authors argue that collapses were caused by insufficient implementation of pluri-national federal principles. for example war started in croatia because the serb population wanted to stay in Yugoslavia (this sentiment also spread to bosnia).

the authors even argue that it is because of these centralising movements and majoritarian policies of the dominant groups that contribute to the wars and conflict of the existing nations that came out of the federations.

wrong historical causation leads to prominent arguments against multinational federation. the authors in this article argues its attempts to unitarise and centralise multinational federations that lead to secession and violence. For Yugoslavia, the successive Serbian-dominated moves against autonomy of the others lead to Kosovo to de facto breakaway. it could be said that federal constitutions with procedural and negotiable secession rules might have avoided violence and even succession better.

the authors of this paper counters the argument that mono-nation-building strategies can be used as an alternative for deeply diverse states is that these strategies have not been successful. UK's civic and unitary state did not prevent the nationalism of its different nations. Therefore the UK had to use a devolution strategy but it still does not quell nationalism.

counterfactuals = relating to or expressing what has not happened or is not the case. authors against multinational federation argue that it was unnecessary to accommodate diversity through federation and that there were democratic civic or unitarists (one nation) alternatives.

the econoic systems within these failed federations eventually proved incapable of providing a reasonable or growing standard of living for citizens. different economic systems of the different regions of the state caused resentment.

colonial federations still were imprinted by the departing metropolitan. s decision to federate instead of the indigenous elites. this case includes Nigeria and Cameroon.

Yugoslavia isn't actually a multinational federation, it was decentralised but that doesn't mean its was democratic, it was held together by the League of Communists. Other "multinational federations" which are more like pseudo-federations include USSR, Czechoslovakia, and Nigeria. they all had weak or no overarching identities and no democratic mechanism for developing those identities

this harks back to an article i read a little bit of (i think it was the Yugoslavia one) where america's motivation relied on ideological differences to further break up that federation. but that's an interpretation. regarding the paragraph, american academic argue that the break-up of former communist federations are due to their implemenation of "ethno-federal" strcutures. Jack Snyder argues that ethnofederalism tends to heighten and politicise ethnic consciousness, creating self-conscious intelligentsia and org strucutres of an ethnic state in waiting. implying that federalism leaves ethnic groups waiting for something they will not receiving leading to nationalism and tensions. additionally snyder notes that nationalist violence happened only where ethnofederal institutions channelled pol activity along ethnic lines (ex: USSR and Yugoslavia).

to manage divisions in thnically heterogenous soc many different authors have suggests that federations can be partly designed to prevent ethnic minorities from becoming local provincial majorities. a balance of power principles, proliferating points of power away from one focal centre, encouraging intra-ethnic divisions, nad creating alignments based on non-ehtnic interests. america suggested this to iraq.

brings in the disagreement that american academy and policy making have with multinational federations that is unlike Jacobins because americans reject the strong state idea. The disagreement is that America's position on federalism is that it shoud be more national than pluri-national. Instead american praise federalism for its ability to diffuse power to multiple points and it protects liberalism and enhances markets. americans even encouraged federalism for post-war germany. additionally, american federalism's goal is the same as Jacobinism which is to contruct a single poeple out of many.

breaksdown where the negativity toward multinational federations are coming from. Specifically from France's Jacobins where he thinks that pol recognition of multiple nations/ethnic comm institutionalizes and reinforces division, endangers national/state unity, and leads to state breka up. Then refers to practical examples which include several eastern euro states that have moved to replacing pluri-naitonal federations with "nationalising" states (tightly centralised, controlled by dom national comm, and intent on honogenisation of deviant identities). these nationalist seek indep as unitary, sovereign and indivisible nation-staets with some able to consider confederation.

highlights the federal principle of the ability for groups to easily secede if they want to. in the case of Yugoslavia is was a part of the full constitutent unit of the USSR were able to break away and break even further into independent states. In the case of succession it begins when the regional gov balme their central counterpart for whatever ails them; this contributes to intergovernmental politics of division

details the accounts were multi-national federations have collapsed or have failed to be durably democratic all around the world.

understanding the term and relevance of "multination/pluri-national" or "ethno-federal" federations instability. mentions Yugoslavia's disintegration during its transition to democracy. (which the authors argues is crucial for the stability of multinational federations, alongside the choices of no democracy and no single state)

argues that multinational federations cna succeed under certain conditions and the arguments against are greatly exagerated, based on majoritarian bias, spurious arguments, and misleading comparisons.

fucking agree!

1. Snacks (I do not really snack a whole lot, but they are nice to have just in case).
2. Books (I love buying new books, but I have so many that I already have and should read).
3. Make up (I do not wear makeup often, so why do I have so much?)

Money Problems and debt.

A big storm brings things into focus

What engineers were doing

social history of the above

Geography, infrastructure, history policy cf Bangkok floodplain - People shoudl be left with an overview of the situation Bkk faces vis-a-vis climate flooding hydrology "broader stakes" of innovation

3 sentences, tight

2 pgs

4+ pgs

4+ pgs

modes of practical expertise

moveable mudbanks

experience,. moving shore, colonial prehistory, design artifact

Q4. Kaitlin Breuchel In concluding their argument, Ball and Loewe assert that there is creativity in all writing, and not just what we term "creative writing." They argue that setting a definition for creativity to be used in some forms of writing makes people imagine and thought that enter ordinary writing. From what I have read, I view "creative writing" differently i think it cannot just be referring to poetry or fiction but any form of writing where a person is deciding, communicating ideas, and speaking with others.

Q1. Kaitlin Breuchel I have authored styles like academic reports and technical reports, which Cheryl E. Ball and Drew M. Loewe have termed weaker in terms of creativity. I used creativity in organizing arguments, picking examples, and crafting my papers yet persuasive explanations in an effort to make complex ideas comprehensible and persuasive.

Q2. Kaitlin Breuchel I'd identify more with being a writer because I like to think about how to communicate my ideas and engage other individuals with them. Most individuals are okay with labeling themselves as readers because reading is natural and simple it's something that we all do every single day without even thinking about it. Writing, is intimidating since people seem to think that it needs to be perfect or "creative." I think that expectation makes a lot of people wonder if they are really writers, even though everyone writes in some capacity.

AI Tools for each phase of analysis

von Wissen, Normen, Gesetzen, Gerichtsurteilen …

Prozesswandel?

Stronger alternatives: The central claim that, "AI-powered systems enable teams collaborate, explore new choices, and develop ideas in real time," is a good argument, however I would end the sentence with a stronger explanation. The ending of their sentence is vague and indistinct.

Consistency: This article is consistent, drawing all conclusion back to the thesis that AI improves firms. It also recognizes that AI needs to be implemented with care. By doing this the article is recognizing the counter claim that AI is "taking over," which improves the logic of the argument.

Ambiguity: The term "operations efficiency" is used quite a bit in this article. An explanation of what operations efficiency really means should be provided so that the reader understands the full context of the argument. Operations efficiency could refer to refer to product development, marketing, or sales.

Fallacies: I couldn't find a fallacy in this article. Evidence is used to support the claim, there aren't emotional/personal attacks, and evidence is broken down and explained. This article is thoroughly written with the intent to inform, not attack.

Validity: This article uses both data and reasoning to support the claim. It gives numerous examples and statistics of how AI betters firms. Each piece of evidence is brought back to the claim that AI is maximizing innovation and creativity.

Soundness: The evidence this article uses is true and relavant. I know it's true because they provide data and statistics from an analysis that support the claim. This evidence connects to the claim that AI increases innovation, which is a prime aspect of the thesis.

Link your ethics to your plans to grow the business sustainably--this allows you to both articulate your values and end with a hopeful note about the future. It's a good move that helps both describe and promote your vision.

Second-order headings (reflected in the Table of Contents) and bulleted lists help break up the clutter of the document.

You may wish to consider some bulleted lists to make the examples stand out more visually.

