Reviewer #3 (Public review):

This work by Du et al. addresses a critical problem in cryo-electron microscopy. To date, there are few ways of generating phase contrast during cryo-EM imaging while remaining in focus. Cryo-EM practitioners today must generate contrast by collecting out-of-focus exposures, a process that introduces aberrations in the resulting image data. Recent work has shown that standing wave lasers are capable of using the ponderomotive effect to shift the phase of electrons in transmission electron microscopy to generate in-focus phase contrast imaging for cryo-EM. A limitation of this 'laser phase plate' is the high laser power required, which can damage optical mirrors and necessitate high laser safety. Thus, alternative approaches are needed for phase contrast imaging in cryo-EM.

In this manuscript, Du et al. exploit their expertise in ultrafast electron microscopy to explore the ability to shift the phase of electrons using pulsed electrons and lasers. The motivation for exploring pulsed laser phase plates stems from the fact that femtosecond pulses from 9W lasers can generate extremely high power (as much as the standing-wave laser phase plate, > 1 gigawatt) at the back focal plane. If successful, this type of instrument will likely be much more affordable and easier to deploy worldwide.

The work outlined here shows a proof of principle, highlighting that an ultrafast scanning electron microscopy beam at 30 kV can have the electron packets phase shift by 430 radians (24637 degrees), which is much greater than the required 1.5 radians (90 degrees) needed for phase contrast imaging. The data presented do not use any biological samples; instead, they measure the spread of the electron beam on a test sample to assess the ability to target pulsed lasers onto electron packets and the amount of electron spread (which relates to the phase shift). They were also able to take their system a step further to measure how changes to the system in terms of laser power affect performance, and show that the system can be stable for 10+ hours.

The only weaknesses relate to the broad readability of the text. Improved textual clarity will help ensure a wider readership.

Overall, this work is an important step toward developing lower-cost alternatives to the standing-wave laser phase plate.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors present the development and characterization of a pulsed ponderomotive phase plate for transmission electron microscopy (TEM). The primary goal is to overcome the long-standing challenge of generating stable, tunable phase contrast for weakly scattering biological specimens - a capability that has remained elusive despite decades of development. While the commercially available Volta Phase Plate offers phase enhancement, it suffers from a lack of control and stability. More recent efforts have focused on continuous-wave (CW) laser phase plates; however, these systems face significant practical hurdles, including extreme optical power requirements, thermal instability of mirrors, and the necessity for high-finesse optical cavities that act as diffraction gratings for the electron beam. The authors aim to demonstrate that a pulsed, free-space laser interaction can circumvent these limitations, offering a more robust path toward practically usable phase plates

Strengths:

The most significant strength of this work is the elegant use of a free-space pulsed interaction, which fundamentally simplifies the hardware requirements compared to cavity-based designs. By utilizing a high-intensity pulsed laser focus rather than a standing wave inside a resonator, the authors eliminate the need for complex locking feedback loops and avoid the thermal mirror deformation that currently limits CW systems.

Furthermore, this approach provides a critical theoretical advantage regarding image quality. Current CW cavity-based designs must grapple with the Kapitza-Dirac effect, where the standing wave creates a diffraction grating that generates unwanted "ghost images," delocalizing the signal. Recent proposals have had to resort to complex crossed-beam geometries to mitigate these artifacts. In contrast, the traveling-wave nature of the pulsed interaction described here inherently avoids the creation of a standing wave grating, thereby eliminating ghost images entirely without requiring elaborate compensation strategies.

The authors successfully demonstrate a proof-of-concept implementation, reporting a pronounced peak phase shift of approximately 430 radians and a stable angular deflection of the electron beam. The stability data, covering a 10-hour period, suggests that this approach is robust enough for data collection sessions typical in structural biology.

Weaknesses:

However, the strength of the evidence is modestly tempered by limitations in data presentation and analysis. The agreement between the experimental data and the theoretical simulation in Figure 2b is imperfect; the simulation underestimates the depth of the central signal trough. While the authors acknowledge this "muted" prediction, the discrepancy suggests that the theoretical model or the estimation of experimental parameters (such as electron beam size or laser intensity) requires refinement to fully describe the interaction.

While the authors claim stability over many hours, the data in Figure 3c reveal a significant drift in the baseline reference signal. Although attributed to a weakening electron beam, this drift complicates the reader's ability to assess the true stability of the laser-induced phase shift. A drift-corrected analysis would have provided more compelling evidence of the "stable angular kick" described.

Despite these specific weaknesses in data presentation, the work represents a fundamental step forward. The authors have effectively demonstrated that the trade-off between beam current and spatiotemporal resolution (driven by space-charge effects) can be managed to achieve significant phase modulation. By moving the field away from the tight constraints of optical cavities and toward free-space pulsed interactions, this work establishes a potentially more viable route for integrating laser phase plates into routine biological imaging workflows. This study will be of high value to biophysicists and microscopists seeking to push the boundaries of contrast in cryo-EM

Reviewer #1 (Public review):

Summary:

Du, Daniel X. et al studied the interaction of the ultrashort electron and laser pulses inside a scanning electron microscopy (SEM), aiming to build a foundation for pulsed laser phase plate electron microscopy, in which the contrast of cryo samples can be significantly increased. The author modified a commercial SEM to accommodate optics to introduce a laser beam inside the instrument to overlap with the electron beam and performed multiple experiments aimed to characterize the electron-light interaction, particularly reaching an extremely high phase shift of >400 rad. Moreover, the authors built a theoretical model for this interaction and estimated the laser beam parameters needed to reach 90 degrees phase shift in transmission electron microscopy (TEM).

Strengths:

The conclusion on the interaction of the electron pulses and laser pulses is well described and supported by the experiment.

The presented instrument can serve as a great tool for studying fundamental interactions of electrons with extremely intense light pulses.

Weaknesses:

The authors motivate the project by using the pulsed electron beam with a phase shift for improving the contrast in cryo-EM, and while they indicate the low current in UEM, they do not discuss the limitations of the laser beam properties.

Such, even for 1 ps electron pulses with the repetition rate of 100 GHz (duty cycle of 10%), they will need to use 100 GHz laser pulses with pulse energies of at least ~1 uJ a second (the lowest pulse energy reported in the simulations in Figure 4), which would mean that ~10 kW of optical power needs to enter the electron microscope and be dumped somewhere after leaving the instrument. This significantly complicates the system and, in my view, makes it harder to use a pulsed laser phase plate in cryo-EM due to either low acquisition rate at lower repetition rates or extreme difficulties to operate multi kW ultrafast laser system.

I would also expect the unscattered electron beam diameter to be <1 micron, which would significantly change the plot in 4b for the 300 keV electron beam.

Adding experimental parameters for a typical cryo-EM experiment with the pulsed phase plate, including the repetition rate, electron pulse duration, number of electrons per pulse, electron beam size, and the parameters of the laser beam (wavelength, laser pulse duration, pulse energy), will help readers better understand technical requirements for the proposed cryo-EM experiments.

Reviewer #1 (Public review):

Review of the revised submission:

I thank the authors for their detailed consideration of my comments and for the additional data, analyses, and clarifications they have incorporated. The new behavioral experiments, quantification of targeted manipulations, and expanded methodological details strengthen the manuscript and address many of my initial concerns. While some questions remain for future work, the authors' careful responses and the additional evidence provided help resolve the main issues I raised, and I am generally satisfied with the revisions.

Review of original submission:

Summary

In this article, Kawanabe-Kobayashi et al., aim to examine the mechanisms by which stress can modulate pain in mice. They focus on the contribution of noradrenergic neurons (NA) of the locus coeruleus (LC). The authors use acute restraint stress as a stress paradigm and found that following one hour of restraint stress mice display mechanical hypersensitivity. They show that restraint stress causes the activation of LC NA neurons and the release of NA in the spinal cord dorsal horn (SDH). They then examine the spinal mechanisms by which LC→SDH NA produces mechanical hypersensitivity. The authors provide evidence that NA can act on alphaA1Rs expressed by a class of astrocytes defined by the expression of Hes (Hes+). Furthermore, they found that NA, presumably through astrocytic release of ATP following NA action on alphaA1Rs Hes+ astrocytes, can cause an adenosine-mediated inhibition of SDH inhibitory interneurons. They propose that this disinhibition mechanism could explain how restraint stress can cause the mechanical hypersensitivity they measured in their behavioral experiments.

Strengths:

(1) Significance. Stress profoundly influences pain perception; resolving the mechanisms by which stress alters nociception in rodents may explain the well-known phenomenon of stress-induced analgesia and/or facilitate the development of therapies to mitigate the negative consequences of chronic stress on chronic pain.

(2) Novelty. The authors' findings reveal a crucial contribution of Hes+ spinal astrocytes in the modulation of pain thresholds during stress.

(3) Techniques. This study combines multiple approaches to dissect circuit, cellular, and molecular mechanisms including optical recordings of neural and astrocytic Ca2+ activity in behaving mice, intersectional genetic strategies, cell ablation, optogenetics, chemogenetics, CRISPR-based gene knockdown, slice electrophysiology, and behavior.

Weaknesses:

(1) Mouse model of stress. Although chronic stress can increase sensitivity to somatosensory stimuli and contribute to hyperalgesia and anhedonia, particularly in the context of chronic pain states, acute stress is well known to produce analgesia in humans and rodents. The experimental design used by the authors consists of a single one-hour session of restraint stress followed by 30 min to one hour of habituation and measurement of cutaneous mechanical sensitivity with von Frey filaments. This acute stress behavioral paradigm corresponds to the conditions in which the clinical phenomenon of stress-induced analgesia is observed in humans, as well as in animal models. Surprisingly, however, the authors measured that this acute stressor produced hypersensitivity rather than antinociception. This discrepancy is significant and requires further investigation.

(2) Specifically, is the hypersensitivity to mechanical stimulation also observed in response to heat or cold on a hotplate or coldplate?

(3) Using other stress models, such as a forced swim, do the authors also observe acute stress-induced hypersensitivity instead of stress-induced antinociception?

(4) Measurement of stress hormones in blood would provide an objective measure of the stress of the animals.

(5) Results:

(a) Optical recordings of Ca2+ activity in behaving rodents are particularly useful to investigate the relationship between Ca2+ dynamics and the behaviors displayed by rodents.

(b) The authors report an increase in Ca2+ events in LC NA neurons during restraint stress: Did mice display specific behaviors at the time these Ca2+ events were observed such as movements to escape or orofacial behaviors including head movements or whisking?

(c) Additionally, are similar increases in Ca2+ events in LC NA neurons observed during other stressful behavioral paradigms versus non-stressful paradigms?

(d) Neuronal ablation to reveal the function of a cell population.

(e) The proportion of LC NA neurons and LC→SDH NA neurons expressing DTR-GFP and ablated should be quantified (Figures 1G and J) to validate the methods and permit interpretation of the behavioral data (Figures 1H and K). Importantly, the nocifensive responses and behavior of these mice in other pain assays in the absence of stress (e.g., hotplate) and a few standard assays (open field, rotarod, elevated plus maze) would help determine the consequences of cell ablation on processing of nociceptive information and general behavior.

(f) Confirmation of LC NA neuron function with other methods that alter neuronal excitability or neurotransmission instead of destroying the circuit investigated, such as chemogenetics or chemogenetics, would greatly strengthen the findings. Optogenetics is used in Figure 1M, N but excitation of LC→SDH NA neuron terminals is tested instead of inhibition (to mimic ablation), and in naïve mice instead of stressed mice.

(g) Alpha1Ars. The authors noted that "Adra1a mRNA is also expressed in INs in the SDH".

(h) The authors should comprehensively indicate what other cell types present in the spinal cord and neurons projecting to the spinal cord express alpha1Ars and what is the relative expression level of alpha1Ars in these different cell types.

(i) The conditional KO of alpha1Ars specifically in Hes5+ astrocytes and not in other cell types expressing alpha1Ars should be quantified and validated (Figure 2H).

(j) Depolarization of SDH inhibitory interneurons by NA (Figure 3). The authors' bath applied NA, which presumably activates all NA receptors present in the preparation.

k) The authors' model (Figure 4H) implies that NA released by LC→SDH NA neurons leads to the inhibition of SDH inhibitory interneurons by NA. In other experiments (Figure 1L, Figure 2A), the authors used optogenetics to promote the release of endogenous NA in SDH by LC→SDH NA neurons. This approach would investigate the function of NA endogenously released by LC NA neurons at presynaptic terminals in the SDH and at physiological concentrations and would test the model more convincingly compared to the bath application of NA.

(l) As for other experiments, the proportion of Hes+ astrocytes that express hM3Dq, and the absence of expression in other cells, should be quantified and validated to interpret behavioral data.

(m) Showing that the effect of CNO is dose-dependent would strengthen the authors' findings.

(n) The proportion of SG neurons for which CNO bath application resulted in a reduction in recorded sIPSCs is not clear.

(o) A1Rs. The specific expression of Cas9 and guide RNAs, and the specific KD of A1Rs, in inhibitory interneurons but not in other cell types expressing A1Rs should be quantified and validated.

(6) Methods:

It is unclear how fiber photometry is performed using "optic cannula" during restraint stress while mice are in a 50ml falcon tube (as shown in Figure 1A).

Reviewer #2 (Public review):

Summary:

This study investigates the role of spinal astrocytes in mediating stress-induced pain hypersensitivity, focusing on the LC (locus coeruleus)-to-SDH (spinal dorsal horn) circuit and its mechanisms. The authors aimed to delineate how LC activity contributes to spinal astrocytic activation under stress conditions, explore the role of noradrenaline (NA) signaling in this process, and identify the downstream astrocytic mechanisms that influence pain hypersensitivity.

The authors provide strong evidence that 1-hour restraint stress-induced pain hypersensitivity involves the LC-to-SDH circuit, where NA triggers astrocytic calcium activity via alpha1a adrenoceptors (alpha1aRs). Blockade of alpha1aRs on astrocytes-but not on Vgat-positive SDH neurons-reduced stress-induced pain hypersensitivity. These findings are rigorously supported by well-established behavioral models and advanced genetic techniques, uncovering the critical role of spinal astrocytes in modulating stress-induced pain.

However, the study's third aim-to establish a pathway from astrocyte alpha1aRs to adenosine-mediated inhibition of SDH-Vgat neurons-is less compelling. While pharmacological and behavioral evidence is intriguing, the ex vivo findings are indirect and lack a clear connection to the stress-induced pain model. Despite these limitations, the study advances our understanding of astrocyte-neuron interactions in stress-pain contexts and provides a strong foundation for future research into glial mechanisms in pain hypersensitivity.

Strengths:

The study is built on a robust experimental design using a validated 1-hour restraint stress model, providing a reliable framework to investigate stress-induced pain hypersensitivity. The authors utilized advanced genetic tools, including retrograde AAVs, optogenetics, chemogenetics, and subpopulation-specific knockouts, allowing precise manipulation and interrogation of the LC-SDH circuit and astrocytic roles in pain modulation. Clear evidence demonstrates that NA triggers astrocytic calcium activity via alpha1aRs, and blocking these receptors effectively reduces stress-induced pain hypersensitivity.

Weaknesses:

The study offers mainly indirect evidence for astrocyte-released adenosine acting on SDH-VGAT neurons. The potential contributions of astrocyte-derived D-serine and adenosine to different spinal neuron subtypes, as well as the transient "dip" in astrocytic calcium following LC optostimulation, merit further clarification in future work once appropriate tools become available.

Comments on revisions:

The authors have thoroughly addressed my previous comments, resolving most of the points I raised except those noted in the "Weaknesses" section above. I understand that some of these aspects will require future tool development.

Reviewer #3 (Public review):

Summary

This is an exciting and timely study addressing the role of descending noradrenergic systems in nocifensive responses. While it is well-established that spinally released noradrenaline (aka norepinephrine) generally acts as an inhibitory factor in spinal sensory processing, this system is highly complex. Descending projections from the A6 (locus coeruleus, LC) and the A5 regions typically modulate spinal sensory processing and reduce pain behaviours, but certain subpopulations of LC neurons have been shown to mediate pronociceptive effects, such as those projecting to the prefrontal cortex (Hirshberg et al., PMID: 29027903).

The study proposes that descending cerulean noradrenergic neurons potentiate touch sensation via alpha-1 adrenoceptors on Hes5+ spinal astrocytes, contributing to mechanical hyperalgesia. This finding is consistent with prior work from the same group (dd et al., PMID:). However, caution is needed when generalising about LC projections, as the locus coeruleus is functionally diverse, with differences in targets, neurotransmitter co-release, and behavioural effects. Specifying the subpopulations of LC neurons involved would significantly enhance the impact and interpretability of the findings.

Strengths

The study employs state-of-the-art molecular, genetic, and neurophysiological methods, including precise CRISPR and optogenetic targeting, to investigate the role of Hes5+ astrocytes. This approach is elegant and highlights the often-overlooked contribution of astrocytes in spinal sensory gating. The data convincingly support the role of Hes5+ astrocytes as regulators of touch sensation, coordinated by brain-derived noradrenaline in the spinal dorsal horn, opening new avenues for research into pain and touch modulation.

Furthermore, the data support a model in which superficial dorsal horn (SDH) Hes5+ astrocytes act as non-neuronal gating cells for brain-derived noradrenergic (NA) signalling through their interaction with substantia gelatinosa inhibitory interneurons. Locally released adenosine from NA-stimulated Hes5+ astrocytes, following acute restraint stress, may suppress the function of SDH-Vgat+ inhibitory interneurons, resulting in mechanical pain hypersensitivity. However, the spatially restricted neuron-astrocyte communication underlying this mechanism requires further investigation in future studies.

Comments on revisions:

One important point remains insufficiently resolved. In Figure S4C, two of the three visible neurons in the A5 example appear to show a white "halo" at the cell border, suggesting a merge of eGFP (green) and TH (magenta) and therefore possible transgene positivity. To draw a confident conclusion about the specificity of the approach for the A6 (LC) population, the authors are kindly asked to provide high-resolution images of several representative A5 sections, presented both as merged and as separate colour channels. Ideally, quantification across multiple rostrocaudal sections of A5, A6 and A7 should be provided. This is essential for determining whether any transgene expression occurs within the A5 nucleus, particularly given its several-millimetre rostrocaudal extent. As the behavioural phenotype arises from manipulation of only a small subset of A6 neurons, ruling out any contribution from A5 (or A7) is critical for validating pathway specificity, especially in light of prior reports showing that similar approaches can label A5 fibres.

Reviewer #2 (Public review):

Zhang et al. have developed an advanced three-dimensional culture system of human endometrial cells, termed a receptive endometrial assembloid, that models the uterine lining during the crucial window of implantation (WOI). During this mid-secretory phase of the menstrual cycle, the endometrium becomes receptive to an embryo, undergoing distinctive changes. In this work, endometrial cells (epithelial glands, stromal cells, and immune cells from patient samples) were grown into spheroid assembloids and treated with a sequence of hormones to mimic the natural cycle. Notably, the authors added pregnancy-related factors (such as hCG and placental lactogen) on top of estrogen and progesterone, pushing the tissue construct into a highly differentiated, receptive state. The resulting WOI assembloid closely resembles a natural receptive endometrium in both structure and function. The cultures form characteristic surface structures like pinopodes and exhibit abundant motile cilia on the epithelial cells, both known hallmarks of the mid-secretory phase. The assembloids also show signs of stromal cell decidualization and an epithelial mesenchymal transition, like process at the implantation interface, reflecting how real endometrial cells prepare for possible embryo invasion.

Although the WOI assembloid represents an important step forward, it still has limitations: the supportive stromal and immune cell populations decrease over time in culture, so only early-passage assembloids retain full complexity. Additionally, the differences between the WOI assembloid and a conventional secretory-phase organoid are more quantitative than absolute; both respond to hormones and develop secretory features, but the WOI assembloid achieves a higher degree of differentiation due to the addition of "pregnancy" signals. Overall, while it's a reinforced model (not an exact replica of the natural endometrium), it provides a valuable in vitro system for implantation studies and testing potential interventions, with opportunities to improve its long-term stability and biological fidelity in the future.

[Editors' note: the authors have responded to the previous round of recommendations.]

Reviewer #1 (Public review):

Summary:

This paper presents maRQup a Python pipeline for automating the quantitative analysis of preclinical cancer immunotherapy experiments using bioluminescent imaging in mice. maRQup processes images to quantify tumor burden over time and across anatomical regions, enabling large-scale analysis of over 1,000 mice. The study uses this tool to compare different CAR-T cell constructs and doses, identifying differences in initial tumor control and relapse rates, particularly noting that CD19.CD28 CAR-T cells show faster initial killing but higher relapse compared to CD19.4-1BB CAR-T cells. Furthermore, maRQup facilitates the spatiotemporal analysis of tumor dynamics, revealing differences in growth patterns based on anatomical location, such as the snout exhibiting more resistance to treatment than bone marrow.

Strengths:

(1) The maRQup pipeline enables the automatic processing of a large dataset of over 1,000 mice, providing investigators with a rapid and efficient method for analyzing extensive bioluminescent tumor image data.

(2) Through image processing steps like tail removal and vertical scaling, maRQup normalizes mouse dimensions to facilitate the alignment of anatomical regions across images. This process enables the reliable demarcation of nine distinct anatomical regions within each mouse image, serving as a basis for spatiotemporal analysis of tumor burden within these consistent regions by quantifying average radiance per pixel.

Weaknesses:

(1) While the pipeline aims to standardize images for regional assessment, the reliance on scaling primarily along the vertical axis after tail removal may introduce limitations to the quantitative robustness of the anatomically defined regions. This approach does not account for potential non-linear growth across dimensions in animals of different ages or sizes, which could result in relative stretching or shrinking of subjects compared to an average reference.

(2) Furthermore, despite excluding severely slanted images, the pipeline does not fully normalize for variations in animal pose during image acquisition (e.g., tucked body, leaning). This pose variability not only impacts the precise relative positioning of internal anatomical regions, potentially making their definition based on relative image coordinates more qualitative than truly quantitative for precise regional analysis, but it also means that the bioluminescent light signal from the tumor will not propagate equally to the camera as photons will travel differentially through the tissue. This differing light path through tissues due to variable positioning can introduce large variability in the measured radiance that was not accounted for in the analysis algorithm. Achieving more robust anatomical and quantitative normalization might require methods that control animal posture using a rigid structure during imaging.

Comments on revisions:

(1) Clarification of 2D Analysis. We strongly recommend that the authors explicitly define maRQup as a 2D spatiotemporal analysis technique. Since optical imaging quantification is inherently dependent on tissue type and signal depth, characterizing this as a 3D or volumetric method without tomographic correction is inaccurate. Please precede "spatiotemporal" with "2D" throughout the text to ensure precision regarding the method's capabilities.

(2) Data Validation and Scaling in Supplemental Figure g currently lacks the units necessary to support the assertion.

Non-Uniform Growth: The authors' method implies that mouse growth is linear and uniform in all directions (isotropic). However, murine growth is not akin to the inflation of a balloon; animals elongate and widen at different rates. The current scaling does not account for these physiological non-linearities.

Pose Variability: The scaling approach appears to neglect significant variability in animal positioning. Even under anesthesia, animal pose is rarely identical across subjects or time points.

Requirement for Evidence: Without quantitative data, there appears to be significant differences between the individual images and the merged image. If the authors assert that this is a "classical setting" where mouse positioning is 100% consistent and growth curves are identical in multiple dimensions, please provide specific references that validate these assumptions. Otherwise, the scaling must be corrected to account for anisotropic growth and pose differences or stated that scaling was only based on one dimension.

(3) Methodology of Spatial Regions The manuscript does not currently indicate how the nine distinct spatial regions were determined. Please expand the methods section to include the specific segmentation algorithms or anatomical criteria used to define these regions, as this is critical for reproducibility.

Reviewer #3 (Public review):

Summary:

The paper "The 1000+ mouse project: large-scale spatiotemporal parametrization and modeling of preclinical cancer immunotherapies" is focused on developing a novel methodology for automatic processing of bioluminescence imaging data. It provides quantitative and statistically robust insights on preclinical experiments that will contribute to optimizing cell-based therapies. There is an enormous demand for such methods and approaches that enable the spatiotemporal evaluation of cell monitoring in large cohorts of experimental animals.

Strengths:

The manuscript is generally well written, and the experiments are scientifically sound. The conclusions reflect the soundness of experimental data. This approach seems to be quite innovative and promising to improve the statistical accuracy of BLI data quantification.<br /> This methodology can be used as a universal quantification tool for BLI data for in vivo assessment of adoptively transferred cells due to the versatility of the technology.

Comments on revisions:

The critiques have been taken care of appropriately.

Reviewer #1 (Public review):

Summary:

This study presents a technically sophisticated intravital two-photon calcium imaging approach to characterize meningeal macrophage Ca²⁺ dynamics in awake mice. The development of a Pf4Cre:GCaMP6s reporter line and the integration of event-based Ca²⁺ analysis represent clear methodological strengths. The findings reveal niche-specific Ca²⁺ signaling patterns and heterogeneous macrophage responses to cortical spreading depolarization (CSD), with potential relevance to migraine and neuroinflammatory conditions. Despite these strengths, several conceptual, technical, and interpretational issues limit the impact and mechanistic depth of the study. Addressing the points below would substantially strengthen the manuscript.

Strengths:

The use of chronic two-photon Ca²⁺ imaging in awake, behaving mice represents a major technical strength, minimizing confounds introduced by anesthesia. The development of a Pf4Cre:GCaMP6s reporter line, combined with high-resolution intravital imaging, enables long-term and subcellular analysis of macrophage Ca²⁺ dynamics in the meninges.

The comparison between perivascular and non-perivascular macrophages reveals clear niche-dependent differences in Ca²⁺ signaling properties. The identification of macrophage Ca²⁺ activity temporally coupled to dural vasomotion is particularly intriguing and highlights a potential macrophage-vascular functional unit in the dura.

By linking macrophage Ca²⁺ responses to CSD and implicating CGRP/RAMP1 signaling in a subset of these responses, the study connects meningeal macrophage activity to clinically relevant neuroimmune pathways involved in migraine and other neurological disorders.

Weaknesses:

The manuscript relies heavily on Pf4Cre-driven GCaMP6s expression to selectively image meningeal macrophages. Although prior studies are cited to support Pf4 specificity, Pf4 is not an exclusively macrophage-restricted marker, and developmental recombination cannot be excluded. The authors should provide direct validation of reporter specificity in the adult meninges (e.g., co-labeling with established macrophage markers and exclusion of other Pf4-expressing lineages). At minimum, the limitations of Pf4Cre-based labeling should be discussed more explicitly, particularly regarding how off-target expression might affect Ca²⁺ signal interpretation.

The manuscript offers an extensive characterization of Ca²⁺ event features (frequency spectra, propagation patterns, synchrony), but the biological significance of these signals is largely speculative. There is no direct link established between Ca²⁺ activity patterns and macrophage function (e.g., activation state, motility, cytokine release, or interaction with other meningeal components). The discussion frequently implies functional specialization based on Ca²⁺ dynamics without experimental validation. To strengthen the conceptual impact, a clearer framing of the study as a foundational descriptive resource, rather than a functional dissection, would improve alignment between data and conclusions.

The GLM analysis revealing coupling between dural perivascular macrophage Ca²⁺ activity and vasomotion is technically sophisticated and intriguing. However, the directionality of this relationship remains unresolved. The current data do not distinguish whether macrophages actively regulate vasomotion, respond to mechanical or hemodynamic changes, or are co-modulated by neural activity. Statements suggesting that macrophages may "mediate" vasomotion are therefore premature. The authors should reframe these conclusions more cautiously, emphasizing correlation rather than causation, and expand the discussion to explicitly outline experimental strategies required to establish causality (e.g., macrophage-specific Ca²⁺ manipulation).

The authors conclude that synchronous Ca²⁺ events across macrophages are driven by extrinsic signals rather than intercellular communication, based primarily on distance-time analyses. This conclusion is not sufficiently supported, as spatial independence alone does not exclude paracrine signaling, vascular cues, or network-level coordination. No perturbation experiments are presented to test alternative mechanisms. The authors can either provide additional experimental evidence or rephrase the conclusion to acknowledge that the source of synchrony remains unresolved.

A major and potentially important finding is that the dominant macrophage response to CSD is a persistent decrease in Ca²⁺ activity, which is independent of CGRP/RAMP1 signaling. However, this phenomenon is not mechanistically explored. It remains unclear whether Ca²⁺ suppression reflects macrophage inhibition, altered viability, homeostatic resetting, or an anti-inflammatory program. Minimally, the discussion should be more deeply engaged with possible interpretations and implications of this finding.

The pharmacological blockade of RAMP1 supports a role for CGRP signaling in persistent Ca²⁺ increases after CSD, but the experiments are based on a relatively small number of cells and animals. The limited sample size constrains confidence in the generality of the conclusions. Pharmacological inhibition alone does not establish cell-autonomous effects in macrophages. The authors should acknowledge these limitations more explicitly and avoid overextension of the conclusions.

Reviewer #2 (Public review):

Using chronic intravital two-photon imaging of calcium dynamics in meningeal macrophages in Pf4Cre:TIGRE2.0-GCaMP6 mice, the study identified heterogeneous features of perivascular and non-perivascular meningeal macrophages at steady state and in response to cortical spreading depolarization (CSD). Analyses of calcium dynamics and blood vessels revealed a subpopulation of perivascular meningeal macrophages whose activity is coupled to behaviorally driven diameter fluctuations of their associated vessels. The analyses also investigated synchrony between different macrophage populations and revealed a role for CGRP/RAMP1 signaling in the CSD-induced increase, but not the decrease, in calcium transients.

This is a timely study at both the technical and conceptual levels, examining calcium dynamics of meningeal macrophages in vivo. The conclusions are well supported by the findings and will provide an important foundation for future research on immune cell dynamics within the meninges in vivo. The paper is well written and clearly presented.

I have only minor comments.

(1) Please indicate the formal definition of perivascular versus non-perivascular macrophages in terms of distance from the blood vessel. This information is not provided in the main text or the Methods. In addition, please explain how the meningeal vasculature was imaged in the main text.

(2) Similarly, the method used to induce acute CSD (pin prick) is not described in the main text and is only mentioned in the figure legends and Methods. Additional background on the neurobiology of acute CSD, as well as the resulting brain activity and neuroinflammatory responses, could be helpful.

Reviewer #3 (Public review):

Summary:

The authors of this report wish to show that distinct populations of meningeal macrophages respond to cortical spreading depolarization (CSD) via unique calcium activity patterns depending on their location in the meningeal sub-compartments. Perivascular macrophages display calcium signaling properties that are sometimes in opposition to non-perivascular macrophages. Many of the meningeal macrophages also displayed synchronous activity at variable distances from one another. Other macrophages were found to display calcium signals in response to dural vasomotion. CSD could induce variable calcium responses in both perivascular and non-perivascular macrophages in the meninges, in part due to RAMP1-dependent effects. Results will inform future research on the calcium responses displayed by macrophages in the meninges under both normal and pathological conditions.

Strengths:

Sophisticated in vivo imaging of meningeal immune cells is employed in the study, which has not been performed previously. A detailed analysis of the distinct calcium dynamics in various subtypes of meningeal macrophages is provided. Functional relevance of the responses is also noted in relation to CSD events.

Weaknesses:

The specificity of the methods used to target both meningeal macrophages and RAMP1 is limited. Additional discussion points on the functional relevance of the two subtypes of meningeal macrophages and their calcium responses are warranted. A section on potential pitfalls should be included.

Reviewer #1 (Public review):

Summary:

In this study, the authors' aim was to determine whether hepatic palmitoylation is a physiologically relevant regulator of systemic metabolism. The data demonstrate that loss of DHHC7 in hepatocytes disrupts Gαi palmitoylation, enhances cAMP-PKA-CREB signaling, and drives transcriptional upregulation and secretion of Prg4. The KO mice display increased body weight, fat mass, and plasma cholesterol, but at 12 weeks on HFD, do not exhibit insulin resistance. The potential mechanism underlying the metabolic phenotype was examined by assessing adipocyte signaling and by exploring whether Prg4 acts through GPR146. Through this pathway, the authors intend to link DHHC7-dependent palmitoylation to the regulation of hepatokines that exert systemic metabolic effects.

Strengths:

(1) Hepatic palmitoylation in systemic metabolic regulation is largely unexplored. The authors demonstrate the role of DHHC7 in vivo using a successful liver-specific knockout mouse model that causes HFD-dependent obesity without insulin resistance.

(2) Several studies were performed on chow and HFD, as well as male and female mice.

(3) Plasma proteomics identified Prg4 as a circulating factor elevated in KO mice. Prg4 overexpression phenocopied the KO mice.

(4) There is solid mechanistic data supporting the hypothesis that hepatic DHHC7 loss selectively increases Prg4 secretion as a hepatokine.

(5) There is convincing evidence for the DHHC7 mechanism in liver: DHHC7 controls cAMP-PKA-CREB via Gαi palmitoylation. The authors recognize that the palmitoylation change is causative rather than correlated, and this needs to be more fully explored in the future.

(6) Strong in vitro data support that Prg4 acts through adipocyte GPR146 via its SMB domain

Weaknesses:

(1) The assessment of liver and adipose tissue responses to DHH7 loss is insufficient to support claims that it alters systemic lipolysis. In this new mouse model, liver histology is necessary, especially given the cholesterol increase in the KO. As this is a newly established mouse line, common assessments of the liver during HFD feeding would be important for interpreting the phenotype.

(2) The data show DHH7 loss causes adipose tissue dysfunction and alterations in lipid metabolism. Beyond that, I suggest not stating more regarding the phenotype of the DHH7 mice for this work. A thorough analysis would be needed to determine which factor drives the obesity and changes in energy balance in the mice. For example, the KO mice had lower oxygen consumption (but no change in CO2 production, which is also usually similarly altered), suggesting a CNS component could drive obesity. However, since the data are not normalized for lean mass and there is no information about locomotor activity, this analysis is incomplete. RER may be informative if available. A broad conservative description of the KO phenotype would be more accurate since Pgr4 has many paracrine targets and likely has autocrine signaling in the liver.

(3) Most references to lipolysis or lipolysis flux systemically would be inaccurate. To suggest a suppression of lipolysis, serum NEFA would need to be measured, and in vivo or in vitro lipolysis assays performed to test the effect of DHH7 loss or the specificity of PGR4 action on adipocytes in vivo. To demonstrate adipose tissue dysfunction, analysis of lipogenesis markers, canonical markers for insulin sensitivity, and mitochondrial dysfunction should be performed/measured.

(4) Line 179: The experiment was performed in brown adipocytes to show that Prg4 does not affect p-CREB Figure S8 under the heading: "DHHC7 controls hepatic PKA-CREB activity through Gαi palmitoylation to regulate Prg4 transcription." Unless repeated using liver lysate, the conclusions stated in the text throughout the paper should be revised.

(5) It appears that the serum and liver proteomics were only assessed for factors that increased in KO mice? Were proteins that were significantly decreased analyzed?

(6) The beige adipocyte culture method is unclear. The methods do not describe the fat pad used, and the protocol suggests the cells would be differentiated into mature white adipocytes. If they are beige cells, a reference for the method, gene expression, and cell images could support that claim.

(7) The use of tamoxifen can confound adipocyte studies, as it increases beigeing and weight gain even after a brief initiation period. Both groups were treated with Tam, but another way to induce Cre would be ideal.

(8) Evidence for the lack of the glucose phenotype is incomplete. One reason could be due to the IP route of glucose administration, which has a large impact on glucose handling during a GTT. To confirm the absence of a glucose tolerance phenotype, an OGTT should be performed, as it is more physiological. In addition, the mice should be fed for 16 weeks. Prg4 affects immune cells, changing how adipose tissue expands, and 12 weeks of HFD feeding is often not long enough to see the effects of adipose tissue inflammation spilling over into the system.

(9) There may be liver-adipose tissue crosstalk in KO mice, but this was not fully assessed in this study and would be difficult to determine in any setting, given the diverse cell types that are targets of Pdg4. The crosstalk claim is unnecessary to share the basic premises; there is the DHH7 mechanism/phenotype and the Pgr4 mechanism/phenotype, and while there is no Pgr4 adipose direct mechanism, the paper can be successfully reframed.

(10) Although the DHH7 loss on the chow diet did not result in a phenotype, did the Pgr4 increase in the KO mice on chow? This would determine whether either i) the expression of Pgr4 is dependent on HFD/obesity, or ii) circulating Pgr4 has effects only in an HFD condition. The receptors may also change on HFD, especially in adipocytes.

Impact:

This work would significantly contribute to the study of liver metabolism, provided it includes data describing the liver. The role of Pgr4 in adipocytes and other cell types is of substantial value to the field of metabolism. By reframing the paper and conducting some key experiments, its quality and impact can be increased.

Reviewer #2 (Public review):

In the current report, Sun and Colleagues sought to determine the liver-specific role that DHHC7, a DHHC palmitoyltransferase protein, plays in regulating whole-body energy balance and hepatic crosstalk with adipose tissues. The authors generated an inducible, liver-specific DHHC7 knockout mouse to determine how altered palmitoylation in hepatocytes alters hepatokine production/secretion, and in turn, systemic metabolism. The ablation of DHHC7 was found to alter the production of proteoglycan 4 (Prg4), a hepatokine previously linked to metabolic regulation. The authors propose that the change in Prg4 production is mediated by the loss of Gαi palmitoylation, due to DHHC7 ablation, thereby augmenting cAMP-PKA-CREB signaling in hepatocytes, which alleviates the 'brake' on Prg4 production. The authors further propose that Prg4 overexpression leads to excessive binding to GPR146 on adipocytes, which in turn suppresses PKA-mediated HSL activation, promoting impairments in lipolysis, leading to obesity. The report is interesting and generally well-written, but it appears to have some clear gaps in additional data that would aid in interpretation. The addition of confirmatory culture studies would be incredibly helpful for testing the hypotheses being explored. My comments, concerns, and/or suggestions are outlined below in no particular order.

(1) Figures: All data should be presented in dot-boxplot format so the reader knows how many samples were analyzed for each assay and group. n=3 for some assays/experiments is incredibly low, particularly when considering the heterogeneity in responsiveness to HFD, food intake, etc....

(2) Figure 1E-F: It is unclear when the food intake measure was performed. Mice can alter their feeding behavior based on a myriad of environmental and biological cues. It would also be interesting to show food intake data normalized to body mass over time. Mice can counterregulate anorexigenic cues by altering neuropeptide production over time. It is not clear if this is occurring in these mice, but the timing of measuring food intake is important. Additionally, the VO2 measure appears to be presented as being normalized to total body mass, when in fact, it would probably be more accurate to normalize this to lean body mass. Normalizing to total body mass provides a denominator effect due to excessive adiposity, but white fat is not as metabolically active as other high-glucose-consuming tissues. If my memory serves me right, several reports have discussed appropriate normalizations in circumstances such as this.

(3) Figure 1J-N: It is not all that surprising that fasting glucose and/or TGs were found to be similar between groups. It is well-established that mice have an incredible ability to become hyperinsulinemic in an effort to maintain euglycemia and lipid metabolism dynamics. A few relatively easy assays can be performed to glean better insights into the metabolic status of the authors' model. First, fasting insulin concentrations will be incredibly helpful. Secondly, if the authors want to tease out which adipose depot is most adversely affected by ablation, they could take an additional set of CON and KO mice, fast them for 5-6 hours, provide a bolus injection of insulin (similar to that provided during an insulin tolerance test), and then quickly harvest the animals ~15 minutes after insulin injections; followed by evaluating AKT phosphorylation. This will really tell them if these issues have impairments in insulin signaling. The gold-standard approach would be to perform a hyperinsulinemic-euglyemic clamp in the CON and KO mice. I now see GTT and ITT data, but the aforementioned assays could help provide insight.

(4) Figure 3A: This looks overexposed to me.

(5) Figures 3-4: It appears that several of these assays could be complemented with culture-based models, which would almost certainly be cleaner. The conditioned media could then be used from hepatocyte cultures to treat differentiated adipocytes.

(6) Figure 4: It is unclear how to interpret the phospho-HSL data because the fasting state can affect this readout. It needs to be made clear how the harvest was done. Moreover, insulin and glucagon were never measured, and these hormones have a significant influence over HSL activity. I suspect the KO mice have established hyperinsulinemia, which would likely affect HSL activity. This provides an example of why performing some of these experiments in a dish would make for cleaner outcomes that are easier to interpret.

Reviewer #3 (Public review):

Summary:

In the current manuscript, Sun et al aimed to determine the metabolic function of hepatocyte DHHC7, one of the key enzymes in protein palmitoylation. They generated inducible liver-specific Dhhc7 knockout mice and discovered that Dhhc7-LKO mice are more prone to gain weight and develop adipose expansion and obesity. Via unbiased proteomic analysis, they identified PRG4 as one of the top secreted factors in the liver of Dhhc7-LKO mice. Hepatic overexpression of PRG4 recapitulates the obesity phenotype observed in Dhh7-LKO mice. At the mechanistic level, PRG4, once secreted from the liver, can bind to GPR146 on adipocytes and inhibit PKA-HSL signaling and lipolysis. Taken together, their findings suggest a novel pathway by which the liver communicates with adipose tissue and impacts systemic metabolism.

Strengths:

(1) The systemic metabolic homeostasis depends on coordination among metabolically active tissues. Thus, active communication between the liver and adipose tissue when facing nutritional challenges (such as high-fat diet feeding) is crucial for achieving metabolic health. The concept that the liver can communicate with adipose tissue and impact the lipolysis process via secreted hepatokines is quite significant but remains poorly understood.

(2) Hepatocyte Dhhc7 knockout mice developed a significant obesity phenotype, which is associated with adipose expansion.

(3) Unbiased proteomic analysis identified PRG4 as one of the top secreted factors in the liver of Dhh7-LKO mice. Hepatic overexpression of PRG4 recapitulates the obesity phenotype observed in Dhh7-LKO mice.

(4) In vitro cell-based assay showed that PRG4 can bind to adipocyte GPR146, inhibit PKA-mediated HSL phosphorylation, and subsequently, the lipolysis process.

Weaknesses:

(1) Lack of a causal-effect study to generate evidence directly linking hepatocyte DHH7 and PRG4 in driving adipose expansion and obesity upon HFD feeding.

(2) Lack of direct evidence to support that PRG4 inhibits adipocyte lipolysis via GPR146. A functional assay demonstrating adipocyte lipolysis is required.

