determines the correct response [1st order]. These operations can be accomplished without dimensionality reduction by simply carrying out the remapping instructed by each element. For example, on a trial beginning with a blue color cue, the subject can use a lookup table to translate this into the rule "square = left; diamond = right". When the shape is subsequently presented, the subject responds according to this rule. This is really no different from any of the several studies that have shown prospective recoding of information in working memory, including the work from the 1990s in nonhuman primates, and several subsequent studies using fMRI in humans beginning in the 2000s. Importantly, this account does not involve dimensionality reduction in any overt way. If it were the case that the more recent computational work indicates that this operation of "prospective recoding" does, in fact, entail dimensionality reduction on this type of task, that would be interesting. However, I don't see evidence that this is the case. Although the authors carry out several analyses of shattering dimensionality, I do not find any that track this measure across epochs within the trial, an approach that would presumably capture epoch-to-epoch dimensionality reduction, if it occurred.

With regard to mischaracterization of a hypothesis, the authors state: "We hypothesised that working memory processes control the dimensionality of neural representations by selecting features for maintenance. We tested this prediction by exploring the learning dynamics of the colour representation." However, what is described here is not a test of a prediction about dimensionality reduction. Rather, it's a test of a prediction that color decoding would not persist after color offset. To describe this as "dimensionality reduction" misrepresents/mischaracterizes what's happening, which is the translation of color (on any trial, a low-dimensional variable) into the rule that was cued by that color. It is a translation of what kind of information is being represented, as opposed to a dimensionality reduction applied to a representation.

With regard to constructs that don't actually exist, it is unclear what the reality is in the study of a "color pair"? I.e., because colors are never presented together, nor associated in some way, this would seem to be a device that's helpful to the authors for thinking about how their task might be solved, rather than a fundamental aspect of the task that the reader needs to understand. Furthermore, the example given here wasn't helpful for this reader. (What WAS helpful was the description of the two possible strategies and accompanying references to Mayr & Kleigel and to Vandierendonck.)

With regard to logical inconsistencies, one is the notion that color is irrelevant. This is not true, in a literal sense, because if every color cue were rendered as the same monochromatic patch, one wouldn't be able to solve the task. What the authors could do to make their point is perhaps refer to Strategy 1, which corresponds to a less efficient way to solve the task.

Also inconsistent is the relation of the present work to a previous study carried out by this group in nonhuman primates. That task did not include a working memory delay, and so this is difficult to reconcile the comparison that the authors draw with this task with the many suggestions that they make that it's something about WM, per se, that allows for the efficient performance of this task.

"Crucially, the irrelevant feature was only discarded during the delay after it entered working memory." This statement is in direct contradiction with the authors' own reporting of the results: "Decoding analyses demonstrated that colour information peaked in the early colour locked period of the trial and then rapidly declined over time to reach chance levels before the delay-locked period, 𝑐𝑙𝑢𝑠𝑡𝑒𝑟 1: 0.082 − 0.484 𝑚𝑠, 𝑝 = 0.006 (Fig. 2c)."

Other areas where I had difficulties include:

(1) "These results suggest that participants rapidly discarded irrelevant colour information. Only information relevant for performance (context) entered working memory and was maintained."<br /> Although this may be the case, each of the four colors also instructed a rule, and so what's being documented in this study is the translation of a cue into a rule, not the transformation of a "meaningless color" into a "meaningful context." It is very possible that if the authors only used two colors, one for each rule (i.e., one for each "context"), they'd get the same decoding results.

(2) "A defining characteristic of low-dimensional task representations is that they can be easily cross-generalised to different sensory instances of the same task."<br /> This result is difficult to reconcile with the loss of color decoding with color offset. Must it not mean that the rule is being represented differently when cued, e.g., by blue vs. by pink, or by green vs. by khaki? If this is true, then this would also argue against the idea of dimensionality reduction during the delay period, because subjects will, in effect, have swapped needing to represent one of four colors with needing to represent one of four rules.

(3) The authors assert that "cross-colour generalisation of context in the delay period is already implied by the significant context decoding combined with the absence of irrelevant colour coding."<br /> This is contradicted, however, by the failure of the direct test of cross-color decoding!

(4) "Taken together, these findings imply that participants constructed abstract representations of task features but that the mechanism responsible for this transformation relied heavily on discarding colour information early in trial time."

This statement does not follow from the data because no mechanism is being directly measured. Rather, it's simply the case that after translating the color to a rule, the color is no longer needed and so is no longer kept in an active state. There is certainly no evidence for "heavy reliance".

Reviewer #3 (Public review):

Summary:

Wójcik and colleagues investigated how the maintenance of task information in working memory influences the dimensionality of task representations. The task required an exclusive-or (XOR) mapping as the output by combining stimulus features separated by a delay period. The authors found that context information invariant to input features (i.e., color) is maintained and enhanced over the course of learning the task.

The significance of this study lies in its demonstration of how learning selectively changes the geometry of task representations. The clear-cut results emphasize that learning promotes the abstraction of task representations for context-dependent computations. It is also important to investigate how working memory mechanisms contribute to the geometry and optimization of task representations, as such studies in humans are scarce.

Strengths:

(1) The task design and analyses are clear.

(2) The theoretical motivation to study low-dimensional representations and temporal decomposition is strong. Understanding how learning changes these qualities is a novel and important question.

Weaknesses:

(1) The specific contribution of working memory maintenance to the dimensionality and abstraction of representations is unclear. While the task likely recruits working memory, there are no direct assessments linking the observed results to particular qualities or mechanisms of working memory. In other words, neural representations observed during the delay period are interpreted as working memory.

(2) The dissociation between XOR and motor representations is ambiguous, as they only become distinguishable during error trials. Additionally, they show similar time courses and learning-related changes.

Reviewer #1 (Public review):

Circannual timing is a phylogenetically widespread phenomenon in long-lived organisms and is central to the seasonal regulation of reproduction, hibernation, migration, fur color changes, body weight, and fat deposition in response to photoperiodic changes. Photoperiodic control of thyroid hormone T3 levels in the hypothalamus dictates this timing. However, the mechanisms that regulate these changes are not fully understood. The study by Stewart et al. reports that hypothalamic iodothyronine deiodinase 3 (Dio3), the major inactivator of the biologically active thyroid hormone T3, plays a critical role in circannual timing in the Djungarian hamster. Overall, the study yields important results for the field and is well-conducted, with the exception of the CRISPR/Cas9 manipulation.

Figure 1 lays the foundation for examining circannual timing by establishing the timing of induction, maintenance, and recovery phases of the circannual timer upon exposure of hamsters to short photoperiod (SP) by monitoring morphological and physiological markers. Measures of pelage color, torpor, body mass, plasma glucose, etc, established that the initiation phase occurred by weeks 4-8 in SP, the maintenance by weeks 12-20, and the recovery after week 20, where all morphological and physiological changes started to reverse back to long photoperiod phenotypes. The statistical analyses look fine, and the results are unambiguous. Their representation could, however, be improved. In Figures 1d and 1e, two different measures are plotted on each graph and differentiated by dots and upward or downward arrowheads. The plots are so small, though, that distinguishing between the direction of the arrows is difficult. Some color coding would make it more reader-friendly. The same comment applies to Figure S4. The authors went on to profile the transcriptome of the mediobasal and dorsomedial hypothalamus, paraventricular nucleus, and pituitary gland (all known to be involved in seasonal timing) every 4 weeks over the different phases of the circannual interval timer. A number of transcripts displaying seasonal rhythms in expression levels in each of the investigated structures were identified, including transcripts whose expression peaks during each phase. This included two genes of particular interest due to their known modulation of expression in response to photoperiod, Dio3 and Sst, found among the transcripts upregulated during the induction and maintenance phases, respectively. The experiments are technically sound and properly analyzed, revealing interesting candidates. Again, my main issues lie with the representation in the figure. In particular, the authors should clarify what the heatmaps on the right of Figures 1f and 1g represent. I suspect they are simply heatmaps of averaged expression of all genes within a defined category, but a description is missing in the legend, as well as a scale for color coding near the figure.