"Another thing" is generic sounding. Use strong beginnings to each sentence, e.g. "Bean There also provides."

For each value, list at least one practice that demonstrates that value. It need not be long, but it does make your commitment clearer to the reader of the document.

The story about COVID can demonstrate qualities like resilience and determination, which you can then describe as qualities that drive your business model.

So, tldr, you are always linking your company's actions and your company's values.

Can you briefly tell us what was wrong with the existing ones that created this opportunity for you to make a better (and more ethical) product? What did the opportunity look like?

Nah, you wanna exceed expectations rather than just meet them.

Use the "insert table of contents" function to create a symmetrical presentation with consistent columns.

eLife Assessment

This fundamental study presents a new method for longitudinally tracking cells in two-photon imaging data that addresses the specific challenges of imaging neurons in the developing cortex. It provides compelling evidence demonstrating reliable longitudinal identification of neurons across the second postnatal week in mice. The study should be of interest to development neuroscientists engaged in population-level recordings using two-photon imaging.

Reviewer #1 (Public review):

Summary:

This manuscript presents a compelling and innovative approach that combines Track2p neuronal tracking with advanced analytical methods to investigate early postnatal brain development. The work provides a powerful framework for exploring complex developmental processes such as the emergence of sensory representations, cognitive functions, and activity-dependent circuit formation. By enabling the tracking of the same neurons over extended developmental periods, this methodology sets the stage for mechanistic insights that were previously inaccessible.

Strengths:

(1) Innovative Methodology:

The integration of Track2p with longitudinal calcium imaging offers a unique capability to follow individual neurons across critical developmental windows.

(2) High Conceptual Impact:

The manuscript outlines a clear path for using this approach to study foundational developmental questions, such as how early neuronal activity shapes later functional properties and network assembly.

(3) Future Experimental Potential:

The authors convincingly argue for the feasibility of extending this tracking into adulthood and combining it with targeted manipulations, which could significantly advance our understanding of causality in developmental processes.

(4) Broad Applicability:

The proposed framework can be adapted to a wide range of experimental designs and questions, making it a valuable resource for the field.

Weaknesses:

None major. The manuscript is conceptually strong and methodologically sound. Future studies will need to address potential technical limitations of long-term tracking, but this does not detract from the current work's significance and clarity of vision

Comments on revisions:

I have no further requests. I think this is an excellent manuscript

Reviewer #2 (Public review):

Summary:

The manuscript by Majnik and colleagues introduces "Track2p", a new tool designed to track neurons across imaging sessions of two-photon calcium imaging in developing mice. The method addresses the challenge of tracking cells in the growing brain of developing mice. The authors showed that "Track2p" successfully tracks hundreds of neurons in the barrel cortex across multiple days during the second postnatal week. This enabled identification of the emergence of behavioral state modulation and desynchronization of spontaneous network activity around postnatal day 11.

Strengths

The authors have satisfactorily addressed the majority of our questions and comments, and the revisions substantially improve the manuscript. The expansion of Track2p to accept general NumPy array inputs makes the tool more accessible to researchers using different analysis pipelines. While the absence of benchmarking standards remains a limitation across the field, the release of the ground-truth dataset is an important step forward that will allow other researchers to evaluate and compare algorithms.

Minor point

(1) The authors tested the robustness of the algorithm across non-consecutive days. As expected, performance drops significantly under these conditions. We agree that this limitation reflects biological constraints due to brain growth rather than shortcomings of the algorithm itself. This is relevant for researchers planning to use Track2p for longitudinal imaging or benchmarking new algorithms, and we recommend including some of this information in the Supplementary Information along with a brief discussion.

Comments on revisions:

We acknowledge the extended documentation for using Track2p and converting between Suite2p outputs and NumPy arrays. This addition is of great utility. We would also suggest further expanding the documentation for the NumPy array implementation, as we ran into some errors when testing this feature using NumPy arrays generated from deltaF traces, TIFF FOVs, and Cellpose masks.

Reviewer #3 (Public review):

Summary:

In this manuscript Majnik et al. developed a computational algorithm to track individual developing interneurons in the rodent cortex at postnatal stages. Considerable development in cortical networks takes place during the first postnatal weeks, however, tools to study them longitudinally at a single cell level are scarce. This paper provides a valuable approach to study both single cell dynamics across days and state-drive network changes. The authors used Gad67Cre mice together with virally introduced TdTom to track interneurons based on their anatomical location in the FOV and AAVSynGCaMP8m to follow their activity across the second postnatal week, a period during which the cortex is known to undergo marked decorrelation in spontaneous activity. Using Track2P, the authors show feasibility to track populations of neurons in the same mice capturing with their analysis previously described developmental decorrelation and uncovering stable representations of neuronal activity, coincident with the onset of spontaneous active movement. The quality of the imaging data is compelling, and the computational analysis is thorough, providing a widely applicable tool for the analysis of emerging neuronal activity in the cortex. Below are some points for the authors to consider.

Major points

The authors use a viral approach to label cortical interneurons. It is unclear how Track2P will perform in dense networks of excitatory cells using GCaMP transgenic mice.

The authors used 20 neurons to generate a ground truth data set. The rational for this sample size is unclear. Figure 1 indicates capability to track ~728 neurons. A larger ground truth data set will increase the robustness of the conclusions.

It is unclear how movement was scored in the analysis shown in Fig 5A. Was the time that the mouse spent moving scored after visual inspection of the videos? Were whisker and muscle twitches scored as movement or was movement quantified as amount of time in which the treadmill was displaced?

The rational for binning the data analysis in early P11 is unclear. As the authors acknowledged, it is likely that the decoder captured active states from P11 onwards. Because active whisking begins around P14, it is unlikely to drive this change in network dynamics at P11. Does pupil dilation in the pups change during locomotor and resting states? Does the arousal state of the pups abruptly change at P11?

Comments on revisions:

The authors have addressed carefully all my comments. This is an interesting paper.

Author response:

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

Reviewer #1 (Public review):

We thank the reviewer for very enthusiastic and supportive comments on our manuscript. 

Summary:

This manuscript presents a compelling and innovative approach that combines Track2p neuronal tracking with advanced analytical methods to investigate early postnatal brain development. The work provides a powerful framework for exploring complex developmental processes such as the emergence of sensory representations, cognitive functions, and activity-dependent circuit formation. By enabling the tracking of the same neurons over extended developmental periods, this methodology sets the stage for mechanistic insights that were previously inaccessible.

Strengths:

(1) Innovative Methodology:

The integration of Track2p with longitudinal calcium imaging offers a unique capability to follow individual neurons across critical developmental windows.

(2) High Conceptual Impact:

The manuscript outlines a clear path for using this approach to study foundational developmental questions, such as how early neuronal activity shapes later functional properties and network assembly.

(3) Future Experimental Potential:

The authors convincingly argue for the feasibility of extending this tracking into adulthood and combining it with targeted manipulations, which could significantly advance our understanding of causality in developmental processes.

(4) Broad Applicability:

The proposed framework can be adapted to a wide range of experimental designs and questions, making it a valuable resource for the field.

Weaknesses:

No major weaknesses were identified by this reviewer. The manuscript is conceptually strong and methodologically sound. Future studies will need to address potential technical limitations of long-term tracking, but this does not detract from the current work's significance and clarity of vision.

Reviewer #2 (Public review):

Summary:

The manuscript by Majnik and colleagues introduces "Track2p", a new tool designed to track neurons across imaging sessions of two-photon calcium imaging in developing mice. The method addresses the challenge of tracking cells in the growing brain of developing mice. The authors showed that "Track2p" successfully tracks hundreds of neurons in the barrel cortex across multiple days during the second postnatal week. This enabled the identification of the emergence of behavioral state modulation and desynchronization of spontaneous network activity around postnatal day 11.