(3) The conclusion is largely based on the correlation evidence.

Reviewer #1 (Public review):

Summary:

This manuscript by Lin et al. presents a timely, technically strong study that builds patient-specific midbrain-like organoids (MLOs) from hiPSCs carrying clinically relevant GBA1 mutations (L444P/P415R and L444P/RecNcil). The authors comprehensively characterize nGD phenotypes (GCase deficiency, GluCer/GluSph accumulation, altered transcriptome, impaired dopaminergic differentiation), perform CRISPR correction to produce an isogenic line, and test three therapeutic modalities (SapC-DOPS-fGCase nanoparticles, AAV9-GBA1, and SRT with GZ452). The model and multi-arm therapeutic evaluation are important advances with clear translational value.

My overall recommendation is that the work undergo a major revision to address the experimental and interpretive gaps listed below.

Strengths:

(1) Human, patient-specific midbrain model: Use of clinically relevant compound heterozygous GBA1 alleles (L444P/P415R and L444P/RecNcil) makes the model highly relevant to human nGD and captures patient genetic context that mouse models often miss.

(2) Robust multi-level phenotyping: Biochemical (GCase activity), lipidomic (GluCer/GluSph by UHPLC-MS/MS), molecular (bulk RNA-seq), and histological (TH/FOXA2, LAMP1, LC3) characterization are thorough and complementary.

(3) Use of isogenic CRISPR correction: Generating an isogenic line (WT/P415R) and demonstrating partial rescue strengthens causal inference that the GBA1 mutation drives many observed phenotypes.

(4) Parallel therapeutic testing in the same human platform: Comparing enzyme delivery (SapC-DOPS-fGCase), gene therapy (AAV9-GBA1), and substrate reduction (GZ452) within the same MLO system is an elegant demonstration of the platform's utility for preclinical evaluation.

(5) Good methodological transparency: Detailed protocols for MLO generation, editing, lipidomics, and assays allow reproducibility

Weaknesses:

(1) Limited genetic and biological replication

(a) Single primary disease line for core mechanistic claims. Most mechanistic data derive from GD2-1260 (L444P/P415R); GD2-10-257 (L444P/RecNcil) appears mainly in therapeutic experiments. Relying primarily on one patient line risks conflating patient-specific variation with general nGD mechanisms.

(b) Unclear biological replicate strategy. It is not always explicit how many independent differentiations and organoid batches were used (biological replicates vs. technical fields of view).

(c) A significant disadvantage of employing brain organoids is the heterogeneity during induction and potential low reproducibility. In this study, it is unclear how many independent differentiation batches were evaluated and, for each test (for example, immunofluorescent stain and bulk RNA-seq), how many organoids from each group were used. Please add a statement accordingly and show replicates to verify consistency in the supplementary data.

(d) Isogenic correction is partial. The corrected line is WT/P415R (single-allele correction); residual P415R complicates the interpretation of "full" rescue and leaves open whether the remaining pathology is due to incomplete correction or clonal/epigenetic effects.

(e) The authors tested week 3, 4, 8, 15, and 28 old organoids in different settings. However, systematic markers of maturation should be analyzed, and different maturation stages should be compared, for example, comparing week 8 organoids to week 28 organoids, with immunofluorescent marker staining and bulk RNAseq.

(f) The manuscript frequently refers to Wnt signaling dysregulation as a major finding. However, experimental validation is limited to transcriptomic data. Functional tests, such as the use of Wnt agonist/inhibitor, are needed to support this claim (see below).

(g) Suggested fixes/experiments

Add at least one more independent disease hiPSC line (or show expanded analysis from GD2-10-257) for key mechanistic endpoints (lipid accumulation, transcriptomics, DA markers)

Generate and analyze a fully corrected isogenic WT/WT clone (or a P415R-only line) if feasible; at minimum, acknowledge this limitation more explicitly and soften claims.

Report and increase independent differentiations (N = biological replicates) and present per-differentiation summary statistics.

(2) Mechanistic validation is insufficient

(a) RNA-seq pathways (Wnt, mTOR, lysosome) are not functionally probed. The manuscript shows pathway enrichment and some protein markers (p-4E-BP1) but lacks perturbation/rescue experiments to link these pathways causally to the DA phenotype.

(b) Autophagy analysis lacks flux assays. LC3-II and LAMP1 are informative, but without flux assays (e.g., bafilomycin A1 or chloroquine), one cannot distinguish increased autophagosome formation from decreased clearance.

(c) Dopaminergic dysfunction is superficially assessed. Dopamine in the medium and TH protein are shown, but no neuronal electrophysiology, synaptic marker co-localization, or viability measures are provided to demonstrate functional recovery after therapy.

(d) Suggested fixes/experiments

Perform targeted functional assays:

(i) Wnt reporter assays (TOP/FOP flash) and/or treat organoids with Wnt agonists/antagonists to test whether Wnt modulation rescues DA differentiation.

(ii)Test mTOR pathway causality using mTOR inhibitors (e.g., rapamycin) or 4E-BP1 perturbation and assay effects on DA markers and autophagy.

Include autophagy flux assessment (LC3 turnover with bafilomycin), and measure cathepsin activity where relevant.

Add at least one functional neuronal readout: calcium imaging, MEA recordings, or synaptic marker quantification (e.g., SYN1, PSD95) together with TH colocalization.

(3) Therapeutic evaluation needs greater depth and standardization

(a) Short windows and limited durability data. SapC-DOPS and AAV9 experiments range from 48 hours to 3 weeks; longer follow-up is needed to assess durability and whether biochemical rescue translates into restored neuronal function.

(b) Dose-response and biodistribution are under-characterized. AAV injection sites/volumes are described, but transduction efficiency, vg copies per organoid, cell-type tropism quantification, and SapC-DOPS penetration/distribution are not rigorously quantified.

(c) Specificity controls are missing. For SapC-DOPS, inclusion of a non-functional enzyme control (or heat-inactivated fGCase) would rule out non-specific nanoparticle effects. For AAV, assessment of off-target expression and potential cytotoxicity is needed.

(d) Comparative efficacy lacking. It remains unclear which modality is most effective in the long term and in which cellular compartments.

(e) Suggested fixes/experiments

Extend follow-up (e.g., 6+ weeks) after AAV/SapC dosing and evaluate DA markers, electrophysiology, and lipid levels over time.

Quantify AAV transduction by qPCR for vector genomes and by cell-type quantification of GFP+ cells (neurons vs astrocytes vs progenitors).

Include SapC-DOPS control nanoparticles loaded with an inert protein and/or fluorescent cargo quantitation to show distribution and uptake kinetics.

Provide head-to-head comparative graphs (activity, lipid clearance, DA restoration, and durability) with statistical tests.

(4) Model limitations not fully accounted for in interpretation

(a) Absence of microglia and vasculature limits recapitulation of neuroinflammatory responses and drug penetration, both of which are important in nGD. These absences could explain incomplete phenotypic rescues and must be emphasized when drawing conclusions about therapeutic translation.

(b) Developmental vs degenerative phenotype conflation. Many phenotypes appear during differentiation (patterning defects). The manuscript sometimes interprets these as degenerative mechanisms; the distinction must be clarified.

(c) Suggested fixes

Tone down the language throughout (Abstract/Results/Discussion) to avoid overstatement that MLOs fully recapitulate nGD neuropathology.

Add plans or pilot data (if available) for microglia incorporation or vascularization to indicate how future work will address these gaps.

(5) Statistical and presentation issues

(a) Missing or unclear sample sizes (n). For organoid-level assays, report the number of organoids and the number of independent differentiations.

(b) Statistical assumptions not justified. Tests assume normality; where sample sizes are small, consider non-parametric tests and report exact p-values.

(c) Quantification scope. Many image quantifications appear to be from selected fields of view, which are then averaged across organoids and differentiations.

(d) RNA-seq QC and deposition. Provide mapping rates, batch correction details, and ensure the GEO accession is active. Include these in Methods/Supplement.

(e) Suggested fixes

Add a table summarizing biological replicates, technical replicates, and statistical tests used for each figure panel.

Recompute statistics where appropriate (non-parametric if N is small) and report effect sizes and confidence intervals.

(6) Minor comments and clarifications

(a) The authors should validate midbrain identity further with additional regional markers (EN1, OTX2) and show absence/low expression of forebrain markers (FOXG1) across replicates.

(b) Extracellular dopamine ELISA should be complemented with intracellular dopamine or TH+ neuron counts normalized per organoid or per total neurons.

(c) For CRISPR editing: the authors should report off-target analysis (GUIDE-seq or targeted sequencing of predicted off-targets) or at least in-silico off-target score and sequencing coverage of the edited locus.

(d) It should be clarified as to whether lipidomics normalization is to total protein per organoid or per cell, and include representative LC-MS chromatograms or method QC.

(e) Figure legends should be improved in order to state the number of organoids, the number of differentiations, and the exact statistical tests used (including multiple-comparison corrections).

(f) In the title, the authors state "reveal disease mechanisms", but the studies mainly exhibit functional changes. They should consider toning down the statement.

(7) Recommendations

This reviewer recommends a major revision. The manuscript presents substantial novelty and strong potential impact but requires additional experimental validation and clearer, more conservative interpretation. Key items to address are:

(a) Strengthening genetic and biological replication (additional lines or replicate differentiations).

(b) Adding functional mechanistic validation for major pathways (Wnt/mTOR/autophagy) and providing autophagy flux data.

(c) Including at least one neuronal functional readout (calcium imaging/MEA/patch) to demonstrate functional rescue.

(d) Deepening therapeutic characterization (dose, biodistribution, durability) and including specificity controls.

(e) Improving statistical reporting and explicitly stating biological replicate structure.

Reviewer #2 (Public review):

Sun et al. have developed a midbrain-like organoid (MLO) model for neuronopathic Gaucher disease (nGD). The MLOs recapitulate several features of nGD molecular pathology, including reduced GCase activity, sphingolipid accumulation, and impaired dopaminergic neuron development. They also characterize the transcriptome in the MLO nGD model. CRISPR correction of one of the GBA1 mutant alleles rescues most of the nGD molecular phenotypes. The MLO model was further deployed in proof-of-principle studies of investigational nGD therapies, including SapC-DOPS nanovesicles, AAV9-mediated GBA1 gene delivery, and substrate-reduction therapy (GZ452). This patient-specific 3D model provides a new platform for studying nGD mechanisms and accelerating therapy development. Overall, only modest weaknesses are noted.

Reviewer #3 (Public review):

Summary:

In this study, the authors describe modeling of neuronopathic Gaucher disease (nGD) using midbrain-like organoids (MLOs) derived from hiPSCs carrying GBA1 L444P/P415R or L444P/RecNciI variants. These MLOs recapitulate several disease features, including GCase deficiency, reduced enzymatic activity, lipid substrate accumulation, and impaired dopaminergic neuron differentiation. Correction of the GBA1 L444P variant restored GCase activity, normalized lipid metabolism, and rescued dopaminergic neuronal defects, confirming its pathogenic role in the MLO model. The authors further leveraged this system to evaluate therapeutic strategies, including: (i) SapC-DOPS nanovesicles for GCase delivery, (ii) AAV9-mediated GBA1 gene therapy, and (iii) GZ452, a glucosylceramide synthase inhibitor. These treatments reduced lipid accumulation and ameliorated autophagic, lysosomal, and neurodevelopmental abnormalities.

Strengths:

This manuscript demonstrates that nGD patient-derived MLOs can serve as an additional platform for investigating nGD mechanisms and advancing therapeutic development.

Comments:

(1) It is interesting that GBA1 L444P/P415R MLOs show defects in midbrain patterning and dopaminergic neuron differentiation (Figure 3). One might wonder whether these abnormalities are specific to the combination of L444P and P415R variants or represent a general consequence of GBA1 loss. Do GBA1 L444P/RecNciI (GD2-10-257) MLOs also exhibit similar defects?

(2) In Supplementary Figure 3, the authors examined GCase localization in SapC-DOPS-fGCase-treated nGD MLOs. These data indicate that GCase is delivered to TH⁺ neurons, GFAP⁺ glia, and various other unidentified cell types. In fruit flies, the GBA1 ortholog, Gba1b, is only expressed in glia (PMID: 35857503; 35961319). Neuronally produced GluCer is transferred to glia for GBA1-mediated degradation. These findings raise an important question: in wild-type MLOs, which cell type(s) normally express GBA1? Are they dopaminergic neurons, astrocytes, or other cell types?

(3) The authors may consider switching Figures 2 and 3 so that the differentiation defects observed in nGD MLOs (Figure 3) are presented before the analysis of other phenotypic abnormalities, including the various transcriptional changes (Figure 2).

Reviewer #1 (Public review):

Summary:

This study uses data from a recent RVFV serosurvey among transhumant cattle in The Gambia to inform the development of an RVFV transmission model. The model incorporates several hypotheses that capture the seasonal nature of both vector-borne RVFV transmission and cattle migration. These natural phenomena are driven by contrasting wet and dry seasons in The Gambia's two main ecoregions and are purported to drive cyclical source-sink transmission dynamics. Although the Sahel is hypothesized to be unsuitable for year-long RVFV transmission, findings suggest that cattle returning from the Gambia River to the Sahel at the beginning of the wet season could drive repeated RVFV introductions and ensuing seasonal outbreaks. Upon review, the authors have removed an additional analysis evaluating the potential impacts of cattle movement bans on transmission dynamics, which was poorly supported by the methodological approach.

Strengths:

Like most infectious diseases in animal systems in low- and middle-income countries, the transmission dynamics of RVFV in cattle in The Gambia are poorly understood. This study harnesses important data on RVFV seroepidemiology to develop and parameterize a novel transmission model, providing plausible estimates of several epidemiological parameters and transmission dynamic patterns.

This study is well written and easy to follow.

The authors consider both deterministic and stochastic formulations of their model, demonstrating potential impacts of random events (e.g. extinctions) and providing confidence regarding model robustness.

The authors use well-established Bayesian estimation techniques for model fitting and confront their transmission model with a seroepidemiological model to assess model fit.

Elasticity analyses help to understand the relative importance of competing demographic and epidemiological drivers of transmission in this system.

Weaknesses:

The model does not include an impact of infection on cattle birth rates, but the authors justify that this parameter should have limited impact on dynamics given predicted low-level circulation patterns, as opposed to explosive outbreaks, in this region.

The importance of the LVFV positivity decay rate is highlighted but loss of immunity is not considered in the SIR model. The authors do discuss uncertainty regarding model structure and a need for future data collection to begin to answer this question.

The model's structure, including homogenous mixing within each ecoregion and step-change seasonality, allows for estimation of generalized transmission rates at a macro scale. However, it greatly simplifies the movement process itself and assumes that transhumant cattle movement is the only mechanism for RVF reintroduction into the Sahel region. The authors discuss that integration of more finely-scaled movement and contact data may help to address this limitation in future work.

This model seems well-suited to be exploited in future work to explore for e.g. impacts of cattle vaccination, and potential differential efficiency when targeting T herds relative to M or L.

Comments on revisions:

I thank the authors for thoughtfully and thoroughly addressing my concerns. I have no further comments.

Reviewer #1 (Public review):

Summary:

In this work by Mohite et al., they have used transcriptomic and metabolic profiling of H. armigera, muscle development, and S. frugiperda to link energy trehalose metabolism and muscle development. They further used several different bioinformatics tools for network analysis to converge upon transcriptional control as a potential mechanism of metabolite-regulated transcriptional programming for muscle development. The authors have also done rescue experiments where trehalose was provided externally by feeding, which rescues the phenotype. Though the study is exciting, there are several concerns and gaps that lead to the current results as purely speculative. It is difficult to perform any genetic experiments in non-model insects; the authors seem to suggest a similar mechanism could also be applicable in systems like Drosophila; it might be possible to perform experiments to fill some missing mechanistic details.

A few specific comments below:

The authors used N-(phenylthio) phthalimide (NPP), a trehalose-6-phosphate phosphatase (TPP) inhibitor. They also find several genes, including enzymes of trehalose metabolism, that change. Further, several myogenic genes are downregulated in bulk RNA sequencing. The major caveat of this experiment is that the NPP treatment leads to reduced muscle development, and so the proportion of the samples from the muscles in bulk RNA sequencing will be relatively lower, which might have led to the results. So, a confirmatory experiment has to be performed where the muscle tissues are dissected and sequenced, or some of the interesting targets could be validated by qRT-PCR. Further to overcome the off-target effects of NPP, trehalose rescue experiments could be useful.

Even the reduction in the levels of ADP, NAD, NADH, and NMN, all of which are essential for efficient energy production and utilization, could be due to the loss of muscles, which perform predominantly metabolic functions due to their mitochondria-rich environment. So it becomes difficult to judge if the levels of these energy molecules' reduction are due to a cause or effect.

The authors have used this transcriptomic data for pathway enrichment analysis, which led to the E2F family of transcription factors and a reduction in the level of when trehalose metabolism is perturbed. EMSA experiments, though, confirm a possibility of the E2F interaction with the HaTPS/TPP promoter, but it lacks proper controls and competition to test the actual specificity of this interaction. Several transcription factors have DNA-binding domains and could bind any given DNA weakly, and the specificity is ideally known only from competitive and non-competitive inhibition studies.

The work seems to have connected the trehalose metabolism with gene expression changes, though this is an interesting idea, there are no experiments that are conclusive in the current version of the manuscript. If the authors can search for domains in the E2F family of transcription factors that can bind to the metabolite, then, if not, a chip-seq is essential to conclusively suggest the role of E2F in regulating gene expression tuned by the metabolites.

Some of the above concerns are partially addressed in experiments where silencing of E2F/Dp shows similar phenotypes as with NPP and dsRNA. It is also notable that silencing any key transcription factor can have several indirect effects, and delayed pupation and lethality could not be definitely linked to trehalose-dependent regulation.

Trehalose rescue experiments that rescue phenotype and gene expression are interesting. But is it possible that the fed trehalose is metabolized in the gut and might not reach the target tissue? In which case, the role of trehalose in directly regulating transcription factors becomes questionable. So, a confirmatory experiment is needed to demonstrate that the fed trehalose reaches the target tissues. This could possibly be done by measuring the trehalose levels in muscles post-rescue feeding. Also, rescue experiments need to be done with appropriate control sugars.

No experiments are performed with non-target control dsRNA. All the experiments are done with an empty vector. But an appropriate control should be a non-target control.

Reviewer #2 (Public review):

Summary:

This study shows that the knockdown of the effects of TPS/TPP in Helicoverpa armigera and Spodoptera frugiperda can be rescued by trehalose treatment. This suggests that trehalose metabolism is necessary for development in the tissues that NPP and dsRNA can reach.

Strengths:

This study examines an important metabolic process beyond model organisms, providing a new perspective on our understanding of species-specific metabolism equilibria, whether conserved or divergent.

Weaknesses:

While the effects observed may be truly conserved across Lepidopterans and may be muscle-specific, the study largely relies on one species and perturbation methods that are not muscle-specific. The technical limitations arising from investigations outside model systems, where solid methods are available, limit the specificity of inferences that may be drawn from the data.

Reviewer #3 (Public review):

The hypothesis is that Trehalose metabolism regulates transcriptional control of muscle development in lepidopteran insects.

The manuscript investigates the role of Trehalose metabolism in muscle development. Through sequencing and subsequent bioinformatics analysis of insects with perturbed trehalose metabolism (knockdown of TPS/TPP), the authors have identified transcription factor E2F, which was validated through RT-PCR. Their hypothesis is that trehalose metabolism regulates E2F, which then controls the myogenic genes. Counterintuitive to this hypothesis, the investigators perform EMSAs with the E2F protein and promoter of the TPP gene and show binding. Their knockdown experiments with Dp, the binding partner of E2F, show direct effect on several trehalose metabolism genes. Similar results are demonstrated in the trehalose feeding experiment, where feeding trehalose leads to partial rescue of the phenotype observed as a result of Dp knockdown. This seems contradictory to their hypothesis. Even more intriguing is a similar observation between paramyosin, a structural muscle protein, and E2F/Dp - they show that paramyosin regulates E2F/Dp and E2F/Dp regulated paramyosin. The only plausible way to explain the results is the existence of a feed-forward loop between TPP-E2F/Dp and paramyosin-E2F/Dp. But the authors have mentioned nothing in this line. Additionally, I think trehalose metabolism impacts amino acid content in insects, and that will have a direct bearing on muscle development. The sequencing analysis and follow-up GSEA studies have demonstrated enrichment of several amino acid biosynthetic genes. Yet authors make no efforts to measure amino acid levels or correlate them with muscle development. Any study aiming to link trehalose metabolism and muscle development and not considering the above points will be incomplete.

The result section of the manuscript is quite concise, to my understanding (especially the initial few sections), which misses out on mentioning details that would help readers understand the paper better. While technical details of the methods should be in the Materials and Methods section, the overall experimental strategy for the experiments performed should be explained in adequate detail in the results section itself or in figure legends. I would request authors to include more details in the results section. As an extension of the comment above, many times, abbreviations have been used without introducing them. A thorough check of the manuscript is required regarding this.

The Spodoptera experiments appear ad hoc and are insufficient to support conservation beyond Helicoverpa. To substantiate this claim, please add a coherent, minimal set of Spodoptera experiments and present them in a dedicated subsection. Alternatively, consider removing these data and limiting the conclusions (and title) to H. armigera.

In order to check the effects of E2F/Dp, a dsRNA-mediated knockdown of Dp was performed. Why was the E2F protein, a primary target of the study, not chosen as a candidate? The authors should either provide justification for this or perform the suggested experiments to come to a conclusion. I would like to point out that such experiments were performed in Drosophila.

Silencing of HaDp resulted in a significant decrease in HaE2F expression. I find this observation intriguing. DP is the cofactor of E2F, and they both heterodimerise and sit on the promoter of target genes to regulate them. I would request authors to revisit this result, as it contradicts the general understanding of how E2F/Dp functions in other organisms. If Dp indeed controls E2F expression, then further experiments should be conducted to come to a conclusion convincingly. Additionally, these results would need thorough discussion with citations of similar results observed for other transcription factor-cofactor complexes.

I consider the overall bioinformatics analysis to remain very poorly described. What is specifically lacking is clear statements about why a particular dry lab experiments were conducted.

In my judgement, the EMSA analysis presented is technically poor in quality. It lacks positive and negative controls, does not show mutation analysis or super shifts. Also, it lacks any competition assays that are important to prove the binding beyond doubt. I am not sure why protein is not detected at all in lower concentrations. Overall, the EMSA assays need to be redone; I find the current results to be unacceptable.

GSEA studies clearly indicate enrichment of the amino acid synthesis gene in TPP knockdown samples. This supports the plausible theory that a lack of Trehalose means a lack of enough nutrients, therefore less of that is converted to amino acids, and therefore muscle development is compromised. Yet the authors make no effort to measure amino acid levels. While nutrients can be sensed through signalling pathways leading to shut shutdown of myogenic genes, a simple and direct correlation between less raw material and deformed muscle might also be possible.

The authors are encouraged to stick to one color palette while demonstrating sequencing results. Choosing a different color palette for representing results from the same sequencing analysis confuses readers.

Expression of genes, as understood from sequencing analysis in Figure 1D, Figure 2F, and Figure 3D, appears to be binary in nature. This result is extremely surprising given that the qRT-PCR of these genes have revealed a checker and graded expression.

In several graphs, non-significant results have been interpreted as significant in the results section. In a few other cases, the reported changes are minimal, and the statistical support is unclear; please recheck the analyses and include exact statistics. In the results section, fold changes observed should be discussed, as well as the statistical significance of the observed change.

Finally, I would add that trehaolse metabolism regulates cell cycle genes, and muscle development genes establish correlation and causation. The authors should ensure that any comments they make are backed by evidence.

Reviewer #1 (Public review):

Summary:

This study evaluates the feasibility of using crispant founder mice, first-generation animals directly edited by CRISPR/Cas9, for initial phenotypic assessments. The authors target seven genes known to produce visible recessive traits to test whether mosaicism in founder animals prevents meaningful phenotype-genotype interpretation. Remarkably, they observe clear null phenotypes in founders for six of the seven genes, with high editing efficiencies. These results demonstrate that crispant mice can, under specific conditions, display recessive phenotypes that are readily interpretable. However, this conclusion should be moderated, as the study addresses only one biological context, visible Mendelian traits, and may not generalize to quantitative, subtle, or late-onset phenotypes. The report also examines attempts at multiplex CRISPR targeting, which reduce viability, underscoring limits for concurrent gene disruptions. Finally, the detailed description of diverse alleles generated by CRISPR provides valuable insight into how allelic series can be exploited to investigate protein function.

Strengths:

(1) The manuscript provides a comprehensive and technically rigorous description of CRISPR/Cas9‑induced mutations across several loci. The accompanying genotyping, sequencing, and analytical approaches are sound, complementary, and well-detailed, providing a resource that will be valuable to researchers using genome editing beyond the specific application of genetic screening.

(2) By documenting a wide diversity of alleles and mutation types, the study contributes to understanding how allelic series generated by CRISPR can be leveraged for dissecting protein function, a perspective that has been less systematically presented in prior literature and could be compared to targeted strategies such as those described by Cassidy et al. (2022, DOI: [10.1016/bs.mie.2022.03.053]) or other relevant studies addressing CRISPR-based allelic series generation.

(3) The work demonstrates technically solid editing and validation workflows, setting a methodological reference point for similar projects across species or trait categories.

Weaknesses:

(1) There is a disconnect between the abstract/introduction and the discussion. While both the abstract and introduction focus on the potential use of crispant founders for phenotypic assessment in the context of genetic screening, with the introduction notably emphasizing this framework through a detailed section on ENU-based screens, the discussion devotes relatively little attention to this aspect. Instead, it primarily examines CRISPR mutagenesis outcomes, mutation detection, and allele characterization. Overall, the study's aims are not clearly or explicitly defined, which contributes to the lack of alignment across sections.

(2) Important limitations of the approach are not sufficiently discussed. For instance, the paper does not address how applicable the findings are beyond visible Mendelian traits, such as for quantitative, late-onset, or more subtle phenotypes, including behavioral ones, or how to interpret wild-type appearing founders. There is little consideration of appropriate experimental controls (e.g., wild-type or mock-edited animals) or of how many animals might be required to robustly establish genotype-phenotype associations.

(3) The conclusion that this strategy will "dramatically reduce time, resources, and animal numbers" is not quantitatively supported by the data presented and should be expressed more cautiously.

Reviewer #2 (Public review):

Summary:

The authors sought to validate the use of genetic screening pipelines that assess phenotypes in founders (F0, referred to as "crispants") obtained from CRISPR/Cas9 gene editing in 1-cell zygotes. The application of this approach in mice has generally been avoided due to concerns that results would be confounded by genetic mosaicism, but benefits to this approach include reducing animal numbers needed to achieve goals of identifying knockout phenotypes, as well as improved efficiency in the use of time and resources. The authors targeted seven genes associated with visible recessive phenotypes and observed the expected null phenotype in up to 100% of founders for each gene. Although mosaicism was common in the crispants, the various alleles were generally all functional null alleles and, in fact, some in-frame deletions with null phenotypes revealed critical functional motifs within the gene products. The rigorous data presented support using crispants to assess knockout phenotypes when guide RNAs with strong on-target and low off-target scores are used for gene editing in 1-cell mouse embryos.

Strengths:

By targeting multiple genes with existing, well-characterized mutations, the authors established a robust system for validating the analysis of crispants to assess gene function.

Cutting-edge technologies were used to carefully assess the spectrum of mutations generated.

Weaknesses:

There could have been some discussion regarding how this approach would be impacted if mutations are dominant or embryonic lethal (for the latter, for example, F0 can be examined as embryos).

Reviewer #3 (Public review):

Summary:

The study assesses whether CRISPR-generated founder (F0) "crispant" mice can be reliably used for initial phenotypic assessment and screening. By targeting seven genes with known visible recessive phenotypes, the authors show that, despite genetic mosaicism, the expected null phenotypes were observed in all targeted genes. These findings demonstrate that the phenotyping and screening of F0 "crispant" mice is a valid (and efficient) approach to selecting candidate alleles for follow-up studies, thereby streamlining mouse breeding and animal husbandry-related costs.

Strengths:

The study is comprehensive, carefully executed, and provides deep insight into the utility of F0 "crispant" mice for phenotypic screening. The authors evaluated the CRISPR/Cas9 editing outcomes across seven genes using multiple sequencing modalities, providing a robust framework for determining and interpreting complex founder genotypes. Importantly, the study examines/highlights the biological insight gained from compound heterozygous founders and naturally arising allelic series, enabling genotype-phenotype associations and functional inferences about protein domains.

More broadly, the authors' thorough evaluation of the CRISPR/Cas9-based gene editing events in the founders can serve as a benchmark for others in the field, engineering their own mouse "crispants."

Weaknesses:

The relationship between the sgRNA/Cas9 concentrations delivered to the zygotes and the resulting editing efficiencies are not explicitly investigated.

Reviewer #1 (Public review):

Summary:

Alveolar macrophages (AMs) are key sentinel cells in the lungs, representing the first line of defense against infections. There is growing interest within the scientific community in the metabolic and epigenetic reprogramming of innate immune cells following an initial stress, which alters their response upon exposure to a heterologous challenge. In this study, the authors show that exposure to extracellular ATP can shape AM functions by activating the P2X7 receptor. This activation triggers the relocation of the potassium channel TWIK2 to the cell surface, placing macrophages in a heightened state of responsiveness. This leads to the activation of the NLRP3 inflammasome and, upon bacterial internalization, to the translocation of TWIK2 to the phagosomal membrane, enhancing bacterial killing through pH modulation. Through these findings, the authors propose a mechanism by which ATP acts as a danger signal to boost the antimicrobial capacity of AMs.

Strengths:

This is a fundamental study in a field of great interest to the scientific community. A growing body of evidence has highlighted the importance of metabolic and epigenetic reprogramming in innate immune cells, which can have long-term effects on their responses to various inflammatory contexts. Exploring the role of ATP in this process represents an important and timely question in basic research. The study combines both in vitro and in vivo investigations and proposes a mechanistic hypothesis to explain the observed phenotype.

Weaknesses:

The authors have revised the manuscript to address the comments raised during the first round of review. However, several figures, figure legends, and methodological sections still require additional adjustments and clarification.

The interpretation of CFU from lysates as 'killing' is unclear; lysate CFUs typically reflect intracellular surviving bacteria and are confounded by differences in uptake. Please include an uptake control (early time point) or time-course to distinguish phagocytosis from intracellular killing. Also, clarify how bacterial burden was calculated (supernatant vs cell-associated vs total). Supernatant alone may not capture adherent bacteria. The normalization as 'fold killing' (mean negative control / sample) is non-standard; please report absolute CFU (log scale) and specify the exact definition of killing/survival.

The Methods section is largely incomplete and requires substantial revision. For instance, the authors report quantification of cytokine concentrations, yet no information is provided regarding how these measurements were performed. It is unclear whether cytokines were measured in BALF by ELISA, or assessed at the mRNA level by qPCR from total lung lysates, or by another method. This information must be clearly specified. In addition, the rationale for selecting the measured cytokines should be justified. While the choice of IL-1β and IL-6 is relatively straightforward, the focus on IL-18 requires explicit justification.

Similarly, the methodology used to quantify immune cell populations presented in Figure 2 is not described. It is not stated how immune cells were isolated and identified (e.g. flow cytometry from lung tissue). No information is provided regarding tissue digestion, cell isolation procedures, or gating strategy (presumably by flow cytometry). These details are essential and should be included, together with the corresponding gating strategy and absolute cell numbers.

Moreover, immune cell quantification would be expected in the context of the challenge experiment as well. Reporting unchanged percentages of lung immune cells following ATP exposure does not support the conclusion of a training effect, particularly one that is specific to alveolar macrophages (AMs). In addition, AMs are not considered recruited immune cells; this should be corrected in the figure legend and throughout the manuscript where applicable.

There are inconsistencies throughout the manuscript. For example, the authors report n = 5 for the survival curves in the figure legend, whereas n = 7 is stated in the Methods section. This discrepancy is unclear and should be clarified.

Supplementary Fig. 1 contains major conceptual errors. The volcano plot represents ATAC-seq peaks (differentially accessible chromatin regions), yet the figure, legend, and color scale repeatedly refer to 'genes' and 'differentially expressed genes'. This conflates chromatin accessibility with gene expression and is misleading. Peaks are secondarily annotated to nearby genes, which should be clearly described as an annotation step rather than the unit of analysis. The figure should be revised to explicitly present peak-level statistics (DARs), with gene names shown only as optional annotations. Additionally, the use of simultaneous P < 0.05 and Q < 0.05 thresholds is non-standard, and the absence of down-regulated regions in the plot requires explanation.

In Figure 7, trained WT and Nlrp3⁻/⁻ mice display similar levels of bacterial clearance. How should this result be interpreted?

Overall, while the study addresses an interesting biological question, the manuscript would benefit from substantial revision prior to publication. In particular, clarifications and improvements regarding the methodology, data presentation, and interpretation are required to strengthen the rigor and reproducibility of the conclusions.

Reviewer #2 (Public review):

Summary:

In this manuscript, Thompson et al. investigate the impact of prior ATP exposure on later macrophage functions as a mechanism of immune training. They describe that ATP training enhances bactericidal functions which they connect to the P2x7 ATP receptor, Nlrp3 inflammasome activation, and TWIK2 K+ movement at the cell surface and subsequently at phagosomes during bacterial engulfment. This is an incremental addition to existing literature, which has previously explored how ATP alters TWIK2 and K+, and linked it to Nlrp3 activation. The novelty here is in discovering the persistence of TWIK2 change and exploring the impact this biology may have on bacterial clearance. Additional experiments could strengthen their hypothesis that the in vivo protective effect of ATP-training on bacterial clearance is mediated by alveolar macrophages.

Strengths:

The authors demonstrate three novel findings: 1) prolonged persistence of TWIK2 at the macrophage plasma membrane following ATP that can translocate to the phagosome during particle engulfment, 2) a persistent impact of ATP exposure on remodeling chromatin around nlrp3, and 3) administering mice intra-nasal ATP to 'train' lungs protects mice from otherwise fatal bacterial infection.

Weaknesses:

(1) Some methods remain unclear including the timing and method by which lung cellularity was assessed in Figure 2. It is also difficult to understand how many mice were used in experiments 1, 2 and 6 and thus how rigorous the design was. A specific number is only provided for 1D and the number of mice stated in legend and methods do not match.

(2) The study design is not entirely ideal for the authors' in vivo question. Overall, the discussion would benefit from a clear summary of study caveats, which are primarily that that 1) in vitro studies attributing ATP training-mediated bacterial killing to persistent TWIK2 relocation, K+ influx, a glycolytic metabolic shift , and epigenetic nlrp3 reprogramming were performed in BMDM or RAW cells and not primary AMs, 2) data does not eliminate the possibility that non-AM immune or non-immune cells in the lung are "trained" and responsible for ATP-mediated protection in vivo; flow data examined total lung digest which may obscure important changes in alveolar recruitment, and 3) in vivo work shows data on acute bacterial clearance but does not explore potential risks that "training" for a more responsive inflammasome may have for the severity of lung injury during infection.

(3) The is some lack of transparency on data and rigor of methods. Clear data is not provided regarding the RNA-sequencing results. Specific identities of DEGs is not provided, only one high-level pathway enrichment figure. It would also be ideal if controls were included for subcellular fractionating to confirm pure fractions and for dye microscopy to show negative background.

(4) In results describing 5A, the text states that "ATP-induced macrophage training effects, as measured by augmented bactericidal activity, were diminished in macrophages treated with protease inhibitors". However, these data are not identified significant in the figure; protease dependence can be described as a trend that supports the authors' hypothesis but should not be stated as significant data in text.

In summary, this work contains some useful data showing how ATP can train macrophages via TWIK2/Nlrp3. Revisions have significantly improved methods reporting, added some data to strengthen the conclusions, and toned down on overstatements to bring conclusions more in line with data presented. The title still overstates what the authors have actually tested, since no macrophage-specific targeting in vivo (no conditional gene deletion, macrophage depletion etc) was performed in infection studies. However, in vitro data provide clear evidence that macrophages can be trained by ATP, and through caveats remain, it is plausible that macrophage training is a key mechanism for the protection observed here in the lung.

Reviewer #1 (Public review):

Summary

The authors set out to define the genomic distribution and potential functional associations of acetylation of histone H3 lysine 115 (H3K115ac), a poorly characterized modification located in the globular domain of histone H3. Using native ChIP-seq and complementary genomic approaches in mouse embryonic stem cells and during differentiation to neural progenitor cells, they report that H3K115ac is enriched at CpG island promoters, active enhancers, and CTCF binding sites, where it preferentially localizes to regions containing fragile or subnucleosomal particles. These observations suggest that H3K115ac marks destabilized nucleosomes at key regulatory elements and may serve as an informative indicator of chromatin accessibility and regulatory activity.

Strengths

A major strength of this study is its focus on a histone post-translational modification in the globular domain, an area that has received far less attention than histone tail modifications despite strong evidence from structural and in vitro studies that such marks can directly influence nucleosome stability. The authors employ a wide range of complementary genomic analyses-including paired-end ChIP-seq, fragment size-resolved analyses, contour (V-) plots, and sucrose gradient fractionation-to consistently support the association of H3K115ac with fragile nucleosomes across promoters, enhancers, and architectural elements. The revised manuscript is careful in its interpretation and provides a coherent and internally consistent picture of how H3K115ac differs from other acetylation marks such as H3K27ac and H3K122ac. The datasets generated will be valuable to the chromatin community as a resource for further exploration of nucleosome dynamics at regulatory elements.

Weaknesses

The conclusions are largely correlative. While the authors provide strong evidence for the localization of H3K115ac to fragile nucleosomes, the work does not directly test whether this modification causally contributes to nucleosome destabilization or regulatory function in vivo. Questions regarding the enzymes responsible for depositing or removing H3K115ac and its direct functional consequences therefore remain open.

Overall assessment and impact

Overall, the authors largely achieve their stated aims by providing a detailed and well-supported characterization of H3K115ac distribution in mammalian chromatin and its association with fragile nucleosomes at regulatory elements. While mechanistic insight remains to be established, the study introduces a compelling new perspective on globular-domain histone acetylation and highlights H3K115ac as a potentially useful marker for identifying functionally important regulatory regions. The work is likely to stimulate further mechanistic studies and will be of broad interest to researchers studying chromatin structure, transcriptional regulation, and genome organization.

Reviewer #2 (Public review):

Summary:

Kumar et al. aimed to assess the role of the understudied H3K115 acetylation mark, which is located in the nucleosomal core. To this end, the authors performed ChIP-seq experiments of H3K115ac in mouse embryonic stem cells as well as during differentiation into neuronal progenitor cells. Subsequent bioinformatic analyses revealed an association of H3K115ac with fragile nucleosomes at CpG island promoters, as well as with enhancers and CTCF binding sites. This is an interesting study, which provides important novel insights into the potential function of H3K115ac. However, the study is mainly descriptive, and functional experiments are missing.

Strengths:

(1) The authors present the first genome-wide profiling of H3K115ac and link this poorly characterized modification to fragile nucleosomes, CpG island promoters, enhancers, and CTCF binding sites.

(2) The study provides a valuable descriptive resource and raises intriguing hypotheses about the role of H3K115ac in chromatin regulation.

(3) The breadth of the bioinformatic analyses adds to the value of the dataset

Comments on revisions:

The authors sufficiently addressed my concerns.

Reviewer #3 (Public review):

Summary:

Kumar et al. examine the H3K115 epigenetic mark located on the lateral surface of the histone core domain and present evidence that it may serve as a marker enriched at transcription start sites (TSSs) of active CpG island promoters and at polycomb-repressed promoters. They also note enrichment of the H3K115ac mark is found on fragile nucleosomes within nucleosome-depleted regions, on active enhancers and CTCF bound sites. They propose that these observations suggest that H3K115ac contributes to nucleosome destabilization and so may servers a marker of functionally important regulatory elements in mammalian genomes.

Strengths:

The authors present novel observations suggesting that acetylation of a histone residue in a core (versus on a histone tail) domain may serve a functional role in promoting transcription in CPG islands and polycomb-repressed promoters. They present a solid amount of confirmatory in silico data using appropriate methodology that supports the idea that H3K115ac mark may function to destabilize nucleosomes and contribute to regulating ESC differentiation. These findings are quite novel.

Weaknesses:

Additional experiments to confirm specificity of the antibodies used have been done, improving confidence in the study.

Reviewer #1 (Public Review):

This study by Ryu et al, provides compelling evidence to demonstrate the distributions of Oxt and Oxtr in the murine brain using an advanced RNAscope technique. Detailed information on the distributions was provided, revealing differences in Oxt and Oxtr expressions between males and females. This study will provide a new platform for investigators to study previously unknown roles of brain-region specific Oxt and Oxtr neurons and signaling in animal behaviors and metabolism, and others.

Reviewer #2 (Public Review):

This an exciting study investigating the role of OXT in central nervous system (CNS) regulation of different behaviors and physiological processes. The study clearly shows the expression level of Oxt and Oxtr in different brain nuclei and regions.

Sex differences in Oxt expression are also well demonstrated.

Extensions of OXT's function in CNS regulation are sufficiently discussed.

Overall, this provides a good direction for further investigate OXT's role in CNS's regulation on different behaviors and physiological processes.

Reviewer #1 (Public Review):

This study extends a previous study by the same group on the generalization of odor discrimination from one nostril to the other. In their earlier study, the group showed that learning to discriminate between two enantiomers does not generalize across nostrils. This was surprising given the Mainland & Sobel 2001 study that found that detecting androstenone in people who do not detect it can generalize across the two nostrils. In this study, they confirmed their previous results and reported that, unlike enantiomers, learning to discriminate odor mixtures generalizes across nostrils, generalizes to other odor mixtures, and is persistent over at least two weeks. This interesting and important result extends our knowledge of this phenomenon and will likely steer more research. It may also help develop new training protocols for people with impairments in their sense of smell.

The main weakness of this study is its scope, as it does not provide substantial insight into why the results differ for enantiomers and why training on odor mixtures generalizes to other odor mixtures.

Reviewer #2 (Public Review):

The manuscript from Chang et al. taps on an important issue in olfactory perceptual plasticity, named the generalization of perceptual learning effect by training using odors. They employed a discrimination training/learning task with either binary odor mixture or odor enantiomers, and tested for post-training effect at several time intervals. Their results showed contrasting patterns of specificity (enantiomers) and transfer (odor mixtures), and the learning effect persisted at 2 weeks post-training. They demonstrated that the effect was independent of task difficulty, olfactory adaptation and gender.