Figure 2 reveals that SP-programmed body mass loss is correlated to increased Dio3-dependent somatostatin (Sst) expression. First, to distinguish whether the body mass loss was controlled by rheostatic mechanisms and not just acute homeostatic changes in energy balance, experiments from hamsters fed ad lib or experiencing an acute food restriction in both LP and SP were tested. Unlike plasma insulin, food restriction had no additional effect on SP-driven epididymal fat mass loss (Figure S7). This clearly establishes a rheostatic control of body mass loss across weeks in SP conditions. Importantly, Sst expression in the mediobasal hypothalamus increased in both ad lib fed or restriction fed SP hamsters and this increase in expression could be reduced by a single subcutaneous injection of active T3, clearly suggesting that increase in Sst expression in SP is due to a decrease of active T3 likely via Dio3 increase in expression in the hypothalamus. The results are unambiguous.

Figure 3 provides a functional test of Dio3's role in the circannual timer. Mediobasal hypothalamic injections of CRISPR-Cas9 lentiviral vectors expressing two guide RNAs targeting the hamster Dio3 led to a significant reduction in the interval between induction and recovery phases seen in SP as measured by body mass, and diminished the extent of pelage color change by weeks 15-20. In addition, hamsters that failed to respond to SP exposure by decreasing their body mass also had undetectable Dio3 expression in the mediobasal hypothalamus. Together, these data provide strong evidence that Dio3 functions in the circannual timer. I noted, however, a few problems in the way the CRISPR modification of Dio3 in the mediobasal hypothalamus was reported in Figure S8. One is in Figure S8b, where the PAM sites are reported to be 9bp and 11bp downstream of sgRNA1 and sgRNA2, respectively. Is this really the case? If so, I would have expected the experiment to fail to show any effect as PAM sites need to immediately follow the target genomic sequence recognized by the sgRNA for Cas9 to induce a DNA double-stranded break. It seems that each guide contains a 3' NGG sequence that is currently underlined as part of sgRNAs in both Fig S8b and in the method section. If this is not a mistake in reporting the experimental design, I believe that the design is less than optimal and the efficiencies of sgRNAs are rather low, if at all functional. The authors report efficiencies around 60% (line 325), but how these were obtained is not specified. Another unclear point is the degree to which the mediobasal hypothalamus was actually mutated. Only one mutated (truncated) sequence in Figure S8c is reported, but I would have expected a range of mutations in different cells of the tissue of interest. Although the authors clearly find a phenotypic effect with their CRISPR manipulation, I suspect that they may have uncovered greater effects with better sgRNA design. These points need some clarification. I would also argue that repeating this experiment with properly designed sgRNAs would provide much stronger support for causally linking Dio3 in circannual timing.

A proposed schematic model for mechanisms of circannual interval timing is presented in Figure S9. I think this represents a nice summary of the findings put in a broader context and should be presented as a main figure in the manuscript itself rather than being relayed in supplementary materials.

Reviewer #2 (Public review):

Summary:

Several animals and plants adjust their physiology and behavior to seasons. These changes are timed to precede the seasonal transitions, maximizing chances of survival and reproduction. The molecular mechanisms used for this process are still unclear. Studies in mammals and birds have shown that the expression of deiodinase type-1, 2, and 3 (Dio1, 2, 3) in the hypothalamus spikes right before the transition to winter phenotypes. Yet, whether this change is required or an unrelated product of the seasonal changes has not been shown, particularly because of the genetic intractability of the animal models used to study seasonality. Here, the authors show for the first time a direct link between Dio3 expression and the modulation of circannual rhythms.

Strengths:

The work is concise and presents the data in a clear manner. The data is, for the most part, solid and supports the author's main claims. The use of CRISPR is a clear advancement in the field. This is, to my knowledge, the first study showing a clear (i.e., causal) role of Dio3 in the circannual rhythms in mammals. Having established a clear component of the circannual timing and a clean approach to address causality, this study could serve as a blueprint to decipher other components of the timing mechanism. It could also help to enlighten the elusive nature of the upstream regulators, in particular, on how the integration of day length takes place, maybe within the components in the Pars tuberalis, and the regulation of tanycytes.

Weaknesses:

Due to the nature of the CRISPR manipulation, the low N number is a clear weakness. This is compensated by the fact that the phenotypes shown here are strong enough. Also, this is the only causal evidence of Dio3's role; thus, additional evidence would have significantly strengthened the author's claims. The use of the non-responsive population of hamsters also helps, but it falls within the realm of correlations. Additionally, the consequences of the mutations generated by CRISPR are not detailed; it is not clear if the mutations affect the expression of Dio3 or generate a truncation or deletion, resulting in a shorter protein.

Reviewer #3 (Public review):

The authors investigated SP-induced physiological and molecular changes in Djungarian hamsters and the endogenous recovery from it after circa half a year. The study aimed to elucidate the intrinsic mechanism and included nice experiments to distinguish between rheostatic effects on energy state and homeostatic cues driven by an interval timer. It also aimed to elucidate the role of Dio3 by introducing a targeted mutation in the MBH by ICV. The experiments and analyses are sound, and the amount of work is impressive. The impact of this study on the field of seasonal chronobiology is probably high.

Even though the general conclusions are well-founded, I have fundamental criticism concerning 3 points, which I recommend revising:

(1) The authors talk about a circannual interval timer, but this is no circannual timer. This is a circa-semiannual timer. It is important that the authors use precise wording throughout the manuscript.

(2) The authors put their results in the context of clocks. For example, line 180/181 seasonal clock. But they have described and investigated an interval timer. A clock must be able to complete a full cycle endogenously (and ideally repeatedly) and not only half of it. In contrast, a timer steers a duration. Thus, it is well possible that a circannual clock mechanism and this circa-semiannual timer of photoperiodic species are 2 completely different mechanisms. The argumentation should be changed accordingly.

(3) The authors chose as animal model the Djungarian hamster, which is a predominantly photoperiodic species and not a circannual species. A photoperiodic species has no circannual clock. That is another reason why it is difficult to draw conclusions from the experiment for circannual clocks. However, the Djungarian hamster is kind of "indifferent" concerning its seasonal timing, since a small fraction of them are indeed able to cycle (Anchordoquy HC, Lynch GR (2000), Evidence of an annual rhythm in a small proportion of Siberian hamsters exposed to chronic short days. J Biol Rhythms 15:122-125.). Nevertheless, the proportion is too small to suggest that the findings in the current study might reflect part of the circannual timing.

Therefore, the authors should make a clear distinction between timers and clocks, as well as between circa-annual and circa-semiannual durations/periods.

Reviewer #1 (Public review):

Summary:

This paper describes an interesting phenotype of C. elegans lite-1 mutants. Previous work showed that lite-1 mutants lose a violet/blue light avoidance response. The authors show here that lite-1 mutants also show a defect in negative diacetyl chemotaxis. While wild-type worms avoid diacetyl at high concentrations, lite-1 mutants are instead *attracted* to it. The authors go on to perform Ca2+ imaging in sensory neurons and find that ADL and ASK neurons show altered Ca2+ responses to diacetyl in lite-1 mutants, suggesting LITE-1 is required for these responses. As unc-13 mutants with defective synaptic transmission show similar diacetyl Ca2+ responses as wild-type, this suggests these neurons respond cell autonomously to diacetyl. However, whether lite-1 also acts cell-autonomously is not discussed. Indeed, because unc-13 and lite-1 mutants show different ADL and ASK Ca2+ responses, it seems the diacetyl response regulated by LITE-1 is likely acting outside of those cells. An interesting result that is not commented on is the switching of the valence of the ASK Ca2+ response in lite-1 mutants. ASK neurons still respond to diacetyl, but instead of a strong increase in Ca2+, diacetyl appears to drive it strongly lower. This may be consistent with the switch in valence in the diacetyl chemotaxis assay. It also argues against the idea that LITE-1 is a low-affinity diacetyl receptor that drives avoidance or the Ca2+ responses in ASK, since it is still present in lite-1 mutants. The authors then use a strain that expresses LITE-1 in the body wall muscles and show this expression is sufficient to engender them with sensitivity to diacetyl, as measured through altered swimming and hypercontractility. The authors interpret this result as LITE-1 may act as a diacetyl receptor. The authors test whether a structurally similar molecule, 2,3-pentanedione, shows similar effects, and they find it does. Alpha-fold modeling and molecular docking analysis show where diacetyl might bind to the LITE-1 protein. They then test whether lite-1 mutants show chemotaxis defects to other molecules, as seen with diacetyl. Generally, they find that the observed diacetyl responses are unique, although lite-1 mutants do lose their avoidance response to 2,3-pentanedione. However, unlike the acquisition of diacetyl attraction in lite-1 mutants, 2,3 pentanedione avoidance is *lost*; it is not switched to attraction. Overall, I felt the description of the results and their implications could have been more in-depth. Further, the evidence that LITE-1 is a chemoreceptor itself, rather than acting in some way to shape chemoreceptor responses (via light or otherwise), remains unclear, as conceded by the authors.