Strengths:

The manuscript is well written, and the analysis pipeline is clearly described. Moreover, the dataset used for validation is of high quality, considering the technical challenges associated with longitudinal two-photon recordings in mouse pups. The authors provide a convincing comparison of both manual annotation and "CellReg" to demonstrate the tracking performance of "Track2p". Applying this tracking algorithm, Majnik and colleagues characterized hallmark developmental changes in spontaneous network activity, highlighting the impact of longitudinal imaging approaches in developmental neuroscience. Additionally, the code is available on GitHub, along with helpful documentation, which will facilitate accessibility and usability by other researchers.

Weaknesses:

(1) The main critique of the "Track2p" package is that, in its current implementation, it is dependent on the outputs of "Suite2p". This limits adoption by researchers who use alternative pipelines or custom code. One potential solution would be to generalize the accepted inputs beyond the fixed format of "Suite2p", for instance, by accepting NumPy arrays (e.g., ROIs, deltaF/F traces, images, etc.) from files generated by other software. Otherwise, the tool may remain more of a useful add-on to "Suite2p" (see https://github.com/MouseLand/suite2p/issues/933) rather than a fully standalone tool.

We thank the reviewer for this excellent suggestion. 

We have now implemented this feature, where Track2p is now compatible with ‘raw’ NumPy arrays for the three types of inputs. For more information, please check the updated documentation: https://track2p.github.io/run_inputs_and_parameters.html#raw-npy-arrays. We have also tested this feature using a custom segmentation and trace extraction pipeline using Cellpose for segmentation.

(2) Further benchmarking would strengthen the validation of "Track2p", particularly against "CaIMaN" (Giovannucci et al., eLife, 2019), which is widely used in the field and implements a distinct registration approach.

This reviewer suggested  further benchmarking of Track2P.  Ideally, we would want to benchmark Track2p against the current state-of-the-art method. However, the field currently lacks consensus on which algorithm performs best, with multiple methods available including CaIMaN, SCOUT (Johnston et al. 2022), ROICaT (Nguyen et al. 2023), ROIMatchPub (recommended by Suite2p documentation and recently used by Hasegawa et al. 2024), and custom pipelines such as those described by Sun et al. 2025. The absence of systematic benchmarking studies—particularly for custom tracking pipelines—makes it impossible to identify the current state-of-the-art for comparison with Track2p. While comparing Track2p against all available methods would provide comprehensive evaluation, such an analysis falls beyond the scope of this paper.

We selected CellReg for our primary comparison because it has been validated under similar experimental conditions—specifically, 2-photon calcium imaging in developing hippocampus between P17-P25 (Wang et al. 2024)—making it the most relevant benchmark for our developmental neocortex dataset.

That said, to support further benchmarking in mouse neocortex (P8-P14), we will publicly release our ground truth tracking dataset.

(3) The authors might also consider evaluating performance using non-consecutive recordings (e.g., alternate days or only three time points across the week) to demonstrate utility in other experimental designs.

Thank you for your suggestion. We have performed a similar analysis prior to submission, but we decided against including it in the final manuscript, to keep the evaluation brief and to not confuse the reader with too many different evaluation methods. We have included the results inAuthor response images 1 and 2 below.

To evaluate performance in experimental designs with larger time spans between recordings (>1 day) we performed additional evaluation of tracking from P8 to each of the consecutive days while omitting the intermediate days (e. g. P8 to P9, P8 to P10 … P8 to P14). The performance for the three mice from the manuscript is shown below:

Author response image 1.

As expected with increasing time difference between the two recordings the performance drops significantly (dropping to effectively zero for 2 out of 3 mice). This could also explain why CellReg struggles to track cells across all days, since it takes P8 as a reference and attempts to register all consecutive days to that time point before matching, instead of performing registration and matching in consecutive pairs of recordings (P8-P9, P9-P10 … P13-P14) as we do.

Finally for one of the three mice we also performed an additional test where we asked how adding an additional recording day might rescue the P8-P14 tracking performance. This corresponds to the comment from the reviewer, answering the question if we can only perform three days of recording which additional day would give the best tracking performance. 

Author response image 2.

As can be seen from the plot, adding the P10 or P11 recording shows the most significant improvement to the tracking performance, however the performance is still significantly lower than when including all days (see Fig. 4). This test suggests that including a day that is slightly skewed to earlier ages might improve the performance more than simply choosing the middle day between the two extremes. This would also be consistent with the qualitative observation that the FOV seems to show more drastic day-to-day changes at earlier ages in our recording conditions.

Reviewer #3 (Public review):

Summary:

In this manuscript, Majnik et al. developed a computational algorithm to track individual developing interneurons in the rodent cortex at postnatal stages. Considerable development in cortical networks takes place during the first postnatal weeks; however, tools to study them longitudinally at a single-cell level are scarce. This paper provides a valuable approach to study both single-cell dynamics across days and state-driven network changes. The authors used Gad67Cre mice together with virally introduced TdTom to track interneurons based on their anatomical location in the FOV and AAVSynGCaMP8m to follow their activity across the second postnatal week, a period during which the cortex is known to undergo marked decorrelation in spontaneous activity. Using Track2P, the authors show the feasibility of tracking populations of neurons in the same mice, capturing with their analysis previously described developmental decorrelation and uncovering stable representations of neuronal activity, coincident with the onset of spontaneous active movement. The quality of the imaging data is compelling, and the computational analysis is thorough, providing a widely applicable tool for the analysis of emerging neuronal activity in the cortex. Below are some points for the authors to consider.

We thank the reviewer for a constructive and positive evaluation of our MS. 

Major points:

(1) The authors used 20 neurons to generate a ground truth dataset. The rationale for this sample size is unclear. Figure 1 indicates the capability to track ~728 neurons. A larger ground truth data set will increase the robustness of the conclusions.

We think this was a misunderstanding of our ground truth dataset analysis which included 192 and not 20 neurons. Indeed, as explained in the methods section, since manually tracking all cells would require prohibitive amounts of time, we decided to generate sparse manual annotations, only tracking a subset of all cells from the first recording day onwards. To do this, we took the first recording (s0), and we defined a grid 64 equidistant points over the FOV and, for each point, identified the closest ROI in terms of euclidean distance from the median pixel of the ROI (see Fig. S3A). We then manually tracked these 64 ROIs across subsequent days. Only neurons that were detected and tracked across all sessions were taken into account and referred to as our ground truth dataset (‘GT’ in Fig. 4). This was done for 3 mice, hence 3X64 neurons and not 20 were used to generate our GT dataset. 

(2) It is unclear how movement was scored in the analysis shown in Figure 5A. Was the time that the mouse spent moving scored after visual inspection of the videos? Were whisker and muscle twitches scored as movement, or was movement quantified as the amount of time during which the treadmill was displaced?

Movement was scored using a ‘motion energy’ metric as in Stringer et al. 2019 (V1) or Inácio et al. 2025 (S1). This metric takes each two consecutive frames of the videography recordings and computes the difference between them by summing up the square of pixelwise differences between the two images. We made the appropriate changes in the manuscript to further clarify this in the main text and methods in order to avoid confusion.

Since this metric quantifies global movements, it is inherently biased to whole-body movements causing more significant changes in pixel values around the whole FOV of the camera. Slight twitches of a single limb, or the whisker pad would thus contribute much less to this metric, since these are usually slight displacements in a small region of the camera FOV. Additionally, comparing neural activity across all time points (using correlation or R²) also favours movements that last longer (such as wake movements / prolonged periods of high arousal) since each time point is treated equally.

As we suggested in the discussion, in further analysis it would be interesting to look at the link between twitches and neural activity, but this would likely require extensive manual scoring. We could then treat movements not as continuous across all time-points, but instead using event-based analysis for example peri-movement time histograms for different types of movements at different ages, which is however outside of the scope of this study.

(3) The rationale for binning the data analysis in early P11 is unclear. As the authors acknowledged, it is likely that the decoder captured active states from P11 onwards. Because active whisking begins around P14, it is unlikely to drive this change in network dynamics at P11. Does pupil dilation in the pups change during locomotor and resting states? Does the arousal state of the pups abruptly change at P11?