Overall this was a well-controlled study and shows novel results. The strength of the study includes the consideration of odor structure and perceptual (dis)similarity and the control training condition. I have two minor issues that hope the authors could address in the next version of the manuscript.

1) The author used a binary odor mixture with a ration 7:9 or 9:11, why is this ratio chosen and used for the experiment?

2) Over the course of training, has the valence of odor (odor mixture) changed, it would be helpful to include these results in the supplements. As the author indicated in the discussion, the potential site underlying the transfer effect is the OFC, which has been found to represent odor valence previously (Anderson, Christoff et al. 2003). It would be nice to see the author replicate the results with odor/odor mixture valence (change) controlled.

Anderson, A. K., K. Christoff, I. Stappen, D. Panitz, D. G. Ghahremani, G. Glover, J. D. Gabrieli and N. Sobel (2003). "Dissociated neural representations of intensity and valence in human olfaction." Nat Neurosci 6(2): 196-202.

Reviewer #1 (Public Review):

The authors tested the hypothesis that at high elevations avian eggs will be adapted to prevent desiccation that might arise from loss of water to surrounding drier air. They used a combination of gas diffusion experiments and scanning electron microscopy to examine water vapour conductance rates and eggshell structure, including thickness, pore size, and pore density among 197 bird species distributed along an elevational gradient in the Andes. While there was a correlation between water vapour conductance and elevation among species, a decrease in water vapour conductance with elevation was not associated with eggshell thickness, pore size, and pore density, suggesting the variation in the structure of the eggshells is unlikely to do with among species differences in water loss along elevational gradients. This study is very interesting and timely, especially with increasing water vapour pressure due to climate warming. It is a very well-written study and easy to read. However, I have some concerns about the conclusions drawn from the results.

There are more than twice as many species in low and medium-elevation sites compared to high-elevation sites, so the amount of variation in low and medium-elevation should be expected to be higher by default. The argument for a wider range of variation in low-elevation species will be stronger if the comparison was a similar sample size. Moreover, the pattern clearly breaks down within families. Note also that for Low and medium elevation there is no difference in the amount of variation in conductance residuals possibly because the sample sizes are similar. The seemingly strong positive correlation between eggshell conductance and egg mass may be driven by the five high and two medium-elevation species with large eggs. There seem to be hardly any high-elevation species with egg mass greater than 12g whereas species in low elevation egg size seem to be as high as 80g (Figure 2a). Since larger eggs (and thus eggs of larger birds) lose more water compared to smaller eggs, the correlation between water vapour conductance and elevation may be more strongly associated with body size distribution along elevational gradients rather than egg structure and function.

Authors argue that the observed variation in the relationship between water vapour conductance and elevation among and within bird families suggests potential differences in the adaptive response to common selective pressures in terms of eggshell thickness and pore density, and size. The evidence for this is generally weak from the data analyses because the decrease in water vapour conductance with elevation was not consistent across taxonomic groups nor were differences associated with specific patterns in eggshell thickness and pore density, and size.

It is not clear how the authors expected the relationship between water vapour conductance and elevation to differ among taxonomic groups and there was no attempt to explain the biological implication of these differences among taxonomic groups based on the specific traits of the species or their families. This missing piece of information is crucial to justify the argument that differences among taxonomic groups may be due to differences in adaptive response.

Reviewer #2 (Public Review):

Many tropical montane species live only within narrow elevational ranges. Rapid climate change has led to considerable interest in determining whether these narrow elevational ranges are the result of physiological specialization: if so, then warming temperatures will have direct fitness consequences. Thus far, studies of tropical montane ectotherms have often found patterns consistent with physiological specialization, while the few field studies of tropical montane birds (endotherms) have not. However, these few studies measured the thermal physiology of adult birds. The early life stages of birds may show physiological specialization, as eggs and nestlings function as ectotherms.

In this paper, Ocampo and colleagues provide the first test of the hypothesis that bird eggs are physiologically specialized to the climatic conditions of certain elevational zones. They use experiments and observations to measure water vapor conductance rates and eggshell traits in a diverse set of 197 species that live from the lowland Amazon to the high Andes. Ocampo and colleagues present two principal results: (1) High-elevation eggs lose less water over time than do low-elevation eggs, high elevations tend to be less humid than low elevations and (2) Eggshell traits do not show consistent patterns along the elevational gradient. The pattern in water loss is consistent with the hypothesis that high-elevation eggs are physiologically specialized for the slightly drier environments they experience. The finding that eggshell traits did not vary with elevation, however, means that the pattern of water loss is not driven by single eggshell traits (thicker eggshells could reduce water loss rates, as could fewer or smaller eggshell pores).

This paper represents a strong advance for two main reasons. First, it provides evidence that egg physiology varies with elevation as predicted by the hypothesis that eggs are physiologically adapted to certain climatic conditions. This means egg physiological adaptation is a factor that could influence species' elevational ranges. Second, it is a proof-of-concept study that shows it is possible to measure eggshell physiology for a large number of species in the field in order to test hypotheses. As such, it should inspire many further tests that examine adaptation in egg physiology in the context of species' distributions along environmental gradients.

There are two caveats that readers should be aware of. First, measuring these traits is difficult, and there remain questions about the efficacy of different methods. For example, the authors note that quantifying eggshell structures is very difficult, with several unresolved questions about their method of using scanning electron microscopy images to measure eggshell pores. Similarly, the authors mention that temperature variation may partially influence their main result that high-elevation eggs lose water at slower rates than low-elevation eggs (temperatures were colder for experiments at high elevations than for low elevations). Second, I regard the analyses of eggshell traits for specific families as exploratory. There are no a priori expectations for how different families might be expected to differ in their patterns. These analyses are fruitful in that they generate additional hypotheses that future work can test. However, it does mean that the statistical significance of eggshell trait relationships with elevation for specific families should be interpreted with caution.

Reviewer #1 (Public Review):

This study aims to develop a new system to analyze genetic determinants of neutrophil function by large-scale genetic screens. For that, the authors use a genetically-engineered ER-Hoxb8 neutrophil progenitor line that expresses Cas9 to perform CRISPR screens and to identify genes required for neutrophil survival and differentiation.

A main strength of this study is that the authors develop a pooled CRISPR sgRNA library applicable to neutrophils and show potential determinants of neutrophil differentiation in vitro using this screening methodology.

A main weakness of this study is that identified candidates associated with neutrophil differentiation, as those indicated in Fig. 4A, were not validated in vivo using neutrophil-specific K.O. models or further characterized in vitro (e.g. transcriptional or epigenetic changes during maturation when compared to non-targeting sgRNA controls).

As secondary strengths, the authors provide evidence of efficient gene editing in Cas9+ER-Hoxb8 neutrophils both in vivo and in vitro and provide evidence of the specificity of this methodology using a Cas9+ER-Hoxb8 immortalized cell line that differentiates into macrophages.

In terms of methodology, this study provides a useful tool to explore gene regulatory networks in neutrophils in large-scale genetic screens. However, it falls short in identifying and validating the true potential of this kind of methodology in the biology of the neutrophil.

Moreover, the technical advances in the field are only incremental as several studies, including those using CRISPR/Cas9 technology in Hoxb-8 immortalized neutrophil progenitor cell lines have been already performed.

Reviewer #2 (Public Review):

In this manuscript, Jong et al. provide and validate a very useful resource for performing CRISPR screenings to study neutrophil differentiation and function. The major strength of the paper lies in its careful validation of many aspects of the Hoxb8-immortalized progenitor cells, including their differentiation capacity, their ability to clear bacteria, and their capacity to differentiate in vivo. The authors succeed at this, with results correctly supporting their conclusions. The major weaknesses are its presentation and writing, some of which are poorly organized. Finally, while the potential impact of this resource in the field could be very large, the CRISPR screening results appear half-baked, almost preliminary, and could be better validated, or at least presented. A few other points that warrant revision are included below:

• The introduction should be better constructed and organized. It should be written with more connectors to present facts in a stream that flows naturally, from the broad general facts to the experimental details implemented in the manuscript. It should also discuss other similar approaches used in the literature, such as LaFleur et al. 2019, and relate in which ways these presented methods could be better.

• The scheme in Figure 4A should more clearly indicate the timings, doublings, numbers of cells, and other aspects of the experimental design.

• The volcano plot in Figure 4B is poorly informative and almost redundant. What does one make of it?

• The representation (normalized reads) of each sgRNA in the library and across multiple experiments, including their correlation, should be checked and plotted, to visualize how robust these replicates are.

• In Figure 4E, the distribution of the hit sgRNAs should be compared to all other targeting guides (instead of just to non-targeting controls). Linear density distribution plots or scatter plots of all guides are usually the best way, but there are others (for example, see Figure 4 of LaFleur et al. 2019). Ideally, each independent sgRNA for each gene in the library, as well as biological replicates, should be separately shown, with hits clearly highlighted.

• While in vivo differentiation is shown as possible with these cell lines, it is unclear whether CRISPR screenings could be performed in vivo too. Would sgRNA representation suffice for genome-wide? At least some of the new hits could be validated by testing differentiation in vivo (i.e. WASH complex).

• In the methods section, the RNA-seq analysis pipeline details are missing (versions, software for alignment, quantification, differential gene expression, and enrichment). Also, parameters for MAGeCK and MAGeCKFlute should be explicit and detailed.

• The discussion is mostly a summary of the results. It is lacking in detail and thoughtful discussion regarding novelty and impact beyond the validation of the cell line. What about potential applications? What about extending screenings to test bacterial-killing, as suggested in Figure 2? What about limitations compared to other similar methods out there? There is little discussion of such important potential matters. Also, a large part of the discussion is dedicated to discussing details about Cebpe that are all well known in the literature and add little value.

• Figure legends are typically too succinct and hard to interpret, especially for non-experts. The text should enable the figure reader to correctly interpret what is shown in each panel.

Reviewer #3 (Public Review):

Primary neutrophils are difficult to modify genetically, whereas the generation of knockout mice to study the role of specific proteins is time-consuming and expensive. CRISPR-Cas 9 genetic modification of neutrophil progenitors in vitro offers a platform to study neutrophil biology. Hoxb8 cells are immortalized neutrophil progenitors that differentiate into neutrophils when cultured in the presence of G-CSF, and have been shown to recapitulate the stages of murine neutrophil differentiation. They have also been shown to be amendable to CRISPR-Cas 9 genetic editing with successful deletion of key transcriptional regulators of neutrophil maturation and function. The authors of this manuscript offer an extension to this technique, by generating Hoxb8 cells that constitutively express Cas9. This may reduce the variation between the generated knock-out cells by avoiding the introduction of Cas9 in a plasmid every time together with a guide RNA.

The first part of the manuscript is dedicated to the characterisation of Cas9+HoxB8 cells throughout their differentiation. Considering the existing body of literature on HoxB8 progenitors and their differentiation into neutrophils ex vivo, it does not significantly further our understanding of these cells, but rather provides a good validation to their Cas9+ modified version of them. Gene editing using Cas9+ Hoxb8 progenitors seems to be highly efficient, which is an important technical point, however, it is hard to assess the degree of improvement in efficiency compared to the published protocols with Cas9 delivery in a plasmid.

As a test, the authors use Cas9+HoxB8 progenitor to generate a knockout of CEBPE, known for its important role in neutrophil development. They convincingly demonstrate its impact on HoxB8 cell differentiation, with in vivo reconstitution of wild-type and CEBPE-deficient HoxB8 progenitors into irradiated mice being especially elegant. However, the transfer into different recipient mice assumed no differences in the recipient environment, while immunophenotyping for mature neutrophils within the HoxB8 progenitor-derived cells did not account for possible differences in numbers of wt and CEBPE KO surviving cells, limiting the conclusions.

Finally, the authors put the system to the test by screening a library of Brie gRNA library of ~80K mouse sgRNAs, targeting almost 20K genes with 4 gRNA per gene coverage, to identify genes that are needed for the differentiation of Cas9+ERHoxb8 progenitors into mature neutrophils. They identify a number of hits, amongst which the WASH complex and CEBPE are highlighted. A comparison of cell numbers prior to differentiation and at 4 days post differentiation indicates that they are indeed required for neutrophil survival. To validate the role of these hits in neutrophil maturation itself, as they stated in the aims, i.e. "to identify genes that modulate the differentiation of Cas9+ERHoxb8 progenitors into mature neutrophils", phenotypic, functional, and morphological characterization of these cell lines could have been appropriate.

Overall, this study has the potential to improve on the established lentiviral CRISPR-Cas9 editing of Hoxb8 cells and be valuable for library-screening approaches for neutrophil modulators, which will benefit the community.

Reviewer #4 (Public review):

I maintain that the images in Figure 12 (new Figure 14) do not support the authors' interpretation that 2-cell embryos resulted from in vitro fertilization (IVF) of Amrc-/- rescued sperm. They are clearly not normal 2-cell embryos and instead look very much like fragmented eggs that can be seen occasionally following in vitro fertilization procedures even when that is done with wild type eggs and sperm. The only portion of current Figure 14 that has normal looking 2-cell embryos is in panel 14A4, where wild type B6D2 sperm were used. Even in that panel, there are some fragmented eggs that the authors identify as 2-cell embryos.

The authors offer the explanation that CD1 eggs fertilized by B6D2F1 hybrid male sperm do not develop beyond the 2-cell stage, citing a 2008 paper published in Biology of Reproduction by Fernandez-Goonzalez et al. I read through that paper very carefully and even had a colleague read through it in case I missed something, but that paper says nothing at all about strain incompatibilities, much less 2-cell arrest due to them. The only crosses done in that paper are CD1 eggs x CD1 sperm and B6D2 eggs x B6D2 sperm, all by intracytoplasmic sperm injection and not standard in vitro fertilization. [Note that the paper does mention performing in vitro fertilization but says nothing about how it was done or what mouse strains were used.] I even searched the literature for information regarding incompatibility between these strains and could find nothing relevant. But even if the authors are correct and there happens to be a strain incompatibility and 2-cell arrest is expected, what the authors are calling 2-cell embryos are clearly not.

A second explanation offered by the authors is that they used collagenase to remove the cumulus cells and that this may have affected the appearance of the embryos. This technique is actually used to remove both the cumulus cells and the zona pellucida and has been described as a gentler way to do so than other standard methods (hyaluronidase treatment followed by acid Tyrodes to remove the zona pellucida) (Yamatoya et al., Reprod Med Biol 2011, DOI 10.1007/s12522-011-0075-8). I think it is highly relevant to the current study that the method they used to remove cumulus cells also dissolves the zona, either partially or completely. Given that many of the eggs, fragmented eggs, and 2-cell embryos (from the WT sperm) in Figure 14A are lacking a zona pellucida, it seems very likely that many of the eggs were either zona-free or had partial zona dissolution from the start. In fact, the authors state in the Methods section that "Cumulus-free and zona-free eggs were collected..." for how IVF was done. Partial zona dissolution is standard in some protocols for performing IVF using frozen mouse sperm, which usually have much lower motility and overall efficacy than fresh sperm. In any case, it would improve transparency if the manuscript made clear somewhere other than buried in the Methods that the IVF procedure was done on eggs with partially or fully removed zonas, to allow proper interpretation.

In the rebuttal, the authors go on to state: "To provide additional functional evidence, we complemented the IVF experiments with ICSI using rescued Armc2-/- sperm and B6D2 oocytes, which allowed embryos to develop to the blastocyst stage. In these experiments, 25% of injected oocytes reached the blastocyst stage with rescued sperm compared to 13% for untreated Armc2-/- sperm (Supplementary Fig. 9) These results support the functional competence of rescued sperm and demonstrate partial recovery of fertilization ability following Armc2 mRNA electroporation."

Their conclusion that the data support partial recovery of fertilization ability following Armc2 mRNA electroporation in my opinion has no basis. This experiment was done only once, and no information is provided regarding how many eggs underwent ICSI or how many reached the blastocyst stage. The authors claim that the rescued sperm were better than the Armc2-/- sperm in producing blastocysts, but this is based on a simple percentage report of 25% vs 13% without any statistical analysis, even on the results from the single experiment presented.

Overall, the paper shows rescue of some sperm motility by the new method they use, and the new title is therefore appropriate. The authors have also dealt reasonably with many of the original concerns regarding documenting that their methodology was effective in producing protein (at least the GFP marker) in spermatogenic cells. In my view the authors have, however, not shown any indication of functional recovery over what is already known for the knockout sperm, that ICSI can support blastocyst stage embryo development. They also have not, in my view, justified the claims at the end of the abstract "These motile sperm were able to produce embryos by IVF..." and that "...mRNA electroporation can restore...partially fertilizing ability..."

Reviewer #5 (Public review):

While the study presents an innovative and potentially impactful mRNA-based approach for addressing monogenic causes of male infertility, several significant weaknesses limit the strength of the authors' central conclusions.

First, the functional evidence for true fertility restoration remains incomplete. Although the authors convincingly demonstrate partial recovery of sperm motility, the downstream reproductive outcomes, particularly for IVF, are weak. Importantly, these concerns are shared by all three reviewers and the former Reviewing Editor, and to my eye they are both thoughtfully articulated and well warranted. The ICSI data show modest improvement, but this rescue is difficult to interpret.

In parallel, significant mechanistic questions persist regarding the unusually prolonged persistence of naked mRNA and reporter protein expression in germ cells, which is not fully reconciled with established mRNA and protein half-life biology and is supported largely by inference rather than by direct decay measurements.

Finally, although the authors have conducted additional cellular analyses, concerns about the extent and specificity of germ-cell targeting versus Sertoli-cell expression remain unresolved. Together, these issues do not negate the technical novelty of the work, but they do constrain the confidence with which the current dataset can support the authors' strongest therapeutic claims.

Reviewer #1 (Public Review):

The heterogeneity within the neutrophil population is becoming clear. However, it was not clear if neutrophil progenitors are also heterogenous. Because neutrophils are short-lived, it is technically challenging to tackle the question. This study used a system to isolate and expand clonal neutrophil progenitors (granulocyte-monocyte progenitors; GMPs) to achieve molecular and functional profiling. In the study, transcriptional profiling was performed by RNAseq and ATACseq. Functional assays were performed ex vivo to examine phagocytosis, ROS production, NET formation, and neutrophil swarming using Candida albicans, as well as C. glabrata and C. auris. The strengths of this study include the use of the neutrophil clone system to track GMPs developing into neutrophils. The clone-based approach made it possible to evaluate the functions of multiple neutrophil subpopulations. Limitations of this study include the dependency on ex vivo approaches and the modest degree of heterogeneity within presented neutrophils. Nevertheless, the finding - the heterogeneity of neutrophils can be traced back to the GMP stage - is significant.

Reviewer #2 (Public Review):

The stated goal of the authors is to establish and characterize an experimental system to study neutrophil heterogeneity in a manner that allows for functional outcomes to be probed. To do so, they start with murine GMPs that are conditionally immortalized by ER-HoxB8 expression and make single-cell clonal populations to ask whether those GMPs or neutrophils derived by differentiating such clonal GMPs harbor heterogeneity. At a conceptual level, this is an innovative approach that could shed light on mechanisms of neutrophil heterogeneity that have been described in both health and disease. They perform bulk multi-omics and functional analyses of both the clonal GMPs and neutrophil-like cells, including transcriptional and epigenetic profiling. However, the major weakness of the study is that the authors do not provide rigorous or convincing data that the cells they derive are truly mature neutrophils. To the contrary, the neutrophil-like cells lack Ly6G expression and so the authors fall back on using CD11b as the primary marker for delineating neutrophils; however, CD11b is expressed by both myeloid progenitors and some premature and mature myeloid lineages that are not neutrophils. They acknowledge this shortcoming, but they make an assumption that Ly6G expression is the only way in which the cells they derive are different from primary neutrophils without presenting any evidence indicating such. The authors use only SCF during the maturation of ER-HoxB8 GMPs into leukocytes, rather than including other cytokines such as G-CSF (or use in vivo maturation) that could have better-induced differentiation and maturation into granulocytes/neutrophils. The authors did not use their transcriptional analyses to further establish that the cells they derive from ER-HoxB8 GMPs are similar/different from primary murine neutrophils. Unfortunately, this shortcoming means that all of the analyses of neutrophil-like cells derived from clonal GMPs may or may not represent the transcriptional, epigenetic, etc. profile of a true mature neutrophil. It is also not rigorously addressed whether what they call PMNs derived from clonal GMPs are a transcriptionally uniform population or if they harbor heterogeneity within the bulk population. Overall, while conceptually intriguing and in pursuit of an experimental system that would be impactful for the field, the study as performed has critical flaws.

Reviewer #1 (Public review):

Summary:

In their study the authors investigated the F. graminearum homologue of the Drosophila Misato-Like Protein DML1 for a function in secondary metabolism and sensitivity to fungicides.

Strengths:

Generally, the topic of the study is interesting and timely and the manuscript is well written, albeit in some cases details on methods or controls are missing.

Weaknesses:

However, a major problem I see is with the core result of the study, the decrease of the DON content associated with deletion of FgDML1: Although some growth data are shown in figure 6 - indicating a severe growth defect - the DON production presented in figure 3 is not related to biomass. Also, the method and conditions for measuring DON are not described. Consequently, it could well be concluded that the decreased amount of DON detected is simply due to a decreased growth and specific DON production of the mutant remains more or less the same.

To alleviate this concern, it is crucial to show the details on the DON measurement and growth conditions and to relate the biomass formation on the same conditions to the DON amount detected. Only then a conclusion as to an altered production in the mutant strains can be drawn.

Comments to the revised manuscript:

The authors carefully revised the manuscript and provided explanations for methods in several cases. However, there are still some problems - probably due to misunderstanding - that need revision.

(1) A major problem of the first version of the manuscript was the lack of appropriate description of biomass analysis and the consideration of the respective results for evaluation of production of DON and other metabolites. Although the authors provide some explanation in the response to reviews, I could not find a corresponding explanation or description in the manuscript. It is not sufficient to explain the problem to me, but a detailed explanation and description of the method has to be provided in the manuscript along with the definition of one "unit of mycelium". It is still not entirely clear to me what such a "unit of mycelium" is.

Please clarify this and any other uncertainties that were commented on by me and other reviewers in the manuscript, not only in the response to reviews. Also adjust the reference list accordingly.

(2) Another problem was, that the authors considered FgDML1 a regulator of DON production. As mentioned by me and reviewer 3, FgDML1 is crucial to numerous functions in F. graminearum and its lack causes a plethora of problems for fungal physiology. Hence, although it is clear that the lack of FgDML1 causes alterations in DON production, it is not appropriate to designate this factor as a "regulator".<br /> It seems to me that the authors are afraid that if FgDML1 would not be a "regulator" that this would decrease the value of their study, which is not the case. This is a matter of correct wording. Therefore, please revise the wording accordingly, starting with the title:

...FgDML1 impacts DON toxin biosynthesis...

Moreover, for sure the manuscript might benefit from more detailed description of the whole cascade leading from FgDML1 to DON biosynthesis and production of the other metabolites that change upon deletion. Such explanation can help the reader grasp the relevance of FgDML for regulatory processes as well as on more general versus specific effects.

Reviewer #2 (Public review):

Summary:

The manuscript entitled "Mitochondrial Protein FgDML1 Regulates DON Toxin Biosynthesis and Cyazofamid Sensitivity in Fusarium graminearum by affecting mitochondrial homeostasis" identified the regulatory effect of FgDML1 in DON toxin biosynthesis and sensitivity of Fusarium graminearum to cyazofamid. The manuscript provides a theoretical framework for understanding the regulatory mechanisms of DON toxin biosynthesis in F. graminearum and identifies potential molecular targets for Fusarium head blight control. The paper in innovative, but there are issues in the writing that need to be added and corrected.

Comments on revisions:

The author has addressed my questions.

Reviewer #2 (Public review):

This paper proposes two changes to classic RSA, a popular method to probe neural representation in neuroimaging experiments: computing RSA at row/column level of RDM, and using linear mixed modeling to compute second level statistics, using the individual row/columns to estimate a random effect of stimulus. The benefit of the new method is demonstrated using simulations and a re-analysis of a prior fMRI dataset on object perception and memory encoding.

The author's claim that tRSA is a promising approach to perform more complete modeling of cogneuro data, and to conceptualize representation at the single trial/event level (cf Discussion section on P42), is appealing.

In their revised manuscript, the authors have addressed some previous concerns, now referencing more literature aiming to improve RSA and its associated statistical inferences, and providing more guidance on methodological considerations in the Discussion. However, I wish the authors had more extensively edited the Introduction to better contextualize the work and clarify the specific settings in which they see the method as being beneficial over classic RSA. For example, some of the limitations of cRSA mentioned on page 6, e.g. related to presenting the same stimuli to multiple subjects, seem to be quite specific to settings where the researcher expects differential responses across subjects to fundamentally alter the interpretation, rather than something that will just average out by repeatedly offering the same stimulus, or combining data across subjects. It's not clear to me how the switch from 'matrix-level' to 'row-level' analysis in tRSA necessarily addresses this problem. I would be very helpful if the authors would more explicitly outline what problem the row-level aspect of tRSA is solving; what problem statistical inference via LMM is solving; and walk the reader through a very specific use case (perhaps a toy version of the real-data experiment which is now at the end of the paper). Explaining the utility of tRSA for experimental settings in which assessing representational strength for a single-events is crucial would clarify the contribution of this new method better.

A few weaknesses mentioned in my previous review were not adequately addressed. To demonstrate the utility of the method on real neural recordings, only a single dataset is used with a quite complicated experimental design; it's not clear if there is any benefit of using tRSA on a simpler real dataset. Moreover, the cells of an RDM/RSM reflect pairwise comparisons between response patterns. Because the response patterns are repeatedly compared, the cells of this matrix are not independent of one another. While the authors show examples that failure to meet independence assumptions do not affect results in their specific dataset, it does not get acknowledged as a problem at a more fundamental level. Finally, while the paper now states that 'simulations and example tRSA code' are publicly available, the link points to the lab's general github page containing many lab repositories, in which I could not identify a specific repository related to this paper. This is disappointing given that the main goal of this manuscript is to provide a new method that they encourage others to use; a clear pointer to available code is only a minimal requirement to achieve that goal. A dedicated repository, including documentation, READMEs and tutorials/demo's to run simulations, compare methods, etc. would greatly enhance the paper's contribution.

Reviewer #1 (Public review):

In this manuscript, Dillard and colleagues integrate cross-species genomic data with a systems approach to identify potential driver genes underlying human GWAS loci and establish the cell type(s) within which these genes act and potentially drive disease.

Specifically, they utilize a large single cell RNA-seq (scRNA-seq) dataset from an osteogenic cell culture model - bone marrow-derived stromal cells cultured under osteogenic conditions (BMSC-OBs) - from a genetically diverse outbred mouse population called the Diversity Outbred (DO) stock to discover network driver genes that likely underlie human bone mineral density (BMD) GWAS loci. The DO mice segregate over 40M single nucleotide variants, many of which affect gene expression levels, therefore making this an ideal population for systems genetic and co-expression analyses.

The current study builds on previous published work from the same group that used co-expression analysis to identify co-expressed "modules" of genes that were enriched for BMD GWAS associations. In this study, the authors utilized a much larger scRNA-seq dataset from 80 DO BMSC-OBs, inferred co-expression based on Bayesian networks for each identified mesenchymal cell type, focused on networks with dynamic expression trajectories that are most likely driving differentiation of BMSC-OBs, and then prioritized genes ("differentiation driver genes" or DDGs) in these osteogenic differentation networks that had known expression or splicing QTLs (eQTL/sQTLs) in any GTEx tissue that co-localized with human BMD GWAS loci. The systems analysis is impressive, the experimental methods are described in detail, and the experiments appear to be carefully done. The computational analysis of the single cell data is comprehensive and thorough, and the evidence presented in support of the identified DDGs, including Tpx2 and Fgfrl1, is for the most part convincing. Some limitations in the data resources and methods hamper enthusiasm somewhat and are discussed below.

Overall, while this study will no doubt be valuable to the BMD community, the cross-species data integration and analytical framework may be more valuable and generally applicable to the study of other diseases, especially for diseases with robust human GWAS data but for which robust human genomic data in relevant cell types is lacking.

Specific strengths of the study include the large scRNA-seq dataset on BMSC-OBs from 80 DO mice, the clustering analysis to identify specific cell types and sub-types, the comparison of cell type frequencies across the DO mice, and the CELLECT analysis to prioritize cell clusters that are enriched for BMD heritability (Figure 1). The network analysis pipeline outlined in Figure 2 is also a strength, as is the pseudotime trajectory analysis (results in Figure 3).

Potential drawbacks of the authors' approach include their focus on genes that were previously identified as having an eQTL or sQTL in any GTEx tissue. The authors rightly point out that the GTEx database does not contain data for bone tissue, but reason that eQTLs can be shared across many tissues - this assumption is valid for many cis-eQTLs, but it could also exclude many genes as potential DDGs with effects that are specific to bone/osteoblasts. Indeed, the authors show that important BMD driver genes have cell-type specific eQTLs. Another issue concerns potential model overfitting in the iterativeWGCNA analysis of mesenchymal cell type-specific co-expression, which identified an average of 76 co-expression modules per cell cluster (range 26-153). Based on the limited number of genes that are detected as expressed in a given cell due to sparse per cell read depth (400-6200 reads/cell) and drop outs, it's surprising that as many as 153 co-expression modules could be distinguished within any cell cluster. I would suspect some degree of model overfitting is responsible for these results.

Overall, though, these concerns are minor relative to the many strengths of the study design and results. Indeed, I expect the analytical framework employed by the authors here will be valuable to -- and replicated by -- researchers in other disease areas.

Comments on revisions:

Thank you for addressing my concerns. This is an impressive study and manuscript that you should be proud of.

Reviewer #2 (Public review):

Summary:

In this manuscript, Farber and colleagues have performed single cell RNAseq analysis on bone marrow derived stem cells from DO Mice. By performing network analysis, they look for driver genes that are associated with bone mineral density GWAS associations. They identify two genes as potential candidates to showcase the utility of this approach.

Strengths:

The study is very thorough and the approach is innovative and exciting. The manuscript contains some interesting data relating to how cell differentiation is occurring and the effects of genetics on this process. The section looking for genes with eQTLs that differ across the differentiation trajectory (Figure 4) was particularly exciting.

Weaknesses:

The manuscript is, in parts, hard to read due to the use of acronyms and there are some questions about data analysis that still need to be addressed.

Comments on revisions:

Dillard et al have made several improvements to their manuscript.

(1) We previously asked the authors to determine whether any cell types were enriched for BMD-related traits since the premise of the paper is that 'many genes impacting BMD do so by influencing osteogenic differentiation or ... adipogenic differentiation'. Given the potential for the cell culture method to skew the cell type distribution non-physiologically, it is important to establish which cell types in their assay are most closely associated with BMD traits. The new CELLECT analysis and Figure 1E address this point nicely. However, it would still be nice to see the correlations between these cell types and BMD traits in the mice as this would provide independent evidence to support their physiological importance more broadly.

(2) Shortening the introduction.

(3) Addressing limitations that arise from not accounting for founder genome SNPs when aligning scRNA-seq data.

(4) The main take-away of this paper is, to us, the development of a single cell approach to studying BMD-related traits. It is encouraging that the cells post-culture appear to be representative of those pre-culture (supplemental figure 3).

However, the authors seem to have neglected several comments made by both reviewers. While we share the authors' enthusiasm for the single cell analytical approach, we do not understand their reluctance to perform further statistical tests. We feel that the following comments have still not been addressed:

(1) The manuscript still contains the following:

"To provide further support that tradeSeq-identified genes are involved in differentiation, we performed a cell type-specific expression quantitative trait locus (eQTL) analysis for each mesenchymal cell type from the 80 DO mice. We identified 563 genes (eGenes) regulated by a significant cis-eQTL in specific cell types of the BMSC-OB scRNA-seq data (Supplementary Table S14). In total, 73 eGenes were also tradeSeq-identified genes in one or more cell type boundaries along their respective trajectories (Supplementary Table S9)."

The purpose of this paragraph is to convince readers that the eGenes approach aligns with the tradeSeq approach (and that their approach can therefore be trusted). It is essential that such claims are supported by statistical reasoning. Given that it would be very simple to perform permutation/enrichment analyses to address this point, and both reviewers requested similar analyses, we do not understand the author's reluctance here. Otherwise, this section should be rewritten so that it does not imply that the identification of these genes provides support for their approach.

(2) Given that a central purpose of this manuscript is to establish a systematic workflow for identifying candidate genes, the manuscript could still benefit from more explanation as to why the authors chose to highlight Tpx2 and Fgfrl1. Tpx2 does already have a role in bone physiology through the IMPC. The authors should comment on why they did not explore Kremen1, for instance, as this gene seems important for the transition to both OB1 and 2.

A final minor comment is that it would be very helpful if the authors could indicate if the DDGs in Table 1 are also eGenes for the relevant cell type. This is much more meaningful than looking through GTEx.

Reviewer #1 (Public review):

Summary:

In this fMRI study, the authors wished to assess neural mechanisms supporting flexible temporal construals. For this, human participants learned a story consisting of fifteen events. During fMRI, events were shown to them, and participants were instructed to consider the event from "an internal" or from "an external" perspective. The authors found distinct patterns of brain activity in the posterior parietal cortex (PPC) and anterior hippocampus for the internal and the external viewpoint. Specifically, activation in the posterior parietal cortex positively correlated with distance during the external-perspective task, but negatively during the internal-perspective task. The anterior hippocampus positively correlated with distance in both perspectives. The authors conclude that allocentric sequences are stored in the hippocampus, whereas egocentric sequences are supported by the parietal cortex.

Strengths:

The research topic is fascinating, and very few labs in the world are asking the question of how time is represented in the human brain. Working hypotheses have been recently formulated, and the work tackles them from the perspective of construals theory.

Weaknesses:

Although the work uses two distinct psychological tasks, the authors do not elaborate on the cognitive operationalization the tasks entail, nor the implication of the task design for the observed neural activation.

Reviewer #2 (Public review):

Summary:

Xu et al. used fMRI to examine the neural correlates associated with retrieving temporal information from an external compared to internal perspective ('mental time watching' vs. 'mental time travel'). Participants first learned a fictional religious ritual composed of 15 sequential events of varying durations. They were then scanned while they either (1) judged whether a target event happened in the same part of the day as a reference event (external condition); or (2) imagined themselves carrying out the reference event and judged whether the target event occurred in the past or will occur in the future (internal condition). Behavioural data suggested that the perspective manipulation was successful: RT was positively correlated with sequential distance in the external perspective task, while a negative correlation was observed between RT and sequential distance for the internal perspective task. Neurally, the two tasks activated different regions, with the external task associated with greater activity in the supplementary motor area and supramarginal gyrus, and the internal condition with greater activity in default mode network regions. Of particular interest, only a cluster in the posterior parietal cortex demonstrated a significant interaction between perspective and sequential distance, with increased activity in this region for longer sequential distances in the external task but increased activity for shorter sequential distances in the internal task. Only a main effect of sequential distance was observed in the hippocampus head, with activity being positively correlated with sequential distance in both tasks. No regions exhibited a significant interaction between perspective and duration, although there was a main effect of duration in the hippocampus body with greater activity for longer durations, which appeared to be driven by the internal perspective condition. On the basis of these findings, the authors suggest that the hippocampus may represent event sequences allocentrically, whereas the posterior parietal cortex may process event sequences egocentrically.

Strengths:

The topic of egocentric vs. allocentric processing has been relatively under-investigated with respect to time, having traditionally been studied in the domain of space. As such, the current study is timely and has the potential to be important for our understanding of how time is represented in the brain in the service of memory. The study is well thought out and the behavioural paradigm is, in my opinion, a creative approach to tackling the authors' research question. A particular strength is the implementation of an imagination phase for the participants while learning the fictional religious ritual. This moves the paradigm beyond semantic/schema learning and is probably the best approach besides asking the participants to arduously enact and learn the different events with their exact timings in person. Importantly, the behavioural data point towards successful manipulation of internal vs. external perspective in participants, which is critical for the interpretation of the fMRI data. The use of syllable length as a sanity check for RT analyses as well as neuroimaging analyses is also much appreciated.

Suggestions:

The authors have done a commendable job addressing my previous comments. In particular, the additional analyses elucidating the potential contribution of boundary effects to the behavioural data, the impact of incorporating RT into the fMRI GLMs, and the differential contributions of RT and sequential distance to neural activity (i.e., in PPC) are valuable and strengthen the authors' interpretation of their findings.

My one remaining suggestion pertains to the potential contribution of boundary effects. While the new analyses suggest that the RT findings are driven by sequential distance and duration independent of a boundary effect (i.e., Same vs. Different factor), I'm wondering whether the same applies to the neural findings? In other words, have the authors run a GLM in which the Same vs. Different factor is incorporated alongside distance and duration?

Reviewer #1 (Public review):

In this study, the authors provide an integrated proteogenomics pipeline to enable the discovery of novel peptides in an Ewing sarcoma cell line (A673). To identify novel full-length resolved isoforms, they performed long-read RNA sequencing (Oxford Nanopore Technology). Then, to increase the chance of detecting Ewing-specific neopeptides, the authors combined two approaches: a multi-protease digestion and a multi-dimensional proteomics approach.

Given the importance of novel isoforms and cryptic sites in neoantigen discovery and its putative applications in immunotherapy, this method and resource paper are of interest for the Ewing community and potentially for a broader cancer audience. The originality of this paper relies mostly on this optimized method to discover novel peptides (long-read sequencing with multiprotease, multi-dimensional trapped ion mobility spectrometry parallel accumulation-serial fragmentation mass spectrometry). Although, to my knowledge, no study combining long-read sequencing and proteomics methods has been published on Ewing Sarcoma, this study appears limited by a few aspects:

(1) The study is restricted to the analysis of a single cell line (A673). The authors should consider extending the analysis to other Ewing cell lines.

(2) The characterization of the 1121 non-canonical transcripts can be improved. How many are just splice variants of known genes, and how many are bona fide neogenes? In this respect, the definition of what the authors call neogene is quite unclear. Is a transcript with a new exon reported as a neogene? Is a transcript with a new start site reported as a neogene? It should be clearly indicated which categories of Figure 4B are reported on Figure 4D. A general flow chart would be very useful to help follow the analysis process.

(3) Similarly, the authors detect 3216 A673 specific proteins with no match in SwissProt. This number decreases to 72 "putative non-canonical proteoforms with unique peptides after BLASTp" against Uniprot. Again, a flow chart would conveniently enable one to follow the step-by-step analysis.

(4) Finally, only 17 spectral matches are suggested to be derived from non-canonical proteoforms. It would be important to compare the spectrum of these detected peptides with that of synthetic peptides. Such an analysis would enable us to assess the number of reliably detected proteoforms that can be expected in an Ewing sarcoma cell line.

(5) It is very unclear what the authors want to highlight in Supplementary Figure 5. Is it that non-canonical transcripts are broadly expressed in normal tissue? Which again raises the question of definitions of neogenes, non-canonical... Apparently, this figure shows that these non-canonical transcripts contain a large part of canonical sequences, which account for the strong signal in many normal tissues. A similar heatmap could be presented, including only the non-canonical sequences of the non-canonical transcripts. This figure should also include Ewing sarcoma samples.

Reviewer #2 (Public review):

The paper from Kulej et al. reports a set of tools for proteogenomic analysis of cancer proteomes. Their approach utilizes modern methods in long-read RNA sequencing to assemble a proteome database that is specific to Ewing sarcoma-derived A673 cells. To maximize proteome coverage and therefore increase the odds of detecting cancer-specific alterations at the protein level, the authors use multiple enzymes (trypsin, gluC, etc.) to digest cellular proteins and then perform multidimensional peptide fractionation. Peptide samples are then analyzed by LC-MS/MS using data-dependent and data-independent schemes on a timstof mass spectrometer. Proteogenomics is an important area of investigation for cancer research and does require new informatics tools.

The authors describe an end-to-end workflow where they claim to have optimized four different steps:

(1) Assembly of a sample-specific protein database using long-read transcriptomic data.

(2) Use of 8 different proteolytic enzymes to maximize diversity of peptides.

(3) Multiple stages of peptide fractionation using SCX and high pH rp chromatography.

(4) Utilize acquisition methods on the timstof mass spec to provide MS/MS data from single-charged peptides and multiply-charged peptides.

The authors published two earlier versions of ProteomeGenerator (versions 1 and 2) in the Journal of Proteome Research. In these earlier versions, 'ProteomeGenerator' was the set of software tools designed to integrate DNA and RNA sequencing to create a sample-specific protein database. To test the performance of each ProteomeGenerator version, the authors generated LC-MS/MS data using a combination of trypsin and LysC, then in the other paper, trypsin, LysC, and GluC. In both papers, they performed some levelof peptide fractionation prior to LC-MS/MS. They acquired LC-MS/MS data on a Thermo Q-Exactive in one paper and a Thermo Orbitrap mass spec in the other paper.

In the current paper, the primary innovation is the use of long-read sequencing to potentially improve the quality of the sample specific protein database. The other three components noted above are incremental compared to the authors' previous two papers and generally accepted practices in the field of proteomics. To note one example, the authors previously digested proteins using three enzymes and now use eight. Similarly, they are now using a timstof Bruker mass spec instead of one from Thermo. The detailed descriptions around the use of many enzymes and peptide fractionation, etc., create a very technically oriented paper, similar to or more so than the authors' earlier papers in J. Proteome Research. So, while there is enthusiasm for the use of long-read sequencing across biomedical research, the impact here for proteogenomic applications is somewhat lost with all of the technical description for experimental details that are not particularly innovative. In this respect, the report is not well matched to a broad readership.

Reviewer #1 (Public review):

Summary:

This manuscript investigates how herbivorous insects, specifically whiteflies and planthoppers, utilize salivary effectors to overcome plant immunity by targeting the RLP4 receptor.

Strengths:

The authors present a strong case for the independent evolution of these effectors and provide compelling evidence for their functional roles.

Reviewer #2 (Public review):

Summary:

The authors tested an interesting hypothesis that white flies and planthoppers independently evolved salivary proteins to dampen plant immunity by targeting a receptor-like protein. Unlike previously reported receptor-like proteins with large ligand-binding domains, the NtRLP4 here has a malectin LRR domain. Interestingly, it also associates with the adaptor SOBIR1. While the function of this protein remains to be further explored, the authors provide strong evidence showing it's the target of salivary proteins as the insects' survival strategy.