Strengths:

Overall, the study follows up on an interesting and useful result. The experiments as presented are generally well-conceived and performed. The authors use a variety of behavioral and imaging approaches to test how LITE-1 mediates diacetyl avoidance.

Weaknesses:

The study is missing experiments needed to resolve whether LITE-1 is doing what they propose. The evidence that LITE-1 is a diacetyl receptor is lacking support since lite-1 mutants have their avoidance and calcium responses flipped, which would not be expected if it were acting solely as an avoidance receptor. Presumably, the authors are concluding that the attractive response that is left in the lite-1 mutant is mediated by ODR-10, but that experiment is not shown. Similarly, the authors concede that "the use of lite-1 point mutants that affect specific LITE-1 function, such as light sensing, channel gating, or binding pocket, could further elucidate LITE-1 mechanisms." This reviewer agrees, and such experiments designed to localize diacetyl binding site(s) would be necessary to conclude definitively that LITE-1 is a diacetyl receptor. The body wall muscle assay used or some other heterologous experimental system could work for such a structure-function analysis. A concern is whether the extensive number of LITE-1 point mutants described in the literature affect cell surface expression vs. receptor function, which might complicate the interpretation of a result showing loss of diacetyl responses.

Reviewer #2 (Public review):

Summary:

Koh and colleagues investigate the broader sensory role of LITE-1, a gustatory receptor previously linked to UV light detection in C. elegans. Their study explores whether LITE-1 also mediates avoidance of specific chemical stimuli-namely, high concentrations of diacetyl and 2,3-pentanedione. They show that LITE-1 is required in the ADL and ASK neurons for calcium responses to diacetyl, and that its expression in body-wall muscles is sufficient to trigger hypercontraction upon odorant exposure. Molecular docking suggests both odorants may directly bind to LITE-1 with micromolar affinity. These findings suggest LITE-1 may act as a multimodal receptor for both light and chemical stimuli.

Strengths:

(1) Methodological Precision: The study is technically strong, with well-executed calcium imaging and quantitative behavioral assays that clearly show neural and muscular responses to chemical stimuli.

(2) Novelty and Scope: The work presents a compelling case for LITE-1 functioning as a multimodal sensor, which is an intriguing expansion of its known role.

(3) Potential Impact: If validated, the findings could significantly advance the understanding of sensory integration in C. elegans, and the tools developed may be broadly useful to the research community.

(4) Relevance to the Field: The study adds to evidence that C. elegans uses non-canonical sensory pathways and may inspire further exploration of multimodal receptor functions in other systems.

Weaknesses:

(1) Lack of Rescue Experiments: The absence of rescue experiments makes it difficult to definitively link the observed phenotypes to loss of lite-1.

(2) Single Loss-of-Function Approach: The reliance on a single genetic mutant limits interpretability. Additional strategies such as RNAi (e.g., neuron-specific knockdown) would provide stronger evidence.

(3) Unclear Neuronal Contribution: While calcium responses in ADL and ASK are reduced, it's unclear which neuron(s) are necessary for behavioral avoidance. Cell-specific rescue or knockdown experiments are needed.

(4) Unvalidated Docking Data: The molecular docking predictions lack experimental validation. Site-directed mutagenesis would be needed to support claims of direct interaction.

(5) Limited Odorant Specificity Testing: Docking analysis does not include non-binding odorants, making it difficult to assess binding specificity.

(6) Incomplete Quantification: Some calcium imaging results (e.g., in AWA neurons of unc-13 mutants) lack statistical comparisons, which limits their interpretive value.

Reviewer #3 (Public review):

In this work, Brown and colleagues report that the photosensor protein LITE-1 of the nematode C. elegans may also be a chemosensor that can be activated by high concentrations of the compound diacetyl. LITE-1 was described as a putative ion channel of the gustatory receptor family, which is mainly constituted by insect odorant receptors. These form tetrameric ion channels that can be activated by odorants. Specificity is achieved by forming heteromeric channels from three copies of the odorant receptor co-receptor (ORCO) and another subunit that resembles ORCO in the pore-forming C-terminus, but brings in a binding site for the respective odorant. LITE-1 has a very similar structure, according to Alphafold3 predictions, and also carries a binding pocket. In LITE-1, this was proposed to be occupied by a light-absorbing molecule that activates the channel when a photon is absorbed. Alternatively, compounds generated by absorption of high-energy photons may be formed in vivo and bound by the LITE-1 binding pocket. Koh et al. now demonstrate that another, non-light-activated compound, diacetyl, at high concentrations, can activate cells expressing LITE-1. Such (chemosensory) cells are also responsible for the avoidance of high concentrations of diacetyl. LITE-1 activation in excitable cells, i.e, muscles, causes strong body contraction and paralysis, and the authors show that this is also the case when diacetyl is presented. The authors further present molecular docking studies showing that diacetyl could occupy the binding pocket of LITE-1. Last, they show that another compound chemically resembling diacetyl, i.e., 2,3-pentanedione, can also induce avoidance in a LITE-1 dependent manner, though not as potently.

The data are intriguing, and the demonstration of LITE-1 being a diacetyl chemosensor is interesting. Yet, there are a few questions arising that the authors should address.

The authors identified mutants lacking diacetyl responses. In their chemotaxis assay (Figures 1A, B), they show that lite-1 mutants do not avoid high concentrations of diacetyl. However, the animals actually showed attraction, as the chemotaxis index was positive. If the lite-1 animals were insensitive, they should be indifferent, and the chemotaxis index should be close to zero. This means, other neurons contribute to the diacetyl response, and the result of these neurons being activated means/remains attraction? If so, the authors need to rule out any effects of these neurons on the effects they attribute to LITE-1 in the other assays.

The effect of diacetyl on muscle cells (Figure 3C) is pretty rapid, i.e., already during 1 minute after application, the animals are almost maximally contracted. How fast is it really? Can the authors provide a time course with more time points during the first minute? This is a relevant question, as the compound would have to either pass the worm cuticle or enter through the gut and diffuse through the body to reach the muscle cells. Can one expect this to occur within (less than) a minute?

In this context, the authors need to rule out that other mechanisms may be at play. E.g., diacetyl may be immediately sensed by ciliated chemosensory neurons that might release a signaling molecule that leads to activation of LITE-1 in muscles, or that sensitizes it somehow, responding to light used for filming animals. The authors should repeat this assay in a lite-1 mutant background. Furthermore, the authors tested unc-13 mutants to rule out indirect effects on the neurons recorded. Likewise, they should eliminate neuropeptide signaling via unc-31 mutants (a recent paper cited by the authors showed involvement of neuropeptide signaling in LITE-1-mediated light avoidance behavior). Last, to demonstrate that effects are not indirect in response to chemosensory neurons, the authors should repeat the contraction or swimming assay in a tax-4 mutant, which largely lacks chemosensation. This also applies to the chemotaxis assay. Animals should exhibit a chemotaxis index to diacetyl of zero, then.

Does diacetyl activate other neurons expressing LITE-1? A number of cells express LITE-1 at high levels, which the authors have not tested (they restricted their analyses to chemosensory neurons). This is important to address because it leaves the possibility that LITE-1 requires a specific partner only present in these chemosensory neurons to detect diacetyl. This partner would have to be present also in muscles, where diacetyl could activate ectopically expressed LITE-1. According to CeNGEN scRNAseq data, cells expressing LITE-1 can be identified. The ADL and ASH neurons actually come up only at the lowest threshold, so some of the other cells showing much higher levels of LITE-1 mRNAs, i.e., AVG, ALM, PLM, ASG, PHA, PHB, AVM, RIF, or some pharyngeal neurons, should be tested. ASG was among the cells the authors recorded from, but this neuron did not show a response.