We agree that P11 does not match any change in mouse behavior that we have been able to capture. However, arousal state in mice does change around postnatal day 11. This period marks a transition from immature, fragmented states to more organized and regulated sleep-wake patterns, along with increasing influence from neuromodulatory and sensory systems. All of these changes have been recently reviewed in Wu et al. 2024 (see also Martini et al. 2021). In addition, in the developing somatosensory system, before postnatal day 11 (P11), wake-related movements (reafference) are actively gated and blocked by the external cuneate nucleus (ECN, Tiriac et al. 2016 and all excellent recent work from the Blumberg lab). This gating prevents sensory feedback from wake movements from reaching the cortex, ensuring that only sleep-related twitches drive neural responses. However, around P11, this gating mechanism abruptly lifts, enabling sensory signals from wake movements to influence cortical processing—signaling a dramatic developmental shift from Wu et al. 2024

Reviewer #1 (Recommendations for the authors):

This manuscript represents a significant advancement in the field of developmental neuroscience, offering a powerful and elegant framework for longitudinal cellular tracking using the Track2p method combined with robust analytical approaches. The authors convincingly demonstrate that this integrated methodology provides an invaluable template for investigating complex developmental processes, including the emergence of sensory representations and higher cognitive functions.

A major strength of this work is its emphasis on the power of longitudinal imaging to illuminate activity-dependent development. By tracking the same neurons over time, the authors open up new possibilities to uncover how early activity patterns shape later functional outcomes and the organization of neuronal assemblies-insights that would be inaccessible using conventional cross-sectional designs.

Importantly, the manuscript highlights the potential for this approach to be extended even further, enabling continuous tracking into adulthood and thus offering an unprecedented window into long-term developmental trajectories. The authors also underscore the exciting opportunity to incorporate targeted perturbation experiments, allowing researchers to causally link early circuit dynamics to later outcomes.

Given the increasing recognition that early postnatal alterations can underlie the etiology of various neurodevelopmental disorders, this work is especially timely. The methods and perspectives presented here are poised to catalyze a new generation of developmental studies that can reveal mechanistic underpinnings of both typical and atypical brain development.

In summary, this is a technically impressive and conceptually forward-looking study that sets the stage for transformative advances in developmental neuroscience.

Thank you for the thoughtful feedback—it's greatly appreciated!

Reviewer #2 (Recommendations for the authors):

Minor points:

(1) Figure 1. Consider merging or moving to Supplemental, as its rationale is well described in the text.

We would like to retain the current figure as we believe it provides an effective visual illustration of our rationale that will capture readers' attention and could serve as a valuable reference for others seeking to justify longitudinal tracking of the developing brain. We hope the reviewer will understand our decision.

(2) Some axis labels and panels are difficult to read due to small font sizes (e.g. smaller panels in Figures 5-7).

Modified, thanks 

(3) Supplementary Figures. The order of appearance in the main text is occasionally inconsistent.

This was modified, thanks

(4) Line 132. Add a reference to the registration toolbox used (elastix). A brief description of the affine transformation would also be helpful, either here or in the Methods section (p. 27).

We have added reference to Ntatsis et al. 2023 and described affine transformation in the main text (lines 133-135): 

Firstly, we estimate the spatial transformation between s0 and s1 using affine image registration (i.e. allowing shifting, rotation, scaling and shearing, see Fig. 2B, the transformation is denoted as T).

(5) Lines 147-151. If this method is adapted from another work, please cite the source.

Computing the intersection over union of two ROIs for tracking is a widely established and intuitive method used across numerous studies, representing standard practice rather than requiring specific citation. We have however included the reference to the paper describing the algorithm we use to solve the linear sum assignment problem used for matching neurons across a pair of consecutive days (Crouse 2016).

(6) Line 218. "classical" or automatic?

We meant “classical” in the sense of widely used. 

(7) Lines 220-231. Did the authors find significant variability of successfully tracked neurons across mice? While the data for successfully tracked cells is reported (Figure 5B), the proportions are not. Could differences in neuron dropout across days and mice affect the analysis of neuronal activity statistics?

We thank the reviewer for raising this important point. We computed the fraction of successfully tracked cells in our dataset and found substantial variability:

Cells detected on day 0: [607, 1849, 2190, 1988, 1316, 2138] 

Proportion successfully tracked: [0.47, 0.20, 0.36, 0.37, 0.41, 0.19]

Notably, the number of cells detected on the first day varies considerably (607–2138 cells). There appears to be a trend whereby datasets with fewer initially detected cells show higher tracking success rates, potentially because only highly active cells are identified in these cases.

To draw more definitive conclusions about the proportion of active cells and tracking dropout rates, we would require activity-independent cell detection methods (such as Cellpose applied to isosbestic 830 nm fluorescence, or ideally a pan-neuronal marker in a separate channel, e.g., tdTomato). We have incorporated the tracking success proportions into the revised manuscript.

(8) Line 260. Please briefly explain, here or in the Methods, the rationale for using data from only 3 mice (rather than all 6) for evaluating tracking performance.

We used three mice for this analysis due to the labor-intensive nature of manually annotating 64 ROIs across several days. Given the time constraints of this manual process, we determined that three subjects would provide adequate data to reliably assess tracking performance.

(9) Line 277. Consider clarifying or rephrasing the phrase "across progressively shorter time intervals"? Do you mean across consecutive days?

This has been rephrased as follows: 

Additionally, to assess tracking performance over time, we quantified the proportion of reconstructed ground truth tracks over progressively longer time intervals (first two days, first three days etc. ‘Prop. correct’ in Fig. 4C-F, see Methods). This allowed us to understand how tracking accuracy depends on the number of successive sessions, as well as at which time points the algorithm might fail to successfully track cells.

(10) Line 306. "we also provide additional resources and documentation". Please add a reference or link.

Done, thanks

Track2p  

(11) Lines 342-344. Specify that the raster plots refer to one example mouse, not the entire sample.

Done, thanks.

(12) Lines 996-1002. Please confirm whether only successfully tracked neurons were used to compute the Pearson correlations between all pairs.

Yes of course, this only applies to tracked neurons as it is impossible to compute this for non-tracked pairs.

(13) Line 1003. Add a reference to scikit-learn.

Reference was added to: 

Pedregosa, F., Varoquaux, G., Gramfort, A., Michel, V., Thirion, B., Grisel, O., Blondel, M., Prettenhofer, P., Weiss, R., Dubourg, V., Vanderplas, J., Passos, A., Cournapeau, D., Brucher, M., Perrot, M., & Duchesnay, E. (2011). Scikit-learn: Machine Learning in Python. Journal of Machine Learning Research, 12, 2825–2830. 

(14) Typos.Correct spacing between numeric values and units.

We did not find many typos regarding spacing between the numerical value and the unit symbol (degrees and percent should not be spaced right?).

Reviewer #3 (Recommendations for the authors):

The font size in many of the figures is too small. For example, it is difficult to follow individual ROIs in Figure S3.

Figure font size has been increased, thanks. In Figure S3 there might have been a misunderstanding, since the three FOV images do not correspond to the FOV of the same mouse across three days but rather to the first recording for each of the three mice used in evaluation (the ROIs can thus not be followed across images since they correspond to a different mouse). To avoid confusion we have labelled each of the FOV images with the corresponding mouse identifier (same as in Fig. 4 and 5).

eLife Assessment

This is a valuable study that explores the role of the conserved transcription factor POU4-2 in the maintenance, regeneration, and function of planarian mechanosensory neurons. The authors present convincing evidence provided by gene expression and functional studies to demonstrate that POU4-2 is required for the maintenance and regeneration of mechanosensory neurons and mechanosensory function in planarians. Furthermore, the authors identify conserved genes associated with human auditory and rheosensory neurons as potential targets of this transcription factor.