Major points:

The authors mixed the concepts of LRR-RLPs with malectin LRR-RLPs. These are two different type of receptors. While LRR-RLPs are well studied, little is known about malectin LRR-RLPs. The authors should not simply apply the mode of function of LRR-RLPs to RLP4 which is a malectin LRR-RLP. In addition, LRR-RLPs that function as ligand-binding receptors typically possess >20 LRRs, whereas RLP4 in this work has a rather small ectodomain. It remains unclear whether it will function as a PRR.

I can't agree with the author's logic of testing uninfested plants for proving a PRR's function. The function of a pattern recognition receptor depends on perceiving the corresponding ligand. As shown by the data provided, RLP4-OE plants have altered transcriptional profile indicating activated defense, suggesting it's unlikely a PRR. An alternative explanation is needed.

More work on BAK1 will also help to clarify the ideas proposed by the authors.

Reviewer #3 (Public review):

Summary:

In this study, Wang et al., investigate how herbivorous insects overcome plant receptor-mediated immunity by targeting plant receptor-like proteins. The authors identify two independently evolved salivary effectors, BtRDP in whiteflies and NlSP694 in brown planthoppers, that promote the degradation of plant RLP4 through the ubiquitin-dependent proteasome pathway. NtRLP4 from tobacco and OsRLP4 from rice are shown to confer resistance against herbivores by activating defense signaling, while BtRDP and NlSP694 suppress these defenses by destabilizing RLP4 proteins.

Strengths:

This work highlights a convergent evolutionary strategy in distinct insect lineages and advances our understanding of insect-plant coevolution at the molecular level.

Two minor comments:

In line 140, yeast two-hybrid (Y2H) was used to screen for interacting proteins in plants. However, it is generally difficult to identify membrane receptors using Y2H. Please provide more methodological details to justify this approach, or alternatively, include a discussion explaining this.

In Figure S12C, the interaction between the two proteins appears to be present in the nucleus as well. Please provide a possible explanation for this observation.

Reviewer #1 (Public review):

Summary:

In this manuscript, Seegren and colleagues demonstrate that in a mouse model of neonatal E. coli meningitis, loss of endothelial toll-like receptor 4 (TLR4) leads to a marked decrease in transcriptional dysregulation across multiple leptomeningeal cell types, a decrease in vascular permeability, and a decrease in macrophage abundance. In contrast, loss of macrophage TLR4 had less pronounced effects. Using cultured wild-type and TLR4-knockout endothelial cells, the authors further demonstrate that TLR4-NF-κB signaling leads to reversible internalization of the tight junction protein claudin-5, establishing a potential mechanism of increased vascular permeability. Finally, the authors use RNA-sequencing of wild-type and TLR4-knockout endothelial cells to define the TLR4-dependent cell-autonomous transcriptional response to E. coli.

Strengths:

(1) The authors address an important, well-motivated hypothesis related to the cellular and molecular mechanisms of leptomeningeal inflammation.

(2) The authors use model systems (mouse conditional knockouts and cultured endothelial cells) that are appropriate to address their hypotheses. The data are of high quality.

Weaknesses:

(1) The authors perform single-nucleus RNA-seq on dissected leptomeninges from control and E. coli-infected mice across three genotypes (WT, Tlr4MKO, and Tlr4ECKO). A major discovery from this experiment, as summarized by the authors, is: "Tlr4ECKO mice exhibited a global attenuation of infection-induced transcriptional responses across all major leptomeningeal cell types, as judged by the positions of cell clusters in the UMAP." This conclusion could be considerably strengthened by improving the qualitative and quantitative analysis.

(2) The authors interpret E. coli infection-induced increases in leptomeningeal sulfo-NHS-biotin as evidence of compromised BBB integrity (i.e., extravasation from the vasculature) (Results, page 7), but another possible route in this context is sulfo-NHS-biotin entry from the dura across a compromised arachnoid barrier. The complete rescue in Tlr4ECKOs is strongly suggestive that the vascular route dominates, but it would strengthen the work if the authors could assess arachnoid barrier fidelity (e.g. via immunohistochemistry). At a minimum, authors should mention that the sulfo-NHS-biotin signal in this context may represent both vascular and arachnoid barrier extravasation.

(3) The authors state that "deletion of TLR4 prevented both NF-κB nuclear translocation and Cldn5 internalization in response to E. coli (Figure 4A-D)" (Results, page 9). In Figures 4C and D, however, there is no indicator of a statistical test directly comparing the two genotypes. A comparison of within-genotype P-values should not be used to support a genotype difference (PMID: 34726155).

(4) In the first paragraph of the Results, the authors summarize the meningeal layers as (1) pia, (2) subarachnoid space, (3) arachnoid, and (4) dura, and then state "The second and third layers constitute the leptomeninges." This definition of leptomeninges seems to omit the pia, which is widely considered part of the leptomeninges (PMID: 37776854).

(5) The Cdh5-CreER/+;Tlr4 fl/- mouse lacks TLR4 in all endothelial cells (i.e., in peripheral organs as well as CNS/leptomeninges), and, as the authors note, the periphery is exposed to E. coli. It would be helpful if the authors could comment in the Discussion on the possibility that peripheral effects (e.g., peripheral endothelial cytokine production, changes to blood composition as a result of changes to peripheral endothelial permeability) may contribute to the observed leptomeningeal phenotypes.

Reviewer #2 (Public review):

Summary:

The authors use a postnatal mouse model of E. coli bacterial meningitis and a mouse brain endothelioma cell line combined with cell-type-specific gene deletion to study the function of endothelial TLR4, a cell surface receptor that recognizes gram positive bacterial wall components, in the local leptomeningeal (LPM) response with a focus on endothelial barrier breakdown mediated by TLR4. Single-cell transcriptional profiling and imaging studies using whole-mount preps of the LPM support that LPM endothelial, CD206+ local macrophage and LPM fibroblast and arachnoid barrier cell inflammatory response and is abrogated in endothelial-specific KO of TLR4, pointing to a role for endothelial TLR4 in local LPM response. Culture studies using Bend3.1 cells (a mouse brain endothelioma cell line) support a direct role for TLR4 in the bacteria-mediated inflammatory response and in internalization of Cldn5 via the endosomal-lysosomal pathway, resulting in loss of barrier integrity

Strengths:

The local LPM cell response in meningitis and the role of specific LPM cells in inflammation and CNS barrier breakdown have not been extensively studied, despite ample evidence for primary immune response in the meninges in human patients and in animal models. The authors employ a robust, multi-model approach using both in vivo and in vitro models with cell-type-specific knockout to study the function of TLR4 in brain endothelial cell response. The authors nicely combine functional barrier assays with IF for junctional localization in their experimental design, and they delve into potential mechanisms of Cldn5 internalization using markers of endosomal-lysosomal pathway localization. The authors also describe a new type of barrier assay using a streptavidin-coated plate upon which barrier-forming cell cultures can be placted, this could be a very useful alternative or complement to other size-selective barrier assays and presumably could work for other barrier forming cells types, likely epithelial cells.

Weaknesses:

(1) There are no measures of bacterial burden in peripheral organs, blood, in the LPM or brain in the TLR4 endothelial cKO mice. Lack of TLR4 in endothelial cells could prevent bacterial 'access' into the LPM and brain, essentially preventing meningitis and leading to a lack of inflammatory responses in the LPM-located cells simply because there is no bacteria present. Bacteremia may also be reduced, as might inflammatory responses in peripheral organs with TLR4-deficient peripheral endothelium. Bacterial counts and inflammatory measures in peripheral organs and blood are important to better understand the mechanism(s) underlying the reduced inflammatory profile in LPM cells and no LPM endothelial breakdown in the Tlr4 endothelial cKO mice. In other words, does deleting TLR4 in EC protect against the development of meningitis by somehow blocking bacteria access to the LPM (this would be supported by low or no CFU counts in infected Tlr4 endothelial cKO) or is it what the authors appear to propose in Figure 1J that TLF4 in EC is the only cell responding to the bacteria to trigger the immune cascade in the LPM? More data is needed to resolve this, as this is a major claim of the paper.

(2) The authors look at the underlying cortical response (cerebral vasculature for ICAM and immune cells) but do not use markers that could identify microglia (Iba1), the primary resident immune cell (CD206 is not useful, at this stage, in perivascular macrophages that are extremely sparse in the postnatal brain). This would be important to better study the impact on CNS resident immune cell morphological activation.

(3) The authors suggest that Cldn5 junctional localization is selectively disrupted upon bacterial exposure, mediated by TLR4 - they suggest this based on studying PECAM, GLUT-1, ZO-1 and B-catenin (all normally junction or cell surface located in cultured Bend3.1) in relationship to Cldn5 localization (normally high) - it is possibly these are also impact by bacteria exposure (maybe through different mechanisms?) - a better measure would be to use the similar cyto/PM measure they do for Cldn5 in Fig. 4D and to evaluate this or to use intensity measurements.

(4) The discussion could benefit from delving more into the prior literature on E.coli-mediated breakdown of junctions in cultured human microvascular brain endothelial cell model and critical host-pathogen interactions of the bacteria with ECs (PMID: 14593586), and how this might involve TLR4.

(5) It would be important to discuss how their results relate to earlier studies on TLR4-/- and TLR2-/- global knockout mice and protection vs vulnerability to development of meningitis (see PMCID: PMC3524395) - this paper showed that TLR4 global KO mice have increased susceptibility to die from meningitis and have much higher CFU counts in the CNS. In this manuscript and their prior work (Wang et al., 2023), this group shown that both global TLR4-/- mutants and their EC-specific KO have reduced barrier permeability, but we don't have any information about CFU or susceptibility to death from meningitis in their models.

Reviewer #3 (Public review):

Summary:

This study investigates the molecular underpinnings of immune responses in the leptomeninges in neonatal bacterial meningitis. Bacterial meningitis is a major disease burden, particularly for neonates, and it has previously been noted that the meningeal immune environment in infants is permissive to opportunistic infection (Kim et al., Sci Immunol, 2023). There is less known about the contribution of the stromal compartment to meningeal immune responses. Seegren et al. interrogate the role of leptomeningeal endothelium in host defence in E. coli infected neonatal mice using mouse genetic tools to delete the LPS receptor Tlr4 from either endothelial cells (using Cdh5-CreER) or macrophages (using LysM-Cre). The authors use snRNAseq, cleared cortical mounts, and in vitro work to define the impact of E. coli infection on leptomeningeal endothelial cells. This study uses a range of innovative techniques to probe the role of the stromal compartment in meningitis.

Strengths:

This study makes excellent use of cleared cortical mounts to examine the biology of the leptomeninges, in particular, changes to the endothelium, with unprecedented detail. In combination with high-quality sequencing data provide new insights into the impact of meningitis on the leptomeninges. The data presented by the authors is of very high quality.

Weaknesses:

The weaknesses of the study were in terms of interpretation and perhaps study design.

(1) Most importantly, the authors need to provide additional validation of their conditional knockout models. The authors need to confirm that the Cdh5-CreER does not impact leptomeningeal fibroblasts and to confirm gene deletion in macrophages.

(2) The authors could also strengthen the paper by providing data on the impact of these conditional knockout models on the course of meningitis and bacterial burden.

(3) Finally, it is perhaps not surprising that Tlr4 is required for meningitis responses with E. coli. However, it is unclear if these findings can be generalised to other, more common, meningitis infections (streptococcal/pneumococcal).

(4) There are additional minor issues; for instance, the arachnoid fibroblast 2 population appears to closely resemble dural border cells.

(5) The cell line model (bEnd.3) is a relatively low-fidelity model of BBB endothelial cells, and this should be acknowledged.

With these caveats, it is difficult to be certain that the endothelium alone is the driver of meningeal immune responses in meningitis, and what the impact of these is.

Reviewer #1 (Public review):

Summary:

In brief, this manuscript addresses a very interesting topic, namely, the impact of the Mediterranean diet on the development of cancer. Using one mouse model and three tumor cell lines, the data show that a Mediterranean diet is sufficient to promote an anti-tumor response mediated by the microbiota, metabolites, and the immune system. Mechanistically, the Mediterranean diet promotes the expansion of Bacteroides thetaiotaomicron (B. theta for short), which converts tryptophan into 3-IAA. Both B. theta and the metabolite are sufficient to phenocopy the effect of the Mediterranean diet on cancer growth in vivo. The manuscript also shows that this effect is mediated by CD8 T cells and suggests, by way of in vitro assays, that 3-IAA sustains the functionality of CD8 T cells, preserving their exhaustion and blocking the ISR pathway.

Strengths:

The conclusions of this manuscript are potentially interesting and of potential clinical relevance.

Weaknesses:

For a full technical evaluation of the strength of the data, I am missing important technical and experimental details (e.g., number of independent experiments, statistics), and found some legends with potential labelling inaccuracies.

Reviewer #2 (Public review):

Summary:

The authors aimed to investigate the mechanistic link between a Mediterranean-mimicking diet (MedDiet)-specifically the synergy between high fiber and fish oil-and its ability to suppress tumor growth. They successfully identify that this dietary combination alters the gut microbiome to favor the expansion of Bacteroides thetaiotaomicron. This bacterium metabolizes dietary tryptophan into indole-3-acetic acid (3-IAA), which then acts systemically to prevent CD8+ T-cell exhaustion.

Strengths:

The study integrates controlled dietary interventions, microbiome perturbation, metabolite profiling, and immune functional analyses into a coherent and well-organized framework, making the overall logic of the work easy to follow. The dietary design is carefully controlled, allowing clear interpretation of which broad dietary features are associated with the observed antitumor effects. The immune dependence of the phenotype is addressed using appropriate experimental approaches, and the results broadly support a role for gut microbiota-derived metabolites in shaping immune cell function. In addition, analyses of human datasets provide important context and enhance the potential relevance and usefulness of the findings for a broader research community.

Weaknesses:

While the manuscript provides strong support for a role of the microbial metabolite indole-3-acetic acid and downstream stress signaling in shaping immune cell function, the upstream mechanism by which this metabolite exerts its effects remains unresolved. In particular, the specific molecular sensor or binding target through which the metabolite acts has not been identified, and this uncertainty limits mechanistic precision. Framing this point more explicitly as an open question would help align the interpretation with the current data.

In addition, at several points, the presentation may imply that a single microbial species is uniquely responsible for the observed effects. However, the experimental evidence more directly demonstrates sufficiency under the tested conditions rather than necessity. A clearer distinction between "sufficient" and "necessary" claims would help readers better assess the generality of the findings and their applicability to more complex microbial communities.

The interpretation of the human data also warrants some caution. The diet-associated score applied to human datasets is derived from gene-expression signatures identified in mouse models and therefore represents an indirect proxy rather than a direct measure of dietary intake. Although the score correlates with clinical outcomes, it does not establish that patient survival is driven by consumption of specific dietary components such as fiber and fish oil.

Reviewer #1 (Public review):

Summary:

In this manuscript, Zhang et al. demonstrate that depletion of the 18S rRNA m6A methyltransferase Mettl5 compromises translation fidelity and consequently increases neoantigen generation, thereby uncovering an unexpected role for Mettl5 in tumor immunity. Mettl5-KO tumors exhibit enhanced CD8⁺ T-cell infiltration and show improved responses to immune checkpoint blockade. Mechanistically, loss of Mettl5 perturbs the local structure of 18S rRNA and disrupts the ribosome's ability to perform accurate translation. Subsequent ribosome profiling and mass spectrometry analyses provide compelling evidence that Mettl5 functions as a previously unrecognized regulator of translation to participate in tumor immune evasion.

Strengths:

This study presents a comprehensive set of experimental data supporting a mechanistic link between rRNA modification, translation fidelity, and neoantigen generation. The observed synergistic effect of Mettl5 depletion and anti-PD-1 therapy highlights the potential translational relevance of targeting rRNA modifications in cancer immunotherapy.

Weaknesses:

(1) In light of the principal function of Mettl5, which is to methylate 18S rRNA within the small ribosomal subunit, the authors focus primarily on translation fidelity, largely associated with elongation, but provide limited exploration of potential effects on translation initiation. Loss of Mettl5 may alter the initiation landscape, potentially promoting alternative or noncanonical initiation events (e.g., initiation at CUG codons), which could also contribute to the observed neoantigen repertoire changes. Further investigation into initiation-level alterations would strengthen the mechanistic interpretation.

(2) Given the broad involvement of rRNA methyltransferases in ribosome function, the authors should incorporate a parallel analysis using another enzyme (e.g., Zcchc4 or Nsun5) as a negative control. Such an experiment is essential to demonstrate that the tumor immunity phenotype observed is specific to Mettl5 rather than a general consequence of perturbing rRNA modification.

Reviewer #2 (Public review):

Summary:

This study demonstrates that METTL5-mediated rRNA m⁶A1832 modification regulates tumor neoantigen generation by maintaining translational fidelity. Loss of METTL5 in tumor cells promotes immune cell infiltration into the tumor microenvironment and enhances the therapeutic efficacy of anti-PD-1 treatment, identifying a novel and potentially important target for cancer immunotherapy.

Strengths:

In murine tumor models, the authors found that Mettl5 depletion increases CD8⁺T cell infiltration and T cell receptor (TCR) repertoire diversity, and revealed a novel mechanism by which reduced ribosomal translation fidelity enhances non-canonical translation, thereby promoting the production of tumor neoantigens.

Weaknesses:

(1) While Mettl5 knockout enhances T-cell infiltration into tumors, it remains unclear whether loss of Mettl5 affects the expression of chemokines involved in immune cell recruitment.

(2) Although the authors report a significant reduction in tumor cell growth as well as tumor volume and weight, direct evidence demonstrating T-cell-mediated cytotoxicity is lacking.

Reviewer #1 (Public review):

This is an excellent paper from Dr. Yokoyama and colleagues. The experiments are technically demanding, given the very low cell numbers and the challenges of working with implantation sites at gestational days 6.5, 10.5, and 14.5. Overall, the impact of TGF-β receptor II deficiency in the NK lineage on uterine trNK cell numbers and litter size is convincing, and the authors' conclusions are well supported by the data. Less convincing, however, is the claim that the decrease in trNK cells is compensated by an increase in cNK cells; rather, the absence of TGF-β receptor II appears to result in an overall reduction of NK/ILC1 cells.

Major Points:

(1) Figure 1A and B

Although a trend is evident, it does not appear that the absolute number of cNK cells at day 14 is significantly changed from day 6.5?

(2) Figure 2E

The authors state, "This reduction of uterine trNK cells was accompanied by a concomitant increase in the absolute number and frequency of CD49b+Eomes+ cNK cells within the pregnant uterus of TGF-βRIINcr1Δ dams (Figure 2 D, E). The number of cNK cells appears relatively low (visually ~1,000-1,300), and although the difference is statistically significant, its physiological relevance is unclear. More importantly, this modest increase does not correlate with the marked decrease in trNK and ILC1 populations, as cNK cells do not appear to accumulate. In my opinion, the conclusion "Collectively, these findings indicate that a TGF-β-driven differentiation pathway directs the conversion of peripheral cNK cells into uterine trNK cells during murine pregnancy" should be slightly toned down.

(3) Figures 2-4

It is unclear whether the littermate controls are floxed mice or floxhet-Ncr1iCre mice? This distinction is important, as Ncr1iCre expression itself could potentially lead to a phenotype.

Reviewer #2 (Public review):

In their manuscript "TGF-β drives the conversion of conventional NK cells into uterine tissue-resident NK cells to support murine pregnancy", Yokoyama and colleagues investigate the role of Tgfbr2 expression by NK cells in the formation of tissue-resident uterine NK cells and subsequent importance in murine pregnancy. By transferring congenic splenic conventional NK cells into pregnant mice, they show conversion of circulating NK cells into uterine ivCD45 negative tissue-resident NK cells. When interfering with the formation of uterine trNK cells, spiral artery remodelling was impaired, fetal resorption rates were increased, and litter sizes were reduced.

Generally, this is a research topic of high interest, yet the manuscript is lacking detailed mechanistic insights, and some questions remain open. At the current state, the data represent an interesting characterisation of the Tgfbr2-fl/fl Ncr1-Cre mice in pregnancy, but considering (a) the recent publication by the group (Reference 17) on the role of Eomes+ cNK cells during pregnancy, (b) the previously described role of Tgfbr2 and autocrine TGFb expression for uterine NK cell differentiation in virgin mice (also cited by the authors), and (c) the well-known relevance of uterine NK cells during pregnancy, additional experiments addressing the specific role of Tgfb during pregnancy would help to improve novelty and significance of the manuscript. To this end, the following aspects should be discussed and, where applicable, experimentally addressed by the authors:

(1) The authors suggest cNK extravasation and local differentiation into iv- trNK.

Can it be estimated how much this process contributes to the trNK pool vs. a potential local proliferation of already existing trNK? How do absolute numbers of CD49a+ Eomes+ trNK change during pregnancies? (In Figure 1A, the cell numbers of CD49a+ Eomes+ trNK seem to go down dramatically between gd 6.5 and 14.5). The plot in 1B could also include absolute numbers of ILC1s and trNKs. Would recruited cNK cells compensate for a potential loss of CD49a+ Eomes+ trNK?

(2) Figure 1C: 2.5

Mio cNK cells have been transferred, but only very few cells can be detected within the uterus (concatenated FACS plot shown). What may represent the limit to generate uterine trNK out of cNK? Is the niche supporting cNK-trNK differentiation limited? Is it only a specific subset of (splenic) cNK capable of differentiating into trNK? Is gd 0.5 the optimal timepoint for the transfer? Is there continuous recruitment of cNK into the uterus and differentiation into trNK, or is it enhanced at specific timepoints of pregnancy? Could there be local proliferation of cNK-derived trNK? This could be studied by proliferation dye dilution of WT cNK cells in this transfer-setup.

(3) The authors should consider inducible Tgfbr2 deletion (e.g. with Tamoxifen-inducible Cre) to enable development of the uterine NK compartment in virgin mice and only ablate trNK differentiation during pregnancy. This could help to estimate the turnover of cNK into trNK, or to understand if constant cNK recruitment is required to form the uterine trNK compartment during pregnancy.

(4) Did the authors consider transfer of Tgfbr2-floxed Ncr1-Cre cNK in the same setup as in Fig. 1C? This experiment could confirm the requirement of Tgfbr-dependent signalling for cNK to trNK conversion during pregnancy versus effects of Tgfb signals on trNK numbers in the uterus at steady state (before pregnancy).

(5) Figures 2D/E

The authors should state that ILC1s are reduced in the virgin uterus of female Tgfbr2-floxed or Tgfb1-floxed Ncr1-Cre mice and cite the relevant work (the Ref #29 discussed in this context did not show that?). It would be helpful to include an analysis of all three uterine ILC subsets in steady state. This could help to answer the question if the cNK cell changes are pregnancy-specific or a general phenomenon in Tgfbr2-floxed Ncr1-Cre mice.

(6) Figure 2E

Please phrase more carefully about the "concomitant increase" of cNKs, since this increase is much less pronounced compared to the very strong reduction (absence) of trNKs in Tgfbr2-floxed Ncr1-Cre mice. Do the authors suggest that cNKs are halted at this stage and cannot differentiate into trNK, based on these data?

(7) Figure 3/4

Can the reduced litter size and the abnormal spiral artery formation be rescued by transfer of WT cNK into Tgfbr2-floxed Ncr1-Cre mice?

Reviewer #1 (Public review):

The manuscript titled "The distinct role of human PIT in attention control" by Huang et al. investigates the role of the human posterior inferotemporal cortex (hPIT) in spatial attention. Using fMRI experiments and resting-state connectivity analyses, the authors present compelling evidence that hPIT is not merely an object-processing area, but also functions as an attentional priority map, integrating both top-down and bottom-up attentional processes. This challenges the traditional view that attentional control is localized primarily in frontoparietal networks.

The manuscript is strong and of high potential interest to the cognitive neuroscience community. Below, I raise questions and suggestions to help with the reliability, methodology, and interpretation of the findings.

(1) The authors argue that hPIT satisfies the criteria for a priority map, but a clearer justification would strengthen this claim. For example, how does hPIT meet all four widely recognized criteria, such as spatial selectivity, attentional modulation, feature invariance, and input integration, when compared to classical regions such as LIP or FEF? A more systematic summary of how hPIT meets these benchmarks would be helpful. Additionally, to what extent are the observed attentional modulations in hPIT independent of general task difficulty or behavioral performance?

(2) The authors report that hPIT modulation is invariant to stimulus category, but there appear to be subtle category-related effects in the data. Were the face, scene, and scrambled images matched not only in terms of luminance and spatial frequency, but also in terms of factors such as semantic familiarity and emotional salience? This may influence attentional engagement and bias interpretation.

(3) The result that attentional load modulates hPIT is important and adds depth to the main conclusions. However, some clarifications would help with the interpretation. For example, were there observable individual differences in the strength of attentional modulation? How consistent were these effects across participants?

(4) The resting-state data reveal strong connections between hPIT and both dorsal and ventral attention networks. However, the analysis is correlational. Are there any complementary insights from task-based functional connectivity or latency analyses that support a directional flow of information involving hPIT? In addition, do the authors interpret hPIT primarily as a convergence hub receiving input from both DAN and VAN, or as a potential control node capable of influencing activity in these networks? Also, were there any notable differences between hemispheres in either the connectivity patterns or attentional modulation?

(5) A few additional questions arise regarding the anatomical characteristics of hPIT: How consistent were its location and size across participants? Were there any cases where hPIT could not be reliably defined? Given the proximity of hPIT to FFA and LOp, how was overlap avoided in ROI definition? Were the functional boundaries confirmed using independent contrasts?

Comments on revisions:

The authors have successfully addressed my previous questions and concerns. The public comments above reflect my views on the initial submission and, in my opinion, will remain helpful for general readers. Given this, I do not have additional public comments and will keep my previous public review unchanged.

Reviewer #2 (Public review):

Summary

This study investigates the role of the human posterior inferotemporal cortex (hPIT) in attentional control, proposing that hPIT serves as an attentional priority map that integrates both top-down (endogenous) and bottom-up (exogenous) attentional processes. The authors conducted three types of fMRI experiments and collected resting-state data from 15 participants. In Experiment 1, using three different spatial attention tasks, they identified the hPIT region and demonstrated that this area is modulated by attention across tasks. In Experiment 2, by manipulating the presence or absence of visual stimuli, they showed that hPIT exhibits strong attentional modulation in both conditions, suggesting its involvement in both bottom-up and top-down attention. Experiment 3 examined the sensitivity of hPIT to stimulus features and attentional load, revealing that hPIT is insensitive to stimulus category but responsive to task load - further supporting its role as an attentional priority map. Finally, resting-state functional connectivity analyses showed that hPIT is connected to both dorsal and ventral attention networks, suggesting its potential role as a bridge between the two systems. These findings extend prior work on monkey PITd and provide new insights into the integration of endogenous and exogenous attention.

Strength

(1) The study is innovative in its use of specially designed spatial attention tasks to localize and validate hPIT, and in exploring the region's role in integrating both endogenous and exogenous attention, as prior works focus primarily on its involvement in endogenous attention.

(2) The authors provided very comprehensive experiment designs with clear figures and detailed descriptions.

(3) A broad range of analyses was conducted to support the hypothesis that hPIT functions as an attentional priority map -- including experiments of attentional modulation under both top-down and bottom-up conditions, sensitivity to stimulus features and task load, and resting-state functional connectivity. These analyses showed consistent results.

(4) Multiple appropriate statistical analyses - including t-tests, ANOVAs, and post-hoc tests-were conducted, and the results are clearly reported.

Comments on revisions:

The authors have addressed our comments in their revised manuscript and in their response to the reviewers. We don't have any further suggestions or comments.

Reviewer #1 (Public review):

Summary:

The article investigates how the Japanese macaque makes gait transitions between quadruped and biped gaits. It presents a compelling neuromechanical simulation that replicates the transition and an interesting analysis based on an inverted pendulum that can explain why some transition strategies are successful and others are not.

Strengths:

I enjoyed reading this article. I think it presents an interesting study and elegant modeling approaches (musculoskeletal + inverted pendulum). The study is well conducted, and the results are interesting. I particularly liked how the success of gait transitions could be predicted based on the inverted pendulum and its saddle node stability. I think it makes a useful and interesting contribution to the state of the art.

Weaknesses:

The article is already in great shape, but could be improved a bit by:

(1) Strengthening the comparison to animal data. In particular, videos of the real animal should be included + snapshots of their gaits (quadruped, biped, and transitions).

(2) Exploring and testing a broader range of conditions. I think it would be very interesting to test gaits and gait transitions on up and down slopes (both with the musculoskeletal model and with the inverted pendulum model). This could be used to make predictions on how the real animal adapts to those conditions. Ideally, this should be tested on the animal as well. I think this could increase (even more) the impact of this work.

(3) Better explaining several aspects of the PSO optimization.

(4) (Ideally) performing a sensitivity analysis on the optimized parameters (e.g. variations of +-5, 10, 20%) in order to determine their respective importance and how much their instantiated values have influenced the results.

(5) Running a spell checker, as there are quite a few typos.

Reviewer #2 (Public review):

Summary:

This article presents a neuromusculoskeletal (NMS) model of the Japanese Macaque. This model is added with a neural feedforward controller based on CPG and synergy that allows for reproducing quadrupedal and bipedal gait as well as the transition between quadrupedal and bipedal gait. The model and controller were validated using experimental data. Results were also compared to an inverted pendulum model to show that the transition between quadrupedal and bipedal in macaque is using this kind of representation for transition and stability. Overall, the article is very interesting, but it sometimes lacks clarity.

Strengths:

The results of the model present impressive results for quadrupedal, bipedal, and transition, validated by experimental data. NMS controllers based on feedforward controllers are very difficult to fine-tune.

Weaknesses:

(1) The movement regulator is not clear and should be better explained. At first, it seems that it is just a new CPG/synergy (feedforward) added, but in the methods, it seems to be a feedback controller.

(2) It is also not clear what is meant by discretizing the weight for the trigger limb from 0 to 1 (page 8).

(3) The controller is mainly using a feedforward controller, allowing only anticipatory movement. Animals are also using a reflex-based feedback controller. A controller with feedback/reflex could reduce failed attempts in training and better represent the transition.

(4) There are small typos throughout the article that should be corrected.

Reviewer #3 (Public review):

Summary:

The purpose of this study was to test the hypothesis that the inverted pendulum mechanism contributes to the gait transition from quadrupedal to bipedal gait in Japanese macaques. The author uses a neuromusculoskeletal model to generate different motor tasks by varying motor command parameters during forward dynamics simulations. After simulations were done, the authors used dynamical system analysis of the inverted pendulum model to reveal the underlying principles of gait transition control. The authors showed that successful gait transition from quadrupedal to bipedal gait mostly depends on increased step length of a hindlimb.

Strengths:

This study is important not only for understanding gait transition, but also to understand stability control of bipedal gaits. Another advantage of this study is that it allows us to estimate the effect of one control mechanism and find its effect and limits. In animal studies, we also have a combination of compensatory stability control mechanisms.

Weaknesses:

Any simulation is not perfect, so discrepancies from experimental data are expected. A 2D model is used, but the advantage of using a 3D model is not clear, and it is much more complicated.

Reviewer #1 (Public review):

Summary:

Hoverflies are known for a striking sexual dimorphism in eye morphology and early visual system physiology. Surprisingly, the male and female flight behaviors show only subtle differences. Nicholas et al. investigate the sensori-motor transformation of sexually dimorphic visual information to flight steering commands via descending neurons. The authors combined intra- and extracellular recordings, neuroanatomy, and behavioral analysis. They convincingly demonstrate that descending neurons show sexual dimorphisms - in particular at high optic flow velocities - while wing steering responses seem relatively monomorphic. The study highlights a very interesting discrepancy between neuronal and behavioral response properties.

More specifically, the authors focused on two types of descending neurons that receive inputs from well-characterized wide-field sensitive tangential cells: OFS DN1, which receives inputs from so-called HS cells, and OFS DN2, which receives input from a set of VS cells. Their likely counterparts in Drosophila connect to the neck, wing, and haltere neuropils. The authors characterized the visual response properties of these two neuronal classes in both male and female hoverflies and identified several interesting differences. They then presented the same set of stimuli, tracked wing beat amplitude, and analyzed the sum and the difference of right and left wing beat amplitude as a readout of lift or thrust, and yaw turning, respectively. Behavioral responses showed little to no sexual dimorphism, despite the observed neuronal differences.

Strengths:

I find the question very interesting and the results both convincing and intriguing. A fundamental goal in neuroscience is to link neuronal responses and behavior. The current study highlights that the transformations - even at the level of descending neurons to motoneurons - are complex and less straightforward than one might expect.

Weaknesses:

The authors investigated two types of descending neurons, but it was not clear to me how many other descending neurons are thought to be involved in wing steering responses to wide-field motion. I would suggest providing a more in-depth overview of what is known about hoverflies and Drosophila, since the conclusions drawn from the study would be different if these two types were the only descending neurons involved, as opposed to representing a subset of the neurons conveying visual information to the wing neuropil.

Both neuronal classes have counterparts in Drosophila that also innervate neck motor regions. The authors filled the hoverfly DNs in intracellular recordings to characterize their arborization in the ventral nerve cord. In my opinion, these anatomical data could be further exploited and discussed a bit more: is the innervation in hoverflies also consistent with connecting to the neck and haltere motor regions? Are there any obvious differences and similarities to the Drosophila neurons mentioned by the authors? If the arborization also supports a role in neck movements, the authors could discuss whether they would expect any sexual dimorphism in head movements.

Reviewer #2 (Public review):

Summary:

Many fly species exhibit male-specific visual behaviors during courtship, while little is known about the circuit underlying the dimorphic visuomotor transformations. Nicholas et al focus on two types of visual descending neurons (DNs) in hoverflies, a species in which only males exhibit high-speed pursuit of conspecifics. They combined electrophysiology and behavior analysis to identify these DNs and characterize their response to a variety of visual stimuli in both male and female flies. The results show that the neurons in both sexes have similar receptive fields but exhibit speed-dependent dimorphic responses to different optic flow stimuli.

Strengths:

Hoverflies, though not a common model system, show very interesting dimorphic behaviors and provide a unique and valuable entry point to explore the brain organization behind sexual dimorphism. The findings here are not only interesting on their own right but will also likely inspire those working in other systems, particularly Drosophila.

The authors employed rigorous morphology, electrophysiology, and behavior methods to deliver a comprehensive characterization of the neurons in question. The precision of the measurements allowed for identifying a subtle and nuanced neuronal dimorphism and set a standard for future work in this area.

Weaknesses:

Cell-typing using receptive field preferred directions (RFPDs): if I understood correctly, this classification method mostly relies on the LPDs near the center of the receptive field (median within the contour in Fig.1). I have two concerns here. First, this method is great if we are certain there are only two types of visual DNs as described in the manuscript. But how certain is this? Given the importance of vision in flight control, I would expect many DNs that transmit optic flow information to the motor center. I'd also like to point out that there are other lobula plate tangential cells (LPTCs) than HS and VS cells, which are much less studied and could potentially contribute to dimorphic behaviors. Second, this method feels somewhat impoverished given the richness of the data. The authors have nicely mapped out the directional tuning for almost the entire visual field. Instead of reducing this measurement to 2 values (center and direction), I was wondering if there is a better method to fully utilize the data at hand to get a better characterization of these DNs. As the authors are aware, local features alone can be ambiguous in characterizing optic flows. What's more, taking into account more global features can be useful for discovering potentially new cell types.

Line 131, it wasn't clear to me why full-screen stimuli were used for comparison here, instead of the full receptive field maps. Male flies exhibit sexual dimorphic behaviors only during courtship, which would suggest that small-sized visual stimuli (mimicking an intruder or female conspecific) would be better suited to elicit dimorphic neuronal responses. A similar comment applies to the later results as well. Based on the receptive field mapping in Figure 1, I'm under the impression that these 2 DN types are more suited to detect wide-field optic flows, those induced by self-motion as mentioned in the manuscript. The results are still very interesting, but it's good to make this point clear early on to help set appropriate expectations. Conversely, this would also suggest that there are other visual DN types that are responsible for the courtship-related sexually dimorphic behaviors.

Reviewer #1 (Public review):

Summary:

The dysgranular retrosplenial cortex (RSD) and hippocampus both encode information related to an animal's navigation through space. Here, the authors study the different ways in which these two brain regions represent spatial information when animals navigate through interconnected rooms. Most importantly, they find that the RSD contains a small fraction of neurons that encode properties of interconnected rooms by firing in different head directions within each room. This direction is shifted by 180 degrees in 2-room environments, and by 90 degrees in 4-room environments. While it cannot be definitively proven that this encoding is not just related to the presence of exits (doors) in each room, this is a noteworthy finding and will motivate further study in more complex and well-controlled environments to understand this coding scheme in the RSD. The recordings and analyses used to identify these multi-directional cells are mostly solid. Additional conclusions regarding the rotational symmetry across rooms seen in the RSD neurons that do not encode direction (representing the majority of RSD neurons) remain incomplete, given the evidence presented thus far. The differences between RSD and hippocampus encoding of space are clear and consistent with prior observations.

Strengths:

(1) Use of tetrode recordings from the RSD to identify multi-direction cells that only encode one direction in each room, but shift the preferred direction by either 180 or 90 degrees depending on the number of rooms in the environment.

(2) Solid controls to show that this multi-direction encoding is stable over time and across some environmental manipulations.

(3) Convincing evidence that these multi-direction cells can co-exist with single-direction head direction cells in the RSD (as both cell types can be simultaneously recorded).

(4) Convincing evidence for clear differences between directional and spatial encoding in the RSD versus hippocampus, consistent with prior observations.

Weaknesses:

(1) The paper mostly uses the term "retrosplenial cortex", but it is important to clarify that the study is only focused on the dysgranular retrosplenial cortex (RSD; Brodmann Area 30) and not the granular retrosplenial cortex (Brodmann Area 29). These are two distinct regions (despite the similar names), each with distinct connectivity and distinct behavioral encoding and function, so it is important to clarify in the abstract and title that the present study is solely about the RSD to prevent confusion in the literature.

(2) The proportion of each observed cell type is not clearly stated, although it is clear that the multi-directional cells are in the minority. Having the proportion of well-isolated neurons in distinct sessions that encode each type of information (e.g., multi vs single direction encoding) would greatly aid the interpretation of the result and help the field know how common each cell type is in the RSD.

(3) The authors state that "MDCs [multi-directional cells] never exhibited multidirectional activity within a single room" - but many of the single room examples from the 4-room environment (shown in Figures 2E and 2F) reveal multi-peaked directional encoding. This suggests that the multi-direction encoding may be more compatible with encoding some property of the number of exits rather than relative room orientations.

(4) The spatial rotation analyses of non-directional cell analyses are considered incomplete. This is impacted by the slower speed at the doors and hence altered firing rates (as evidenced in spatial rate plots). The population rate is not relevant as the correlational analyses are done on a single cell level. Since some cells fire more with increasing speed and others fire less, that will necessarily result in a population rate map that minimizes firing rate differences near the doorway, where the animals move more slowly. But on a single cell level, that reduced speed is having a big effect, as evidenced by individual rate map examples, and the rooms will need to be rotated to obtain a higher correlation by overlapping the doorway regions. This does not necessarily say anything about spatial coding across the two or four interconnected rooms being rotationally symmetric, and it would appear difficult to draw any conclusions related to spatial encoding from those analyses.

Reviewer #2 (Public review):

Summary:

Laurent et al. perform in vivo electrophysiological recordings in the retrosplenial cortex of rats foraging in multi-compartment environments with either identical or unique visual features. The authors characterize two types of directional signals in the area that they have previously reported: classic head direction cells anchored to the global allocentric reference frame and multi-direction cells (MDCs), which have a rotationally preserved directional field anchored to local compartments. The primary finding of this work is that MDCs seem sensitive to local environmental geometry rather than visual context. They also show that MDC tuning persists in the absence of hippocampal place field repetition, further dissociating the RSC local directional signal from the broader allocentric representation of space. A novel observation is that RSC non-directional spatial signals are anchored to the local environment, which could and should be explored further. While the data is solid and the analyses are mostly appropriate, the primary findings are incremental, and more interesting novel claims are not explored in detail or not explicitly tested.

Strengths:

The environmental manipulations clearly demonstrate that tuning is not modulated by complex visual information.

The finding that RSC two-dimensional spatial responses are stable and anchored to environmental features is novel and can be further explored in future work.

Weaknesses:

The observation that BDCs and MDCs are insensitive to visual context builds upon the author's previous work (and replicates aspects of Zhang et al., 2022) but leaves many open questions that are not addressed with the current set of experiments. Specifically, what exactly are MDCs anchoring to? The primary theory is that they anchor to environmental geometry, but there are no explicit experimental manipulations to test this theory. It is important to note that 2- and 4-compartment environments share many features, including the same cardinal axes, making any differences/similarities in these two conditions difficult to interpret.

The main finding presented with respect to BDC/MDs tuning is that they are not sensitive to visual context as manipulated by distinct visual patterns on the wall and floor in multicompartment environments. One could argue that the individual rooms are, in actuality, quite similar in low-level visual features - each possesses a large white background square visual feature on a single wall with a fixed relationship to the door(s). How can the authors rule out that i) BDC/MDC responses are modulated by these low-level features rather than geometry and/or ii) that the rats are not paying attention to any visual features at all? There is no task requiring them to indicate which room they are in. Furthermore, the doorways themselves are prominent visual features that are present in each context. It would be interesting to see if MDC/BDC tuning persisted in a square room where the number of doorways was manipulated to rule out this possibility.

A strong possibility is that the rotational symmetry of both MDCs and non-directional spatial neurons is related to i) door-related firing, 2) stereotyped movement, and 3) stereotyped directional sampling. In Supplemental Figure 8, the authors begin to address this by comparing a 'population ratemap' to a 'population speed map.' I do not think this is sufficient and is difficult to interpret. Instead, the authors should assess whether MDC and BDCs fire more at doorways and what the overlap is with the speed-modulated cells they report. Moreover, they should assess whether the spatial speed profile itself is rotationally symmetric within each session. It would also be useful to look at the confluence of the variables simultaneously using some form of regression analysis. The authors could generate a directional predictor that captures the main response property of these cells and see if it accounts for greater variability in spiking than speed or x,y position. Finally, rotationally symmetric directional sampling biases could arise from the doors being present on the same two walls in each room. The authors should assess whether MDC tuning is still present if directional sampling is randomly downsampled to match directional observations in each compartment.