The authors need to show that diacetyl responses of ADL and/or ASK can be rescued by expressing LITE-1 specifically in these neurons in a lite-1 mutant background.

Molecular docking studies are not described in detail. How was this done? Diacetyl is a very small molecule. How well can docking algorithms assess this at all? Did the authors preselect the binding pocket, or did the algorithm sample the entire molecular surface of the LITE-1 model and end up with the binding pocket? The latter would be very convincing. The authors should provide control docking experiments with other molecules that caused avoidance in their hands (i.e. benzaldehyde, 2,4,5,trimethlythiazole, isoamyl alcohol, nonanone, octanone), but did not activate LITE-1. Also, they should try docking molecules related to diacetyl, and if there are some that do not dock under the same conditions, such molecules should be used in a behavioral experiment. Ideally, they should also not activate LITE-1. Examples could be, e.g., diacetyl monoxime or 2,4-pentanedione.

Last, the authors should provide a PDB file with the docked diacetyl to allow readers to assess the binding for themselves. Since a large number of mutations of LITE-1 have been reported, it may be that amino acids shown to be essential for LITE-1 function are also required for diacetyl binding. If so, this could be backed up with an experiment.

Reviewer #1 (Public review):

The axonal membrane periodic skeleton (MPS) comprises axially aligned tetramers of α and β spectrins that are attached to evenly distributed radial F-actin rings, which maintain a<br /> typical spacing of 180 - 190 nm. The exact molecular mechanisms underlying the early organization have been unclear. The focus of this study is on those mechanisms.

This is a comprehensive and professionally carried out study. It brings convincing evidence that intact actin and microtubules are required for normal development of MPS and that the actin-binding and lipid-interacting domains of βII-spectrin are critical for its subplasmalemmal confinement and, subsequently, MPS maturation. However, whilst the study does bring new insights, we are still missing the overall understanding of how everything comes together.

The study describes, using spectrin mutations, that the membrane and actin binding of spectrin are required for the proper organization of MPS. However, it is unclear how everything could come together mechanistically.

The authors follow how the MPS is organized by looking at spectrin. Latrunculin affects actin polymerization, as well as CK666 and formin inhibition, but it remains unclear which actin structures are affected. The same is true for microtubules; while they are affected, we don't know how they are affected.

Reviewer #2 (Public review):

Summary:

In their manuscript, Bodas et al present a chronological analysis of the development of the axonal MPS in embryonic DRG neurons, using a series of biochemical assays coupled with STED nanoscopy. Several interesting conclusions, well supported by the data presented, are drawn that further our understanding of bII-spectrin axonal recruitment and on the role of microtubules and actin dynamics during the early MPS formation and at the latter stages of neuronal maturation.

Strengths:

The assays presented are well-designed, and the results obtained clearly support the main conclusions drawn by the authors. Their findings highlight important aspects of cytoskeleton regulation and dynamics required for MPS formation/maintenance, i.e, during different stages of neuronal development, that remained undocumented.

Weaknesses:

The study is mostly limited to biochemical assays followed by STED microscopy to analyse MPS periodicity and (in certain cases) axonal diameter. Functional implications of the manipulations done are lacking, as well as analyses of axonal integrity/degeneration. This is a relevant aspect, as some of the effects observed may be a secondary effect of decreased neuronal/axonal viability.

Reviewer #3 (Public review):

Summary:

In this study, Shivani Bodas et al. investigate the role of actin, actin-binding proteins, and microtubules in regulating the membrane-associated periodic skeleton (MPS) in neuronal axons. The MPS, first reported by Ke Xu et al. in 2013 (Science), has since been implicated in various neuronal functions, including mechanical support, axonal diameter control, axonal degeneration regulation, and spatial organization of signaling molecules. Given its biological importance, further elucidation of MPS assembly mechanisms is of considerable interest. However, I have concerns regarding the novelty and strength of the conclusions presented in this work. Many of the findings largely reiterate previously published observations, and the most novel conclusions are not fully substantiated by the data.

Strengths:

(1) The MPS represents a structurally and functionally important cytoskeletal system in neurons. Studies aimed at understanding its developmental mechanisms are biologically meaningful and potentially impactful.

(2) The authors attempt to dissect MPS assembly during early neuronal development, a process that could offer mechanistic insight into how the MPS is established and maintained.

Weaknesses:

(1) Limited Novelty Across Results Sections:

Of the seven Results sections, only one (Figure 6) and part of another (Figure 9) present data leading to relatively novel interpretations, specifically, the authors' claim that βII-spectrin is recruited to the axonal cortex via F-actin interactions as early as DIV1, followed by rearrangement into a periodic structure by DIV4. However, this conclusion is not fully supported (see below). The remaining results (Figures 1-5, 7, and 8) largely recapitulate findings reported in earlier studies and thus add limited new knowledge.

(2) Insufficient Evidence for Early Recruitment and Rearrangement of βII-spectrin:

The claim that βII-spectrin is recruited to the axonal cortex via F-actin interactions as early as at DIV 1 and subsequently reorganized into a periodic structure during DIV1-4 is central to the manuscript but lacks robust experimental support.

On Page 17, Line 526, the authors the authors state that " To exclude cytoplasmic spectrin resulting from overexpression, only axons with low expression of βII spectrin-GFP were selected for the analysis". However, selecting for low expression alone does not guarantee the absence of cytoplasmic signal. Without volumetric imaging (e.g., 3D super-resolution imaging to see the cross section of axons), it is difficult to definitively conclude that the FRAP data (Figures 6 and 9) reflect cortical rather than cytoplasmic localization.

Prior FRAP studies (Zhong et al., eLife 2014) observed minimal fluorescence recovery over 1800 seconds in axons expressing βII-spectrin-GFP at low levels, with faster recovery (~200-300 seconds) only evident under high expression conditions. The fast recovery kinetics (tens of seconds) reported in this manuscript could plausibly result from free diffusion of cytoplasmic βII-spectrin-GFP rather than cortical turnover.

Furthermore, on Page 10, Line 310, the authors assert that endogenous βII-spectrin "is recruited early to the axonal cortex, followed by progressive establishment of periodic order". However, the STED images shown in Figure 1 do not convincingly distinguish between cortical and cytoplasmic pools.

As such, the observed disordered βII-spectrin molecules, whether overexpressed or endogenous, could still represent a diffuse cytoplasmic population. An alternative and perhaps more parsimonious interpretation is that βII-spectrin is initially cytoplasmic and only later recruited and arranged into periodic structures at the cortex.

(3) Use of Pharmacological Perturbations:

Like many earlier studies, this manuscript relies heavily on pharmacological perturbation (e.g., cytoskeletal drugs) to assess the roles of actin, actin-binding proteins, and microtubules in MPS assembly. While this approach is widely used, it is important to acknowledge that such agents may have off-target effects. The manuscript would benefit from greater caution in interpreting these results, or better yet, the inclusion of genetic or optogenetic approaches to independently validate these findings.

Reviewer #1 (Public review):

Summary:

This study delineates a highly specific role for the pPVT in unconditioned defensive responses. The authors use a novel, combined SEFL and SEFR paradigm to test both conditioned and unconditioned responses in the same animal. Next, a c-fos mapping experiment showed enhanced PVT activity in the stress group when exposed to the novel tone. No other regions showed differences. Fiber photometry measurements in pPVT showed enhancement in response to the novel tone in the stressed but not non-stressed groups. Importantly, there were also no effects when calcium measurements were taken during conditioning. Using DREADDS to bidirectionally manipulate global pPVT activity, inhibition of the PVT reduced tone freezing in stressed mice while stimulation increased tone freezing in non-stressed mice.

Strengths:

A major strength of this research is the use of a multi-dimensional behavioral assay that delineates behavior related to both learned and non-learned defensive responses. The research also incorporates high-resolution approaches to measure neuronal activity and provide causal evidence for a role for PVT in a very narrow band of defensive behavior. The data are compelling, and the manuscript is well-written overall.