Reviewer #1 (Public review):

Summary:

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

Strengths:

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

Reviewer #2 (Public review):

Summary:

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

Strengths:

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

Author response:

The following is the authors’ response to the original reviews

Reviewer #1 (Public review): 

Summary: 

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

Strengths: 

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

Weaknesses: 

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

Reviewer #2 (Public review): 

Summary: 

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

Strengths: 

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

Weaknesses: 

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

Reviewing Editor Comments: 

(1) Quantification of pou4-2(+) cells that express (or do not express) hmcn-1-L and/or pkd1L-2(-) is a common suggestion amongst reviewers. It is recognized that Ross et al. (2018) showed that pkd1L-2 and hmcn-1L expression is detected in separate cells by double FISH, and the analysis presented in Supplementary Figure S3 is helpful in showing that some cells expressing pou4-2 (magenta) are not labeled by the combined signal of pkd1L-2 and hmcn-1-L riboprobes (green). However, I am not sure that we can conclude that pkd1L-2 and hmcn-1-L are effectively detected when riboprobes are combined in the analysis. Therefore, quantification of labeled cells as proposed by Reviewers 1 and 2 would help.

Combining riboprobes is a standard approach in the field, and we chose this method as a direct way to determine which cells lack expression of both genes. We agree that providing the raw quantification data would be helpful for readers, and we included this data in Supplementary File S7; the file contains the quantification information for this dFISH experiment represented in Supplementary Figure 3.

(2) It may be helpful to comment on changes (or lack of changes) in atoh gene RNA levels in RNAseq analyses of pou4-2 animals. As mentioned by one of the reviewers, in situs that don't show signal are inconclusive in this regard. 

We fully agree with both reviewers. Two of the planarian atonal homologs are difficult to detect and produce background signals, which we attempted and previously reported in Cowles et al. Development (2013). We conceived performing reciprocal RNAi/in situ experiments, born out of curiosity given the reported role of atonal in the pou4 cascade in other organisms. However, these exploratory experiments lacked a strong rationale for inclusion, particularly given that pou4-2 and the atonal homologs do not share expression patterns, co-expression, or differential expression in our RNA-seq dataset. Therefore, we decided to omit the atonal in situs following pou4-2 RNAi. We retained the experiments showing that knockdown of the atonal genes does not show robust effects on the mechanosensory neuron pattern, as expected. We thank the reviewing editor and reviewers for pinpointing the concern. We agree that additional experiments, such as qPCR experiments, would be needed. We reasoned that while these additional experiments could be informative, they are unlikely to alter the key conclusions of this study substantially.

(3) There seem to be typos at bottom of Figure 10 and top of page 11 when referencing to Figure 4B (should be to 5B instead): "While mechanosensory neuronal patterned expression of Eph1 was downregulated after pou4-2 and soxB1-2 inhibition, low expression in the brain branches of the ventral cephalic ganglia persisted (Figure 4B)." 

Thank you! We have fixed those.

(4) Typo (page 13; kernel?): "...to test to what extent the Pou4 gene regulatory kernel is conserved among these widely divergent animals." 

Regulatory kernels are defined as the minimal sets of interacting genes that drive developmental processes and are the core circuits within a gene regulatory network, but we recognize that this might not be as well known, so we have changed the term to “network” for clarity.

Reviewer #1 (Recommendations for the authors): 

(1) The authors indicate that they are interested in finding out whether POU4-2 is important in the creation of mechanosensory neurons in adulthood as well as in embryogenesis (in other words, whether the mechanism is "reused during adult tissue maintenance and regeneration"). The manuscript clearly shows that planarian POU4 -2 is important in adult neurogenesis in planarians, but there is no evidence presented to show that this is a recapitulation of embryogenesis. Is pou4-2 expressed in the planarian embryo? This might be possible to examine by ISH or through the evaluation of sequencing data that already exists in the literature. 

We agree that these statements should be precise. We have clarified when we make comparisons to the role of Pou4 in sensory system development in other organisms versus its role in the adult planarian. We examined its expression using the existing database of embryonic gene expression. Thanks for hinting at this idea. We performed BLAST in Planosphere (Davies et al., 2017) to cross-reference our clone matching dd_Smed_v6_30562_0_1, which is identical to SMED30002016. The embryonic gene expression for SMED30002016 indicates this gene is expressed at the expected stages given prior knowledge of the timing of organ development in Schmidtea mediterranea (a positive trend begins at Stage 5, with a marked increase by Stage 6 that remains comparable to the asexual expression levels shown). We thank the reviewer for pointing out this oversight. We have incorporated this result in the paper as a Supplementary Figure and discuss how we can only speculate that it has a similar role as we detect in the adult asexual worms.

(2) Can it be determined whether the punctate pou4-2+ cells outside of the stripes are progenitors or other neural cell types? Are there pou4-2+ neurons that are not mechanosensory cell types? Could there be other roles for POU4-2 in the neurogenesis of other cell types? It might help to show percentages of overlap in Figure 4A and discuss whether the two populations add up to 100% of cells. 

These are good questions that arise in part from other statements that need clarification in the text (pointed out by Reviewer 2). We think some of the dorsal pou4-2⁺ might represent progenitor cells undergoing terminal differentiation (see Supplementary Figure 4). We attempted BrdU pulse chase experiments but were not successful in consistently detecting pou4-2 at sufficient levels with our protocol. In response to this helpful comment, we have included this question as a future direction in the revised Discussion. Finally, we have edited our description of the expression pattern. We already pointed out that there are other cells on the ventral side that are not affected when soxB1-2 is knocked down. We attempted to resolve the potential identity of those cells working with existing scRNA-seq data in collaboration with colleagues, but their low abundance made it difficult to distinguish other populations. While we acknowledge this interesting possibility, we have chosen to focus this report on the role of pou4-2 downstream of soxB1-2, as this represents the most well-supported aspect of the dataset and was positively highlighted by both the reviewer and editor.

(3) The authors discuss many genes from their analysis that play conserved roles in mechanosensation and hearing. Were there any conserved genes that came up in the analysis of pou4-2(RNAi) planarians that have not yet been studied in human hearing and neurodevelopment? I am wondering the extent to which planarians could be used as a discovery system for mechanosensory neuron function and development, and discussion of this point might increase the impact of this paper or provide critical rationale for expanding work on planarian mechanosensation. 

Indeed, we agree that planarians could be used to identify conserved genes with roles in mechanosensation and have included this point in the Discussion. In this study, we have focused on demonstrating the conservation of gene regulation. While this study was initially based on a graduate thesis project, we have since generated a more comprehensive dataset from isolated heads, which we are currently analyzing. This has been emphasized in the revised Discussion.

Minor: 

(1) For Figure 6E, the authors could consider showing data along a negative axis to indicate a decrease in length in response to vibration and to more clearly show that this decrease doesn't occur as strongly after pou4-2(RNAi). 

We displayed this behavior as the percent change, as this is a standard way to represent this data. As the percent change is a positive value, we represent the data as these positive values.

(2) The authors should consider quantifying the decrease of pou4-2 mRNA after atonal(RNAi) conditions, either by RT-qPCR or cell quantification. Visually, the signal in the stripes after atoh8-2(RNAi) seems lower, particularly in the tail. The punctate pattern outside the stripes may also be decreased after atoh8-1(RNAi). But quantification might strengthen the argument. 

We agree with the reviewer and acknowledge that we should have been more cautious in interpreting these results. Those two genes are difficult to detect and did not show specific patterns in Cowles et al. (2013). The reviewer is correct that additional experiments are necessary before reaching conclusions, but we do not think as discussed earlier we do not think new experiments would provide insights for the major conclusions. These experiments were exploratory in nature and tangential to our main conclusions, especially in the absence of reciprocal evidence (e.g., shared expression patterns, co-expression, or differential expression in our RNA-seq data. Therefore, we decided to eliminate the atonal in situs following pou4-2 RNAi.