Recent work has demonstrated that neurons with egocentric corner or boundary tuning are observed in RSC. The authors do not address whether egocentric tuning contributes to MDC signals. An explicit analysis of the relationship and potential overlap of MDC and egocentric populations is warranted.

Many of the MDCs presented in the main figures are not especially compelling. This includes alterations to MDC tuning in Figure 2, which is a key datapoint. The authors should show significantly more (if not all) examples of MDCs in each environment. It would similarly be useful to see all/more examples of non-directional spatially tuned neurons with rotationally symmetric firing patterns.

"One might hypothesize that specific environmental cues, such as door orientation or landmark positioning, drive these tuning shifts. However, our results argue against this interpretation. In four-room environments, each room had multiple entry points, yet MDCs never exhibited multidirectional activity within a single room."

I do not understand the logic here. Can the authors unpack this? Also, it is clear that some of the example cells have more than one peak in individual compartments. How is this quantified?

Reviewer #3 (Public review):

Summary:

The authors examine firing of dysgranular retrosplenial cortex (dRSC) neurons in relation to head orientation and location for rats exploring open-field environments. One environment utilized was a square arena with high walls that is split into two rectangular spaces connected by a doorway. Another environment is a square arena split into quadrants connected by doors near the center. For each, the different sub-spaces of the environments are either identical in terms of visual and tactile cues or different. For head direction neurons, the authors present one population where each neuron maintains a single tuning direction for the two or four sub-compartments of the two environments. A second population exhibits what is termed multi-directional firing, wherein neurons exhibit (overall) two or four head direction peaks in firing. For such neurons, firing in each of the sub-compartments is associated with only a single preferred direction, but the directions across compartments are shown to be at 180-degree (two-compartment environment) or 90-degree offsets. The offsets evidence tuning to the "same" orientation for the sub-compartments that are, in the global reference frame, oriented at 180 or 90 degree offsets. The results are similar whether or not the sub-compartments have the same or different tactile and visual cues. Thus, the first population is said to be global in its head direction tuning, while the second relates to each local environment in a way that is systematic across sub-compartments. Spatially-specific activity of another population of non-direction-tuned RSC neurons is examined, and comparisons of sub-compartment spatial firing maps suggest that spatial tuning in RSC also repeats across compartments when the firing maps for the compartments are rotated to match each other (as in physical space). Finally, a population of hippocampal "place" cells exhibited different location mapping across sub-compartments. The findings are interpreted to indicate that RSC can simultaneously map orientation in both local and global reference frames, possibly forming a mechanism whereby the sub-compartments' shared geometry (given by the boundary shapes and the door locations) can be related to each other and to the global space they share.

Strengths:

This paper addresses an interesting problem and expands how the field will think about directional tuning.

Weaknesses:

It is not clear that the experimental design allows for a clear interpretation of the data. Rates for preferred turning are low, as are ratemap correlations for spatially-tuned neurons.

(1) It is concerning that the neurons with head direction tuning have fairly low peak firing rates (mean close to 5 Hz), where prior studies examining head direction tuning in dRSC found head direction-tuned neurons with peak rates more than an order of magnitude higher (100 Hz or more). Under circumstances where neurons are tuned well to variables other than head direction (for example, angular velocity of movement), weak head direction tuning may be observed if those other variables are not sampled equally across head directions. The manuscript contains no rigorous control for this possibility. One place to start to address this issue would be to map out variables such as angular velocity by head orientation, and to test whether such relationships also carry 90 and 180 degree offsets.

(2) There is some question as to whether dRSC neurons (spatial or directional) following the sub-compartment "geometry" is appropriate in terms of interpreting the data. In the condition with sub-compartments carrying different tactile and visual cues, it seems that such cues pertain only to the floor of the environments. The distal visual space of the boundaries appears to be identical. One is left to wonder whether distinguishing environments according to boundary wall visual cues would lead to different results. The CA1 data does not help to rule this possibility out. A second reason to doubt the "shared geometry" interpretation is that there is no condition where sub-compartment geometry is varied. It is also the case that the sub-compartment doorways may stand as the only salient distal visual cue linking the environments. Local sensory cues and geometry seem not so disentangled in this study, but this is a major claim in the abstract.

(3) There is some concern with the interpretation that the spatial tuning of some dRSC neurons repeats in rotated form across sub-compartments. The firing rate map correlations are very low on average (~0.2), and far lower than the population of CA1 having repeating fields across the same vs different visual/tactile cue conditions. The authors should define the chance level of ratemap correlation by shuffling neuron identities. Apologies if this is indeed the current approach, but it seems not to be (I was left a bit lost by the description in the methods). For any population of hippocampal place cells, the cross-neuron correlations of firing rate maps are typically not zero, and correlations at 0.2 would normally be evidence for remapping.

(4) A somewhat picky point here that is not meant to claim that multi-compartment studies are not useful - the introduction states that real-world environments typically consist of multi-compartment rooms. This is certainly not true for rodents and is only sometimes true in humans.

(5) The discussion lacks a consideration of how such dRSC output might impact the target structures of dRSC.

(6) The discussion speaks to the idea that multi-directional neurons may aid in transitioning between contexts (sub-compartments). But it is notable that none of the multidirectional neurons have multi-directional tuning in all sub-compartments, but such firing was seen in the 2017 Nature Neuroscience study by Jacob/Jeffery. The discussion should address this difference and perhaps posit a means by which the firing of global and local head direction neurons can be related to each other to yield navigation that depends on both scales.

(7) The authors should provide the size of the smoothing function for spatial firing rate maps.

(8) The authors should devise a measure to define directional tuning in 4 directions (with 90-degree offsets).

(9) Figures 2D and 2H - The offsets in preferred tuning across sub-compartments are rather variable.

Reviewer #1 (Public review):

Summary:

This manuscript presents a tunable Bessel-beam two-photon fluorescence microscopy (tBessel-TPFM) platform that enables high-speed volumetric imaging with stable axial focus. The work is technically strong and broadly significant, as it substantially improves the flexibility and practicality of Bessel-beam-based two-photon microscopy. The demonstrations are generally strong and bridge a wide range of neuroimaging applications, namely vascular dynamics, neurovascular coupling, optogenetic perturbation, and microglial responses. These convincingly show that the approach enables biological measurements that are difficult or impractical with existing methods.

The evidence supporting the technical and biological claims is generally strong. The optical design is carefully motivated, clearly described, and validated through a combination of simulations and experimental characterization. The biological applications are diverse and well chosen to highlight the strengths of the proposed method, and the data are of high quality, with appropriate controls and comparative measurements where relevant.

Strengths:

(1) The optical innovation addresses a well-recognized limitation of existing Bessel-TPFM implementations, namely axial focus drift during tuning, and does so using a relatively simple, light-efficient, and cost-effective design.

(2) The manuscript provides convincing experimental evidence for this being a versatile platform to map flow dynamics across diverse vessel sizes and orientations in both healthy and pathological states.

(3) Biological demonstrations are comprehensive and span multiple domains such as hemodynamics, neurovascular coupling, and neuroimmune responses.

(4) Quantitative analyses of blood flow across vessel sizes and orientations, including kilohertz line scanning, are particularly compelling and clearly beyond the reach of standard Gaussian TPFM.

(5) Particular advantages are that higher blood slow speeds become measurable up to 23mm/sec (20x more than conventional frame scanning), and that simultaneous (Bessel-)imaging and (Gaussian-)perturbation are possible because of the stable axial focus.

Weaknesses:

(1) At present, the paper does not properly position the new Bessel-beam method against previous work, and fails to compare it to alternative fast volumetric imaging methods without Bessel beams.

(2) The cost-effectiveness of the proposed method is not well described or supported by evidence; it would be useful to include more detail or remove this claim.

(3) Some biological conclusions, e.g., regarding novel features of microglial dynamics (i.e., the observed two-wave responses and coordinated extension-retraction), are based on relatively limited sample size and would benefit from clearer discussion of variability across animals and fields of view.

(4) The use of neural network-based denoising for microglial imaging is reasonable but introduces potential concerns about trustworthiness; additional clarification of validation or failure modes would strengthen confidence in these results.

To conclude, most of the authors' claims are well supported by the data. The central conclusion, namely that tBessel-TPFM provides tunable volumetric imaging enabling experiments not feasible with existing two-photon approaches, is justified. Some biological interpretations would benefit from a more cautious framing, but they do not undermine the main technical and methodological contributions of the study. This is a strong and technically rigorous manuscript that makes a substantial methodological advance with clear relevance to neuroscience and intravital imaging. Minor clarifications and a slightly more measured discussion of certain biological findings are recommended.

Reviewer #2 (Public review):

Summary:

The authors describe a tunable Bessel beam two-photon microscope (tBessel-TPFM) designed to overcome a common limitation of Bessel-based volumetric imaging: axial shifts of the effective focus during Bessel beam parameter tuning. Their optical design allows independent control of axial beam length and resolution while keeping the axial center fixed. This is extensively validated through simulations and experiments.

Strengths:

A major strength of the work is the breadth of validation combined with the level of technical detail provided. The authors carefully characterize the optical performance of the system and clearly explain the design choices and underlying derivations, which will make it easier for others to understand and implement. The authors demonstrate the utility of the method across several in vivo applications, including neurovascular imaging, blood flow measurements, optogenetic stimulation, and microglial dynamics.

Weaknesses:

In the in vivo demonstrations, the authors employ different Bessel beam configurations across experiments, but the beam parameters are not dynamically tuned during live imaging. A video example showing continuous or interactive tuning of the Bessel beam within a single in vivo imaging sequence would further highlight the practical advantages of this platform and strengthen the case for its potential applications. In addition, while excitation powers are reported, the manuscript does not place these values in the broader context of known photodamage thresholds for two-photon microscopy, which would be helpful to the readers. Denoising/image restoration are applied in one of the in vivo examples, but it is unclear why this step was used specifically for this dataset and whether it was necessary to achieve adequate SNR or primarily included as an additional demonstration.

Reviewer #3 (Public review):

Summary:

The manuscript presents an elegant and cost-effective approach for generating a tunable Bessel beam on a conventional two-photon microscope. The authors assemble a compact optical module comprising three axicons and a series of lenses that permits rapid adjustment of both lateral resolution and axial extent without modifying the focal plane. This flexibility enables the system to be readily adapted to a variety of biological preparations. As a proof of concept, the authors employ the device to record blood flow velocities in cortical microcapillaries, arterioles, and venules, thereby directly visualizing vasodilatation and vasoconstriction dynamics and permitting quantitative analysis of neurovascular coupling across cortical layers in awake mice.

The authors demonstrate that the tunability of the Bessel beam can be exploited to match the numerical aperture to the vessel type: a high NA configuration, albeit slower scan, is optimal for resolving flow in capillaries, whereas a low NA setting provides faster acquisition suitable for arterioles and venules. By implementing a one-dimensional line scan with the Bessel beam, they achieve an imaging speed that is twentyfold faster than conventional frame-by-frame scanning, which proves sufficient to capture hemodynamic transients before and after an induced ischemic stroke.

In addition to pure observation, the authors integrate a co-propagating Gaussian line to the system, allowing simultaneous imaging and photostimulation within the same focal plane. This capability addresses a common limitation of other Bessel beam implementations, in which the observation and perturbation planes often become misaligned when the Bessel beam is altered. The manuscript also emphasizes the advantage of Bessel beam excitation for calcium imaging after a perturbation, because it captures neuronal activity in planes both above and below the nominal focal plane, signals that would be missed with a standard Gaussian focus. Finally, the authors apply the technique to investigate the neuroimmune response following targeted microglial ablation; they report that adjacent microglia extend processes toward the injury site while retracting processes in the opposite direction.

Overall, the work offers a technically straightforward yet powerful extension to existing two-photon platforms, providing high-speed, volumetric imaging and stimulation capabilities that are well-suited to a broad range of neurovascular and neuroimmune studies. The experimental validation is quite thorough, and the presented data convincingly illustrates the benefits of the approach.

Strengths:

The authors present a truly clever and inexpensive optical module that can be integrated into almost any two-photon microscope, providing a tunable Bessel beam with a minimal modification of the existing system. The experimental data and accompanying quantitative analysis convincingly demonstrate that the system can reveal physiological events, such as capillary flow, calcium transients across multiple axial planes, and microglial process dynamics, that are difficult or impossible to capture with a conventional Gaussian beam. The breadth of experiments chosen for the manuscript illustrates the practical utility of the device and supports the authors' conclusions that it extends the functional repertoire of standard two-photon microscopy.

Weaknesses:

The manuscript would benefit from a more detailed contextualisation of the claimed speed advantage. Although the authors mention other techniques in the introduction, they do not provide any direct comparison with other state-of-the-art high-speed two-photon approaches such as light beads microscopy (Demas et al., Nat. Methods 2021), temporal multiplexing schemes (Weisenburger et al., Cell 2019), or random access microscopy (Villette et al., Cell 2019). A brief comparison of imaging speed, spatial resolution, and instrumental complexity would enable readers to assess the relative merits of the present method.

A second limitation that warrants discussion is the inherent trade off between volumetric coverage and image specificity. Because the Bessel beam excites fluorescence throughout an extended axial range, the detector inevitably integrates signal from a three dimensional volume into a two dimensional image. In densely labelled tissue, this can lead to significant signal crosstalk, reducing contrast and complicating quantitative interpretation. A brief analysis of how labeling density affects the fidelity of flow or calcium measurements, or suggestions for mitigating crosstalk (e.g., computational deconvolution, adaptive excitation shaping, or combinatorial sparse labeling), would broaden the applicability of the technique.

Reviewer #1 (Public review):

Summary:

The study investigates the Drosophila non-visual light receptor rhodopsin7 with regard to its role in light information processing and resulting consequences for behavioral patterns and circadian clock function. Using behavioral, in situ staining, and receptor activation assays together with different fly mutants, the authors show that rhodopsin7 is an important determinant of activity under and response to darkness, which likely signals via a pathway distinct from other, visual Drosophila rhodopsins. Based on phylogenetic analysis, the authors further discuss a potentially conserved functional role of non-visual photoreceptors like rhodopsin7 and the mammalian melanopsin light information processing and circadian clock modulation.

Strengths:

The manuscript follows a very clear structure with all investigations logically building onto each other. Background information and methodology are provided in appropriate detail so that readers can fully understand why and how experiments were conducted. It is further praiseworthy that the authors provide the details that allow also non-experts in the field to fully understand their approaches. Experimental work was conducted in a highly standardized manner, and also considered potential "side-aspects" like the consequences of temperature cycles and changed photoperiods. The detailed and clear description of the obtained results makes them very convincing, with (almost) all observable patterns being addressed.

By highlighting the evolutionary old phylogenetic position of rhodopsin7 and its conservation across numerous clades, the authors provide strong reasoning for the relevance of their work, also pointing out the similarities to the mammalian melanopsin. The postulated hypothesis regarding protein structure and functioning, as well as the role in light information processing and behavioral and circadian clock modulation are well based on the authors' observations, and speculative aspects are correctly pointed out.

Weaknesses:

Where the manuscript still has potential for improvement is the discussion, which in its current form does seem slightly self-contained and does not fully integrate the findings of previous studies on Drosophila rhodopsin7. As the introduction specifically points out that previous findings have been contradictory, this seems like a missed opportunity. Further details on this are provided in the recommendations below.

Similarly, the manuscript currently lacks a discussion of the possible relevance of rhodopsin7 (and other non-visual light receptors in other organisms) in the context of a species' environment and lifestyle, i.e., what is the relevance/benefit of having rhodopsin7 in the fly's everyday life? While this clearly involves speculation, when done carefully, it can elevate the paper's relevance from a primarily academic to a societal one.

An additional point concerns the title and abstract, which postulate rhodopsin7 roles in contrast vision as well as motion and brightness perception. Contrast remains poorly defined in the text, leaving it ambiguous whether it refers to bright/dark contrasts, e.g., along edges, or the temporal contrast that results from dark pulses (startle response). While the latter seems to apply here, the former is likely more intuitive. Thus, this aspect should be rephrased (also in the title) or properly clarified early on. Regarding motion detection, this is backed up by the optomotor response results, but the findings stand somewhat isolated from the other results, lacking a clear connection aside from general visual processing. Lastly, brightness perception is mentioned in the abstract, but never again, possibly due to inconsistent phrasing throughout the manuscript.

Reviewer #2 (Public review):

Summary:

This is a very interesting paper bringing new and important information about the poorly understood rhodopsin 7 photoreceptive molecule. The very ancient origin of the gene is revealed in addition to data supporting a signaling pathway that is different from the one known for the canonical rhodopsins. Precise expression data, particularly in the optic lobe of the fly, as well as clear behavioral phenotypes in responses to light changes, make this study a strong contribution to the understanding of the still-debated function of rhodopsin 7.

Specific comments

(1) Title and abstract: Contribution of Rh7 to circadian clock regulation

(a) It is not that clear to me what rhodopsin does in terms of circadian regulation (even though its function might be circadianly regulated). The clear role in the light/dark distribution of activity might not be circadian per se, but mostly light/dark-driven, and there is no evidence here for a role in the entrainment of the clock.

(b) The authors should cite Lazopulo, which nicely shows that Rh7 has an important role in peripheral neurons to allow flies to escape from blue light (see below).

(2) Figure 2 C

The finding showing that Galphaz but not Galphaq can trigger signaling from light-excited Rh7 is a very intriguing finding to better understand Rh7 function. Since Galphaz is related to Gi/o, it would be interesting to test those, for example, by expressing RNAi with Rh7-gal4 and testing the Light-dark or light-off response behavior.

(3) Figures 3-4

The change in the locomotor activity distribution between light and dark in LD conditions provides a nice assay for Rh7 function. Since Lazopulo et al. (2019) have shown that wild-type but not Rh7 mutants do escape from blue light, it would be important to compare and discuss these LD behavior data with the Lazopulo results. Precisely, is this nighttime preference linked to blue light?

The expression data are really nice and show that Rh7 is mostly a non-retinal photoreceptor. However, the paper would be strongly reinforced by correlating this with the LD behavior. The LD phenotype should be tested in flies with Rh7 expression rescued under Rh7gal4 control (as done for the startle response). This is important to show whether the expression pattern is likely responsible for the described Rh7 function in LD. If L5 and or M11 drivers are available, they should be used to rescue Rh7? Since expression in some clock neurons is shown, the rescue experiment should also be done with a clock neuron driver.

In the same line, can the LD phenotype (or startle response phenotype of Figure 4) be restored by expressing Rh7 under ppk control, as shown for the blue light avoidance phenotype by Lazopulo et al?

Finally, the Rh7 "darkfly" rescued flies should be tested in LD.

Reviewer #3 (Public review):

Summary:

While our knowledge regarding visual opsins is largely very good, a lot more uncertainty exists around the role of non-visual opsins. Using the power of the Drosophila melanogaster model system, Kirsh et al. investigate the role of the non-visual opsin Rhodopsin7 (Rh7). Expression analysis, based on Rh7-Gal4>UAS-GFP and HRC in situ staining, reveals strong expression in the optic lobes and somewhat weaker, but nevertheless extensive expression in the brain. An investigation of motor activity reveals that loss of function leads to an altered day and night rhythm, specifically decreasing activity during the dark phase. These flies were also less sensitive, but still responsive to a light-induced startle response and showed deficiencies in the optomotor response. To further investigate how Rh7 may modulate these responses, inspired by the Dark line of flies (which were kept in the dark for ~1400 generations) and which has accumulated C-terminal related losses, the authors conducted rescues with an intact and a C-terminal-deficient Rh7 and were able to pinpoint that region as an important driver of related behavioral shifts. These findings are particularly intriguing as Rh7 represents an ancient opsin with phylogenetic and mechanistic parallels to mammalian melanopsin.

Strengths:

The paper is well-written and contains high-quality data with appropriate sample sizes, and the conclusions are well supported.

Weaknesses:

No weaknesses were identified by this reviewer, but the following recommendations are made:

(1) The authors should clarify exactly what tissues were taken for the comparative qPCR. This is particularly interesting in terms of the retina. Since Rh7 appears not to be expressed within the photoreceptor cells of the retina, this raises the important question as to which cells it is expressed in. To address this important question, it would also be helpful to include an expression analysis of the retina itself (by extending the RH7-GFP expression patterns and/or adding HCR in situ of the ommatidia array). The cell types of the retina are very well classified, and some evidence already exists for Rh7 expression in support cells (e.g., Charlton-Perkins et al., (2017); PMID: 28562601). This study has a unique opportunity to investigate this further by adding these critical data for a more complete picture of Rh7.

(2) Mammalian opsins should be included in the phylogenetic analysis illustrated in Figure 2A and indicate their position on the tree. This will allow readers to better put the authors' statements regarding the intermediate position of Rh7 into perspective. In addition, note that the distinction between red and deep red is easy to miss regarding the Rh7 cluster. Perhaps the authors could use a more distinct colour scheme, for example, orange and deep red.

(3) More details should be provided on the optomotor response experiments. Specifically, specifications of the frequencies used for the optomotor response are needed. Results show a relatively large level of variation, which may be due to different angular perspectives that flies may have had while viewing the stimulus. If possible, provide videos as examples, as they will make it clearer to viewers how much flies could move around in the setup (from the methods, it seems they could move within the 2.2 of the 3 cm diameter of the arena, which would lead to substantial differences in the visual angle of the viewed grating.

Reviewer #1 (Public review):

Summary:

Using a computational modeling approach based on the Drift and Diffusion Model (DDM) introduced by Ratcliff and McKoon in 2008, the article by Shevlin and colleagues investigates whether there are differences between neutral and negative emotional states in:

(1) The timings of the integration in food choices of the perceived healthiness and tastiness of food options in individuals with bulimia nervosa (BN) and healthy participants (2) The weighting of the perceived healthiness and tastiness of these options.

Strengths:

By looking at the mechanistic part of the decision process, the approach has potential to improve the understanding of pathological food choices.

Weaknesses:

I thank the authors for revising their manuscript.

I still notice that the authors did not go through their manuscript to look for wordings refering to a prediction interpretation of their results while I already highlighted the inappropriateness of this wording in my two first rounds of reviews: e.g. there is still "we used zero-inflated negative binomial models to predict the three-month frequency" and I can find other statements like this. The design of their study does not allow such claims.

The authors answered my major concern regarding the experimental induction towards a negative or a neutral state before running the food decision task. My concern is: BN patients already seemed to be already in a high negative state before undergoing the neutral induction, while these patients are in a lower negative state before undergoing the negative induction. It is therefore not surprising that patients seem to report a similar level of negative state after the two inductions (according to the figure of the authors' previous article). Of note is that the additional analysis the authors ran within the BN group only provides a significant result: this result shows that there has been an induction but does not rule out that patients were in the exact same magnitude of negative state to perform the task as the figure in their previously published article suggests it. The major issue is to show that:

(1) As compared to the neutral induction, there has been a higher variation in negative state after as compared to before the negative induction.

(2) The magnitude of the negative state after the negative induction is higher than the magnitude of the negative state after the neutral induction.

The first point shows that the induction worked. The second point shows that the participants are in two distinct states. Without showing the second point, it may be possible that one induction increases the negative state of participants to the same level as the one of the second induction that has not increased anything.

Within this context, how is it possible to associate, in patients, a difference in the DDM between the two sessions to a negative state (which is one of the main focus of the article) rather than to another parameter that has not been captured? A similar situation would be in an experiment studying the consequence of stress, a stressfull induction over relaxed participants attending the lab has high chances to raise the level of stress of those participants to the same level as the one that the same participants would experience after a neutral induction when these participants attend the lab with an already high level of stress. In that case, would it be approrpiate to claim that a difference at a task performed after the induction would be related to stress while the participants would be at the same level of stress when performing the task despite the fact that the induction worked ?

In the experiment performed by the authors, the additional analysis to perform would be a paired sample t-test (or the appropriate non-parametric test) to check whether the magnitude of negative state of BN patients was different between the negative and neutral conditions after the induction only. If not, associating the difference at the DDM with negative states in BN is highly misleading.

I read carefully the authors' answer related to mixed models: they claim that mixed models take into account correlations within their repeated data. The specification of the structure of the covariance matrix allows to control only partly for that. I notice that the authors did not specify the structure of that matrix: the article they refer to to justify the appropriatness of their analyses is not adapted. The specification of the structure of the covariance matrix needs to address, in a mixed model, the difference in handling 4 repeated data per participants that cannot be paired as compared to 4 repeated data that can be paired (two per session with one before and one after the neutral or negative priming sessions, if I count right). Of note is that a covariance structure that is left free of constraint for the fit of the model does not capture appropriately the pairing of the data: it has all chances to capture the covariance in a different way. And a covariance structure that has constraints has more chances to lead to a model that cannot be estimated because of an absence of convergence of the algorithms.

By the way, a single two-sample t-test (or a Mann-Whitney test if appropriate), and not a set of multiple paired-sample t-test as the authors suggest, would answer the goal of the authors to test for what they call the three-way interaction in their comment. This test would be performed between the two groups of participants (BN/controls) with the computation for each participant separately: (assessment after neutral induction-assessment before neutral induction)-(assessment after negative induction-assessment before negative induction). This analysis answers points 1, 2 and 4 they raise together with my point of controlling for the paired data. I would have agreed with their choice of a mixed model if they had an unbalanced dataset within each participant.

Reviewer #2 (Public review):

Summary:

Binge eating is often preceded by heightened negative affect, but the specific processes underlying this link are not well-understood. The purpose of this manuscript was to examine whether affect state (neutral or negative mood) impacts food choice decision-making processes that may increase likelihood of binge eating in individuals with bulimia nervosa (BN). The researchers used a randomized crossover design in women with BN (n=25) and controls (n=21), in which participants underwent a negative or neutral mood induction prior to completing a food-choice task. The researchers found that despite no differences in food choices in the negative and neutral conditions, women with BN demonstrated a stronger bias toward considering the 'tastiness' before the 'healthiness' of the food after the negative mood induction.

Strengths:

The topic is important and clinically relevant and methods are sound. The use of computational modeling to understand nuances in decision-making processes and how that might relate to eating disorder symptom severity is a strength of the study.

Weaknesses:

Sample size was relatively small, and participants were all women with BN, which limits generalizability of findings to the larger population of individuals who engage in binge eating. It is likely that the negative affect manipulation was weak and may not have been potent enough to change behavior. These limitations are adequately noted in the discussion.

Reviewer #1 (Public review):

Summary:

This useful study provides incomplete evidence of an association between atovaquone-proguanil use (as well as toxoplasmosis seropositivity) and reduced Alzheimer's dementia risk. The study reinforces findings that VZ vaccine lowers AD risk and suggests that this vaccine may be an effect modifier of A-P's protective effect. Strengths of the study include two extremely large cohorts, including a massive validation cohort in the US. Statistical analyses are sound, and the effect sizes are significant and meaningful. The CI curves are certainly impressive.

Weaknesses include the inability to control for potentially important confounding variables. In my view, the findings are intriguing but remain correlative / hypothesis generating rather than causative. Significant mechanistic work needs to be done to link interventions which limit the impact of Toxoplasmosis and VZV reactivation on AD.

Weaknesses:

Major:

(1) Most of the individuals in the study received A-P for malaria prophylaxis as it is not first line for Toxo treatment. Many (probably most) of these individuals were likely to be Toxo negative (~15% seropositive in the US), thereby eliminating a potential benefit of the drug in most people in the cohort. Finally, A-P is not a first line treatment for Toxo because of lower efficacy.

(2) A-P exposure may be a marker of subtle demographic features not captured in the dataset such as wealth allowing for global travel and/or genetic predisposition to AD. This raises my suspicion of correlative rather than casual relationships between A-P exposure and AD reduction. The size of the cohort does not eliminate this issue, but rather narrows confidence intervals around potentially misleading odds ratios which have not been adjusted for the multitude of other variables driving incident AD.

(3) The relationship between herpes virus reactivation and Toxo reactivation seems speculative.

(4) A direct effect on A-P on AD lesions independent on infection is not considered as a hypothesis. Given the limitations above and effects on metabolic pathways, it probably should be. The Toxo hypothesis would be more convincing if the authors could demonstrate an enhanced effect of the drug in Toxo positive individuals without no effect in Toxo negative individuals.

Minor:

(5) "Clinically meaningful" should be eliminated from the discussion given that this is correlative evidence.

Reviewer #2 (Public review):

Summary:

This manuscript examines the association between atovaquone/proguanil use, zoster vaccination, toxoplasmosis serostatus and Alzheimer's Disease, using 2 databases of claims data. The manuscript is well written and concise. The major concerns about the manuscript center around the indications of atovaquone/proguanil use, which would not typically be active against toxoplasmosis at doses given, and the lack of control for potential confounders in the analysis.

Strengths:

(1) Use of 2 databases of claims data.

(2) Unbiased review of medications associated with AD, which identified zoster vaccination associated with decreased risk of AD, replicating findings from other studies.

Weaknesses:

(1) Given that atovaquone/proguanil is likely to be given to a healthy population who is able to travel, concern that there are unmeasured confounders driving the association.

(2) The dose of atovaquone in atovaquone/proguanil is unlikely to be adequate suppression of toxo (much less for treatment/elimination of toxo), raising questions about the mechanism.

(3) Unmeasured bias in the small number of people who had toxoplasma serology in the TriNetX cohort.

Reviewer #1 (Public review):

The current manuscript investigates a regulatory axis containing Prmt1, which methylates RNA binding proteins and alters intron splicing outcomes and expression of matrix genes. Authors test the effects of deficient Prmt1, Sfpq, and various other factors, using a combination of bioinformatic analyses and wet-lab validation approaches. Authors show that intron retention often triggers NMD, contributing to aberrant gene expression regulation and craniofacial development. The revised manuscript introduces several complementary experiments that help to strengthen conclusions. For example, authors directly investigate NMD-mediated transcript turnover to better understand how retention contributes to expression changes in genes of interest, and they assess several additional factors downstream of Prmt1 to justify a centralized interested in the PRMT1/SFPQ axis.

Weaknesses:

However, some points remain unaddressed or unexplored, which could bolster conclusions. For example, the transcriptome data from knockdown experiments indicate robust exon skipping, suggesting that analysis of these patterns in parallel with intron retention could provide additional insights into the responsive gene programs. Given that SFPQ is known to have multiple regulatory roles, a more thorough investigation of its possible mechanisms of action during craniofacial development would allow for definitive conclusions about the isolated impact of SFPQ-dependent splicing. Although authors employ CUT&Tag analysis of Pol II binding at the promoters and across the gene body, at the current scope, no change in Pol II association (i.e., absence of transcriptional repression) does not directly indicate a lack of transcriptional regulation by other means (pause release, elongation rate or processivity, transcription termination, etc.). Without a more thorough investigation of these mechanisms, this confounds definitive claims about their relative contributions to the gene expression landscape.

Reviewer #2 (Public review):

Summary:

The manuscript by Lima et al examines the role of Prmt1 and SFPQ in craniofacial development. Specifically, the authors test the idea that Prmt1 directly methylates specific proteins that results in intron retention in matrix proteins. The protein SFPQ is methylated by Prmt1 and functions downstream to mediate Prmt1 activity. The genes with retained introns activate the NMD pathway to reduce the RNA levels. This paper describes an interesting mechanism for the regulation of RNA levels during development.

Strengths:

The phenotypes support what the authors claim that Prmt1 is involved in craniofacial development and splicing. They use of state of the art sequencing to determine the specific genes that have intron retention and changes in gene expression is a strength.

Weaknesses:

The results now support the conclusions;however, it is still unclear how direct the relationship is between Prmt1 and SFPQ.

Reviewer #1 (Public review):

Summary

The authors determine the phylogenetic relation of the roughly two dozen wtf elements of 21 S. pombe isolates and show that none of them in the original S. pombe are essential for robust mitotic growth. It would be interesting to test their meiotic function by simply crossing each deletion mutant with the parent and analyzing spores for non-Mendelian inheritance. If this has been reported already, that information should be added to the MS. If not, I suggest the authors do these simple experiments and add this information.

Strengths:

The most interesting data (Fig. 4) show that one recombinant (wtfC4) between wtf18 and wtf23 produces in mitotic growth a poison counteracted by its own antidote but not by the parental antidotes. Again, it would be interesting to test this recombinant in a more natural setting - meiosis between it and each of the parents.

Weaknesses:

Some minor rewriting is needed.

Comments on Revision:

(1) The parameter for "maximum growth rate" in Figure 2D needs to be defined and put on the graph.

(2) On page 8, line 182, the authors should consider testing the hybrid wtf in meiosis using strain 975 of Leupold, which is h+, or another standard h+ strain. I don't think the antidote allele is needed; rather, it seems to me it would counter the lethality of the poison protein and should be omitted to test drive of the hybrid wtf. This is a simple experiment and would add considerably to the paper.

Reviewer #3 (Public review):

Summary:

In this manuscript, Wang and colleagues explore factors contributing to the diversification of wtf meiotic drivers. wtf genes are autonomous, single-gene poison-antidote meiotic drivers that encode both a spore-killing poison (short isoform) and an antidote to the poison (long isoform) through alternative transcriptional initiation. There are dozens of wtf drivers present in the genomes of various yeast species, yet the evolutionary forces driving their diversification remain largely unknown. This manuscript is written in a straightforward and effective manner, and the analyses and experiments are easy to follow and interpret. While I find the research question interesting and the experiments persuasive, they do not provide any deeper mechanistic understanding of this gene family.

Revision update:

Having read the response to the reviewers, I believe the major issues have been addressed. However, I would strongly suggest toning down the claim regarding the chimeric WTF element in the abstract, which currently reads

"As proof-of-principle, we generate a novel meiotic driver through artificial recombination between wtf drivers, and its encoded poison cannot be detoxified by the antidotes encoded by their parental wtf genes but can be detoxified by its own antidote."

As the author reports in their response, despite various attempts, it was not possible to show that this chimeric WTF element was indeed capable of meiotic drive in a natural context (not transgenic overexpression experiment). thus the authors should not claim they generated "a novel meiotic driver"

Strengths:

(1) The authors present a comprehensive compendium and analysis of the evolutionary relationships among wtf genes across 21 strains of S. pombe

(2) The authors found that a synthetic chimeric wtf gene, combining exons 1-5 of wtf23 and exon 6 of wtf18, behaves like a meiotic driver that could only be rescued by the chimeric antidote but neither of the parental antidotes. This is a very interesting observation that could account for their inception and diversification.

Weaknesses:

(1) Deletion strains

The authors separately deleted all 25 Wtf genes in the S. pombe ference strain. Next, the authors performed spot assay to evaluate the effect of wtf gene knockout on the yeast growth. They report no difference to the WT and conclude that the wtf genes might be largely neutral to the fitness of their carriers in the asexual life cycle at least in normal growth condition.

The authors could have conducted additional quantitative growth assays in yeast, such as growth curves or competition assays, which would have allowed them to detect subtle fitness effects that cannot be quantified with a spot assay. Furthermore, the authors do not rule out simpler explanations, such as genetic redundancy. This could have been addressed by crossing mutants of closely related paralogs or editing multiple wtf genes in the same genetic background.

Another concern is the lack of detailed information about the 25 knockout strains used in the study. There is no information provided on how these strains were generated or, more importantly, validated. Many of these wtf genes have close paralogs and are flanked by repetitive regions, which could complicate the generation of such deletion strains. As currently presented, these results would be difficult to replicate in other labs due to insufficient methodological details

Revision update:

The authors measured the fitness of the deletion strains using growth curves (Fig. 2C and D) and no significant differences were found, further supporting their claims. The requested information (details on the generation of the deletion strains) is now available in the methods section.

(2) Lack of controls

The authors found that a synthetic chimeric wtf gene, constructed by combining exons 1-5 of wtf23 and exon 6 of wtf18, behaves as a meiotic driver that can be rescued only by its corresponding chimeric antidote, but not by either of the parental antidotes (Figure 4F). In contrast, three other chimeric wtf genes did not display this property (Figure 4C-E). No additional experiments were conducted to explain these differences, and basic control experiments, such as verifying the expression of the chimeric constructs, were not performed to rule out trivial explanations. This should be at the very least discussed. Also, it would have been better to test additional chimeras.

Revision update:

The authors report that the expression of the construct was measured. However, they do not make reference to any specific figure or section of the main text. It would be very useful if the authors explicitly referenced where exactly changes were made (this is true for all changed made)

(3) Statistical analyses

In line 130 the authors state that: "Given complex phylogenetic mixing observed among wtf genes (Figure 1E), we tested whether recombination occurred. We detected signals of recombination in the 25 wtf genes of the S. pombe reference genome (p = 0) and in the wtf genes of the 21 S. pombe strains (p = 0) using pairwise homoplasy index (HPI) test. "<br /> Reporting a p-value of 0 is not appropriate. Please report exact P-values.

Revision update:

This has been addressed.

Joint Public Review:

While DNA sequence divergence, differential expression and differential methylation analysis have been conducted between humans and the great apes to study changes that "make us human", the role of lncRNAs and their impact on the human genome and biology has not been fully explored. In this study the authors computationally predict HSlncRNAs as well as their DNA Binding sites using a method they have developed previously and then examine these predicted regions with different types of enrichment analyses. Broadly the analysis are straightforward and after identifying these regions/HSlncRNAs they examined their effects using different external datasets.

Comments on the latest version from Reviewer #2:

I think this is as good as it is going to get, and I do appreciate that the authors are still engaging in good faith after all these rounds of revision, so I am happy to stop here! I do think the paper is significantly improved from the last time around, and the conclusions have been tempered significantly.

Reviewer #1 (Public review):

Summary:

The present study evaluates the role of visual experience in shaping functional correlations between human extrastriate visual cortex and frontal regions. The authors used fMRI to assess "resting-state" temporal correlations in three groups: sighted adults, congenitally blind adults, and neonates. Previous research has already demonstrated differences in functional correlations between visual and frontal regions in sighted compared to early blind individuals. The novel contribution of the current study lies in the inclusion of an infant dataset, which allows for an assessment of the developmental origins of these differences.

The main results of the study reveal that correlations between prefrontal and visual regions are more prominent in the blind and infant groups, with the blind group exhibiting greater lateralization. Conversely, correlations between visual and somato-motor cortices are more prominent in sighted adults. Based on these data, the authors conclude that visual experience shapes these cortical networks through activity-dependent plasticity. This study provides novel insights into the impact of visual experience on the development of temporal correlations in the brain.

Strengths:

The dissociations in functional correlations observed among the sighted adult, congenitally blind, and neonate groups provide strong support for the main conclusion regarding postnatal experience-driven shaping of visual-frontal connectivity.

The neonatal data offers a unique and valuable developmental anchor for interpreting divergence between blind and sighted adults. This is a major advance over prior studies limited to adult comparisons.

Convergence with prior findings in the blind and sighted adult groups reinforces the reliability and external validity of the present results.

The split-half reliability analysis in the infant and adult data increases confidence in the robustness of the reported group differences.

Weaknesses:

The methodology cannot determine whether group differences in correlations reflect direct changes in communication between visual and frontal regions or indirect effects mediated by other structures.

The cross-sectional design cannot reveal the timecourse over which visual experience shapes connectivity between infancy and adulthood.

Whether the infant resting-state patterns imply similar functional capacity to blind adults (e.g., cross-modal task responses) remains untested.

Comments on revisions:

The authors have done a fantastic job addressing my remaining questions.

Reviewer #2 (Public review):

Summary:

Tian et al. explore the developmental origins of cortical reorganization in blindness. Previous work has found that a set of regions in the occipital cortex show different functional responses and patterns of functional correlations in blind vs. sighted adults. Here, Tian et al. explore how this organisation arises over development, asking whether the infant brain looks more like the blind adult pattern, or more like the sighted adult pattern. Their analyses reveal that the answer depends on the particular networks investigated. Some functional connections in infants look more like blind than sighted adults; other functional connections look more like sighted than blind adults; and others fall somewhere in the middle, or show an altogether different pattern in infants compared with both sighted and blind adults.

Strengths:

The paper addresses very important questions about the "starting state" in the developing visual cortex, and how cortical networks are shaped by experience. Another clear strength lies in the unequivocal nature of many results. Many results have very large effect sizes, critical interactions between regions and groups are tested and found, and infant analyses are replicated in split halves of the data.

Weaknesses:

While potential roles of experience (e.g., visual, cross-modal) are discussed in detail, little consideration is given to the role of experience-independent maturation. The infants scanned are extremely young, only 2 weeks old. It is possible that the sighted adult pattern may still emerge later in infancy or childhood, regardless of infant visual experience. If so, the blind adult pattern may depend on blindness-related experience only (which may or may not reflect "visual" experience per se). In short, it is not clear that the age range studied is a clear-cut "starting point" for development, after which all change can be attributed to experience.

Reviewer #3 (Public review):

Summary

This study aimed to investigate whether the differences observed in the organization of visual brain networks between blind and sighted adults result from a reorganization of an early functional architecture due to blindness, or whether the early architecture is immature at birth and requires visual experience to develop functional connections. This question was investigated through the comparison of 3 groups of subjects with resting-state functional MRI (rs-fMRI). Based on convincing analyses, the study suggests that: 1) secondary visual cortices showed higher connectivity to prefrontal cortical regions (PFC) than to non-visual sensory areas (S1/M1 and A1) in infants like in blind adults, in contrast to sighted adults; 2) the V1 connectivity pattern of infants lies between that of sighted adults (showing stronger functional connectivity with non-visual sensory areas than with PFC) and that of blind adults (showing stronger functional connectivity with PFC than with non-visual sensory areas); 3) the laterality of the connectivity patterns of infants resembled those of sighted adults more than those of blind adults, but infants showed a less differentiated fronto-occipital connectivity pattern than adults.

Strengths

- The question investigated in this article is important for understanding the mechanisms of plasticity during typical and impaired development, and the approach considered, which compares different groups of subjects including, neonates/infants and blind adults, is highly original.

- Overall, the presented analyses are solid and well-detailed, and the results and discussion are convincing.

Weaknesses

- While it is informative to compare the "initial" state (close to birth) and the "final" states in blind and sighted adults to study the impact of post-natal and visual experience, this study does not analyze the chronology of this development and when the specialization of functional connections is completed. This would require investigating the evolution of functional connectivity of the visual system as a function of visual experience and thus as a function of age, at least during toddlerhood given the early and intense maturation of the visual system after birth. This could be achieved by analyzing different developmental periods using open databases such as the Baby Connectome Project.