Weaknesses:

Figure 1 shows a small, but looks to be, statistically significant, increase in freezing in response to the novel tone in the no-stress group relative to baseline freezing. This observation was also noticed in Figures 2 and 7. The tone presented is relatively high frequency (9 kHz) and high dB (90), making it a high-intensity stimulus. Is it possible that this stimulus is acting as an unconditioned stimulus? In addition, in the final experiment, the tone intensity was increased to 115 dB, and the freezing % in the non-stressed group was nearly identical (~20%) to the non-stressed groups in Figures 1-2 and Figure 7. It seems this manipulation was meant as a startle assay (Pantoni et al., 2020). Because the auditory perception of mice is better at high frequencies (best at ~16 kHz), would the effect seen be evident at a lower dB (50-55) at 9 kHz? If the tone was indeed perceived as "neutral," there should be no freezing in response to the tone. This complicates the interpretation of the results somewhat because while the authors do admit the stimulus is loud, would a less loud stimulus result in the same effect? Could the interaction observed in this set of studies require not a novel tone, but rather a high-intensity tone that elicits an unconditioned response? Along these same lines, it appears there may be an elevation in c-fos in the PVT in the non-stress tone test group versus the no-stress home cage control, and overall it appears that tone increases c-fos relative to homecage. Could PVT be sensitive to the tone outside of stress? Would there be the same results with a less intense stimulus? I would also be curious to know what mice in the non-stressed group were doing upon presentation of the tone besides freezing. Were any startle or orienting responses noticed?

Reviewer #2 (Public review):

Summary:

Nishimura and colleagues present findings of a behavioral and neurobiological dissociation of associative and nonassociative components of Stress Enhanced Fear Responding (SEFR).

Strengths:

This is a strong paper that identifies the PVT as a critical brain region for SEFR responses using a variety of approaches, including immunohistochemistry, fiber photometry, and bidirectional chemogenetics. In addition, there is a great deal of conceptual innovation. The authors identify a dissociable behavior to distinguish the effects of PVT function (among other brain regions).

Weaknesses:

(1) The authors find a lack of difference between the Stress and No Stress groups in pPVT activity during SEFL conditioning with fiber photometry but an increase in freezing with Gq DREADD stimulation. How do authors reconcile this difference in activity vs function?

(2) Because the PVT plays a role in defensive behaviors, it would be beneficial to show fiber photometry data during freezing bouts vs exclusively presented during tone a shock cue presentations.

(3) Similar to the above point, were other defensive behaviors expressed as a result of footshock stress or PVT manipulations?

(4) Tone attenuation in Figure 8 seems to be largely a result of minimal freezing to a 115-dB tone. While not a major point of the paper, a more robust fear response would be convincing.

(5) In the open field test, the authors measure total distance. It would be beneficial to also show defensive behavioral (escape, freezing, etc) bouts expressed.

(6) The authors, along with others, show a behavioral and neural dissociation of footshock stress on nonassociative vs associative components of stress; however, the nonassociative components as a direct consequence of the stress seem to be necessary for enhancement of associative aspects of fear. Can authors elaborate on how these systems converge to enhance or potentiate fear?

(7) In the discussion, authors should elaborate on/clarify the cell population heterogeneity of the PVT since authors later describe PVT neurons as exclusively glutamatergic.

Reviewer #3 (Public review):

Summary:

The manuscript by Nishimura et al. examines the behavioural and neural mechanisms of stress-enhanced fear responding (SEFR) and stress-enhanced fear learning (SEFL). Groups of stressed (4 x shock exposure in a context) vs non-stressed (context exposure only) animals are compared for their fear of an unconditioned tone, and context, as well as their learning of new context fear associations. Shock of higher intensity led to higher levels of unlearned stress-enhanced fear expression. Immediate early gene analysis uncovered the PVT as a critical neural locus, and this was confirmed using fiber photometry, with stressed animals showing an elevated neural signal to an unconditioned tone. Using a gain and loss of function DREADDs methodology, the authors provide convincing evidence for a causal role of the PVT in SEFR.

Strengths:

(1) The manuscript uses critical behavioural controls (no stress vs stress) and behavioural parameters (0.25mA, 0.5mA, 1mA shock). Findings are replicated across experiments.

(2) Dissociating the SEFR and SEFL is a critical distinction that has not been made previously. Moreover, this dissociation is essential in understanding the behavioural (and neural) processes that can go awry in fear.

(3) Neural methods use a multifaceted approach to convincingly link the PVT to SEFR: from Fos, fiber photometry, gain and loss of function using DREADDs.

Weaknesses:

No weaknesses were identified by this reviewer; however, I have the following comments:

A closer examination of the Test data across time would help determine if differences may be present early or later in the session that could otherwise be washed out when the data are averaged across time. If none are seen, then it may be worth noting this in the manuscript.

Given the sex/gender differences in PTSD in the human population, having the male and female data points distinguished in the figures would be helpful. I assume sex was run as a variable in the statistics, and nothing came as significant. Noting this would also be of value to other readers who may wonder about the presence of sex differences in the data.

Reviewer #1 (Public review):

Summary:

This study investigates how mice make defensive decisions when exposed to visual threats and how those decisions are influenced by reward value and social hierarchy. Using a naturalistic foraging setup and looming stimuli, the authors show that higher threat leads to faster escape, while lower threat allows mice to weigh reward value. Dominant mice behave more cautiously, showing higher vigilance. The behavioral findings are further supported by a computational model aimed at capturing how different factors shape decisions.

Strengths:

(1) The behavioral paradigm is well-designed and ethologically relevant, capturing instinctive responses in a controlled setting.

(2) The paper addresses an important question: how defensive behaviors are influenced by social and value-based factors.

(3) The classification of behavioral responses using machine learning is a solid methodological choice that improves reproducibility.

Weaknesses:

(1) Key parts of the methods are hard to follow, especially how trials are selected and whether learning across trials is fully controlled for. For example, it is unclear whether animals are in the nest during the looming stimulus presentations. The main text and methods should clarify whether multiple mice are in the nest simultaneously and whether only one mouse is in the arena during looming exposure. From the description, it seems that all mice may be freely exploring during some phases, but only one is allowed in the arena at a time during stimulus presentation. This point is important for understanding the social context and potential interactions, and should be clearly explained in both the main text and methods.

(2) It is often unclear whether the data shown (especially in the main summary figures) come from the first trial or are averages across several exposures. When is the cut-off for trials of each animal? How do we know how many trial presentations were considered, and how learning at different rates between individuals is taken into account when plotting all animals together? This is important because the looming stimulus is learned to be harmless very quickly, so the trial number strongly affects interpretation.

(3) The reward-related effects are difficult to interpret without a clearer separation of learning vs first responses.

(4) The model reproduces observed patterns but adds limited explanatory or predictive power. It does not integrate major findings like social hierarchy. Its impact would be greatly improved if the authors used it to predict outcomes under novel or intermediate conditions.

(5) Some conclusions (e.g., about vigilance increasing with reward) are counterintuitive and need stronger support or alternative explanations. Regarding the interpretation of social differences in area coverage, it's also possible that the observed behavioral differences reflect access to the nesting space. Dominant mice may control the nest, forcing subordinates to remain in the open arena even during or after looming stimuli. In this case, subordinates may be choosing between the threat of the dominant mouse and the external visual threat. The current data do not distinguish between these possibilities, and the authors do not provide evidence to support one interpretation over the other. Including this alternative explanation or providing data that addresses it would strengthen the conclusions.

(6) While potential neural circuits are mentioned in the discussion, an earlier introduction of candidate brain regions and their relevance to threat and value processing would help ground the study in existing systems neuroscience.

(7) Some figures are difficult to interpret without clearer trial/mouse labeling, and a few claims in the text are stronger than what the data fully support. Figure 3H is done for low contrast, but the interesting findings will be to do this experiment with high contrast. Figure 4H - I don't understand this part. If the amount of time in the center after the loom changes for subordinate mice, how does this lead to the conclusion that they spend most of their time in the reward zone?. Figure 3A - The example shown does not seem representative of the claim that high contrast stimuli are more likely to trigger escape. In particular, the 10% sucrose condition appears to show more arena visits under low contrast than high contrast, which seems to contradict that interpretation. Also, the plot currently uses trials on the Y-axis, but it would be more informative to show one line per animal, using only the first trial for each. This would help separate initial threat responses from learning effects and clarify individual variability.