Reviewer #2 (Recommendations for the authors): 

A. Expression of pou4-2 in ciliated mechanosensory neurons: 

(1) The conclusion that pou4-2 is expressed in ciliated mechanosensory neurons is primarily based on co-expression analysis using a published single-cell dataset. Although the authors later show that a subset of pou4-2 cells also express pkd1L-2 (Figure 4A), a known marker of ciliated mechanosensory neurons, this finding is not properly quantified. I recommend moving Figure 4A to earlier in the manuscript (e.g., to Figure 2) and expanding the analysis to include additional known markers of this cell type. Proper quantification of the extent of co-localization is necessary to support the claim robustly. 

As pointed out by the reviewer, there is substantive evidence from our lab and other reports. King et al. also showed pou4-2 and pkd1L-2 ‘regulation’ by their scRNA-seq data, and this function is conserved in the acoel Hofstenia miamia (Hulett et al., PNAS 2024 ). Our analysis shows convincing co-localization by scRNA-seq and expression of soxB1-2 and neural markers in the respective populations. Furthermore, we included colocalization of pou4-2 with mechanosensory genes using fluorescence in situ hybridization (Figure 3B, Supplementary Figure 4, and Supplementary File S7). We are confident the data conclusively show pou4-2 regulates pkd1L-2 expression in a subset of mechanosensory neurons. Given the strength of existing observations and previously published data, we believe that additional staining experiments are not essential to support this conclusion. 

(2) There appears to be a conceptual inconsistency in the interpretation of pou4-2 expression dynamics. On one hand, the authors suggest that delayed pou4-2 expression indicates a role in late-stage differentiation (p.6). On the other hand, they propose that pou4-2 may be expressed in undifferentiated progenitors to initiate downstream transcriptional programs (p.8). These interpretations should be reconciled. Additionally, claims regarding pou4-2 expression in progenitor populations should be supported by co-localization with established stem cell or progenitor markers, rather than inferred from signal intensity alone. 

This is an excellent point, and we agree with the reviewer that this section requires editing. As described in response to Reviewer 1, we attempted BrdU pulse chase experiments but were not successful in consistently detecting pou4-2 at sufficient levels with our protocol. Furthermore, we could not obtain strong signals in double labeling experiments in pou4-2 in situs combined with piwi-1 or PIWI-1 antibodies. We will include those experiments as a future direction and amend our conclusions accordingly.

(3) The expression pattern shown in Figure 1B raises questions about the precise anatomical localization of pou4-2 cells. It is unclear whether these cells reside in the subepidermal plexus or the deeper submuscular plexus, which represent distinct neuronal layers (Ross et al., 2017). The observed signals near the ventral nerve cords could suggest submuscular localization. To clarify this, higher-resolution imaging and co-staining with region-specific neural markers are recommended. 

In Ross et al. (2018), we showed that the pkd1L-2⁺ cells are located submuscularly. The pkd1L-2 cells express pou4-2, thus the pou4-2⁺ cells are located in the same location. Based on co-expression data and co-expression with PKD genes, we are confident it is submuscular.

B. The functional requirements of pou4-2 in the maintenance of mechanosensory neurons: 

(1) To evaluate the functional role of pou4-2 in maintaining mechanosensory neurons, the authors performed whole-animal RNA-seq on pou4-2(RNAi) and control animals, identifying a significant downregulation of genes associated with mechanosensory neuron expression. However, the presentation of these findings is fragmented across Figures 3, 4, and 5. I recommend consolidating the RNA-seq results (Figure 3) and the subsequent validation of downregulated genes (Figures 4 and 5) into a single, cohesive figure. This would improve the logical flow and clarity of the manuscript. 

As suggested by the reviewer, we have combined Figures 3 and 4 (new Figure 3), which we believe improves the flow. We decided to keep Figure 5 (new Figure 4) as a standalone because it focuses on the characterization of new genes revealed by RNAseq and scRNA-seq data mining that were not previously reported in Ross et al. 2018 and

2024.

(2) In pou4-2(RNAi) animals, pkd1L-2 expression appears to be entirely lost, while hmcn-1-L shows faint expression in scattered peripheral regions. The authors suggest that an extended RNAi treatment might be necessary to fully eliminate hmcn-1-L expression. However, an alternative explanation is that pou4-2 is not essential for maintaining all hmcn-1-L cells, particularly if pou4-2 expression does not fully overlap with that of hmcn-1-L. This possibility should be acknowledged and discussed. 

We agree and have acknowledged this point in the revised text.

(3) On page 9, the section title claims that "Smed-pou4-2 regulates genes involved in ciliated cell structure organization, cell adhesion, and nervous system development." While some differentially expressed genes are indeed annotated with these functions based on homology, the manuscript does not provide experimental evidence supporting their roles in these biological processes in planarians. The title should be revised to avoid overstatement, and the limitations of extrapolating a function solely from gene annotation should be acknowledged. 

Excellent point. We have edited the text to indicate that the genes were annotated or implicated.

(4) The cilia staining presented in Figure 6B to support the claim that pou4-2 is required for ciliated cell structure organization is unconvincing. Improved imaging and more targeted analysis (e.g., co-labeling with mechanosensory markers) are needed to support this conclusion. 

We have addressed this concern by adjusting the language to be more precise and indicate that the stereotypical banded pattern is disrupted with decreased cilia labeling along the dorsal ciliated stripe. Indeed, our conclusion overstated the observations made with the staining and imaging resolution. Thank you.

C. The functional requirements of pou4-2 in the regeneration of mechanosensory neurons: 

To evaluate the role of pou4-2 in the regeneration of mechanosensory neurons, the authors performed amputations on pou4-2(RNAi) and control(RNAi) animals and assessed the expression of mechanosensory markers (pkd1L-2, hmcn-1-L) alongside a functional assay. However, the results shown in Figure 4B indicate the presence of numerous pkd1L-2 and hmcn-1-L cells in the blastema of pou4-2(RNAi) animals. This observation raises the possibility that pou4-2 may not be essential for the regeneration of these mechanosensory neurons. The authors should address this alternative interpretation. 

Our interpretation is that there were very few cells expressing the markers compared to controls. The pattern was predominantly lost, which is consistent with other experiments shown in the paper. However, we have added the additional caveat suggested by the reviewer.

Minor points: 

(1) On p.8, the authors wrote "every 12 hours post-irradiation". However, this is not consistent with the figure, which only shows 0, 3, 4, 4.5, 5, and 5.5 dpi. 

We corrected this. Thank you for catching the mistake!

(2) On p.12, the authors wrote "Analysis of pou4-2 RNAi data revealed differentially expressed genes with known roles in mechanosensory functions, such as loxhd-1, cdh23, and myo7a. Mutations in these genes can cause a loss of mechanosensation/transduction". This is misleading because, to my knowledge, the role of these genes in planarians is unknown. If the authors meant other model systems, they should clearly state this in the text and include proper references. 

The reviewer is correct that we are referencing findings from other organisms. We have clarified this point in the revised text. The appropriate references were included and cited in the first version.

(3) On p.7, the authors wrote, "conversely, the expression of atonal genes was unaffected in pou4-2 RNAi-treated regenerates (Supplementary Figure S2B)". However, it is unclear whether the Atoh8-1 and Atoh8-2 signals are real, as the quality of the in situ results is too low to distinguish between real signals and background noise/non-specific staining. 

This valid concern was addressed in our response to Reviewer 1. We have adjusted the figure and the text accordingly.

(4) On p.6 the authors wrote "pinpointed time points wherein the pou4-2 transcripts were robustly downregulated". However, the current version of the manuscript does not provide data explaining why Pou4-2 transcripts are robustly downregulated on day 12. 

Yes, we determined the appropriate time points using qPCR for all sample extractions. As an example, see the figure for qPCR validation at day 12 showing that pou4-2 and pkd1L2 are down.

Author response image 1.

In this graph, samples labeled “G” represent four biological controls of gfp(RNAi) control animals, and samples labeled “P” represent four biological controls of pou4-2(RNAi)animals at day 12 in the RNAi protocol.