- The rationale for grouping full-term neonates and preterm infants (scanned at term-equivalent age) is not understandable when seeking to perform comparisons with adults. Even if the study results do not show differences between full-terms and preterms in terms of functional connectivity differences between regions and of connectivity patterns, preterms group had different neurodevelopment and post-natal (including visual) experiences (even a few weeks might have an impact). And actually they show reduced connectivity strength systematically for all regions compared with full-terms (Sup Fig 7). Considering a more homogeneous group of neonates would have strengthened the study design.

- The rationale for presenting results on the connectivity of secondary visual cortices before the one of primary cortices (V1) could be clarified.

- The authors acknowledge the methodological difficulties for defining regions of interest (ROIs) in infants in a similar way as adults. Since the brain development is not homogeneous and synchronous across brain regions (in particular with the frontal and parietal lobes showing a delayed growth), this poses major problems for registration. This raises the question of whether the study findings could be biased by differences in ROI positioning across groups.

Comments on revisions:

The authors have addressed my specific recommendations, but some weaknesses in the study remain, particularly the inclusion of preterm infants alongside full-term neonates.

Reviewer #1 (Public review):

Summary:

Lesser et al provide a comprehensive description of Drosophila wing proprioceptive sensory neurons at the electron microscopy resolution. This "tour-de-force", provides a strong foundation for future structural and functional research aimed at understanding wing motor control in Drosophila with implications to understanding wing control across other insects.

Strengths:

(1) Authors leverage previous research that described many of the fly wing proprioceptors, and combine this knowledge with EM connectome data such that they now provide a near-complete morphological description of all wing proprioceptors.

(2) Authors cleverly leverage genetic tools and EM connectome data to tie the location of proprioceptors on the wings with axonal projections in the connectome. This enables them to both align with previous literature as well as make some novel claims.

(3) In addition to providing a full description of wing proprioceptors, authors also identified a novel population of sensors on the wing tegula that make direct connections with the B1 wing motor neurons implicating the role of tegula in wing movements that was previously underappreciated.

(4) Despite being the most comprehensive description so far, it is reassuring that authors clearly state the missing elements in the discussion.

Weaknesses:

(1) Authors do their main analysis on data from FANC connectome but provide corresponding IDs for sensory neurons in the MANC connectome. I wonder how the connectivity matrix compares across FANC and MANC if the authors perform similar analysis as they have done in Fig. 2. This could be a valuable addition and potentially also pick up any sexual dimorphism.

(2) Authors speculate about presence of gap junctions based on density of mitochondria. I'm not convinced about this given mitochondrial densities could reflect other things that correlate with energy demands in sub-compartments.

Overall, I consider this an exceptional analysis which will be extremely valuable to the community.

Reviewer #2 (Public review):

Summary:

Lesser et al. present an atlas of Drosophila wing sensory neurons. They proofread the axons of all sensory neurons in the wing nerve of an existing electron microscopy dataset, the female adult fly nerve cord (FANC) connectome. These reconstructed sensory axons were linked with light microscopy images of full-scale morphology to identify their origin in the periphery of the wing and encoded sensory modalities. The authors described the morphology and postsynaptic targets of proprioceptive neurons as well as previously unknown sensory neurons.

Strengths:

The authors present a valuable catalogue of wing sensory neurons, including previously undescribed sensory axons in the Drosophila wing. By providing both connectivity information with linked genetic drive lines, this research facilitates future work on the wing motor-sensory network and applications relating to Drosophila flight. The findings were linked to previous research as well as their putative role in the proprioceptive and nerve cord circuitry, providing testable hypotheses for future studies.

Weaknesses:

With future use as an atlas, it should be noted that the evidence is based on sensory neurons on only one side of the nerve cord. Fruit flies have stereotyped left/right hemispheres in the brain and left/right hemisegments in the nerve cord. Comparison of left and right neurons of the nervous system can give a sense of how robust the morphological and connectivity findings are. Unfortunately, this dataset has damage to the right side, making such comparisons unreliable.

Reviewer #3 (Public review):

Summary:

The authors aim to identify the peripheral end organ origin in the fly's wing of all sensory neurons in the Anterior Dorsal Mesothoracic nerve. They reconstruct the neurons and their downstream partners in an electron microscopy volume of a female ventral nerve cord, analyse the resulting connectome and identify their origin with review of the literature and imaging of genetic driver lines. While some of the neurons were already known through previous work, the authors expand on the identification and create a near complete map of the wing mechanosensory neurons at synapse resolution.

Strengths:

The authors elegantly combine electron microscopy neuron morphology, connectomics and light microscopy methods to bridge the gap between fly wing sensory neuron anatomy and ventral nerve cord morphology. Further, they use EM ultrastructural observations to make predictions on the signaling modality of some of the sensory neurons and thus their function in flight.

The work is as comprehensive as state of the art methods allow to create a near complete map of the wing mechanosensory neurons. This work will be of importance to the field of fly connectomics and modelling of fly behavior as well as a useful resource to the Drosophila research community.

Through this comprehensive mapping of neurons to the connectome the authors create a lot of hypotheses on neuronal function partially already confirmed with the literature and partially to be tested in the future. The authors achieved their aim of mapping the periphery of the fly's wing to axonal projections in the ventral nerve cord, beautifully laying out their results to support their mapping.

The authors identify the neurons in a previously published connectome of a male fly ventral nerve cord to enable cross-individual analysis of connections and find no indication of sexual dimorphism at the sensory neuron level. Further, together with their companion paper Dhawan et al., 2025 describing the haltere sensory neurons in the same EM dataset, they cover the entire mechanosensory space involved in Drosophila flight.

Reviewer #1 (Public review):

The authors show experimentally that, in 2D, bacteria swim up a chemotactic gradient much more effectively when they are in the presence of lateral walls. Systematic experiments identify an optimum for chemotaxis for a channel width of ~8µm, a value close to the average radius of the circle trajectories of the unconfined bacteria in 2D. These chiral circles impose that the bacteria swim preferentially along the right-side wall, which indeed yields chemotaxis in the presence of a chemotactic gradient. These observations are backed by numerical simulations and a geometrical analysis.

Reviewer #3 (Public review):

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

The experimental aspects of this study are well executed. The design of the microfluidic system is clever in that it allows a kind of "multiplexing" in which all the different channel widths are available to a given sample of bacteria.<br /> The authors have included a useful intuitive explanation of their results via a geometric model of the trajectories. In future work it would be interesting to analyze further the voluminous data on the trajectories of cells by formulating the mathematical problem in terms of a suitable Fokker-Planck equation for the probability distribution of swimming directions. In particular, this might help understand how incipient circular trajectories are interrupted by collisions with the walls and how this relates to enhanced chemotaxis.

The authors argue that these findings may have relevance to a number of physiological and ecological contexts. As these would be characterized by significant heterogeneity in pore sizes and geometries, further work will be necessary to translate the present results to those situations.

Reviewer #1 (Public review):

Summary:

This is a careful and comprehensive study demonstrating that effector-dependent conformational switching of the MT lattice from compacted to expanded deploys the alpha tubulin C-terminal tails so as to enhance their ability to bind interactors.

Strengths:

The authors use 3 different sensors for the exposure of the alpha CTTs. They show that all 3 sensors report exposure of the alpha CTTs when the lattice is expanded by GMPCPP, or KIF1C, or a hydrolysis-deficient tubulin. They demonstrate that expansion-dependent exposure of the alpha CTTs works in tissue culture cells as well as in vitro.

Appraisal:

The authors have gone to considerable lengths to test their hypothesis that microtubule expansion favours deployment of the alpha tubulin C-terminal tail, allowing its interactors, including detyrosinase enzymes, to bind. There is a real prospect that this will change thinking in the field. One very interesting possibility, touched on by the authors, is that the requirement for MAP7 to engage kinesin with the MT might include a direct effect of MAP7 on lattice expansion.

Impact:

The possibility that the interactions of MAPS and motors with a particular MT or region feed forward to determine its future interaction patterns is made much more real. Genuinely exciting.

Reviewer #2 (Public review):

The unstructured α- and β-tubulin C-terminal tails (CTTs), which differ between tubulin isoforms, extend from the surface of the microtubule, are post-translationally modified, and help regulate the function of MAPs and motors. Their dynamics and extent of interactions with the microtubule lattice are not well understood. Hotta et al. explore this using a set of three distinct probes that bind to the CTTs of tyrosinated (native) α-tubulin. Under normal cellular conditions, these probes associate with microtubules only to a limited extent, but this binding can be enhanced by various manipulations thought to alter the tubulin lattice conformation (expanded or compact). These include small-molecule treatment (Taxol), changes in nucleotide state, and the binding of microtubule-associated proteins and motors. Overall, the authors conclude that microtubule lattice "expanders" promote probe binding, suggesting that the CTT is generally more accessible under these conditions. Consistent with this, detyrosination is enhanced. Mechanistically, molecular dynamics simulations indicate that the CTT may interact with the microtubule lattice at several sites, and that these interactions are affected by the tubulin nucleotide state.

Strengths and weaknesses:

Key strengths of the work include the use of three distinct probes that yield broadly consistent findings, and a wide variety of experimental manipulations (drugs, motors, MAPs) that collectively support the authors' conclusions, alongside a careful quantitative approach.

The challenges of studying the dynamics of a short, intrinsically disordered protein region within the complex environment of the cellular microtubule lattice, amid numerous other binders and regulators, should not be understated. While it is very plausible that the probes report on CTT accessibility as proposed, the possibility of confounding factors (e.g., effects on MAP or motor binding) cannot be ruled out. Sensitivity to the expression level clearly introduces additional complications. Likewise, for each individual "expander" or "compactor" manipulation, one must consider indirect consequences (e.g., masking of binding sites) in addition to direct effects on the lattice; however, this risk is mitigated by the collective observations all pointing in the same direction.

The discussion does a good job of placing the findings in context and acknowledging relevant caveats and limitations. Overall, this study introduces an interesting and provocative concept, well supported by experimental data, and provides a strong foundation for future work. This will be a valuable contribution to the field.

Reviewer #3 (Public review):

Summary:

In this study, the authors investigate how the structural state of the microtubule lattice influences the accessibility of the α-tubulin C-terminal tail (CTT). By developing and applying new biosensors, they reveal that the tyrosinated CTT is largely inaccessible under normal conditions but becomes more accessible upon changes to the tubulin conformational state induced by taxol treatment, MAP expression, or GTP-hydrolysis-deficient tubulin. The combination of live imaging, biochemical assays, and simulations suggests that the lattice conformation regulates the exposure of the CTT, providing a potential mechanism for modulating interactions with microtubule-associated proteins. The work addresses a highly topical question in the microtubule field and proposes a new conceptual link between lattice spacing and tail accessibility for tubulin post-translational modification. Future work is required to distinguish CTT exposure in the microtubule lattice is sensitive to additional factors present in vivo but not in vitro.

Strengths:

(1) The study targets a highly relevant and emerging topic-the structural plasticity of the microtubule lattice and its regulatory implications.

(2) The biosensor design represents a methodological advance, enabling direct visualization of CTT accessibility in living cells.

(3) Integration of imaging, biochemical assays, and simulations provides a multi-scale perspective on lattice regulation.

(4) The conceptual framework proposed lattice conformation as a determinant of post-translational modification accessibility is novel and potentially impactful for understanding microtubule regulation.

[Editors' note: the authors have responded to the reviewers and this version was assessed by the editors.]

Reviewer #1 (Public review):

Summary:

This manuscript uses primarily simulation tools to probe the pathway of cholesterol transport with the smoothened (SMO) protein. The pathway to the protein and within SMO is clearly discovered and interactions deemed important are tested experimentally to validate the model predictions.

Strengths:

The authors have clearly demonstrated how cholesterol might go from the membrane through SMO for the inner and outer leaflets of a symmetrical membrane model. The free energy profiles, structural conformations and cholesterol-residue interactions are clearly described.

Weaknesses:

None. I find the revised manuscript strong and the work should be published.

Reviewer #2 (Public review):

Summary:

In this work, the authors applied a range of computational methods to probe the translocation of cholesterol through the Smoothened receptor. They test whether cholesterol is more likely to enter the receptor straight from the outer leaflet of membrane or via a binding pathway in the inner leaflet first. Their data reveal that both pathways are plausible but that the free energy barriers of pathway 1 is lower suggesting this route is preferable. They also probe the pathway of cholesterol transport from the transmembrane region to the cysteine-rich domain (CRD).

Strengths:

A wide range of computational techniques are used, including potential of mean force calculations, adaptative sampling, dimensionality reduction using tICA, and MSM modelling. These are all applied in a rigorous manner and the data are very convincing. The computational work is an exemplar of a well-carried out study.

Their computational predictions are experimentally supported using mutagenesis, with an excellent agreement between their PMF and mRNA fold change data.

The data are described clearly and coherently, with excellent use of figures. They combine their findings into a mechanism for cholesterol transport, which on the whole seems sound.

Their methods are described well, and much of their analysis methods have been made available via GitHub, which is an additional strength.

Reviewer #3 (Public review):

This manuscript presents a study combining molecular dynamics simulations and Hedgehog (Hh) pathway assays to investigate cholesterol translocation pathways to Smoothened (SMO), a G protein-coupled receptor central to Hedgehog signal transduction. The authors identify and characterize two putative cholesterol access routes to the transmembrane domain (TMD) of SMO and propose a model whereby cholesterol traverses through the TMD to the cysteine-rich domain (CRD), which is presented as the primary site of SMO activation.

The MD simulations and biochemical experiments are carefully executed and provide useful data.

Comments on revisions:

I appreciate the authors' detailed response and the substantial revisions made to the manuscript. The changes addressing Comments 3.1-3.5 have significantly improved the balance and framing of the work, and my primary concerns regarding overstatement and selective interpretation have been satisfactorily addressed.

The authors' rebuttal to my initial review includes extended argumentation regarding specific interpretations of prior studies and broader models of SMO regulation. These issues represent longstanding differences in interpretation that have already been discussed extensively in the literature and are not essential to evaluating the quality or conclusions of the present study.

For readers seeking a comprehensive and balanced overview of cholesterol-dependent SMO activation that integrates both CRD- and TMD-centered models, I would point to recent review articles (e.g., Zhang and Beachy, Nat Rev Mol Cell Biol2023). I do not feel it is productive to rehash these debates further in the context of this review, and I have no additional substantive concerns with the revised manuscript.

Reviewer #1 (Public review):

Willeke et al. hypothesize that macaque V4, like other visual areas, may exhibit a topographic functional organization. One challenge to studying the functional (tuning) organization of V4 is that neurons in V4 are selective for complex visual stimuli that are hard to parameterize. Thus, the authors leverage an approach comprising digital twins and most exciting stimuli (MEIs) that they have pioneered. This data-driven, deep-learning framework can effectively handle the difficulty of parametrizing relevant stimuli. They verify that the model-synthesized MEIs indeed drive V4 neurons more effectively than matched natural image controls. They then performed psychophysics experiments (on humans) along with the application of contrastive learning to illustrate that anatomically neighboring neurons often care about similar stimuli. Importantly, the weaknesses of the approach are clearly appreciated and discussed.

Comments:

(1) The correlation between predictions and data is 0.43. I'd agree with the authors that this is "reliable" and would recommend that they discuss how the fact that performance is not saturated influences the results.

(2) Modeling V4 using a CNN and claiming that the identified functional groups look like those found in artificial vision systems may be a bit circular.

(3) No architecture other than ResNet-50 was tested. This might be a major drawback, since the MEIs could very well be reflections of the architecture and also the statistics of the dataset, rather than intrinsic biological properties. Do the authors find the same result with different architectures as the basis of the goal-driven model?

(4) The closed-loop analysis seems to be using a much smaller sample of the recorded neurons - "resulting in n=55 neurons for the analysis of the closed-loop paradigm".

(5) A discussion on adversarial machine learning and the adversarial training that was used is lacking.

Reviewer #2 (Public review):

This is an ambitious and technically powerful study, investigating a long-standing question about the functional organization of area V4. The project combined large-scale single-unit electrophysiology in macaque V4 with deep learning-based activation maximization to characterize neuronal tuning in natural image space. The authors built predictive encoding models for V4 neurons and used these models to synthesize most exciting images (MEIs), which are subsequently validated in vivo using a closed-loop experimental paradigm.

Overall, the manuscript advances three main claims:

(1) Individual V4 neurons showed complex and highly structured selectivity for naturalistic visual features, including textures, curvatures, repeating patterns, and apparently eye-like motifs.

(2) Neurons recorded along the same linear probe penetration tended to have more similar MEIs than neurons recorded at different cortical locations (this similarity was supported by human psychophysics and by distances in a learned, contrastive image embedding space).

(3) MEIs clustered into a limited number of functional groups that resembled feature visualizations observed in deep convolutional neural networks.

Strengths:

(1) The study is important in that it is the first to apply activation maximization to neurons sampled at such fine spatial resolution. The authors used 32-channel linear silicon probes, spanning approximately 2 mm of cortical depth, with inter-contact spacing of roughly 60 µm. This enabled fine sampling across most of the cortical thickness of V4, substantially finer resolution than prior Utah-array or surface-biased approaches.

(2) A key strength is the direct in vivo validation of model-derived synthetic images by stimulating the same neurons used to build the models, a critical step often absent in other neural network-based encoding studies.

(3) More broadly, the study highlights the value of probing neuronal selectivity with rich, naturalistic stimulus spaces rather than relying exclusively on oversimplified stimuli such as Gabors.

Weaknesses:

(1) A central claim is that neurons sampled within the same penetration shared MEI tuning properties compared to neurons sampled in different penetrations because of functional organization. I am concerned about technical correlations in activity due to technical or methodology-related approaches (for example, shared reference or grounding) instead of functional organization alone. These recordings were obtained with linear silicon probes, and there have been observations that neuronal activity along this type of probe (including neuropixels probes) may be correlated above what prior work showed, using manually advanced single electrodes. For example, Fujita et al. (1992) showed finer micro-domains and systematic changes in selectivity along a cortical penetration, and it is not clear if that is true or detectable here. I think that the manuscript would be strengthened by a more thorough and explicit characterization of lower-level response correlations (at the neuronal electrophysiology level) prior to starting with fitting models. In particular, the authors could examine noise correlations along the electrode shaft (using the repeated test images, for example), as well as signal correlations in tuning, both within and across sessions. It would also be helpful to clarify whether these correlations depended on penetration day, recording chamber hole (how many were used?), or spatial separation between penetrations, and whether repeated use of the same hole yielded stable or changing correlations. Illustrations of the peristimulus time histogram changes across the shaft and across penetrations would also help. All of this would help us understand if the reports of clustering were technically inevitable due to the technique.

(2) It is difficult to understand a story of visual cortex neurons without more information about their receptive field locations and widths, particularly given that the stimulus was full-screen. I understand that there was a sparse random dot stimulus used to find the population RF, so it should be possible to visualize the individual and population RFs. Also, the investigators inferred the locations of the important patches using a masking algorithm, but where were those masks relative to the retinal image, and how distributed were they as a function of the shaft location? This would help us understand how similar each contact was.

(3) A major claim is that V4 MEIs formed groups that were comparable to those produced by artificial vision systems, "suggesting potential shared encoding strategies." The issue is that the "shared encoding strategy" might be the authors' use of this same class of models in the first place. It would be useful to know if different functional groups arise as a function of other encoding neural network models, beyond the robust-trained ResNet-50. I am unsure to what extent the reported clustering, depth-wise similarity, and correspondence to artificial features depended on architectural and training bias. It would substantially strengthen the manuscript to test whether a similar organizational structure would emerge using alternative encoding models, such as attention-based vision transformers, self-supervised visual representations, or other non-convolutional architectures. Another important point of contrast would be to examine the functional groups encoded by the ResNet architecture before its activations were fit to V4 neuronal activity: put simply, is ResNet just re-stating what it already knows?

(4) Several comparisons to prior work are presented largely at a qualitative level, without quantitative support. For example, the authors state that their MEIs are consistent with known tuning properties of macaque V4, such as selectivity for shape, curvature, and texture. However, this claim is not supported by explicit image analyses or metrics that would substantiate these correspondences beyond appeal to visual inspection. Incorporating quantitative analyses, for instance, measures of curvature, texture statistics, or comparisons to established stimulus sets, would strengthen these links to prior literature and clarify the relationship between the synthesized MEIs and previously characterized V4 tuning properties.

Reviewer #1 (Public review):

The author presents a new method for microRNA target prediction based on (1) a publicly available pretrained Sentence-BERT language model that the author fine-tunes using MeSH information and (2) downstream classification analysis for microRNA target prediction. In particular, the author's approach, named "miRTarDS", attempts to solve the microRNA target prediction problem by utilizing disease information (i.e., semantic similarity scores) from their language model. The author then compares the prediction performance with other sequence- and disease-based methods and attempts to show that miRTarDS is superior or at least comparable to existing methods. The author's general approach to this microRNA target prediction problem seems promising, but fails to demonstrate concrete computational evidence that miRTarDS outperforms other existing methods. The author's claim that disease information-based language models are sufficient is unfounded. The manuscript requires substantial rewriting and reorganization for readers with a strong background in biomedical research.

A major issue related to the author's claim of computational advance of miRTarDS: The author does not introduce existing biomedical-specific language models, and does not compare them against miRTarDS's fine-tuned model. The performance of miRTarDS is largely dependent on the semantic embedding of disease terms. The author shows in Figure 5 that MeSH-based fine-tuning leads to a substantial improvement in MeSH-based correlation compared to the publicly available pretrained SBERT model "multi-qa-MiniLM-L6-cos-v1" without sacrificing a large amount of BIOSSES-based correlation. However, the author does not compare the performance of MeSH- and BIOSSES-based correlation with existing language models such as ChatGPT, BioBERT, PubMedBERT, and more. Also, the substantial improvement in MeSH-based correlation is a mere indication that the MeSH-based fine-tuning strategy was reasonable and not that it's superior to the publicly available pretrained SBERT model "multi-qa-MiniLM-L6-cos-v1".

Another major issue is in the author's claim that disease-information from miRTarDS's language model is "sufficient" for accurate microRNA target prediction. Available microRNA targets with experimental evidence are largely biased for those with disease implications that have been reported in the biomedical literature. It's possible that their language model is biased by existing literature that has also been used to build microRNA target databases. Therefore, it is important that the author provides strong evidence that excludes the possibility of data leakage circularity. Similar concerns are prevalent across the manuscript, and so I highly recommend that the author reassess the evaluation frameworks and account for inflated performance, biased conclusions, and self-confirming results.

Last but not least, the manuscript requires a deeper and careful description and computational encoding of microRNA biology. I'd advise the author to include an expert in microRNA biology to improve the quality of this manuscript. For example, the author uses the pre-miRNA notation and replaces the mature miRNA notation to maintain computational encoding consistency across databases. However, the mature microRNA notation "the '-3p' or '-5p' is critical as the 3p and 5p mature microRNAs have different seed sequences and thus different mRNA targets. The 3p mature microRNA would most likely not target an mRNA targeted by the 5p mature microRNA.

Reviewer #2 (Public review):

Summary:

This study introduces a novel knowledge-driven approach, miRTarDS, which enables microRNA-Target Interaction (MTI) prediction by leveraging the disease association degree between a miRNA and its target gene. The core hypothesis is that this single feature is sufficient to distinguish experimentally validated functional MTIs from computationally predicted MTIs in a binary classification setting. To quantify the disease association, the authors fine-tuned a Sentence-BERT (SBERT) model to generate embeddings of disease descriptions and compute their semantic similarity. Using only this disease association feature, miRTarDS achieved an F1 score of 0.88 on the test set.

Strengths:

The primary strength is the innovative use of the disease association degree as an independent feature for MTI classification. In addition, this study successfully adapts and fine-tunes the Sentence-BERT (SBERT) model to quantify the semantic similarity between biomedical texts (disease descriptions). This approach establishes a critical pathway for integrating powerful language models and the vast growth in clinical/disease data into biochemical discovery, like MTI prediction.

Weaknesses:

The main weakness lies in its definition of the ground-truth dataset, which serves as a foundation for methodological evaluation. The study defines the Negative Set as computationally predicted MTIs that lack experimental evidence. However, the absence of experimental validation does not equate to non-functionality. Similarly, the miRAW sets are classified by whether the target and miRNA could form a stable duplex structure according to RNA structure prediction. This definition is biologically irrelevant, as duplex stability does not fully encapsulate the complex in vivo binding of miRNAs within the AGO protein complex.

Reviewer #1 (Public review):

In this study, Ursu, Centeno, and Leblois record from the cerebellum of zebra finches and analyze neurons for auditory and song-related activity. The paper covers a lot of ground, ranging from lesions of the deep nuclei to song and white noise playback inside and outside of singing, and some level of survey of response types across cerebellar lobules, to provide foundational information on cerebellar relationships with song. There are a number of interesting observations in the study, to me most notably, the lack of responsivity of song-related activity in lobule IV to distorted auditory feedback. This observation is interesting in light of the perennial idea that the cerebellum may participate in rapid error corrections in other somatic control domains. If such a role were relevant for song, it stands to reason that some alteration of activity could be found there. Of course, on the other hand, zebra finches do not show rapid corrections during DAF, so perhaps the null result does not resolve much. Nevertheless, these data are important steps forward in establishing the involvement or lack of involvement in a broader set of brain structures beyond the song control system typically studied. While the study presents some interesting and important inroads, in my opinion, there was a general lack of 'polish' to the study that led to ambiguity in the report and confusing displays. This detracted from rigorous reporting of the findings.

Reviewer #2 (Public review):

In this paper, the authors investigate the role of the cerebellum in song production in the zebra finch. First, they replicate prior studies to show that lesions of the lateral deep cerebellar nuclei (latDCN, primarily lobules IV-VII and IX) result in shorter duration syllables and song motifs than sham controls. The authors then record neural activity from the cerebellum during both passive auditory exposure in anesthetized birds and in freely singing animals. The authors claim that across multiple lobules, the cerebellum receives "non-selective" auditory inputs locked to syllable boundaries (based on acute recordings) and that cerebellar neurons display song-locked responses that are unaffected by auditory feedback perturbations (in chronic recordings). Moreover, the authors emphasized the distinct properties of lobule IV, which they argue is tightly locked to the onset and offset of syllables, and conclude that the cerebellum might contribute to the duration of song elements.

This paper presents novel and useful descriptions of song-related neural activity in the cerebellum. However, there are multiple serious issues. First, there are major issues with the design and presentation of the analysis of the electrophysiological data; based on these, it is unclear whether the authors are justified in some of their conclusions about neural tuning or are entitled to any of their claims about the specific tuning or function of neurons in particular lobules. Second, because the authors' conceptual framework seems to ignore possible non-auditory inputs to the cerebellum, their results on (minimal) effects of auditory manipulation during singing are over-interpreted with respect to providing evidence of a forward model. Third, the paper's central assertion - that the songbird cerebellum may contribute to the duration of vocal events during song - was firmly established by a prior lesion study (Radic et al., 2024). Although the authors do cite this prior study with respect to longer-term postlesion changes after cerebellar lesions, this paper also showed a large change in syllable duration immediately after cerebellar lesion (Figure 5 in Radic et al). The electrophysiological results in the present paper could provide valuable insights into the neural mechanisms underlying this already-described role of the songbird cerebellum; however, given the other concerns above, it is not clear that the authors have done so.

Reviewer #1 (Public review):

Summary:

In this study, the authors investigated the detailed structural mechanism of activation of ABHD5 upon interaction with lipid structures (bilayer and LD). The authors used an elaborate multiscale computational workflow, incorporating coarse-grained, all-atom, and enhanced-sampling molecular dynamics simulations, to propose a structural mechanism for the interaction and activation of ABHD5, as well as its specific interaction with TAG in LD. The authors then corroborated these observations with experimental studies involving hydrogen-deuterium exchange coupled with mass spectrometry of wild-type ABHD5 to assess the structural and conformational changes in ABHD5 upon binding, as well as mutagenesis with cell-based and in vitro assays monitoring membrane association, defining specific interactions that infer ABHD5 to localize LD.

Strengths:

The manuscript is well-written, and the data are reported in high-quality figures. The experimental design and data analysis are rigorous and support the conclusion. One major strength is the multiscale computational work that reveals a mechanism for the insertion of ABHD5 into lipid bilayers and LD involving the insertion of the N-term portion and the lid helix motif. The design of the computational workflow was very elaborate, and the undertaking was quite extensive, with multiple strategies to (GC, all-atom MD and GaMD). The authors then elegantly generate a hypothesis from these observations to experimentally corroborate the proposed mechanism. Particularly, the HDX-MS data support the engagement of the two regions upon binding, and the fluorescence microscopy data show the role of specific residues in localization/specificity to LD.

Weaknesses:

The following limitation is noted. Central to this manuscript is the model, as observed computationally, that initial lipid interaction by the N-term insertion is followed by the insertion of lid-helix in the membrane, which undergoes a conformational switch in the process. However, HDX-MS reveals that, in the unbound form, the lid helix region displays a bimodal isotopic envelope, revealing two species, one with low uptake, suggesting a structured species and one with high uptake, suggesting a less structured species. It is unclear from the manuscript whether the authors think the bimodality fits EX1 regime kinetics or not. Regardless, the model of unbound ABHD5 shows a lid-helix region devoid of secondary structure (Figure 5A), which is more consistent with the unprotected species. The authors also mention that previous modeling had pointed to the high flexibility of the insertion domain. Upon binding, the lid-helix region seems to be ordered from computational observations and loses bimodality by HDX-MS with a deuterium uptake consistent with the protected species of the bimodal envelop in the unbound form. The authors fall short of interpreting or even discussing what the bimodality of the lid-helix represents in the unbound form. What does the protected species in the bimodal envelope represent? Is it a transition representing lid-helix formation and unfolding? Does it imply that interaction and insertion into the lipid structures are governed by conformational selection? This issue should be at the very least acknowledged and discussed, or optimally investigated by performing more integrative studies of the HDX-MS data with the extensive computational data at hand, using existing protection factor calculations or HDX-guided ensemble refinement methods.

Reviewer #2 (Public review):

Summary:

The manuscript describes a combined computational and experimental approach to investigate the ABHD5 binding to and insertion into membranes.

Strengths:

Mutational experiments support computational findings obtained on ABHD5 membrane insertion with enhanced-sampling atomistic simulations.

Weaknesses:

While the addressed problem is interesting, I have several concerns, which fall into two categories:

(A) I see statements throughout the manuscript, e.g. on PNPLA activation, that are not supported by the results.

(B) The presentation of the computational and experimental results lacks in part clarity and detail.

Comments and questions on (A):

(1) I think the following statements in the abstract, which go beyond ABHD5 membrane binding, are not supported by the presented data:

the addition "to control lipolytic activation" in the 3rd sentence of the abstract.

further below ".... transforming ABHD5 into an active and membrane-localized regulator".

(2) The authors state in the Introduction (page numbers and line numbers are missing to be more specific):

"We hypothesize that binding of ABHD5 alters the nanoscale chemical and biophysical properties of the LD monolayer, which, combined with direct protein-protein interactions, enables PNPLA paralogs to access membrane-restricted substrates. This regulatory mechanism represents a paradigm shift from conventional enzyme-substrate interactions to sophisticated allosteric control systems that operate at membrane interfaces."

This hypothesis and the suggested paradigm shift are not supported by the data. Protein-protein interactions are not considered. What is meant by "sophisticated allosteric control"?

(3) The authors state in the Results section:

"We hypothesize that this TAG nanodomain is critical for ABHD5-activated TAG hydrolysis by PNPLA2." In previous pages, the authors state the location of the nanodomain: "TAG nanodomain under ABHD5".

If the nanodomain is located under ABHD5, how can it be accessible to PNPLA2? To my understanding, ABHD5 then sterically blocks access of PNPLA2 to the TAG nandomain.

(4) Another statement: "Our findings suggest that ABHD5-mediated membrane remodeling regulates lipolysis in part by regulating PNPLA2 access to its TAG substrate."

I don't see how the reported results support this statement (see point 3 above).

Comments and questions on (B):

(1) The authors state that the GaMD simulations started "from varying conformations observed during CGMD".

What is missing is a clear description of the CGMD simulation conformations, and the CG simulations as a whole, prior to the results section on GaMD. The authors use standard secondary and tertiary constraints in the Martini CG simulations. Do the authors observe some (constrained) conformational changes of ABHD5 already in the CG simulations (depending on the strength of the constraints)? Or do the conformational changes occur exclusively in the GaMD simulations? Both are fine, but this needs to be described.

(2) The authors write: "Three replicas of GaMD were performed."

Do these replicas lead to similar, or statistically identical, membrane-bound ABHD5 conformations? Is this information, i.e. a statistical analysis of differences in the replica runs, already included in the manuscript?

(3) The authors state on the hydrogen exchange results:

"HDX-MS provided orthogonal experimental evidence for the dynamics of the lid. In solution, a peptide (residues 200-226) spanning the lid helix displayed a bimodal isotopic distribution (Fig. S4), indicating the coexistence of different conformations. Upon LD binding, this distribution shifted to a single, low-exchange peak, demonstrating stabilization of the membrane-bound conformation with reduced solvent accessibility. These experimental observations corroborate our MD simulations."

I find this far too short to be understandable. Also, there are no computational results of ABHD5 in solution that show a bimodal conformational distribution of the lid helix, which is observed in the hydrogen exchange experiments. Which aspects of the MD simulations are corroborated?

Reviewer #1 (Public review):

Summary:

The goal of the study was to address the question of the degree to which social position in a group is a stable trait that persists across conditions. Reinwald et al. use a custom-built cage system with automated tracking and continuous testing for social dominance that does not require intervention by the experimenter. Remixing of individuals from different groups revealed that social position was rather stable and not really predictable from other measures that were taken. The authors conclude that social position is multifaceted but dependent on characteristics like personality traits.

Strengths:

(1) Reductionistic, highly controlled setting that allows for the control of many confounding variables.

(2) Very interesting and important question.

(3) Confirms the emergence of inter-individual behavior-driven differences in inbred mice in a shared environment.

(4) Innovative paradigm and experimental setup.

(5) Fresh perspective on an old question that makes the best use of modern technology.

(6) Intelligent use of behavioral and cognitive covariables to generate a non-social context.

(7) Bold and almost provocative conclusion, inviting discussion and further elaboration.

Weaknesses:

(1) Reductionistic, highly controlled setting that blends out much of the complexity of social behavior in a community.

(2) The motivation to enter the test tube is not "trait" (or at least not solely a trait) but the basic need to reach food and water; chasing behavior would be less dependent on this stimulus.

(3) Dominance is only one aspect of sociality, social structure is reduced to rank. The information that might lie in the chasing behavior is not optimally used to explain social behavior beyond the rank measure.

(4) Focus on rank bears the risk of overgeneralization for readers not familiar with the context.

(5) Conclusion only valid for the reductionistic setting, in which environment, social and non-social changes only within narrow limits, and in which the mouse population does not face challenges

(6) Animals are not naive at the beginning of the experiment, but are already several weeks old.

In summary, this is a wonderful study, but not one that is easy to interpret. The bold conclusion is valid only within the constraints of the study, but nevertheless points in an important direction. The paradigm is clever and could be used for many interesting follow-ups.

To define social position as a personality trait will elicit strong opposition and much debate; the nuances of the paper might be lost on many readers and call for the (re)-consideration of many concepts that are touched. I find this attitude a strength of the paper, but the approach bears the risk of misunderstanding.

Reviewer #2 (Public review):

Summary:

This manuscript presents the "NoSeMaze", a novel automated platform for studying social behavior and cognitive performance in group-housed male mice. The authors report that mice form robust, transitive dominance hierarchies in this environment and that individual social rank remains largely stable across multiple group compositions. They further demonstrate that social dominance and aggressive behaviors, like chasing, are partially dissociable and that dominance traits are independent of non-social cognitive performance. The study includes a genetic manipulation of oxytocin receptor expression in the anterior olfactory nucleus, which showed only transient effects on social rank.

Strengths:

(1) Innovative Methodology:<br /> The NoSeMaze platform is a technically elegant and conceptually well-integrated system that enables fully automated, long-term monitoring of both social and cognitive behaviors in large groups of group-housed mice. It combines tube-test-like dominance contests, voluntary chase-escape interactions, and an embedded operant olfactory discrimination task within a single, ethologically relevant environment. This modular design allows for high-throughput, minimally invasive behavioral assessment without the need for repeated handling or artificial isolation.

(2) Experimental Scale and Rigor:<br /> The study includes 79 male mice and over 4,000 mouse-days of observation across multiple group reshufflings. The use of RFID-based identification, automated data logging, and longitudinal design enables robust quantification of individual trait stability and group-level social structure.

(3) Multidimensional Behavioral Profiling:<br /> The integration of social (tube dominance, proactive chasing), physical (body weight), and cognitive (olfactory learning task) measures offers a rich, multi-dimensional profile of each individual mouse. The authors' finding that social dominance traits and non-social cognitive performance are largely uncorrelated reinforces emerging models of orthogonal behavioral trait axes or "animal personalities".

(4) Clarity and Data Analysis:<br /> The analytical framework is well-suited to the study's complexity, with appropriate use of dominance metrics, mixed-effects models, and permutation tests. The analyses are clearly explained, statistically rigorous, and supported by transparent supplementary materials.

Weaknesses:

(1) Conceptual Novelty and Prior Work:<br /> While the study is carefully executed and methodologically innovative, several of its core findings reaffirm concepts already established in the literature. The emergence of stable, transitive social hierarchies, the persistence of individual differences in social behavior, and the presence of non-despotic social structures have all been previously reported in mice, including under semi-naturalistic conditions (e.g., Fan et al., 2019; Forkosh et al., 2019). Although this work extends those findings with greater behavioral resolution and scale, the manuscript would benefit from a clearer articulation of what is genuinely novel at the conceptual level, beyond the technological advance.

(2) Role of OXTR Deletion:<br /> The inclusion of the OXTR manipulation feels somewhat disconnected from the manuscript's central aims. The effects were minimal and transient, and the authors defer full interpretation to a separate study.

(3) Scope Limitations (Sex and Age):<br /> The study is limited to male mice, and although this is acknowledged, the title and overall framing imply broader generalizability. This sex-specific focus represents a common but problematic bias. Additionally, results from the older mouse cohort are under-discussed; if age had no effect, this should be explicitly stated.

(4) Ambiguity of Dominance as a Construct:<br /> While the study robustly quantifies social rank and hierarchy structure, the broader functional meaning of "dominance" remains unclear. As in prior work (e.g., Varholick et al., 2019), dominance rank here shows only weak associations with physical attributes (e.g., body weight), cognitive strategy, or neuromodulatory manipulation (OXTR deletion). This recurring pattern, where rank metrics are reliably established yet poorly predictive of other behavioral or biological traits, raises important questions about what such measures actually capture. In particular, it challenges the assumption that outcomes in paradigms like the tube test or chase frequency necessarily reflect dominance per se, rather than other constructs.

Reviewer #3 (Public review):

Reinwald et al. present the NoSeMaze, a semi-natural behavioral system designed to track social behaviors alongside reinforcement-learning in large groups of mice. Accumulating more than 4,000 days of behavioral monitoring, the authors demonstrate that social rank (determined by tube competitions) is a stable trait across shuffled cohorts and correlated with active chasing behaviors. The system also provides a solid platform for long-term measurements of reinforcement learning, including flexibility, response adaptation, and impulsiveness. Yet, the authors show that social ranking and chasing are mostly independent of these cognitive traits, and both seem mostly independent of oxytocin signaling in the AON.

Strengths:

(1) The neuroethological approach for automated tracking of several mice under semi-natural conditions is still rare in social behavioral research and should be encouraged.

(2) The assessment of dominance by two independent measures, i.e., spontaneous tube competitions and proactive chasing, is innovative and valuable.

(3) The integration of a long-term reinforcement-learning module into the semi-natural system provides novel opportunities to combine cognitive traits into social personality assessments.

(4) The open-source system provides a valuable resource for the scientific community.

Limitations:

(1) Apparent ambiguity and inconsistency in age structure and cohort participation across rounds, raising concerns about uncontrolled confounds.

(2) Chasing behavior appears more stable than tube-test competitions (Figure 4D vs. Figure 3D), which challenges the authors' decision to treat tube competitions as the primary basis for hierarchy determination.

Major concerns:

(1) Unclear and inconsistent handling of age groups and repeated sampling. The manuscript repeatedly refers to "younger" and "older" adults, but it is unclear whether age was ever controlled for or included in models. Some mice completed only one round, others 2-5 rounds, without explanation of the criteria or balancing.

(2) Stability of chasing appears stronger than the stability of tube competitions. Figure 4D shows highly consistent chasing behavior across weeks, while Figure 3D shows weaker and more variable correlations for tube-based David scores. This is also evident from Figure 5A-B,D. Thus, it appears that chasing, which serves to quantify dominance in similar semi-natural setups, may be a more reliable and behaviorally meaningful measure of dominance than the incidental tube competitions.

(3) Unbalanced participation across rounds compromises stability analyses. Stability analyses (e.g., ICCs, round-to-round correlations) assume comparable sampling across individuals. However, some mice contribute 1 round, others 2, 3, 4, and even 5 rounds. This imbalance may inflate stability estimates or confound group reshuffling effects, and the rationale for variable participation is not explained.

Reviewer #1 (Public review):

Summary:

This manuscript uses serological data to quantify the effects of imprinting on subsequent influenza antibody responses. While this is an admirable goal, the HI dataset sounds impressive, and the authors developed a number of models, the manuscript came off as very dense and technical. One of the biggest pitfalls is that it is not easy to understand the lessons learned. The two Results section headers make clear statements - there was an imprinting signal in the HI titers, but much of this signal could also be seen in an imprinting-free simulation - and then the Discussion states a number of limitations. This is fine, but it leaves the reader wondering exactly how large an error would be introduced by ignoring imprinting effects altogether; alternatively, if imprinting is purposefully added, what would the expected effect size be? The comments below will provide some concrete steps to help clarify these points.

Major comments:

(1) Lines 107-133: The first Results section is a dense slog of information, and the reader is never given a good overview of what the imprinting coefficients exactly are. As the paper currently stands, if you do not start by reading the Methods, you will take away very little. I suggest adding a schematic for any of your models, showing what HI titers would be expected with/without imprinting effects. or age effects, or both, to tie in your modeling coefficients with quantities that all readers are familiar with.