(8) The analysis does not explore individual variability in behavior, which could be an important source of structure in the data. Without this, it is difficult to know whether social hierarchy alone explains behavioral differences or if other stable traits (e.g., anxiety level, prior experiences) also contribute.

(9) The study shows robust looming responses in group-housed animals, which contrasts with other studies that often require single housing to elicit reliable defensive responses. It would be valuable for the authors to discuss why their results differ in this regard and whether housing conditions might interact with social rank or habituation.

Reviewer #2 (Public review):

Zhe Li and colleagues investigate how mice exposed to visual threats and rewards balance their decisions in favour of consuming rewards or engaging in defensive actions. By varying threat intensity and reward value, they first confirm previous findings showing that defensive responses increase with threat intensity and that there is habituation to the threat stimulus. They then find that water-deprived mice have a reduced probability of escaping from low contrast visual looming stimuli when water or sucrose are offered in the environment, but that when the stimulus contrast is high, the presence of sucrose or water increases the probability of escape. By analysing behaviour metrics such as the latency to flee from the threat stimulus, they suggest that this increase in threat sensitivity is due to increased vigilance. Analysis of this behaviour as a function of social hierarchy shows that dominant mice have higher threat sensitivity, which is also interpreted as being due to increased vigilance. These results are captured by a drift diffusion model variant that incorporates threat intensity and reward value.

The main contribution of this work is to quantify how the presence of water or sucrose in water-deprived mice affects escape behaviour. The differential effects of reward between the low and high contrast conditions are intriguing, but I find the interpretation that vigilance plays a major role in this process is not supported by the data. The idea that reward value exerts some form of graded modulation of the escape response is also not supported by the data. In addition, there is very limited methodological information, which makes assessing the quality of some of the analyses difficult, and there is no quantification of the quality of the model fits.

(1) The main measure of vigilance in this work is reaction time. While reaction time can indeed be affected by vigilance, reaction times can vary as a function of many variables, and be different for the same level of vigilance. For example, a primate performing the random dot motion task exhibits differences in reaction times that can be explained entirely by the stimulus strength. Reaction time is therefore not a sound measure of vigilance, and if a goal of this work is to investigate this parameter, then it should be measured. There is some attempt at doing this for a subset of the data in Figure 3H, by looking at differences in the action of monitoring the visual field (presumably a rearing motion, though this is not described) between the first and second trials in the presence of sucrose. I find this an extremely contrived measure. What is the rationale for analysing only the difference between the first and second trials? Also, the results are only statistically significant because the first trial in the sucrose condition happens to have zero up action bouts, in contrast to all other conditions. I am afraid that the statistics are not solid here. When analysing the effects of dominance, a vigilance metric is the time spent in the reward zone. Why is this a measure of vigilance? More generally, measuring vigilance of threats in mice requires monitoring the position of the eyes, which previous work has shown is biased to the upper visual field, consistent with the threat ecology of rodents.

(2) In both low and high contrast conditions, there are differences in escape behaviour between no reward and water or sucrose presence, but no statistically significant differences between water and sucrose (eg, Figure 3B). I therefore find that statements about reward value are not supported by the data, which only show differences between the presence or absence of reward. Furthermore, there is a confound in these experiments, because according to the methods, mice in the no-reward condition were not water deprived. It is thus possible that the differences in behaviour arise from differences in the underlying state.

(3) There is very little methodological information on behavioural quantification. For example, what is hiding latency? Is this the same are reaction time? Time to reach the safe zone? What exactly is distance fled? I don't understand how this can vary between 20 and 100cm. Presumably, the 20cm flights don't reach the safe place, since the threat is roughly at the same location for each trial? How is the end of a flight determined? How is duration measured in reward zone measures, e.g., from when to when? How is fleeing onset determined?

(4) There is little methodological information on how the model was fit (for example, it is surprising that in the no reward condition, the r parameter is exactly 0. What this constrained in any way), and none of the fit parameters have uncertainty measures so it is not possible to assess whether there are actually any differences in parameters that are statistically significant.

Reviewer #3 (Public review):

Male mice were tested in a classic behavioral "flee the looming stimulus" paradigm. This is a purely behavioral study; no neural analyses were done. Mice were housed socially, but faced the looming stimulus individually. Drift-diffusion modeling found that reward-level interacted with threat level such that at low-threat levels, reward contrasted with threat as classically expected (high reward overwhelms low threat, low threat overwhelms low reward), but that reward aligned with threat at higher threat levels.

Note that they define threat level by the darkness of the looming stimulus. I am not sure that darker stimuli are more threatening to mice. But maybe. Figure 3 shows that mice react more quickly to high contrast looming stimuli, but can the authors distinguish between the ability to detect the visual signal from considering it a more dangerous threat? (The fact that vigilance makes a difference in the high contrast condition, not the low contrast condition, actually supports the author's hypotheses here.)

The drift-diffusion model (DDM) is fine. I note that the authors included a "leakage rate", which is not a standard DDM parameter (although I like including it). I would have liked to see more about the parameters. What were the distributions? What did the parameters correlate with behaviorally? I would have liked to see distributions of the parameters under the different conditions and different animals. Figure 2C shows the progression of learning. How do the fit parameters change over time as mice shift from choice to choice? How do the parameters change over mice? How do the parameters change over distance to the threat/distance to safety (as per Fanselow and Lester 1988)? They did a supplemental experiment where the threat arrived halfway along the corridor - we could get a lot more detail about that experiment - how did it change the modeling?

Overall, this is a reasonable study showing mostly unsurprising results. I think the authors could do more to connect the vigilance question to their results (which seems somewhat new to me).

Although the data appear generally fine and the modeling reasonable, the authors do not do the necessary work to set themselves within the extensive literature on decision-making in mice retreating from threats.

First of all, this is not a new paradigm; variants of this paradigm have been used since at least the 1980s. There is an *extensive* literature on this, including extensive theoretical work on the relation of fear and other motivational factors. I recommend starting with the classic Fanselow and Lester 1988 paper (which they cite, but only in passing), and the reviews by Dean Mobbs and Jeansok Kim, and by Denis Paré and Greg Quirk, which have explicit theoretical proposals that the authors can compare their results to. I would also recommend that the authors look into the "active avoidance" literature. Moreover, to talk about a mouse running from a looming stimulus without addressing the other "flee the predator" tasks is to miss a huge space for understanding their results. Again, I would start with the reviews above, but also strongly urge the authors to look at the Robogator task (work by June-Seek Choi and Jeansok Kim, work by Denis Paré, and others).

Similarly, in their anatomical review, they do not mention the amygdala. Given the extensive literature on the role of the amygdala in retreating from danger, both in terms of active avoidance and in terms of encoding the danger itself, it would surprise me greatly if this behavior does not involve amygdala processing. (If there is evidence that the amygdala does not play a role here, but that the superior colliculus does, then that would be a *very* important result that needs to be folded into our understanding of decision-making systems and neural computational processing.)

Second, there is an extensive economic literature on non-human animals in general and on rodents in particular. Again, the authors seem unaware of this work, which would provide them with important data and theories to broaden the impact of their results (by placing them within the literature). First, there are explicit economic literatures in terms of positively-valenced conflicts (e.g., neuroeconomics within the primate literature, sequential foraging and delay-discounting tasks within the rodent literature), but also there is a long history within the rodent conditioning world, such as the classic work by Len Green and Peter Shizgal. I would strongly urge the authors to explore the motivational conflict literature by people like Gavin McNally, Greg Quirk, and Mark Andermann. Again, putting their results into this literature will increase the impact of their experiment and modeling.

Reviewer #1 (Public review):

Summary:

The authors tested two competing mechanisms of expectation: (1) a sharpening model that suppresses unexpected information via center-surround inhibition; (2) a cancelation model that predicts a monotonic gradient response profile. Using two psychophysical experiments manipulating feature space distance between expected and unexpected stimuli, the results consistently supported the sharpening model. Computational modeling further showed that expectation effects were explained by either sharpened tuning curves or tuning shifts. Finally, convolutional neural network simulations revealed that feedback connections critically mediate the observed center-surround inhibition.