(5) On p.13, the authors wrote "collecting RNA from how animals." Is this a typo? 

Thanks for catching the typo. It should read “whole” animals. We have corrected this.

(6) On p.14, the authors wrote "but the expression patterns of planarian atonal genes indicated that they represent completely different cell populations from pou4-2-regulated mechanosensory neurons". However, this is unclear from the images, as the in situ staining of Atoh8-1 and Atoh82 are potentially failed stainings. 

We agree. We have edited accordingly.

eLife Assessment

This valuable manuscript presents an open-source and low-cost acoustic system for quantifying biting and chewing in mice. The approach is carefully validated against human observers, demonstrating strong methodological reliability and enabling high-resolution analysis of feeding microstructure. The tool has broad relevance for studies of appetite circuits and pharmacological interventions. A significant contribution is the identification of previously unrecognized "meal-related" neurons in the lateral hypothalamus, providing novel biological insight into food consumption. While the support for the methodological advances is compelling and robust, some circuit-level conclusions are preliminary or incomplete, relying on small pilot samples and manual classification, and should be interpreted with caution. This paper will be of interest to those interested in ingestive behavior and/or hypothalamus.

Reviewer #1 (Public review):

This is an interesting and valuable paper by Gil-Lievana, Arroyo et al. that presents an open-source method (the "Crunchometer") for quantifying biting and chewing behavior in mice using audio detection. The work addresses an important and unmet need in the field: quantitative measures of feeding behavior with solid foods, since most prior approaches have been limited to liquids. The authors make a clear and compelling case for why this problem is important, and I fully agree with their motivation.

The system is carefully validated against human-scored video data and is shown to be at least as accurate, and in some cases more accurate, than human observers. This is a major strength of the study. I also particularly appreciate the demonstration of the technology in the context of LHA circuitry, which nicely illustrates its utility and importance for mechanistic studies of feeding. I also appreciate the ability to readily time-lock neural data to individual crunches. Overall, the manuscript is well-executed and represents a useful contribution to the field.

The comments I have are largely minor and should be straightforward to address:

(1) The authors should report sample sizes for all mouse cohorts, either alongside the statistics or in the figure legends for mean data.

(2) Clarification is needed as to whether crunch detection fidelity is influenced by the hardness or softness of the food. The focus here is on standard pellets, with some additional high-fat pellet data, but it would be useful to know how generalizable the method is across different textures.

(3) The authors should comment on how susceptible the Crunchometer is to background noise. For example, how well does it perform in the presence of white noise, experimenter movement, or other task-related sounds?

(4) Chemogenetic activation of LHA GABAergic neurons is used. DREADD-based activation may strongly drive these neurons in a way that is not directly comparable to optogenetic or more physiological manipulations. While I do not think additional experiments are required, it would strengthen the discussion to briefly acknowledge this limitation.

Reviewer #2 (Public review):

Summary:

This manuscript introduces the Crunchometer, a low-cost, open-source acoustic platform for monitoring the microstructure of solid food intake in mice. The Crunchometer is designed to overcome the limitations of existing methods for studying feeding behavior in rodents. The goal was to provide a tool that could precisely capture the microstructure of solid food intake, something often overlooked in favor of liquid-based assays, while being affordable, scalable, and compatible with neural recording techniques. By doing so, the authors aimed to enable detailed analysis of how physiological states, drugs, and specific neural circuits shape naturalistic feeding behaviors.

Strengths:

The study's strengths lie in its clear innovation, methodological rigor in validation against human annotation, and demonstration of broad utility across behavioral and neuroscience paradigms. The approach addresses a significant methodological gap in the field by moving beyond liquid-based feeding assays and provides an accessible tool for precisely dissecting ingestive behavior. The system is validated across multiple contexts, including physiological state (fed vs. fasted), pharmacological manipulation (semaglutide), and circuit-level interventions (chemogenetic activation of LH neurons), and is further shown to integrate seamlessly with both electrophysiology and calcium imaging.

(1) Introduces a low-cost, open-source acoustic tool for measuring solid food intake, filling a critical gap left by expensive and proprietary systems.

(2) Makes the method easily adoptable across labs with detailed setup instructions and shared benchmark datasets.

(3) Provides high temporal precision for detecting bite events compared to human observers.

(4) Successfully distinguishes feeding microstructure (bites, bouts, IBIs, gnawing vs. consumption) with greater objectivity than manual annotation.

(5) Demonstrates compatibility with electrophysiology and calcium imaging, enabling fine-scale alignment of neural activity with feeding behavior.

(6) Effectively discriminates between fed vs. fasted states, validating physiological sensitivity.

(7) Captures the pharmacological effects of semaglutide, although this is really just reduced feeding and associated readouts (bouts, latency, etc).

(8) Has potential to distinguish consummatory vs. non-consummatory behaviors (e.g., food spillage, gnawing); however, the current SVM model struggles to separate biting from gnawing due to similar acoustic profiles, and manual validation is still required.

(9) Provides potential for closed-loop experiments.

Weaknesses:

Several limitations temper the strength of the conclusions: the supervised classifier still requires manual correction for gnawing, generalizability across different setups is limited, and the neuroscience findings, particularly calcium imaging of GABAergic and glutamatergic neurons, are based on small pilot samples. These issues do not undermine the value of the tool, but mean that the neural circuit findings should be interpreted as preliminary.

(1) Some neuroscience findings (calcium imaging of GABAergic vs. glutamatergic neurons) are based on small pilot samples (n=2 mice per condition), limiting generalizability.

(2) Chemogenetic and pharmacological experiments used small cohorts, raising statistical power concerns.

(3) Correlation with actual food intake is modest and sometimes less accurate than human observers.

(4) Sensitive to hoarding behavior, which can reduce detection accuracy and requires manual correction for misclassifications (e.g., tail movements, non-food noises). However, these limitations are discussed and not ignored.

Conclusion:

Overall, this is an exciting and impactful methodological advance that will likely be widely adopted in the field. I recommend minor revisions to clarify the limits of classifier generalizability, better contextualize the small-sample neuroscience findings as pilot data, and discuss future directions (e.g., real-time closed-loop applications).

Reviewer #3 (Public review):

Summary:

The manuscript provides detailed information on the construction of open-source systems to monitor ingestive behavior with low-cost equipment. Overall, this is a welcome addition to the arsenal of equipment that could be used to make measurements. The authors show interesting applications with data that reveal important neurophysiological properties of neurons in the lateral hypothalamus. The identification of previously unknown "meal-related" neurons in the LH highlights the utility of the device and is a novel insight that should spark further investigation on the LH. This manuscript and videos provide a wealth of useful information that should be a must-read for anyone in the ingestive behavior or hypothalamus fields.

A scholarly introduction to the history and utility of various ways feeding is measured in rodents is provided. One point - the microstructure of eating solid food - has been studied extensively (for one of many studies, see https://doi.org/10.1371/journal.pone.0246569 ). However, I agree that the crunchometer will allow for more people to access recordings during food intake and temporally lock consummatory behavior to neural activity.

Questions on results:

(1) It is unclear why 10% sucrose solution was used as a liquid instead of water, given that the study is focusing on the solid food source.

(2) It is unclear how essential the human verification is in the pipeline - results for Figure 1 keep referring to the verification as essential. Is that dispensable once the ML algorithms have been trained?

(3) The ability to extrapolate food quantity consumed is limited, with high variability. This limitation does not undercut the utility of the crunchometer, but should be highlighted as one of the parameters that are not suitable for this system. This limitation should be added to the limitations section.

(4) The ability to discriminate between gnawing and consummatory behavior is a strength (Figure 5), and these findings are important. However, it is unclear what can be made of mice that have 'gnawing' behavior in the fasted state (like in Figure 3). It seems they would need to be eliminated from the analysis with this tool?