(1.1) Clarify what the imprinting coefficient (y-axis in Figure 1A) looks like in this schematic.

(1.2) Another aspect that I missed: In addition to stating which models were best by BIC, what is the absolute effect size in the HI titers? During my initial reading, I had hoped that Figure 3 would answer this question, but it turned out to be just an overview of the dataset. I strongly suggest having such a figure to show the imprinting effect inferred by different models. What would the expected effect be if you kept someone's birth year constant but tuned their age? What if you kept their age at collection constant but tuned their birth year?

(1.3) It would also help to explain in your schematic what the x-axis labels (H1, H2, H1/H3) would look like in these scenarios, and what imprinting relative to H3 means.

(2) As mentioned above, it was hard to understand the takeaway messages, such as:

(2.1) A similar question would be: If you model antibody titers without imprinting, how far off would you be from the actual measurements (2x off, 4x off...)? If you add the imprinting effect, how much closer do you get?

(2.2) Are there specific age groups that require imprinting to be taken into consideration, since otherwise HI analyses will be markedly off?

(2.3) Are there age groups where imprinting can be safely ignored?

(3) HI titers against multiple H1 and H3 variants were measured, but it is unclear how these are used, and why titers against a single variant each season would not have worked equally well.

Reviewer #2 (Public review):

Summary:

In this study, the authors were testing the hypothesis that hemagglutination inhibition antibody titers, measured later in life, might be higher against influenza viruses that belong to the same hemagglutinin classification group as the influenza virus that a person was likely first exposed to early in life. This is one conceptualization of a phenomenon termed immune imprinting, which may explain previously observed differences in susceptibility to severe influenza infection between cohorts that were likely first exposed to different hemagglutinin groups. The results of the analysis provide some support for this analysis. However, support for the hypothesis is not consistently observed across sensitivity analyses, and a simulation study finds that antibody patterns consistent with immune imprinting may arise due to other factors in the absence of true imprinting effects. Therefore, overall support for the hypothesis is weak. Nonetheless, this study is important in that it provides guidance and has developed an analytic methodology for additional studies in this area of research. These findings and methods may also be useful for other infectious diseases for which patterns consistent with immune imprinting have been observed.

Strengths:

The strengths of this study include the relatively large cohort data source with broad age representation, rigorous statistical methods, and the use of sensitivity and simulation analyses to assess the robustness of the results.

Weaknesses:

The model outcome includes antibody titers measured against many different viruses, and the imprinting parameter was defined at the subtype level. This may obscure specific imprinting effects related to finer structural similarities between first and subsequent virus exposures. This analysis focuses only on one component of the immune response to influenza; immune imprinting may also involve other immune mechanisms. The analysis was carried out in a Chinese cohort, and vaccination status of the cohort is not discussed; the results may not be generalizable to other populations, particularly if vaccination patterns differ.

Reviewer #1 (Public review):

Summary:

This study employed a saccade-shifting sequential working memory paradigm, manipulating whether a saccade occurred after each memory array to directly compare retinotopic and transsaccadic working memory for both spatial location and color. Across four participant groups (young and older healthy adults, and patients with Parkinson's disease and Alzheimer's disease), the authors found a consistent saccade-related cost specifically for spatial memory - but not for color - regardless of differences in memory precision. Using computational modeling, they demonstrate that data from healthy participants are best explained by a complex saccade-based updating model that incorporates distractor interference. Applying this model to the patient groups further elucidates the sources of spatial memory deficits in PD and AD. The authors then extend the model to explain copying deficits in these patient groups, providing evidence for the ecological validity of the proposed saccade-updating retinotopic mechanism.

Strengths:

Overall, the manuscript is well written, and the experimental design is both novel and appropriate for addressing the authors' key research questions. I found the study to be particularly comprehensive: it first characterizes saccade-related costs in healthy young adults, then replicates these findings in healthy older adults, demonstrating how this "remapping" cost in spatial working memory is age-independent. After establishing and validating the best-fitting model using data from both healthy groups, the authors apply this model to clinical populations to identify potential mechanisms underlying their spatial memory impairments. The computational modeling results offer a clearer framework for interpreting ambiguities between allocentric and retinotopic spatial representations, providing valuable insight into how the brain represents and updates visual information across saccades. Moreover, the findings from the older adult and patient groups highlight factors that may contribute to spatial working memory deficits in aging and neurological disease, underscoring the broader translational significance of this work.

Comments on revisions:

The authors have addressed my earlier concerns.

Reviewer #2 (Public review):

Summary:

Zhao et al investigate how object location and colour are degraded across saccadic eye movements. They employ an eye-tracking task that requires participants to remember two sequentially presented items and subsequently report the colour and position of either one of these. Through counterbalancing of the presence or absence of saccades across items, the authors endeavour to dissect the impact of saccades independently on item location or colour. These behavioural findings form the basis of generative models designed to test competing, nested accounts of how stored information is stored and updated across saccades.

Strengths:

The combination of eye-tracking and generative modelling is a strength of the paper, which opens new perspectives into the impact of Alzheimer's and Parkinson's disease on the performance of visuospatial cognitive tests. The finding that the model parameters covary with clinical performance on the ROCF test is a nice example of a "computational assay" of disease.

Comments on revisions:

I thank the authors for their detailed responses and revisions arising from my feedback on the original manuscript. The revised manuscript adequately addresses all of my concerns.

Reviewer #3 (Public review):

Summary:

The manuscript introduces a visual paradigm aimed at studying tran-saccadic memory.

The authors observe how memory of object location is selectively impaired across eye movements, whereas object colour memory is relatively immune to intervening eye movements.<br /> Results are reported for young and elderly healthy controls, as well as PD and AD participants.

A computational model is introduced to account for these results, indicating how early differences in memory encoding and decay (but not tran-saccadic updating per se) can account for the observed differences between healthy controls and clinical groups.

In the revised manuscript, the authors have addressed most of my initial concerns. The dataset is generally compelling, as it includes healthy younger and older adults as well as clinical populations. In addition, the authors propose an interesting modelling approach designed to isolate and characterize the key components underlying the observed patterns of results.

It is important to acknowledge potential limitations of the modelling approach, particularly the differences in the number of parameters across the tested models. As models with more parameters typically achieve better fit, this issue warrants careful consideration. The authors have substantially addressed this point in their rebuttal.

Concerns regarding the specificity of the findings were also raised and have been adequately discussed in the authors' response. Specifically, they clarified the selective impact of saccade-related costs on spatial working memory updating across eye movements-without affecting feature‑based memory (e.g., color) -as well as the specificity of the updating effects observed with the Rey-Osterrieth Complex Figure.

Reviewer #2 (Public review):

Summary:

The authors of this paper note that although polyphosphate (polyP) is found throughout biology, the biological roles of polyP have been under-explored, especially in multicellular organisms. The authors created transgenic Drosophila that expressed a yeast enzyme that degrades polyP, targeting the enzyme to different subcellular compartments (cytosol, mitochondria, ER, and nucleus, terming these altered flies Cyto-FLYX, Mito-FLYX, etc.). The authors show the localization of polyP in various wild-type fruit fly cell types and demonstrate that the targeting vectors did indeed result in expression of the polyP degrading enzyme in the cells of the flies. They then go on to examine the effects of polyP depletion using just one of these targeting systems (the Cyto-FLYX). The primary findings from depletion of cytosolic polyP levels in these flies is that it accelerates eclosion and also appears to participate in hemolymph clotting. Perhaps surprisingly, the flies seemed otherwise healthy and appeared to have little other noticeable defects. The authors use transcriptomics to try to identify pathways altered by the cyto-FLYX construct degrading cytosolic polyP, and it seems likely that their findings in this regard will provide avenues for future investigation. And finally, although the authors found that eclosion is accelerated in pupae of Drosophila expressing the Cyto-FLYX construct, the reason why this happens remains unexplained.

Strengths:

The authors capitalize on the work of other investigators who had previously shown that expression of recombinant yeast exopolyphosphatase could be targeted to specific subcellular compartments to locally deplete polyP, and they also use a recombinant polyP binding protein (PPBD) developed by others to localize polyP. They combine this with the considerable power of Drosophila genetics to explore the roles of polyP by depleting it in specific compartments and cell types to tease out novel biological roles for polyP in a whole organism. This is a substantial advance.

Weaknesses:

Page 4 of Results (paragraph 1): I'm a bit concerned about the specificity of PPBD as a probe for polyP. The authors show that the fusion partner (GST) isn't responsible for the signal, but I don't think they directly demonstrate that PPBD is binding only to polyP. Could it also bind to other anionic substances? A useful control might be to digest the permeabilized cells and tissues with polyphosphatase prior to PPBD staining, and show that the staining is lost.

In the hemolymph clotting experiments, the authors collected 2 ul of hemolymph and then added 1 ul of their test substance (water or a polyP solution). They state that they added either 0.8 or 1.6 nmol polyP in these experiments (the description in the Results differs from that of the Methods). I calculate this will give a polyP concentration of 0.3 or 0.6 mM. This is an extraordinarily high polyP concentration, and is much in excess of the polyP concentrations used in most of the experiments testing the effects of polyP on clotting of mammalian plasma. Why did the authors choose this high polyP concentration? Did they try lower concentrations? It seems possible that too high a polyP concentration would actually have less clotting activity than the optimal polyP concentration.

In the revised version of the manuscript, the authors have productively responded to the previous criticisms. Their new data show stronger controls regarding the specificity of PPBD with regard to its interaction with polyP. The authors also have repeated their hemolymph clotting experiments with lower polyP concentrations, which are likely to be more physiological.

Reviewer #3 (Public review):

Summary:

Sarkar, Bhandari, Jaiswal and colleagues establish a suite of quantitative and genetic tools to use Drosophila melanogaster as a model metazoan organism to study polyphosphate (polyP) biology. By adapting biochemical approaches for use in D. melanogaster, they identify a window of increased polyP levels during development. Using genetic tools, they find that depleting polyP from the cytoplasm alters the timing of metamorphosis, accelerationg eclosion. By adapting subcellular imaging approaches for D. melanogaster, they observe polyP in the nucleolus of several cell types. They further demonstrate that polyP localizes to cytoplasmic puncta in hemocytes, and further that depleting polyP from the cytoplasm of hemocytes impairs hemolymph clotting. Together, these findings establish D. melanogaster as a tractable system for advancing our understanding of polyP in metazoans.

Strengths:

• The FLYX system, combining cell type and compartment-specific expression of ScPpx1, provides a powerful tool for the polyP community.

• The finding that cytoplasmic polyP levels change during development and affect the timing of metamorphosis is an exciting first step in understanding the role of polyP in metazoan development, and possible polyP-related diseases.

• Given the significant existing body of work implicating polyP in the human blood clotting cascade, this study provides compelling evidence that polyP has an ancient role in clotting in metazoans.

Limitations:

• While the authors demonstrate that HA-ScPpx1 protein localizes to the target organelles in the various FLYX constructs, the capacity of these constructs to deplete polyP from the different cellular compartments is not shown. This is an important control to both demonstrate that the GTS-PPBD labeling protocol works, and also to establish the efficacy of compartment-specific depletion. While not necessary to do for all the constructs, it would be helpful to do this for the cyto-FLYX and nuc-FLYX.

• The cell biological data in this study clearly indicates that polyP is enriched in the nucleolus in multiple cell types, consistent with recent findings from other labs, and also that polyP affects gene expression during development. Given that the authors also generate the Nuc-FLYX construct to deplete polyP from the nucleus, it is surprising that they test how depleting cytoplasmic but not nuclear polyP affects development. However, providing these tools is a service to the community, and testing the phenotypic consequences of all the FLYX constructs may arguably be beyond the scope of this first study.

Editors' note: The authors have satisfactorily responded to our most major concerns related to the specificity of PPDB and the physiological levels of polyPs in the clotting experiments. We also recognise the limitations related to the depletion of polyP in other tissues and hope that these data will be made available soon.

Reviewer #1 (Public review):

Summary:

This paper introduces a dual-pathway model for reconstructing naturalistic speech from intracranial ECoG data. It integrates an acoustic pathway (LSTM + HiFi-GAN for spectral detail) and a linguistic pathway (Transformer + Parler-TTS for linguistic content). Output from the two components are later merged via CosyVoice2.0 voice cloning. Using only 20 minutes of ECoG data per participant, the model achieves high acoustic fidelity and linguistic intelligibility.

Strengths:

(1) The proposed dual-pathway framework effectively integrates the strengths of neural-to-acoustic and neural-to-text decoding and aligns well with established neurobiological models of dual-stream processing in speech and language.

(2) The integrated approach achieves robust speech reconstruction using only 20 minutes of ECoG data per subject, demonstrating the efficiency of the proposed method.

(3) The use of multiple evaluation metrics (MOS, mel-spectrogram R², WER, PER) spanning acoustic, linguistic (phoneme and word), and perceptual dimensions, together with comparisons against noise-degraded baselines, adds strong quantitative rigor to the study.

Comments on revisions:

I thank the authors for their thorough efforts in addressing my previous concerns. I believe this revised version is significantly strengthened, and I have no further concerns.

Reviewer #2 (Public review):

Summary:

The study by Li et al. proposes a dual-path framework that concurrently decodes acoustic and linguistic representations from ECoG recordings. By integrating advanced pre-trained AI models, the approach preserves both acoustic richness and linguistic intelligibility, and achieves a WER of 18.9% with a short (~20-minute) recording.

Overall, the study offers an advanced and promising framework for speech decoding. The method appears sound, and the results are clear and convincing. My main concerns are the need for additional control analyses and for more comparisons with existing models.

Strengths:

• This speech-decoding framework employs several advanced pre-trained DNN models, reaching superior performance (WER of 18.9%) with relatively short (~20-minute) neural recording.

• The dual-pathway design is elegant, and the study clearly demonstrates its necessity: The acoustic pathway enhances spectral fidelity while the linguistic pathway improves linguistic intelligibility.

Comments on revisions:

The authors have thoughtfully addressed my previous concerns about the weaknesses. I have no further concerns.

Reviewer #1 (Public review):

Summary:

Chen et al. engineered and characterized a suite of next-generation GECIs for the Drosophila NMJ that allow for the visualization of calcium dynamics within the presynaptic compartment, at presynaptic active zones, and in the postsynaptic compartment. These GECIs include ratiometric presynaptic Scar8m (targeted to synaptic vesicles), ratiometric active zone localized Bar8f (targeted to the scaffold molecule BRP), and postsynaptic SynapGCaMP8m. The authors demonstrate that these new indicators are a large improvement on the widely used GCaMP6 and GCaMP7 series GECIs, with increased speed and sensitivity. They show that presynaptic Scar8m accurately captures presynaptic calcium dynamics with superior sensitivity to the GCaMP6 and GCaMP7 series and with similar kinetics to chemical dyes. The active-zone targeted Bar8f sensor was assessed for the ability to detect release-site specific nanodomain changes, but the authors concluded that this sensor is still too slow to accurately do so. Lastly, the use of postsynaptic SynapGCaMP8m was shown to enable the detection of quantal events with similar resolution to electrophysiological recordings. Finally, the authors developed a Python-based analysis software, CaFire, that enables automated quantification of evoked and spontaneous calcium signals. These tools will greatly expand our ability to detect activity at individual synapses without the need for chemical dyes or electrophysiology.

Strengths:

In this study, the authors rigorously compare their newly engineered GECIs to those previously used at the Drosophila NMJ, highlighting improvements in localization, speed, and sensitivity. These comparisons appropriately substantiate the authors claim that their GECIs are superior to the ones currently in use.

The authors demonstrate the ability of Scar8m to capture subtle changes in presynaptic calcium resulting from differences between MN-Ib and MN-Is terminals and from the induction of presynaptic homeostatic potentiation (PHP), rivaling the sensitivity of chemical dyes.

The improved postsynaptic SynapGCaMP8m is shown to approach the resolution of electrophysiology in resolving quantal events.

The authors created a publicly available pipeline that streamlines and standardizes analysis of calcium imaging data.

Reviewer #2 (Public review):

Summary:

Calcium ions play a key role in synaptic transmission and plasticity. To improve calcium measurements at synaptic terminals, previous studies have targeted genetically encoded calcium indicators (GECIs) to pre- and postsynaptic locations. Here, Chen et al. improve these constructs by incorporating the latest GCaMP8 sensors and a stable red fluorescent protein to enable ratiometric measurements. Extensive characterization in the Drosophila neuromuscular junction demonstrates favorable performance of these new constructs relative to previous genetically encoded and synthetic calcium indicators in reporting synaptic calcium events. In addition, they develop a new analysis platform, 'CaFire', to facilitate automated quantification. Impressively, by positioning postsynaptic GCaMP8m near glutamate receptors, the authors show that their sensors can report miniature synaptic events with speed and sensitivity approaching that of intracellular electrophysiological recordings. These new sensors and the analysis platform provide a valuable tool for resolving synaptic events using all-optical methods.

Strength:

The authors present rigorous characterization of their sensors using well-established assays. They employ immunostaining and super-resolution STED microscopy to confirm correct subcellular targeting. Additionally, they quantify response amplitude, rise and decay kinetics, and provide side-by-side comparisons with earlier-generation GECIs and synthetic dyes. Importantly, they show that the new sensors can reproduce known differences in evoked Ca²⁺ responses between distinct nerve terminals. Finally, they present what appears to be the first simultaneous calcium imaging and intracellular mEPSP recording to directly assess the sensitivity of different sensors in detecting individual miniature synaptic events.

The revised version contains extensive new data and clarification that fully addressed my previous concerns. In particular, I appreciate the side-by-side comparison with synthetic calcium indicator OGB-1 and the cytosolic version of GCaMP8m (now presented in Figure 3), which compellingly supports the favorable performance of their new sensors.

Weakness:

I have only one remaining suggestion about the precision of terminology, which I do think is important. The authors clarified in the revision that they "define SNR operationally as the fractional fluorescence change (ΔF/F).", and basically present ΔF/F values whenever they mentioned about SNR. However, if the intention is to present ΔF/F comparisons, I would strongly suggest replacing all mentions of "SNR" in the manuscript with "ΔF/F" or "fractional/relative fluorescence change".

SNR and ΔF/F are fundamentally different quantities, each with a well-defined and distinct meaning: SNR measures fluorescence change relative to baseline fluctuations (noise), whereas ΔF/F measures fluorescence change relative to baseline fluorescence (F₀). While larger ΔF/F values often correlate with improved detectability, SNR also depends on additional factors such as indicator brightness, light collection efficiency, camera noise, and the stability of the experimental preparation. Referring to ΔF/F as SNR can therefore be misleading and may cause confusion for readers, particularly those from quantitative imaging backgrounds. Clarifying the terminology by consistently using ΔF/F would improve conceptual accuracy without requiring any reanalysis of the data.

Reviewer #3 (Public review):

Genetically encoded calcium indicators (GECIs) are essential tools in neurobiology and physiology. Technological constraints in targeting and kinetics of previous versions of GECIs have limited their application at the subcellular level. Chen et al. present a set of novel tools that overcome many of these limitations. Through systematic testing in the Drosophila NMJ, they demonstrate improved targeting of GCaMP variants to synaptic compartments and report enhanced brightness and temporal fidelity using members of the GCaMP8 series. These advancements are likely to facilitate more precise investigation of synaptic physiology. This manuscript could be improved by further testing the GECIs across physiologically relevant ranges of activity, including at high frequency and over long imaging sessions. Moreover, while the authors provide a custom software package (CaFire) for Ca2+ imaging analysis, comparisons to existing tools and more guidance for broader usability are needed.

In this revised version, Chen et al. answered most of our concerns. The tools developed here will be useful for the community.

Reviewer #1 (Public review):

Summary:

This study presents a system for delivering precisely controlled cutaneous stimuli to freely moving mice by coupling markerless real-time tracking to transdermal optogenetic stimulation, using the tracking signal to direct a laser via galvanometer mirrors. The principal claims are that the system achieves sub-mm targeting accuracy with a latency of <100 ms. Due to the nature of mouse gait, this enables accurate targeting of forepaws even when mice are moving.

Strengths:

The study is of high quality and the evidence for the claims is convincing. There is increasing focus in neurobiology in studying neural function in freely moving animals, engaged in natural behaviour. However, a substantial challenge is how to deliver controlled stimuli to sense organs under such conditions. The system presented here constitutes notable progress towards such experiments in the somatosensory system and is, in my view, a highly significant development that will be of interest to a broad readership.

My comments on the original submission have been fully addressed.

Reviewer #2 (Public review):

Parkes et al. combined real-time keypoint tracking with transdermal activation of sensory neurons to examine the effects of recruitment of sensory neurons in freely moving mice. This builds on the authors' previous investigations involving transdermal stimulation of sensory neurons in stationary mice. They illustrate multiple scenarios in which their engineering improvements enable more sophisticated behavioral assessments, including 1) stimulation of animals in multiple states in large arenas, 2) multi-animal nociceptive behavior screening through thermal and optogenetic activation, and 3) stimulation of animals running through maze corridors. Overall, the experiments and the methodology, in particular, is written clearly. The revised manuscript nicely demonstrates a state-dependence in the behavioral response to activation of TrpV1 sensory neurons, which is a nice demonstration of how their real-time optogenetic stimulation capabilities can yield new insights into complex sensory processing.

Comments on revisions:

I agree that your revisions have substantially improved the clarity and quality of the work.

Reviewer #3 (Public review):

Summary:

To explore the diverse nature of somatosensation, Parkes et al. established and characterized a system for precise cutaneous stimulation of mice as they walk and run in naturalistic settings. This paper provides a framework for real-time body part tracking and targeted optical stimuli with high precision, ensuring reliable and consistent cutaneous stimulation. It can be adapted in somatosensation labs as a general technique to explore somatosensory stimulation and its impact on behavior, enabling rigorous investigation of behaviors that were previously difficult or impossible to study.

Strengths:

The authors characterized the closed-loop system to ensure that it is optically precise and can precisely target moving mice. The integration of accurate and consistent optogenetic stimulation of the cutaneous afferents allows systematic investigation of somatosensory subtypes during a variety of naturalistic behaviors. Although this study focused on nociceptors innervating the skin (Trpv1::ChR2 animals), this setup can be extended to other cutaneous sensory neuron subtypes, such as low-threshold mechanoreceptors and pruriceptors. This system can also be adapted for studying more complex behaviors, such as the maze assay and goal-directed movements.

Weaknesses:

Although the paper has strengths, its weakness is that some behavioral outputs could be analyzed in more detail to reveal different types of responses to painful cutaneous stimuli. For example, paw withdrawals were detected after optogenetically stimulating the paw (Figures 3E and 3F). Animals exhibit different types of responses to painful stimuli on the hindpaw in standard pain assays, such as paw lifting, biting, and flicking, each indicating a different level of pain. The output of this system is body part keypoints, which are the standard input to many existing tools. Analyzing these detailed keypoints would greatly strengthen this system by providing deeper biological insights into the role of somatosensation in naturalistic behaviors. Additionally, if the laser spot size could be reduced to a diameter of 2 mm², it would allow the activation of a smaller number of cutaneous afferents, or even a single one, across different skin types in the paw, such as glabrous or hairy skin.

Comments on revisions:

The authors successfully addressed all of my questions and concerns.

Reviewer #1 (Public review):

Summary:

The study by Druker et al. shows that siRNA depletion of PHD1, but not PHD2, increases H3T3 phosphorylation in cells arrested in prometaphase. Additionally, the expression of wild-type RepoMan, but not the RepoMan P604A mutant, restored normal H3T3 phosphorylation localization in cells arrested in prometaphase. Furthermore, the study demonstrates that expression of the RepoMan P604A mutant leads to defects in chromosome alignment and segregation, resulting in increased cell death. These data support a role for PHD1-mediated prolyl hydroxylation in controlling progression through mitosis. This occurs, at least in part, by hydroxylating RepoMan at P604, which regulates its interaction with PP2A during chromosome alignment.

Strengths:

The data support most of the conclusions made.

Comments on revisions:

Actually, I am still concerned that PHD1 binds to RepoMan endogenously and directly. Furthermore, the authors have not yet provided genetic evidence demonstrating that PHD1 controls progression through mitosis by catalyzing the hydroxylation of RepoMan.

Reviewer #2 (Public review):

Summary:

This is a concise and interesting article on the role of PHD1-mediated proline hydroxylation of proline residue 604 on RepoMan and its impact on RepoMan-PP1 interactions with phosphatase PP2A-B56 complex leading to dephosphorylation of H3T3 on chromosomes during mitosis. Through biochemical and imaging tools, the authors delineate a key mechanism in regulation of progression of the cell cycle. The experiments performed are conclusive with well-designed controls.

Strengths:

The authors have utilized cutting edge imaging and colocalization detection technologies to infer the conclusions in the manuscript.

Weaknesses:

Lack of in vitro reconstitution and binding data.

Comments on revisions:

Thank you, authors, for providing the statistics and siRNA validations. While I maintain that the manuscript's claims can benefit a lot from the in vitro experiments and that a Pro-Ala mutation may not be a good mimic for Pro-hydroxylation, I understand the authors' reservations and restrictions regarding the new experiments. Despite the lacunae, the manuscript is a good advance for the field.

Reviewer #3 (Public review):

Summary:

The manuscript is a comprehensive molecular and cell biological characterisation of the effects of P604 hydroxylation by PHD1 on RepoMan, a regulatory subunit of the PPIgamma complex. Conclusions are generally supported by results. Overall, a timely study that demonstrates the interplay between hydroxylase signalling and the cell cycle. The study extends the scope of prolyl hydroxylase signalling beyond canonical hypoxia pathways, providing a concrete example of hydroxylation regulating PP1 holoenzyme composition and function during mitosis.

The work would benefit from additional biochemical validation of direct targeting to characterise the specificity and mode of recognition, but this is beyond the scope of the study.

Strengths:

Compelling data, characterisation of how P604 hydroxylation induces the interaction between RepoMan and a phosphatase complex, resulting in loading of RepoMan on Chromatin. Knockdown of PHD1 mimics the disruption of the complex and loss of the regulation of the hydroxylation site by PHD1, resulting in mitotic defects.

Reviewer #1 (Public review):

Summary:

In their paper entitled "Alpha-Band Phase Modulates Perceptual Sensitivity by Changing Internal Noise and Sensory Tuning," Pilipenko et al. investigate how pre-stimulus alpha phase influences near-threshold visual perception. The authors aim to clarify whether alpha phase primarily shifts the criterion, multiplicatively amplifies signals, or changes the effective variance and tuning of sensory evidence. Six observers completed many thousands of trials in a double-pass Gabor-in-noise detection task while an EEG was recorded. The authors combine signal detection theory, phase-resolved analyses, and reverse correlation to test mechanistic predictions. The experimental design and analysis pipeline provide a clear conceptual scaffold, with SDT-based schematic models that make the empirical results accessible even for readers who are not specialists in classification-image methods.

Strengths:

The study presents a coherent and well-executed investigation with several notable strengths. First, the main behavioral and EEG results in Figure 2 demonstrate robust pre-stimulus coupling between alpha phase and d′ across a substantial portion of the pre-stimulus interval, with little evidence that the criterion is modulated to a comparable extent. The inverse phasic relationship between hit and false-alarm rates maps clearly onto the variance-reduction account, and the response-consistency analysis offers an intuitive behavioral complement: when two identical stimuli are both presented at the participant's optimal phase, responses are more consistent than when one or both occur at suboptimal phases. The frontal-occipital phase-difference result suggests a coordinated rather than purely local phase mechanism, supporting the central claim that alpha phase is linked to changes in sensitivity that behave like changes in internal variability rather than simple gain or criterion shifts. Supplementary analyses showing that alpha power has only a limited relationship with d′ and confidence reassure readers that the main effects are genuinely phase-linked rather than a recasting of amplitude differences.

Second, the reverse-correlation results in Figure 3 extend this story in a satisfying way. The classification images and their Gaussian fits show that at the optimal phase, the weighting of stimulus energy is more sharply concentrated around target-relevant spatial frequencies and orientations, and the bootstrapped parameter distributions indicate that the suboptimal phase is best described by broader tuning and a modest change in gain rather than a pure criterion account. The authors' interpretation that optimal-phase perception reflects both reduced effective internal noise and sharpened sensory tuning is reasonable and well-supported. Overall, the data and figures largely achieve the stated aims, and the work is likely to have an impact both by clarifying the interpretation of alpha-phase effects and by illustrating a useful analytic framework that other groups can adopt.

Weaknesses:

The weaknesses are limited and relate primarily to framing and presentation rather than to the substance of the work. First, because contrast was titrated to maintain moderate performance (d′ between 1.2 and 1.8), the phase-linked changes in sensitivity appear modest in absolute terms, which could benefit from explicit contextualization. Second, a coding error resulted in unequal numbers of double-pass stimulus pairs across participants, which affects the interpretability of the response-consistency results. Third, several methodological details could be stated more explicitly to enhance transparency, including stimulus timing specifications, electrode selection criteria, and the purpose of phase alignment in group averaging. Finally, some mechanistic interpretations in the Discussion could be phrased more conservatively to clearly distinguish between measurement and inference, particularly regarding the relationship between reduced internal noise and sharpened tuning, and the physiological implementation of the frontal-occipital phase relationship.

Reviewer #2 (Public review):

Summary:

The study of Pilipenko et al evaluated the role of alpha phase in a visual perception paradigm using the framework of signal detection theory and reverse correlation. Their findings suggest that phase-related modulations in perception are mediated by a reduction in internal noise and a moderate increase in tuning to relevant features of the stimuli in specific phases of the alpha cycle. Interestingly, the alpha phase did not affect the criterion. Criterion was related to modulations in alpha power, in agreement with previous research.

Strengths:

The experiment was carefully designed, and the analytical pipeline is original and suited to answer the research question. The authors frame the research question very well and propose several models that account for the possible mechanisms by which the alpha phase can modulate perception. This study can be very valuable for the ongoing discussion about the role of alpha activity in perception.

Weaknesses:

The sample size collected (N = 6) is, in my opinion, too small for the statistical approach adopted (group level). It is well known that small sample sizes result in an increased likelihood of false positives; even in the case of true positives, effect sizes are inflated (Button et al., 2013; Tamar and Orban de Xivry, 2019), negatively affecting the replicability of the effect.

Although the experimental design allows for an accurate characterization of the effects at the single-subject level, conclusions are drawn from group-level aggregated measures. With only six subjects, the estimation of between-subject variability is not reliable. The authors need to acknowledge that the sample size is too small; therefore, results should be interpreted with caution.

Conclusion:

This study addresses an important and timely question and proposes an original and well-thought-out analytical framework to investigate the role of alpha phase in visual perception. While the experimental design and theoretical motivation are strong, the very limited sample size substantially constrains the strength of the conclusions that can be drawn at the group level.

Bibliography:

Button, K., Ioannidis, J., Mokrysz, C. et al. Power failure: why small sample size undermines the reliability of neuroscience. Nat Rev Neurosci 14, 365-376 (2013). https://doi.org/10.1038/nrn3475

Tamar R Makin, Jean-Jacques Orban de Xivry (2019) Science Forum: Ten common statistical mistakes to watch out for when writing or reviewing a manuscript eLife 8:e48175 https://doi.org/10.7554/eLife.48175

Reviewer #1 (Public review):

Summary:

King and colleagues generated a mouse with a point mutation in IL21R and investigated the influence on IL-21-mediated T and B cell activation and differentiation. They found that mutant mice show a reduced T and B cell response, with CD4 T cell differentiation into T follicular helper cells being primarily affected.

Strengths:

The authors combined in vitro and in vivo analysis, including bone-marrow chimeric mice.

Weaknesses:

The effect of the IL21R EINS mutant does not specifically affect STAT1, as clearly shown in Figure 1 H, I. Particularly at lower doses of IL21, which may be more relevant in vivo, the effects are very similar. A second key weakness is the very small Tfh response, a not very clear PD-1 and CXCR5 staining to identify Tfh, and a lack of a steady-state (prior to immunisation) comparison of Tfh numbers in the different mouse strains. The latter makes it impossible to know what fraction of the response is antigen-specific.

Reviewer #2 (Public review):

Summary:

In the manuscript, "An IL-21R hypomorph circumvents functional redundancy to define STAT1 signaling in germinal center responses," Cecile King and colleagues identify a cytoplasmic site of the IL-21 receptor that differentially regulates STAT1 and STAT3 activation upon IL-21 stimulation. They further examine the immunological consequences of this site-specific alteration on Tfh differentiation and Tfh-dependent humoral immunity, raising important questions about how gene-knockout models may obscure nuanced functional roles of signaling molecules.

Strengths:

The study convincingly highlights a non-redundant role for STAT1 downstream of IL-21-IL-21R signaling in the Tfh differentiation pathway. This conclusion is supported by in vitro analyses of STAT1 and STAT3 activation in CD4 T cells stimulated with IL-21 or IL-6; by in vivo assessments of Tfh and germinal center B cell responses in WT and IL21R-EINS mutant mice, including bone-marrow chimera systems; and by investigating the expression of Tfh-related molecules in WT versus IL21R-EINS CD4 T cells.

Weaknesses:

Although the experiments were carefully executed with appropriate controls, a key question remains unresolved: whether the Tfh differentiation defect in IL21R-EINS mice is directly attributable to reduced STAT1 activation. Rescue experiments that restore STAT1 signaling in IL21R-EINS TCR-transgenic CD4 T cells would provide strong evidence linking the mutation to impaired STAT1 activation and, consequently, defective Tfh differentiation. Without such evidence, it remains formally possible that additional, uncharacterized mutations introduced during ENU mutagenesis contribute to the phenotypes observed, particularly given the discrepancies between IL21R knockout and IL21R-EINS mutant mice.

Reviewer #1 (Public review):

Summary:

This study presents evidence that the addition of the two GTPases EngA and ObgE to reactions comprised of rRNAs and total ribosomal proteins purified from native bacterial ribosomes can bypass the requirements for non-physiological temperature shifts and Mg⁺² ion concentrations for in vitro reconstitution of functional E. coli ribosomes.

Strengths:

This advance allows ribosome reconstitution in a fully reconstituted protein synthesis system containing individually purified recombinant translation factors, with the reconstituted ribosomes substituting for native purified ribosomes to support protein synthesis. This work potentially represents an important development in the long-term effort to produce synthetic cells.

Weaknesses:

While much of the evidence is solid, the analysis is incomplete in certain respects that detract from the scientific quality and significance of the findings:

(1) The authors do not describe how the native ribosomal proteins (RPs) were purified, and it is unclear whether all subassemblies of RPs have been disrupted in the purification procedure. If not, additional chaperones might be required beyond the two GTPases described here for functional ribosome assembly from individual RPs.

(2) Reconstitution studies in the past have succeeded by using all recombinant, individually purified RPs, which would clearly address the issue in the preceding comment and also eliminate the possibility that an unknown ribosome assembly factor that co-purifies with native ribosomes has been added to the reconstitution reactions along with the RPs.

(3) They never compared the efficiency of the reconstituted ribosomes to native ribosomes added to the "PURE" in vitro protein synthesis system, making it unclear what proportion of the reconstituted ribosomes are functional, and how protein yield per mRNA molecule compares to that given by the PURE system programmed with purified native ribosomes.

(4) They also have not examined the synthesized GFP protein by SDS-PAGE to determine what proportion is full-length.

(5) The previous development of the PURE system included examinations of the synthesis of multiple proteins, one of which was an enzyme whose specific activity could be compared to that of the native enzyme. This would be a significant improvement to the current study. They could also have programmed the translation reactions containing reconstituted ribosomes with (i) total native mRNA and compared the products in SDS-PAGE to those obtained with the control PURE system containing native ribosomes; (ii) with specifc reporter mRNAs designed to examine dependence on a Shine-Dalgarno sequence and the impact of an in-frame stop codon in prematurely terminating translation to assess the fidelity of initiation and termination events; and (iii) an mRNA with a programmed frameshift site to assess elongation fidelity displayed by their reconstituted ribosomes.

Reviewer #2 (Public review):

This study presents a significant advance in the field of in vitro ribosome assembly by demonstrating that the bacterial GTPases EngA and ObgE enable single-step reconstitution of functional 50S ribosomal subunits under near-physiological conditions-specifically at 37 {degree sign}C and with total Mg²⁺ concentrations below 10 mM.

This achievement directly addresses a long-standing limitation of the traditional two-step in vitro assembly protocol (Nierhaus & Dohme, PNAS 1974), which requires non-physiological temperatures (44-50 {degree sign}C), and high Mg²⁺ concentrations (~20 mM). Inspired by the integrated Synthesis, Assembly, and Translation (iSAT) platform (Jewett et al., Mol Syst Biol 2013), leveraging E. coli S150 crude extract, which supplies essential assembly factors, the authors hypothesize that specific ribosome biogenesis factors-particularly GTPases present in such extracts-may be responsible for enabling assembly under mild conditions. Through systematic screening, they identify EngA and ObgE as the minimal pair sufficient to replace the need for temperature and Mg²⁺ shifts when using phenol-extracted (i.e., mature, modified) rRNA and purified TP70 proteins.

However, several important concerns remain:

(1) Dependence on Native rRNA Limits Generalizability

The current system relies on rRNA extracted from native ribosomes via phenol, which retains natural post-transcriptional modifications. As the authors note (lines 302-304), attempts to assemble active 50S subunits using in vitro transcribed rRNA, even in the presence of EngA and ObgE, failed. This contrasts with iSAT, where in vitro transcribed rRNA can yield functional (though reduced-activity, ~20% of native) ribosomes, presumably due to the presence of rRNA modification enzymes and additional chaperones in the S150 extract. Thus, while this study successfully isolates two key GTPase factors that mimic part of iSAT's functionality, it does not fully recapitulate iSAT's capacity for de novo assembly from unmodified RNA. The manuscript should clarify that the in vitro assembly demonstrated here is contingent on using native rRNA and does not yet achieve true bottom-up reconstruction from synthetic parts. Moreover, given iSAT's success with transcribed rRNA, could a similar systematic omission approach (e.g., adding individual factors) help identify the additional components required to support unmodified rRNA folding?

(2) Imprecise Use of "Physiological Mg²⁺ Concentration"

The abstract states that assembly occurs at "physiological Mg²⁺ concentration" (<10 mM). However, while this total Mg²⁺ level aligns with optimized in vitro translation buffers (e.g., in PURE or iSAT systems), it exceeds estimates of free cytosolic [Mg²⁺] in E. coli (~1-2 mM). The authors should clarify that they refer to total Mg²⁺ concentrations compatible with cell-free protein synthesis, not necessarily intracellular free ion levels, to avoid misleading readers about true physiological relevance.

In summary, this work elegantly bridges the gap between the two-step method and the extract-dependent iSAT system by identifying two defined GTPases that capture a core functionality of cellular extracts: enabling ribosome assembly under translation-compatible conditions. However, the reliance on native rRNA underscores that additional factors - likely present in iSAT's S150 extract - are still needed for full de novo reconstitution from unmodified transcripts. Future work combining the precision of this defined system with the completeness of iSAT may ultimately realize truly autonomous synthetic ribosome biogenesis.

Reviewer #1 (Public review):

Summary:

In this article by Xiao et al., the authors aimed to identify the precise targets by which magnesium isoglycyrrhizinate (MgIG) functions to improve liver injury in response to ethanol treatment. The authors found through a series of in vivo and molecular approaches that MgIG treatment attenuates alcohol-induced liver injury through a potential SREBP2-IdI1 axis. This manuscript adds to a previous set of literature showing MgIG improves liver function across a variety of etiologies, and also provides mechanistic insight into its mechanism of action.

Strengths:

(1) The authors use a combination of approaches from both in-vivo mouse models to in-vitro approaches with AML12 hepatocytes to support the notion that MgIG does improve liver function in response to ethanol treatment.

(2) The authors use both knockdown and overexpression approaches, in vivo and in vitro, to support most of the claims provided.

(3) Identification of HSD11B1 as the protein target of MgIG, as well as confirmation of direct protein-protein interactions between HSD11B1/SREBP2/IDI1, is novel.

Weaknesses:

Major weaknesses can be classified into 3 groups:

(1) The results do not support some claims made.

(2) Qualitative analyses of some of the lipid measures, as opposed to more quantitative analyses.

(3) There are no appropriate readouts of Srebp2 translocation and/or activity.

More specific comments:

(1) A few of the claims made are not supported by the references provided. For instance, line 76 states MgIG has hepatoprotective properties and improved liver function, but the reference provided is in the context of myocardial fibrosis.

(2) MgIG is clinically used for the treatment of liver inflammatory disease in China and Japan. In the first line of the abstract, the authors noted that MgIG is clinically approved for ALD. In which countries is MgIG approved for clinical utility in this space?

(3) Serum TGs are not an indicator of liver function. Alterations in serum TGs can occur despite changes in liver function.

(4) There are discrepancies in the results section and the figure legends. For example, line 302 states Idil is upregulated in alcohol fed mice relative to the control group. The figure legend states that the comparison for Figure 2A is that of ALD+MgIG and ALD only.

(5) Oil Red O staining provided does not appear to be consistent with the quantification in Figure 1D. ORO is nonspecific and can be highly subjective. The representative image in Figure 1C appears to have a much greater than 30% ORO (+) area.

(6) The connection between Idil expression in response to EtOH/PA treatment in AML12 cells with viability and apoptosis isn't entirely clear. MgIG treatment completely reduces Idi1 expression in response to EtOH/PA, but only moderate changes, at best, are observed in viability and apoptosis. This suggests the primary mechanism related to MgIG treatment may not be via Idi1.

(7) The nile red stained images also do not appear representative with its quantification. Several claims about more or less lipid accumulation across these studies are not supported by clear differences in nile red.

(8) The authors make a comment that Hsd11b1 expression is quite low in AML12 cells. So why did the authors choose to knockdown Hsd11b1 in this model?

(9) Line 380 - the claim that MGIG weakens the interaction between HSD11b1 and SREBP2 cannot be made solely based on one Western blot.

(10) It's not clear what the numbers represent on top of the Western blots. Are these averages over the course of three independent experiments?

(11) The claim in line 382 that knockdown of Hsd11b1 resulted in accumulation of pSREBP2 is not supported by the data provided in Figure 6D.

(12) None of the images provided in Figure 6E support the claims stated in the results. Activation of SREBP2 leads to nuclear translocation and subsequent induction of genes involved in cholesterol biosynthesis and uptake. Manipulation of Hsd11b1 via OE or KD does not show any nuclear localization with DAPI.