Strengths:

The manuscript provides compelling and convergent evidence from both psychophysical experiments and computational modeling to robustly support the sharpening model of expectation, demonstrating clear center-surround inhibition of unexpected information.

Weaknesses:

The manuscript could directly validate the experimental manipulations and address how these results reconcile with existing literature on expectation effects.

Reviewer #2 (Public review):

Summary:

This is a compelling and methodologically rich manuscript. The authors used a variety of methods, including psychophysics, computational modeling, and artificial neural networks, to reveal a non-monotonic, center-surround "Mexican-hat" profile of expectation in orientation space. Their data convincingly extend analogous findings in attention and working memory, and the modeling nicely teases apart sharpening vs. shift mechanisms.

Strengths:

The findings are novel and important in elucidating the potential neural mechanisms by which expectation shapes perception. The authors conducted a series of well-designed psychophysical experiments to careful examination of the profile of expectation's modulation. Computational modeling also provides further insights, linking the neural mechanisms of expectation to behavioral results.

Weaknesses:

There are several aspects that could be strengthened or clarified.

(1) The sharpening model of expectation can predict surround suppression. The authors could further clarify how the cancellation model predicts a monotonic profile of expectation (Figure 1C) with the highest response at the expected orientation, while the cancellation model suggests a suppression of neurons tuned toward the expected stimulus.

(2) I'm a bit concerned about whether the profile solely arises from modulation of expectation. The two auditory cues are each associated with a fixed orientation, which may be confounded by other cognitive processes like visual working memory or attention (which I think the authors also discussed). Although the authors tried to use SFD task to render orientation task-irrelevant, luminance edges (i.e., orientation) and spatial frequency in gratings are highly intertwined and orientation of the gratings may help recall the first grating's SF (fixed at 0.9 c/{degree sign}), especially given the first and second grating's orientations are not very different (4.8{degree sign}).

(3) For each of the expected orientations (20{degree sign} or 70{degree sign}), the unexpected ones are linearly separable (i.e., all unexpected ones lie on one side of the expected angle). This might further encourage people to shift their attended or expected orientation, according to the optimal tuning hypothesis. Would this provide an alternative explanation to the tuning shift that the authors found?

(4) It is great that the authors conducted computational modeling to elucidate the potential neuronal mechanisms of expectation. But I think the sharpening hypothesis (e.g., reviewed in de Lange, Heilbron & Kok, 2018) focuses on the neural population level, i.e., narrowing of population tuning profile, while the authors conducted the sharpening at the neuronal tuning level. However, the sharpening of population does not necessarily rely on the sharpening of individual neuronal tuning. For example, neuronal gain modulation can also account for such population sharpening. I think similar logic applies to the orientation adjustment experiment. The behavioral level shift does not necessarily suggest a similar shift at the neuronal level. I would recommend that the authors comment on this.

(5) If the orientation adjustment experiment suggests that both sharpening and shifting are present at the same time, have the authors tried combining both in their computational model?

Reviewer #1 (Public review):

This is a theoretical study addressing the problem of constructing integrator networks for which the activity state and integrated variables display non-trivial topologies. Historically, researchers in theoretical neuroscience have focused on models with simple underlying geometries (e.g., circle, torus), for which analytical models could be more easily constructed. How these models can be generalised to complex scenarios is, however, a non-trivial question. This is furthermore a time-sensitive issue, as population recordings from the brain in complex tasks and environments increasingly require the ability to construct such models.

I believe the authors do a good job of explaining the challenges related to this problem. They also propose a class of models that, although not fully general, overcome many of these difficulties while appearing solid and well-functioning. This requires some non-trivial mathematics, which is nevertheless conveyed in a reasonably accessible form. The manuscript is well written, and both the methodology and the code are well documented.

That said, I believe the manuscript has two major limitations, which could be addressed in a revision. First, some of the assumptions underlying this class of models are somewhat restrictive but are not sufficiently discussed. Second, although the stated goal of the manuscript is to provide practical recipes for constructing integrator networks, the methods section is not very explicit about the specific steps required for different geometries. I elaborate on these limitations below.


(1) The authors repeatedly describe MADE as a technique for constructing integrators of specified "topologies and geometries." What do they mean by "geometries"? Intuitively, I would associate geometry with properties beyond topology, such as embedding dimensionality or curvature. However, it is unclear to me to what extent these aspects are explicitly specified or controlled in MADE. It seems that geometry is only indirectly defined via the connectivity kernel, which itself obeys certain constraints (e.g., limited spatial scale; see below). I believe it is important for the authors to clarify what they mean by "geometry." They should also specify which aspects are under their control, and whether, in fact, all geometries can be realized.


(2) The authors make two key assumptions: that connectivity is purely inhibitory and that the connectivity kernel has a small spatial scale. They state that under these conditions, the homogeneous fixed point becomes unstable, leading to a non-periodic state. However, it seems to me that they do not demonstrate that this emergent state is necessarily a bump localized in all manifold dimensions -- although this is assumed throughout the manuscript. Are other solutions possible or observed? For example, might the network converge to states that are localized in one dimension but extended in another, yielding e.g., stripe-like activity in the plane rather than bumps? In other words, does the proposed recipe guarantee convergence to bumps? This is a critical point and should be clarified.


(3) Related to the question above: What are the failure modes when these two assumptions are violated? Does the network always exhibit runaway activity (as suggested in the text), or can other types of solutions emerge? It would be useful if the authors could briefly discuss this.


(4) Again, related to the question above: can this formalism be extended to activity profiles beyond bumps? For example, periodic fields as seen in grid cells, or irregular fields as observed in many biological datasets -- particularly in naturalistic environments? These activity profiles are of key importance to neuroscientists, so I believe this is an important point that should at least be addressed in the Discussion. Can MADE be naturally extended to these scenarios? What are the challenges involved?


(5) Line 119: "Since σ is the only spatial scale being introduced in the dynamics, we qualitatively expect that a localized bump state within the ball will have a spatial scale of O(σ)."
Is this statement always true? I understand that the spatial scale of the synaptic inputs exchanged via recurrent interactions (i.e., the argument of the function f in Equation 1) is characterised by the spatial scale σ. But the non-linear function f could modify that spatial scale -- for example, by "cutting" the bump close to its tip. Where am I wrong? Could the authors clarify?


(6) The authors provide beautiful intuition about the problem of constructing integrators on non-trivial topologies and propose a mathematically grounded solution using Killing vectors. Of course, solutions based on Killing vectors are more complex than those with constant offsets, which raises the question: Is the brain capable of learning and handling such complex structures? Perhaps the authors could speculate in the Discussion about the biological plausibility of these mechanisms.


(7) A great merit of this paper is that it provides mathematical tools for neuroscience researchers to build integrators on non-trivial geometries. I found that, although all the necessary information is present in the Methods, the authors could improve the presentation by schematizing the steps required to build each type of model. It would be extremely useful if, for each considered geometry, the authors provided a short list of required components: the manifold P, the choice of distance, and the connectivity offsets defined by the Killing vectors. Currently, this information is presented, but scattered (not grouped by geometry).

Reviewer #2 (Public review):

Summary:

The work by Claudi et al. presents a framework for constructing continuous attractor neural networks (CANs) with user-defined topologies and integration capabilities. The framework unifies and generalizes classical attractor models and includes simulations across a range of topologies, including ring, torus, sphere, Möbius band, and Klein bottle. A key contribution of the paper is the introduction of Killing vectors to enable integration on non-parallelizable manifolds. However, the need for Killing vectors currently appears hypothetical, as biologically discovered manifolds-such as rings and tori-do not require them.

Moreover, throughout the manuscript, the authors claim to be addressing "biologically plausible" attractor networks, yet the constraints required by their construction - such as exact symmetry, fine-tuning of weights, and idealized geometry-seem incompatible with biological variability. It appears that "biologically plausible" is effectively used to mean "capable of integration." While these issues do not diminish the contributions of the work, they should be acknowledged and addressed more explicitly in the text. I applaud the authors for their interesting work. Below are my major and minor concerns.