(13) The entire manuscript is focused on this axis of MgIG-Hsd11b1-Srebp2, but no Srebp2 transcriptional targets are ever measured.

(14) Acc1 and Scd1 are Srebp1 targets, not Srebp2.

(15) A major weakness of this manuscript is the lack of studies providing quantitative assessments of Srebp2 activation and true liver lipid measurements.

Reviewer #2 (Public review):

Summary:

In this manuscript, the authors investigated magnesium isoglycyrrhizinate (MgIG)'s hepatoprotective actions in chronic-binge alcohol-associated liver disease (ALD) mouse models and ethanol/palmitic acid-challenged AML-12 hepatocytes. They found that MgIG markedly attenuated alcohol-induced liver injury, evidenced by ameliorated histological damage, reduced hepatic steatosis, and normalized liver-to-body weight ratios. RNA sequencing identified isopentenyl diphosphate delta isomerase 1 (IDI1) as a key downstream effector. Hepatocyte-specific genetic manipulations confirmed that MgIG modulates the SREBP2-IDI1 axis. The mechanistic studies suggested that MgIG could directly target HSD11B1 and modulate the HSD11B1-SREBP2-IDI1 axis to attenuate ALD. This manuscript is of interest to the research field of ALD.

Strengths:

The authors have performed both in vivo and in vitro studies to demonstrate the action of magnesium isoglycyrrhizinate on hepatocytes and an animal model of alcohol-associated liver disease.

Weaknesses:

The data were not well-organised, and the paper needs proofreading again, with a focus on the use of scientific language throughout.

Here are several comments:

(1) In Supplemental Figure 1A, all the treatment arms (A-control, MgIG-25 mg/kg, MgIG-50 mg/kg) showed body weight loss compared to the untreated controls. However, Figure 1E showed body weight gain in the treatment arms (A-control and MgIG-25 mg/kg), why? In Supplemental Figure 1A, the mice with MgIG (25 mg/kg) showed the lowest body weight, compared to either A-control or MgIG (50 mg/kg) treatment. Can the authors explain why MgIG (25 mg/kg) causes bodyweight loss more than MgIG (50 mg/kg)? What about the other parameters (ALT, ALS, NAS, etc.) for the mice with MgIG (50 mg/kg)?

(2) IL-6 is a key pro-inflammatory cytokine significantly involved in ALD, acting as a marker of ALD severity. Can the authors explain why MgIG 1.0 mg/ml shows higher IL-6 gene expression than MgIG (0.1-0.5 mg/ml)? Same question for the mRNA levels of lipid metabolic enzymes Acc1 and Scd1.

(3) For the qPCR results of Hsd11b1 knockdown (siRNA) and Hsd11b1 overexpression (plasmid) in AML-12 cells (Figure 5B), what is the description for the gene expression level (Y axis)? Fold changes versus GAPDH? Hsd11b1 overexpression showed non-efficiency (20-23, units on Y axis), even lower than the Hsd11b1 knockdown (above 50, units on Y axis). The authors need to explain this. For the plasmid-based Hsd11b1 overexpression, why does the scramble control inhibit Hsd11b1 gene expression (less than 2, units on the Y axis)? Again, this needs to be explained.

Reviewer #1 (Public review):

Summary:

This manuscript addresses an important question: how do circadian clocks adjust to a complex rhythmic environment with multiple daily rhythms? The focus is on the temperature and light cycles (TC and LD) and their phase relationship. In nature, TC usually lags the LD cycle, but the phase delay can vary depending on seasonal and daily weather conditions. The authors present evidence that circadian behavior adjusts to different TC/LD phase relationships, that temperature-sensitive tim splicing patterns might underlie some of these responses, and that artificial selection for preferential evening or morning eclosion behavior impacts how flies respond to different LD/TC phase relationship

Strength:

Experiments are conducted on control strains and strains that have been selected in the laboratory for preferential morning or evening eclosion phenotypes. This study is thus quite unique as it allows us to probe whether this artificial selection impacted how animals respond to different environmental conditions, and thus gives hints on how evolution might shape circadian oscillators and their entrainment. The authors focused on circadian locomotor behavior and timeless (tim) splicing because warm and cold-specific transcripts have been described as playing an important role in determining temperature-dependent circadian behavior. Not surprisingly, the results are complex, but there are interesting observations. In particular, the "late" strain appears to be able to adjust more efficiently its evening peak in response to changes in the phase relationship between temperature and light cycles, but the morning peak seems less responsive in this strain. Differences in the circadian pattern of expression of different tim mRNA isoforms are found under specific LD/TC conditions.

Weaknesses:

These observations are interesting, but in the absence of specific genetic manipulations, it is difficult to establish a causative link between tim molecular phenotypes and behavior. The study is thus quite descriptive. It would be worth testing available tim splicing mutants, or mutants for regulators of tim splicing, to understand in more detail and more directly how tim splicing determines behavioral adaptation to different phase relationships between temperature and light cycles. Also, I wonder whether polymorphisms in or around tim splicing sites, or in tim splicing regulators, were selected in the early or late strains.

I also have a major methodological concern. The authors studied how the evening and morning phases are adjusted under different conditions and different strains. They divided the daily cycle into 12h morning and 12h evening periods, and calculated the phase of morning and evening activity using circular statistics. However, the non-circadian "startle" responses to light or temperature transitions should have a very important impact on phase calculation, and thus at least partially obscure actual circadian morning and evening peak phase changes. Moreover, the timing of the temperature-up startle drifts with the temperature cycles, and will even shift from the morning to the evening portion of the divided daily cycle. Its amplitude also varies as a function of the LD/TC phase relationship. Note that the startle responses and their changes under different conditions will also affect SSD quantifications.

For the circadian phase, these issues seem, for example, quite obvious for the morning peak in Figure 1. According to the phase quantification on panel D, there is essentially no change in the morning phase when the temperature cycle is shifted by 6 hours compared to the LD cycle, but the behavior trace on panel B clearly shows a phase advance of morning anticipation. Comparison between the graphs on panels C and D also indicates that there are methodological caveats, as they do not correlate well.

Because of the various masking effects, phase quantification under entrainment is a thorny problem in Drosophila. I would suggest testing other measurements of anticipatory behavior to complement or perhaps supersede the current behavior analysis. For example, the authors could employ the anticipatory index used in many previous studies, measure the onset of morning or evening activity, or, if more reliable, the time at which 50% of anticipatory activity is reached. Termination of activity could also be considered. Interestingly, it seems there are clear effects on evening activity termination in Figure 3. All these methods will be impacted by startle responses under specific LD/TC phase relationships, but their combination might prove informative.

Reviewer #2 (Public review):

Summary:

The authors aimed to dissect the plasticity of circadian outputs by combining evolutionary biology with chronobiology. By utilizing Drosophila strains selected for "Late" and "Early" adult emergence, they sought to investigate whether selection for developmental timing co-evolves with plasticity in daily locomotor activity. Specifically, they examined how these diverse lines respond to complex, desynchronized environmental cues (temperature and light cycles) and investigated the molecular role of the splicing factor Psi and timeless isoforms in mediating this plasticity.

Major strengths and weaknesses:

The primary strength of this work is the novel utilization of long-term selection lines to address fundamental questions about how organisms cope with complex environmental cues. The behavioral data are compelling, clearly demonstrating that "Late" and "Early" flies possess distinct capabilities to track temperature cycles when they are desynchronized from light cycles.

However, a significant weakness lies in the causal links proposed between the molecular findings and these behavioral phenotypes. The molecular insights (Figures 2, 4, 5, and 6) rely on mRNA extracted from whole heads. As head tissue is dominated by photoreceptor cells and glia rather than the specific pacemaker neurons (LNv, LNd) driving these behaviors, this approach introduces a confound. Differential splicing observed here may reflect the state of the compound eye rather than the central clock circuit, a distinction highlighted by recent studies (e.g., Ma et al., PNAS 2023).

Furthermore, while the authors report that Psi mRNA loses rhythmicity under out-of-sync conditions, this correlation does not definitively prove that Psi oscillation is required for the observed splicing patterns or behavioral plasticity. The amplitude of the reported Psi rhythm is also low (~1.5 fold) and variable, raising questions about its functional significance in the absence of manipulation experiments (such as constitutive expression) to test causality.

Appraisal of aims and conclusions:

The authors successfully demonstrate the co-evolution of emergence timing and activity plasticity, achieving their aim on the behavioral level. However, the conclusion that the specific molecular mechanism involves the loss of Psi rhythmicity driving timeless splicing changes is not yet fully supported by the data. The current evidence is correlative, and without spatial resolution (specific clock neurons) or causal manipulation, the mechanistic model remains speculative.

This study is likely to be of significant interest to the chronobiology and evolutionary biology communities as it highlights the "enhanced plasticity" of circadian clocks as an adaptive trait. The findings suggest that plasticity to phase lags - common in nature where temperature often lags light - may be a key evolutionary adaptation. Addressing the mechanistic gaps would significantly increase the utility of these findings for understanding the molecular basis of circadian plasticity.

Reviewer #3 (Public review):

Summary:

This study attempts to mimic in the laboratory changing seasonal phase relationships between light and temperature and determine their effects on Drosophila circadian locomotor behavior and on the underlying splicing patterns of a canonical clock gene, timeless. The results are then extended to strains that have been selected over many years for early or late circadian phase phenotypes.

Strengths:

A lot of work, and some results showing that the phasing of behavioral and molecular phenotypes is slightly altered in the predicted directions in the selected strains.

Weaknesses:

The experimental conditions are extremely artificial, with immediate light and temperature transitions compared to the gradual changes observed in nature. Studies in the wild have shown how the laboratory reveals artifacts that are not observed in nature. The behavioral and molecular effects are very small, and some of the graphs and second-order analyses of the main effects appear contradictory. Consequently, the Discussion is very speculative as it is based on such small laboratory effects

Reviewer #1 (Public review):

Summary:

The manuscript by Hao Jiang et al described a systematic approach to identify proline hydroxylation proteins. The authors implemented a proteomic strategy with HILIC-chromatographic separation and reported an identification with high confidence of 4993 sites from HEK293 cells (4 replicates) and 3247 sites from RCC4 cells with 1412 sites overlapping between the two cell lines. A small fraction of about 200 sites from each cell line were identified with HyPro immonium ion. The authors investigated the conditions and challenges of using HyPro immonium ions as a diagnostic tool. The study focused the validation analysis of Repo-man (CDCA2) proline hydroxylation comparing MS/MS spectra, retention time and diagnostic ions of purified proteins with corresponding synthetic peptides. Using SILAC analysis and recombinant enzyme assay, the study evaluated Repo-man HyPro604 as a target of PHD1 enzyme.

Strengths:

The study involved extensive LCMS runs for in-depth characterization of proline hydroxylation proteins including four replicated analysis of 293 cells and three replicated analysis of RCC4 cells with 32 HILIC fractions in each analysis. The identification of over 4000 confident proline hydroxylation sites from the two cell lines would be a valuable resource for the community. The characterization of Repo-man proline hydroxylation is a novel finding.

Weaknesses:

As a study mainly focused on methodology, there are some potential technical weaknesses discussed below.

(1) The study applied HILIC-based chromatographic separation with a goal to enrich and separate hydroxyproline containing peptides. The separation effects for peptides from 293 cells and RCC4 cells seems somewhat different (Figure 2A and Figure S1A), which may indicate that the application efficiency of the strategy may be cell line dependent.

(2) The study evaluated the HyPro immonium ion as a diagnostic ion for HyPro identification showcasing multiple influential factors and potential challenges. It is important to note that with only around 5% of the identifications had HyPro immonium ion, it would be very challenging to implement this strategy in a global LCMS analysis to either validate or invalidate HyPro identifications. In comparison, acetyllysine immonium ion was previously reported to be a useful marker for acetyllysine peptides (PMID: 18338905) and the strategy offered a sensitivity of 70% with a specificity of 98%.

(3) The authors aimed to identify potential PHD targets by comparing the HyPro proteins identified with or without PHD inhibitor FG-4592 treatment. The workflow followed a classification strategy, rather than a typical quantitative proteomics approach for comprehensive analysis.

(4) The authors performed inhibitor treatment and in vitro PHD1 enzyme assay to validate that Repo-man can be hydroxylated by PHD1. It remains unknown if PHD1 expression in cells is sufficient to stimulate Repo-man hydroxylation.

Reviewer #2 (Public review):

Summary:

In this manuscript, Jiang et al. developed a robust workflow for identifying proline hydroxylation sites in proteins. They identified proline hydroxylation sites in HEK293 and RCC4 cells, respectively. The authors found that the more hydrophilic HILIC fractions were enriched in peptides containing hydroxylated proline residues. These peptides showed differences in charge and mass distribution compared to unmodified or oxidized peptides. The intensity of the diagnostic hydroxyproline iminium ion depended on parameters including MS collision energy, parent peptide concentration, and the sequence of amino acids adjacent to the modified proline residue. Additionally, they demonstrate that a combination of retention time in LC and optimized MS parameter settings reliably identifies proline hydroxylation sites in peptides, even when multiple proline residues are present

Strengths:

Overall, the manuscript presents an advanced, standardized protocol for identifying proline hydroxylation. The experiments were well designed, and the developed protocol is straightforward, which may help resolve confusion in the field.

Comments on revisions:

All of my concerns have been resolved by the authors. It is ready for publication.

Reviewer #3 (Public review):

Summary:

The authors present a new method for detecting and identifying proline hydroxylation sites within the proteome. This tool utilizes traditional LC-MS technology with optimized parameters, combined with HILIC-based separation techniques. The authors show that they pick up known hydroxy-proline sites and also validate a new site discovered through their pipeline.

Strengths:

The manuscript utilizes state-of-the-art mass spectrometric techniques with optimized collision parameters to ensure proper detection of the immonium ions, which is an advance compared to other similar approaches before. The use of synthetic control peptides on the HILIC separation step clearly demonstrates the ability of the method to reliably distinguish hydroxy-proline from oxidized methionine - containing peptides. Using this method, they identify a site on CDCA2, which they go on to validate in vitro and also study its role in regulation of mitotic progression in an associated manuscript.

Weaknesses:

Despite the authors claim about the specificity of this method in picking up the intended peptides, there is a good amount of potential false positives that also happen to get picked (owing to the limitations of MS-based readout), and the authors' criteria for downstream filtering of such peptides requires further clarification. In the same vein, greater and more diverse cell-based validation approach will be helpful to substantiate the claims regarding enrichment of peptides in the described pathway analyses. Experiments must show reproducibility and contain appropriate controls wherever necessary.

Comments on revisions:

I thank the authors for their clarifications and opinions on my questions and suggestions. Based on the response, the following points are important while considering the significance of this manuscript:

- The manuscript provides a novel method to detect and identify proline hydroxylation residues in the proteome. While this provides several advances over previous methods, the probability of false positives, loss of true positives and incomplete removal of the interference of methionine oxidation in this strategy need to be addressed clearly in the discussion section of the manuscript, so that the strengths and limitations of this method are made aware to the reader.

- Going by the standards of publication in eLife, reproducibility is very important in the experiments done. Hence, I strongly recommend that the authors perform the experiments in triplicate with error bars to confirm reproducibility. Graphs with single data points do not convey that, and this is very important for eLife.

- As for Figure 9C, the authors have rejected the request for a control lane in the figure. It may sound trivial to the authors, but for completeness of the experiment, all applicable controls must be performed and shown alongside the main data. It is essential to show the PHD1 only control to rule out the possibility of the contribution of any non-specific signal in the dot blot by PHD1.

Reviewer #1 (Public review):

Summary:

This study aimed to determine whether bacterial translation inhibitors affect mitochondria through the same mechanisms. Using mitoribosome profiling, the authors found that most antibiotics, except telithromycin, act similarly in both systems. These insights could help in the development of antibiotics with reduced mitochondrial toxicity.

They also identified potential novel mitochondrial translation events, proposing new initiation sites for MT-ND1 and MT-ND5. These insights not only challenge existing annotations but also open new avenues for research on mitochondrial function.

Strengths:

Ribosome profiling is a state-of-the-art method for monitoring the translatome at very high resolution. Using mitoribosome profiling, the authors convincingly demonstrate that most of the analyzed antibiotics act in the same way on both bacterial and mitochondrial ribosomes, except for telithromycin. Additionally, the authors report possible alternative translation events, raising new questions about the mechanisms behind mitochondrial initiation and start codon recognition in mammals.

Weaknesses:

All the weaknesses I previously highlighted were adequately addressed.

Reviewer #3 (Public review):

Summary:

Recently, the off-target activity of antibiotics on human mitoribosome has been paid more attention in the mitochondrial field. Hafner et al applied mitoribosome profilling to study the effect of antibiotics on protein translation in mitochondria as there are similarities between bacterial ribosome and mitoribosome. The authors conclude that some antibiotics act on mitochondrial translation initiation by the same mechanism as in bacteria. On the other hand, the authors showed that chloramphenicol, linezolid and telithromycin trap mitochondrial translation in a context-dependent manner. More interesting, during deep analysis of 5' end of ORF, the authors reported the alternative start codon for ND1 and ND5 proteins instead of previously known one. This is a novel finding in the field and it also provide another application of the technique to further study on mitochondrial translation.

Strengths:

This is the first study which applied mitoribosome profiling method to analyze mutiple antibiotics treatment cells. The mitoribosome profiling method had been optimized carefully and has been suggested to be a novel method to study translation events in mitochondria. The manuscript is constructive and well-written.

Weaknesses:

This is a novel and interesting study, however, most of conclusion comes from mitoribosome profiling analysis, as the result, the manuscript lacks the cellular biochemical data to provide more evidence and support the findings.

Comments on revisions:

The authors addressed most of my concerns and comments, although there is still no biochemical assay which should be performed to support mitoribsome profiling data.

The author also carefully investigated the structure of complex I, however, I am surprised that the author chose to analyse a low resolution structure (3.7 A). Recently, there are more high resolution structures of mammalian complex I published (7R41, 7V2C, 7QSM, 9I4I). Furthermore, the authors should not only respond to the reviewers but also (somehow) discuss these points in the manuscript.

Reviewer #1 (Public review):

Summary:

In this manuscript, Yamazaki et al. conducted multiple microscopy-based GFP localization screens, from which they identified proteins that are associated with PM/cell wall damage stress response. Specifically, the authors identified that bud-localized TMD-containing proteins and endocytotic proteins are associated with PM damage stress. The authors further demonstrated that polarized exocytosis and CME are temporally coupled in response to PM damage, and CME is required for polarized exocytosis and the targeting of TMD-containing proteins to the damage site. From these results, the authors proposed a model that CME delivers TMD-containing repair proteins between the bud tip and the damage site.

Strengths:

Overall, this is a well-written manuscript, and the experiments are overall well-conducted. The authors identified many repair proteins and revealed the temporal coordination of different categories of repair proteins. Furthermore, the authors demonstrated that CME is required for targeting of repair proteins to the damage site, as well as cellular survival in response to stress related to PM/cell wall damage. Although the roles of CME and bud-localized proteins in damage repair are not completely new to the field, this work does have conceptual advances by identifying novel repair proteins and proposing the intriguing model that the repairing cargoes are shuttled between the bud tip and the damaged site through coupled exocytosis and endocytosis.

Weaknesses:

While the results presented in this manuscript are convincing, they might not be sufficient to support some of the authors' claims. Especially in the last two result sessions, the authors claimed CME delivers TMD-containing repair proteins from the bud tip to the damage site. The model is no doubt highly possible based on the date, but caveats still exist. For example, the repair proteins might not be transported from one localization to another localization, but are degraded and re-synthesized. Although the Gal-induced expression system can further support the model to some extent, I think more direct verification (such as FLIP or photo-convertible fluorescence tags to distinguish between pre-existing and newly synthesized proteins) would significantly improve the strength of evidence.

Review on revised version:

The authors addressed most of concerns that were originally raised, primarily by revising the text and figures and expanding the discussion, which improves the clarity of the manuscript. Although the authors did not address my major concern on the shuttling/trafficking model experimentally, I do understand the limitation of resources and time. The authors noted that they planned to do these experiments for their future work, and such studies would be more definitive evaluations for the proposed model. Overall I think this is a very interesting and well-conducted study and I enjoyed reading this manuscript. I look forward to their following research of this study.

Reviewer #2 (Public review):

This paper remarkably reveals the identification of plasma membrane repair proteins, revealing spatiotemporal cellular responses to plasma membrane damage. The study highlights a combination of sodium dodecyl sulfate (SDS) and lase for identifying and characterizing proteins involved in plasma membrane (PM) repair in Saccharomyces cerevisiae. From 80 PM, repair proteins that were identified, 72 of them were novel proteins. The use of both proteomic and microscopy approaches provided a spatiotemporal coordination of exocytosis and clathrin-mediated endocytosis (CME) during repair. Interestingly, the authors were able to demonstrate that exocytosis dominates early and CME later, with CME also playing an essential role in trafficking transmembrane-domain (TMD) containing repair proteins between the bud tip and the damage site.

Weaknesses/limitations:

- Still, there is a lack of clarity about mentioning Pkc1 as the best characterized repair protein, or why is Pkc1 mentioned only as it is changing the localization?!

- The use of a C-terminal GFP-tagged library for the laser damage assay may have limited the identification of proteins whose localization or function depends on an intact N-terminus. N-terminal regions might contain targeting or regulatory elements; therefore, some relevant repair factors may have been missed. Analysis of endogenously N-terminally tagged strains, at least for selected candidates, could help address this limitation.

- The authors appropriately discuss the limitations of SDS- and laser-induced plasma membrane damage, including the possibility that these approaches may not capture proteins involved in other forms of membrane injury, such as mechanical or osmotic stress.

Reviewer #3 (Public review):

Summary:

This work aims to understand how cells repair damage to the plasma membrane (PM). This is important as failure to do so will result in cell lysis and death. Therefore, this is an important fundamental question with broad implications for all eukaryotic cells. Despite this importance, there are relatively few proteins known to contribute to this repair process. This study expands the number of experimentally validated PM from 8 to 80. Further, they use precise laser-induced damage of the PM/cell wall and use live-cell imaging to track the recruitment of repair proteins to these damage sites. They focus on repair proteins that are involved in either exocytosis or clathrin-mediated endocytosis (CME) to understand how these membrane remodeling processes contribute to PM repair. Through these experiments, they find that while exocytosis and CME both occur at the sites of PM damage, exocytosis predominates the early stages of repairs, while CME predominates in the later stages of repairs. Lastly, they propose that CME is responsible for diverting repair proteins localized to the growing bud cell to the site of PM damage.

Strengths:

The manuscript is very well written and the experiments presented flow logically. The use of laser-induced damage and live-cell imaging to validate the proteome-wide screen using SDS induced damage strengthen the role of the identified candidates in PM/cell wall repair.

Comments on revisions:

The authors have very nicely addressed my previous comments and I have no further concerns.

Reviewer #2 (Public review):

Summary:

Feng, Jing-Xin et al. studied the hemogenic capacity of the endothelial cells in the adult mouse bone marrow. Using Cdh5-CreERT2 in vivo inducible system, though rare, they characterized a subset of endothelial cells expressing hematopoietic markers that were transplantable. They suggested that the endothelial cells need the support of stromal cells to acquire blood-forming capacity ex vivo. These endothelial cells were transplantable and contributed to hematopoiesis with ca. 1% chimerism in a stress hematopoiesis condition (5-FU) and recruited to the peritoneal cavity upon Thioglycolate treatment. Ultimately, the authors detailed the blood lineage generation of the adult endothelial cells in a single cell fashion, suggesting a predominant HSPCs-independent blood formation by adult bone marrow endothelial cells, in addition to the discovery of Col1a2+ endothelial cells with blood-forming potential, corresponding to their high Runx1 expressing property.

The conclusion regarding the characterization of hematopoietic-related endothelial cells in adult bone marrow is well supported by data. However, the paper would be more convincing, if the function of the endothelial cells were characterized more rigorously.

(1) Ex vivo culture of CD45-VE-Cadherin+ZsGreen EC cells generated CD45+ZsGreen+ hematopoietic cells. However, given that FACS sorting can never achieve 100% purity, there is a concern that hematopoietic cells might arise from the ones that got contaminated into the culture at the time of sorting. The sorting purity and time course analysis of ex vivo culture should be shown to exclude the possibility.

(2) Although it was mentioned in the text that the experimental mice survived up to 12 weeks after lethal irradiation and transplantation, the time-course kinetics of donor cell repopulation (>12 weeks) would add a precise and convincing evaluation. This would be absolutely needed as the chimerism kinetics can allow us to guess what repopulation they were (HSC versus progenitors). Moreover, data on either bone marrow chimerism assessing phenotypic LT-HSC and/or secondary transplantation would dramatically strengthen the manuscript.

(3) The conclusion by the authors, which says "Adult EHT is independent of pre-existing hematopoietic cell progenitors", is not fully supported by the experimental evidence provided (Figure 4 and Figure S3). More recipients with ZsGreen+ LSK must be tested.

Strengths:

The authors used multiple methods to characterize the blood-forming capacity of the genetically - and phenotypically - defined endothelial cells from several reporter mouse systems. The polylox barcoding method to trace the adult bone marrow endothelial cell contribution to hematopoiesis is a strong insight to estimate the lineage contribution.

Weaknesses:

It is unclear what the biological significance of the blood cells de novo generated from the adult bone marrow endothelial cells is. Moreover, since the frequency is very rare (<1% bone marrow and peripheral blood CD45+), more data regarding its identity (function, morphology, and markers) are needed to clearly exclude the possibility of contamination/mosaicism of the reporter mice system used.

Reviewer #1 (Public review):

Summary:

This manuscript by Feng et al. uses mouse models to study the embryonic origins of HSPCs. Using multiple types of genetic lineage tracing, the authors aimed to identify whether BM-resident endothelial cells retain hematopoietic capacity in adult organisms. Through an important mix of various labeling methodologies (and various controls), they reach the conclusion that BM endothelial cells contribute up to 3% of hematopoietic cells in young mice.

Strengths:

The major strength of the paper lies in the combination of various labeling strategies, including multiple Cdh5-CreER transgenic lines, different CreER lines (col1a2), and different reporters (ZsGreen, mTmG), including a barcoding-type reporter (PolyLox). This makes it highly unlikely that the results are driven by a rare artifact due to one random Cre line or one leaky reporter. The transplantation control (where the authors show no labeling of transplanted LSKs from the Cdh5 model) is also very supportive of their conclusions.

Weaknesses:

We believe that the work of ruling out alternative hypotheses, though initiated, was left incomplete. We specifically think that the authors need to properly consider whether there is specific, sparse labeling of HSPCs (in their native, non-transplant, model, in young animals). Polylox experiments, though an exciting addition, are also incomplete without additional controls. Some additional killer experiments are suggested.

Reviewer #1 (Public review):

Summary:

Morgan et al. studied how paternal dietary alteration influenced testicular phenotype, placental and fetal growth using a mouse model of paternal low protein diet (LPD) or Western Diet (WD) feeding, with or without supplementation of methyl-donors and carriers (MD). They found diet- and sex-specific effects of paternal diet alteration. All experimental diets decreased paternal body weight and the number of spermatogonial stem cells, while fertility was unaffected. WD males (irrespective of MD) showed signs of adiposity and metabolic dysfunction, abnormal seminiferous tubules, and dysregulation of testicular genes related to chromatin homeostasis. Conversely, LPD induced abnormalities in the early placental cone, fetal growth restriction, and placental insufficiency, which were partly ameliorated by MD. The paternal diets changed the placental transcriptome in a sex-specific manner and led to a loss of sexual dimorphism in the placental transcriptome. These data provide a novel insight into how paternal health can affect the outcome of pregnancies, which is often overlooked in prenatal care.

Strengths:

The authors have performed a well-designed study using commonly used mouse models of paternal underfeeding (low protein) and overfeeding (Western diet). They performed comprehensive phenotyping at multiple timepoints, including the fathers, the early placenta, and the late gestation feto-placental unit. The inclusion of both testicular and placental morphological and transcriptomic analysis is a powerful, non-biased tool for such exploratory observational studies. The authors describe changes in testicular gene expression revolving around histone (methylation) pathways that are linked to altered offspring development (H3.3 and H3K4), which is in line with hypothesised paternal contributions to offspring health. The authors report sex differences in control placentas that mimic those in humans, providing potential for translatability of the findings. The exploration of sexual dimorphism (often overlooked) and its absence in response to dietary modification is novel and contributes to the evidence-base for the inclusion of both sexes in developmental studies.

Weaknesses:

The data are overall consistent with the conclusions of the authors. The paternal and pregnancy data are discussed separately, instead of linking the paternal phenotype to offspring outcomes. Some clarifications regarding the methods and the model would improve the interpretation of the findings.

(1) The authors insufficiently discuss their rationale for studying methyl-donors and carriers as micronutrient supplementation in their mouse model. The impact of the findings would be better disseminated if their role were explained in more detail.

(2) It is unclear from the methods exactly how long the male mice were kept on their respective diets at the time of mating and culling. Male mice were kept on the diet between 8 and 24 weeks before mating, which is a large window in which the males undergo a considerable change in body weight (Figure 1A). If males were mated at 8 weeks but phenotyped at 24 weeks, or if there were differences between groups, this complicates the interpretation of the findings and the extrapolation of the paternal phenotype to changes seen in the fetoplacental unit. The same applies to paternal age, which is an important known factor affecting male fertility and offspring outcomes.

(3) The male mice exhibited lower body weights when fed experimental diets compared to the control diet, even when placed on the hypercaloric Western Diet. As paternal body weight is an important contributor to offspring health, this is an important confounder that needs to be addressed. This may also have translational implications; in humans, consumption of a Western-style diet is often associated with weight gain. The cause of the weight discrepancy is also unaddressed. It is mentioned that the isocaloric LPD was fed ad libitum, while it is unclear whether the WD was also fed ad libitum, or whether males under- or over-ate on each experimental diet.

(4) The description and presentation of certain statistical analyses could be improved.

(i) It is unclear what statistical analysis has been performed on the time-course data in Figure 1A (if any). If one-way ANOVA was performed at each timepoint (as the methods and legend suggest), this is an inaccurate method to analyse time-course data.

(ii) It is unclear what methods were used to test the relative abundance of microbiome species at the family level (Figure 2L), whether correction was applied for multiple testing, and what the stars represent in the figure. 3) Mentioning whether siblings were used in any analyses would improve transparency, and if so, whether statistical correction needed to be applied to control for confounding by the father.

Reviewer #2 (Public review):

Summary:

The authors investigated the effects of a low-protein diet (LPD) and a high sugar- and fat-rich diet (Western diet, WD) on paternal metabolic and reproductive parameters and feto-placental development and gene expression. They did not observe significant effects on fertility; however, they reported gut microbiota dysbiosis, alterations in testicular morphology, and severe detrimental effects on spermatogenesis. In addition, they examined whether the adverse effects of these diets could be prevented by supplementation with methyl donors. Although LPD and WD showed limited negative effects on paternal reproductive health (with no impairment of reproductive success), the consequences on fetal and placental development were evident and, as reported in many previous studies, were sex-dependent.

Strengths:

This study is of high quality and addresses a research question of great global relevance, particularly in light of the growing concern regarding the exponential increase in metabolic disorders, such as obesity and diabetes, worldwide. The work highlights the importance of a balanced paternal diet in regulating the expression of metabolic genes in the offspring at both fetal and placental levels. The identification of genes involved in metabolic pathways that may influence offspring health after birth is highly valuable, strengthening the manuscript and emphasizing the need to further investigate long-term outcomes in adult offspring.

The histological analyses performed on paternal testes clearly demonstrate diet-induced damage. Moreover, although placental morphometric analyses and detailed histological assessments of the different placental zones did not reveal significant differences between groups, their inclusion is important. These results indicate that even in the absence of overt placental phenotypic changes, placental function may still be altered, with potential consequences for fetal programming.

Weaknesses:

Overall, this manuscript presents a rich and comprehensive dataset; however, this has resulted in the analysis of paternal gut dysbiosis remaining largely descriptive. While still valuable, this raises questions regarding why supplementation with methyl donors was unable to restore gut microbial balance in animals receiving the modified diets.

Reviewer #1 (Public review):

Meiotic recombination at chromosome ends can be deleterious, and its initiation-the programmed formation of DSBs-has long been known to be suppressed. However, the underlying mechanisms of this suppression remained unclear. A bottleneck has been the repetitive sequences embedded within chromosome ends, which make them challenging to analyze using genomic approaches. The authors addressed this issue by developing a new computational pipeline that reliably maps ChIP-seq reads and other genomic data, enabling exploration of previously inaccessible yet biologically important regions of the genome.

In budding yeast, chromosome ends (~20 kb) show depletion of axis proteins (Red1 and Hop1) important for recruiting DSB-forming proteins. Using their newly developed pipeline, the authors reanalyzed previously published datasets and data generated in this study, revealing heretofore unseen details at chromosome ends. While axis proteins are depleted at chromosome ends, the meiotic cohesin component Rec8 is not. Y' elements play a crucial role in this suppression. The suppression does not depend on the physical chromosome ends but on cis-acting elements. Dot1 suppresses Red1 recruitment at chromosome ends but promotes it in interior regions. Sir complex renders subtelomeric chromatin inaccessible to the DSB-forming machinery.

The high-quality data and extensive analyses provide important insights into the mechanisms that suppress meiotic DSB formation at chromosome ends. To fully realise this value, several aspects of data presentation and interpretation should be clarified to ensure that the conclusions are stated with appropriate precision and that remaining future issues are clearly articulated.

(1) To assess the chromosome fusion effects on overall subtelomeric suppression, authors should guide how to look at the data presented in Figure 2b-c. Based on the authors' definition of the terminal 20 kb as the suppressed region, SK1 chrIV-R and S288c chrI-L would be affected by the chromosome fusion, if any. In addition, I find it somewhat challenging to draw clear conclusions from inspecting profiles to compare subtelomeric and internal regions. Perhaps, applying a quantitative approach - such as a bootstrap-based analysis similar to those presented earlier-would be easier to interpret.

(2) The relationship between coding density and Red1 signal needs clarification. An important conclusion from Figure 3 is that the subtelomeric depletion of Red1 primarily reflects suppression of the Rec8-dependent recruitment pathway, whereas Rec8-independent recruitment appears similar between ends and internal regions. Based on the authors' previous papers (referencess 13, 16), I thought coding (or nucleosome) density primarily influences the Rec8-independent pathway. However, the correlations presented in Figure 2d-e (also implied in Figure 3a) appear opposite to my expectation. Specifically, differences in axis protein binding between chromosome ends and internal regions (or within chromosome ends), where the Rec8-dependent pathway dominates, correlate with coding density. In contrast, no such correlation is evident in rec8Δ cells, where only the Rec8-independent pathway is active and end-specific depletion is absent. One possibility is that masking coding regions within Y' elements influences the correlation analysis. Additional analysis and a clearer explanation would be highly appreciated.

(3) The Dot1-Sir3 section staring from L266 should be clarified. I found this section particularly difficult to follow. It begins by stating that dot1∆ leads to Sir complex spreading, but then moves directly to an analysis of Red1 ChIP in sir3∆ without clearly articulating the underlying hypothesis. I wonder if this analysis is intended to explain the differences observed between dot1∆ and H3K79R mutants in the previous section. I also did not get the concluding statement - Dot1 counteracts Sir3 activity. As sir3Δ alone does not affect subtelomeric suppression, it is unclear what Dot1 counteracts. Perhaps, explicitly stating the authors' working model at the outset of this section would greatly clarify the rationale, results, and conclusions.

Reviewer #2 (Public review):

Summary:

In this manuscript, Raghavan and his colleagues sought to identify cis-acting elements and/or protein factors that limit meiotic crossover at chromosome ends. This is important for avoiding chromosome rearrangements and preventing chromosome missegregation.

By reanalyzing published ChIP datasets, the researchers identified a correlation between low levels of protein axis binding - which are known to modulate homologous recombination - and the presence of cis-acting elements such as the subtelomeric element Y' and low gene density. Genetic analyses coupled with ChIP experiments revealed that the differential binding of the Red1 protein in subtelomeric regions requires the methyltransferase Dot1. Interestingly, Red1 depletion in subtelomeric regions does not impact DSB formation. Another surprising finding is that deleting DOT1 has no effect on Red1 loading in the absence of the silencing factor Sir3. Unlike Dot1, Sir3 directly impacts DSB formation, probably by limiting promoter access to Spo11. However, this explains only a small part of the low levels of DSBs forming in subtelomeric regions.

Strengths:

(1) This work provides intriguing observations, such as the impact of Dot1 and Sir3 on Red1 loading and the uncoupling of Red1 loading and DSB induction in subtelomeric regions.

(2) The separation of axis protein deposition and DSB induction observed in the absence of Dot1 is interesting because it rules out the possibility that the binding pattern of these proteins is sufficient to explain the low level of DSB in subtelomeric regions.

(3) The demonstration that Sir3 suppresses the induction of DSBs by limiting the openness of promoters in subtelomeric regions is convincing.

Weaknesses:

(1) The impact of the cis-encoded signal is not demonstrated. Y' containing subtelomeres behave differently from X-only, but this is only correlative. No compelling manipulation has been performed to test the impact of these elements on protein axis recruitment or DSB formation.

(2) The mechanism by which Dot1 and Sir3 impact Red1 loading is missing.

(3) Sir3's impact on DSB induction is compelling, yet it only accounts for a small proportion of DSB depletion in subtelomeric regions. Thus, the main mechanisms suppressing crossover close to the ends of chromosomes remain to be deciphered.

Reviewer #3 (Public review):

Summary:

The paper by Raghavan et. al. describes pathways that suppress the formation of meiotic DNA double-strand breaks (DSBs) for interhomolog recombination at the end of chromosomes. Previously, the authors' group showed that meiotic DSB formation is suppressed in a ~20kb region of the telomeres in S. cerevisiae by suppressing the binding of meiosis-specific axis proteins such as Red1 and Hop1. In this study, by precise genome-wide analysis of binding sites of axis proteins, the authors showed that the binding of Red1 and Hop1 to sub-telomeric regions with X and Y' elements is dependent on Rec8 (cohesin) and/or Hop1's chromatin-binding region (CBR). Furthermore, Dot1 functions in a histone H3K79 trimethylation-independent manner, and the silencing proteins Sir2/3 also regulate the binding of Red1 and Hop1 and also the distribution of DSBs in sub-telomeres.

Strengths:

The experiments were conducted with high quality and included nice bioinformatic analyses, and the results were mostly convincing. The text is easy to read.

Weaknesses:

The paper did not provide any new mechanistic insights into how DSB formation is suppressed at sub-telomeres.

Reviewer #1 (Public review):

Summary:

In this study, Nagao and Mochizuki examine the fate of germline chromosome ends during somatic genome differentiation in the ciliate Tetrahymena thermophila. During sexual reproduction, a new somatic genome is created from a zygotic, germline-derived genome by extensive programmed DNA elimination events. It has been known for some time that the termini of the germline chromosomes are eliminated, but the exact process and kinetics of the elimination events have not been thoroughly investigated. The authors first use germline-specific telomere probes to show that the loss of these chromosome ends occurs with similar timing as other DNA elimination events. By comparative analysis of the assembled germline and somatic genomes, the authors find that the ends of each of the germline chromosomes are composed of a few hundred kilobases of micronuclear limited sequences (MLS) that are removed starting around 14 hours after the start of conjugation, which initiates sexual development. They then develop an in situ hybridization assay to track the fate of one end of chromosome 4 while simultaneously following the adjacent macronuclear destined sequence (MDS) retained in the new somatic genome. This allows the authors to more clearly show that these adjacent chromosomal segments are initially amplified in the developing genome before the terminal MLS is eliminated. Finally, they mutate the chromosome breakage sequence (CBS) that normally separates the MLS terminus from the adjacent MDS region, to show that strains that develop with only one mutant chromosome can produce viable sexual progeny, but it appears that both the MLS and the MDS from the mutant chromosome are lost. If both chromosome copies have the CBS mutation, the cells arrest during development and do not eliminate many germline-limited sequences and fail to produce viable progeny. Overall, this study provides many new insights into the fate of germline chromosome ends during somatic genome remodeling and suggests extensive coordination of different DNA elimination events in Tetrahymena.

Strengths:

Overall, the experiments were well executed with appropriate controls. The findings are generally robust. Importantly, the study provides several novel findings. First, the authors provide a fairly comprehensive characterization of the size of the MLS at the end of each germline chromosome. I'm not sure whether this has been published elsewhere. Second, the authors develop a novel method to study the fate of chromosome termini during development and use it to conclusively track the elimination of these termini. Third, the authors show that the elimination of these termini appears to occur concurrently with most other DNA elimination events during somatic genome differentiation. And fourth, the authors show that failure to separate these eliminated sequences from the normally retained chromosome alters the fate of these adjacent MDS and the loss of the cells' ability to produce viable progeny.

Weaknesses:

It appears the authors did extensive analysis of the MLS chromosome ends, but did not provide too much information related to their composition. If this has not been published elsewhere, it would be useful to describe the proportion of unique and repetitive sequences and provide more information about the general composition of the chromosome ends. Such information would help the reader understand the nature of these MLS and how they may or may not differ from other eliminated sequences. Although the development of the novel FISH probes for large chromosome ends allowed for these novel discoveries, the signal in several images was visible, but often quite faint. I'm not sure there is anything the authors could do to improve the signal-to-noise ratio, but one needs to stare at the images carefully to understand the findings. One main weakness in the opinion of this reviewer is that the authors did very little to understand why, when a terminal MLS and the adjacent MDS fail to get separated because of failure in chromosome breakage, both segments are eliminated. The authors propose that possibly essential genes in the MDS get silenced, and the resulting lack of gene expression is the issue, but this and other possibilities were not tested. The study would provide more mechanistic insight if they had tried to assess whether the MDS on the CBS mutant chromosome becomes enriched in silencing modifications (e.g., H3K9me3). Alternatively, the authors could have examined changes in gene expression for some of the loci on the neighbouring MDS. The other main weakness is that since the authors only mutated the end of one germline chromosome, it is not clear whether the elimination of the MDS adjacent to the terminal MLS on chromosome 4 when the CBS is mutated is a general phenomenon, i.e., would happen at all chromosome ends, or is unique to the situation at Chromosome 4R. Knowing whether it is a general phenomenon or not would provide important insight into the authors' findings.