Strengths:

(1) Theoretical framework for integrating CANs<br /> The paper introduces a systematic method for constructing continuous attractor networks (CANs) with arbitrary topologies. This goes beyond classical models and includes novel topologies such as the Möbius band, sphere, and Klein bottle. The approach generalizes well-known ring and torus attractor models and provides a unified view of their construction, dynamics, and integration capabilities.

(2) Novel use of killing vector fields<br /> A key theoretical innovation is the introduction of Killing vectors to support velocity integration on non-parallelizable manifolds. This is mathematically elegant and extends the domain of tractable attractor models.

(3) Insightful simulations across manifolds<br /> The paper includes detailed simulations demonstrating bump attractor dynamics across a range of topologies.

Weaknesses:

(1) Biological plausibility is overstated<br /> Despite frequent use of the term "biologically plausible," the models rely on assumptions (e.g., symmetric connectivity, perfect geometries, fine-tuning) that are not consistent with known biological networks, and the authors do not incorporate heterogeneity, noise, or constraints like Dale's law.

(2) Continuum of states not directly demonstrated<br /> The authors claim to generate a continuum of stable states but do not provide direct evidence (e.g., Jacobian analysis with zero eigenvalues along the manifold). This weakens the central claim about the nature of the attractor.

(3) Lack of clarity around assumptions<br /> Several assumptions and analyses (e.g., symmetry breaking, linearity, stability conditions) are introduced without justification or overstated. The analytical rigor in discussing alternative solutions and bifurcation behavior is limited.

(4) Scalability to high dimensions<br /> The authors claim their method scales better than learning-based approaches. This should be better discussed.

Major Concerns

(1) Biological plausibility

The claim that the proposed framework is "biologically plausible" is misleading, as it is unclear what the authors mean by this term. Biological plausibility could include features such as heterogeneity in synaptic weights, randomness in tuning curves, irregular geometries, or connectivity constraints consistent with known biological architectures (e.g., Dale's law, multiple cell types). None of these elements is implemented in the current framework. Furthermore, it is not clear whether the framework can be extended to include such features-for example, CANs with heterogeneous connections or tuning curves. The connectivity matrix is symmetric to allow an energy-based description and analytical tractability, which is fine, but not a biologically realistic constraint. I recommend removing or significantly qualifying the use of the term "biologically plausible."

(2) Continuum of stable states<br /> While the authors claim their model generates a continuum of stable states, this is not demonstrated directly in their simulations or in a stability analysis (though there are some indirect hints). One way to provide evidence would be to compute the Jacobian at various points along the manifold and show that it possesses (approximately) zero eigenvalues in the tangent/on-manifold directions at each point (e.g., see Ságodi et al. 2024 and others). It would be especially valuable to provide such analysis for the more complex topologies illustrated in the paper.

(3) Assumptions, limitations, and analytical rigor<br /> Some assumptions and derivations lack justification or are presented without sufficient detail. Examples include:

• Line 126: "If the homogeneous state (all neurons equally active) were unstable, there must exist some other stable state, with broken symmetry." Is this guaranteed? In the ring model with ReLU activation, there could also be unbounded solutions-not just bump solutions-and, in principle, there could also be oscillatory or other solutions. In general, multiple states can co-exist, with differing stability. It appears the authors only analyze the homogeneous case and do not study the stability or bifurcations of other solutions, limiting their theoretical work.

• Line 122: "The conditions for the formation..." What are these conditions, precisely? A citation or elaboration would be helpful. Why is the assumption σ≪L necessary, and how does it impact the construction or conclusions?

• The theory relies heavily on exact symmetries and fine-tuned parameters. Indeed, in line 106, the authors write: "We seek interaction weights consistent with the formation, through symmetry breaking." Is this symmetry-breaking necessary for all CANs? Or is it a limitation specific to hand-crafted models (see also below)? There is insufficient discussion of such limitations. For example, it is difficult to envision how the authors' framework might form attractor manifolds with different geometries or heterogeneous tuning curves.

(4) Comparison with models of learned attractors<br /> While the connectivity patterns of learned attractors often resemble classical hand-crafted models (e.g., see also Vafidis et al. 2022), this is not always the case. If initial conditions include randomness or if the geometry of the attractor deviates from standard forms, the solutions can diverge significantly from hand-designed architectures. Such biologically realistic conditions highlight the limitations the hand-crafted CANs like those proposed here. I suggest updating the discussion accordingly.

(5) High-Dimensional Manifolds<br /> The authors argue that their method scales better than training-based approaches in high dimensions and that it is straightforward to extend their framework to generate high-dimensional CANs. It would be useful for the authors to elaborate further. First, it is unclear what k refers to in the expression k^M used in the introduction. Second, trained neural networks seem to exhibit inductive bias (e.g., Cantar et al. 2021; Bordelon & Pehlevan 2022; Darshan & Rivkind 2022), which may mitigate such scaling issues. To support their claim, the authors could also provide an example of a high-dimensional manifold and show that their framework efficiently supports a (semi-)continuum of stable states.

Reviewer #1 (Public review):

Summary of the paper:

The paper presents an elegant task designed to investigate humans' ability to generalize knowledge of learned graph structures to new experiences that share the same structure but are built from different stimuli. Using behavior and MEG recordings, the authors test evidence for neural representation and application of structural knowledge.

Review overview:

While the task design is elegant, it isn't clear to me that the data support all the claims made in the paper. I have detailed my concerns below.

Major concerns

(1) The authors claim that their findings reveal "striking learning and generalization abilities based on factorization of complex experiences into underlying structural elements, parsing these into distinct subprocesses derived from past experience, and forming a representation of the dynamical roles these features play within distinct subprocesses." And "neural dynamics that support compositional generalisation, consistent with a structural scaffolding mechanism that facilitates efficient adaption within new contexts".

a. First, terms used in these example quotes (but also throughout the paper) do not seem to be well supported by data or the task design. For example, terms such as 'compositional generalisation' and 'building blocks' have important relevance in other papers by (some of) the same authors (e.g., Schwartenbeck et al., 2023), but in the context of this experiment, what is 'composition'? Can the authors demonstrate clear behavioural or neural evidence for compositional use of multiple graph structures, or alternatively remove reference to these terms? In the current paper, it seems to me that the authors are investigating abstract knowledge for singular graph structures (together with the influence of prior learning), as opposed to knowledge for the compound, more complex graph formed from the product of two simpler graphs.

b. While I would like to be convinced that this data provides evidence for the transfer of abstract, structural knowledge, I think the authors either need to provide more convincing evidence or tone down their claims.

Specifically:

(i) Can the increase in neural similarity between stimuli mapping to the same abstract structural sub-process not be explained by temporal proximity in experiencing the transitions (e.g., Cai et al., 2016)? Indeed, behavior seems to be dominated by direct experience of the structure as opposed to applying abstract knowledge of equivalent structures (and, as a result, there is little difference in behavioural performance between experience and inference probes).

(ii) The strongest evidence for neural representation of abstract task structures seems to be the increase in similarity by transition type. But this common code for 'transition type' is only observed for 6-bridge graphs and only for experienced transitions. There was no significant effect in inference probes. Therefore, there doesn't seem to be evidence for the application of a knowledge scaffold to facilitate transfer learning. Instead, the data reflects learning from direct experience and not generalisation.

(iii) The authors frequently suggest that they are providing insight into temporal dynamics, but there is no mention of particular oscillations or particular temporal sequences of neural representation that support task performance.

(2) Regardless of point (b), can the authors provide more convincing evidence for a graph structure being represented per se (regardless of whether this representation is directly experienced or inferred)? From Figure 3C, it seems that the model RDM doesn't account for relative distance within the graph. Do they see evidence for distance coding? Can they reconstruct the graph from representational patterns using MDS?

(3) In general, the figures are not very clear, and the outcome from statistical tests is not graphically shown. The paper would be easier to digest if, for example, Figures 1-2 were made clearer and statistical significance relative to chance were indicated throughout. To give two examples: (i) Figure 1 should clearly indicate what is meant by observed and held-out transitions and whether it is just the transition or also the compound that is new to the participant. (ii) Figure 2D-E could be shown with relevant comparisons and simpler statistical comparisons. Currently, it is hard to follow without carefully reading the legend.