(5) Why is there a post-semaglutide fed group and not a fasted group in Figure 4? It seems both would have been interesting, as one could expect an effect on feeding even 24h after semaglutide treatment. This would help parse the preference better because the animals eat such a small amount on semaglutide, that it is hard to compare to the fasted condition with saline treatment.

(6) The identification of 'meal-related' neurons in the LH is another strength of the manuscript. Although there is currently insufficient data, could similar recordings be used to give a neurophysiological definition of a 'meal' duration/size? Typically, these were somewhat arbitrarily defined behaviorally. Having a neural correlate to a 'meal' would be a powerful tool for understanding how meals are involved in overall caloric intake.

(7) The conclusion in the title of Figure 8 is premature, given the pilot nature and small number of neurons and mice sampled.

Conclusion:

Overall, this report on the Crunchometer is well done and provides a valuable tool for all who study food intake and the behaviors around food intake. Clarification or answers to the points above will only further the utility and understanding of the tool for the research community. I am excited to see the future utility of this tool in emerging research.

Yes - the oral history project incorporated the 5Rs. I particularly think the "reach" element was present. By recording the interviews and sharing them on a website - it allows students in class (and beyond) to hear these stories.

Yes - definitely. The oral history project helped to share stories of people and their lived experiences. This was an innovative way to share those stories with a broader audience. I enjoyed (and learned) from listening to some.

I really liked the Oral History project that was used in the HIST 150 class at JMU. I enjoyed listening to some of the stories and reminded me of NPR's Story Corps. I can see using something like this in my Population Health Determinants Class to capture student reflections from their service-learning experiences.

I’m thinking about how open pedagogy connects really naturally with some of the things I already do in my classes. In Population Health Determinants, for example, there’s a service-learning component where students engage with local organizations to understand social determinants of health. I can see opportunities for students to create open educational materials or community resources from those experiences—something that lives beyond the course and benefits others.

Similarly, in U.S. and Global Health Care Systems, my students do a podcast project comparing different countries’ health systems. That assignment already emphasizes collaboration and knowledge sharing, but I’d like to take it further by having them publish their work openly so future students (or even community members) can learn from it.

eLife Assessment

This paper is an important overview of the currently published literature on low-intensity focused ultrasound stimulation (TUS) in humans, providing a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects. The overall synthesis is convincing. The database proposed by the paper has the potential to become a key community resource if carefully curated and developed.

Reviewer #1 (Public review):

This paper is a relevant overview of the currently published literature on low-intensity focused ultrasound stimulation (TUS) in humans, with a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects.

The pool of papers to draw from is small, which is not surprising given the nascent technology. It seems, nevertheless, relevant to summarise the current field in the way done here, not least to mitigate and prevent some of the mistakes that other non-invasive brain stimulation techniques have suffered from, most notably the theory- and data free permutation of the parameter space.

A database summarising the literature and allowing for quantitative assessment of these studies is a key contribution of the paper. If curated well, it can become a valuable community resource.

Comments on revisions:

The paper is much improved. There remain a few caveats the authors may want to address.

I'm not going to dwell on this if the authors don't agree, but remain critical about the inclusion of TPS in the discussion. It's comparing apples and oranges, and unless there's a personal interest the authors have in TPS, it remains puzzling why it is included in the first place. As per my previous review, the literature on TPS, and especially the main example cited, has been highly criticised, including national patient and medical associations. A mere disclaimer that more work is needed isn't enough, in this reviewer's opinion - I simply don't understand why the authors go out on a limb here when the rest of the paper is done so well and thoroughly.

Author response:

The following is the authors’ response to the previous reviews

Reviewer #1 (Public review):

Summary:

This paper is a relevant overview of the currently published literature on lowintensity focused ultrasound stimulation (TUS) in humans, with a meta-analysis of this literature that explores which stimulation parameters might predict the directionality of the physiological stimulation effects.

The pool of papers to draw from is small, which is not surprising given the nascent technology. It seems nevertheless relevant to summarize the current field in the way done here, not least to mitigate and prevent some of the mistakes that other non-invasive brain stimulation techniques have suffered from, most notably the theory- and data-free permutation of the parameter space.

The meta-analysis concludes that there are, at best, weak trends toward specific parameters predicting the direction of the stimulation effects. The data have been incorporated into an open database that will ideally continue to be populated by the community and thereby become a helpful resource as the field moves forward.

Strengths:

The current state of human TUS is concisely and well summarized. The methods of the meta-analysis are appropriate. The database is a valuable resource.

We thank the reviewer for their positive assessment of the revised manuscript and the potential importance of the resource to the TUS community. 

Suggestions:

The paper remains lengthy and somewhat unfocused, to the detriment of readability. One can understand that the authors wish to include as much information as possible, but this reviewer is sceptical that this will aid the use of the databank, or help broaden the readership. For one, there is a good chunk of repetition throughout. The intro is also somewhat oscillating between TMS, tDCS and TUS. While the former two help contextualizing the issue, it doesn't seem necessary. In the section on clinical applications of TUs and possible outcomes of TUS, there's an imbalance of the content across examples. That's in part because of the difference in knowledge base but some sections could probably be shortened, eg stroke. In any case, the authors may want to consider whether it is worth making some additional effort in pruning the paper

We thank the reviewer for these suggestions. We have checked for redundancy and that the clinical review section is more balanced, although some of the sections have more TUS studies than others, therefore some imbalance is unavoidable. As some examples, we have condensed the “Stroke and neuroprotection in brain injury” section (lines 624-647). This helps to improve the clarity and readability of the manuscript.

The terms or concept of enhancement and suppression warrant a clearer definition and usage. In most cases, the authors refer to E/S of neural activity. Perhaps using terms such as "neural enhancement" etc helps distinguish these from eg behavioural or clinical effects. Crucially, how one maps onto the other is not clear. But in any case, a clear statement that the changes outlined on lines 277ff do not

We thank the reviewer for this point and agree that it is important to distinguish neural E/S, as we had intended, from behavioral effects. In the first instance and in several places we add ‘neural’ before enhancement/suppression.  Also see Lines 276-279: “Probable net neural enhancement versus suppression was characterised as follows. Note that our use of the terms enhancement and suppression refers exclusively to the increase or decrease of neural activity, respectively, as measured by, neurophysiological methods (EEG-ERPs, BOLD fMRI, etc.) and does not imply equivalent changes in behavioural responses” 

Please see also lines 108-116.

Re tb-TUS (lines 382ff), it is worth acknowledging here that independent replication is very limited (eg Bao et al 2024; Fong et al bioRxiv 2024) and seems to indicate rather different effects

We have updated this section by referencing Bao et al. and Fong et al., as examples of the limited independent replication of tbTUS results. Please see lines 392-396. “However, independent replication of these findings remains limited. For example, Bao, found reduced motor cortex excitability – measured as decreased TMS-MEP amplitude in M1 -- that lasted up to 30 minutes post-sonication (Bao et al., 2024). Whereas Fong reported no significant effects between tbTUS and sham conditions in M1 excitability (Fong et al., 2024).”

The comparison with TPS is troublesome. For one, that original study was incredibly poorly controlled and designed. Cherry-picking individual (badly conducted) proof-of-principle studies doesn't seem a great way to go about as one can find a match for any desired use or outcome. Moreover, other than the concept of "pulsed" stimulation, it is not clear why that original study would motivate the use of TUS in the way the authors propose; both types of stimulation act in very different ways (if TPS "acts" at all). But surely the cited TPS study does not "demonstrate the capability for TUS for pre-operative cognitive mapping". As an aside, why the authors feel the need to state the "potential for TPS... to enhance cognitive function" is unclear, but it is certainly a non-sequitur. This review feels quite strongly that simplistic analogies such as the one here are unnecessary and misleading, and don't reflect the thoughtful discussion of the rest of the paper. In the other clinical examples, the authors build their suggestions on other TUS studies, which seems more sensible.

This is an excellent point, and we have removed that statement replacing it with: “However, TPS effects studies remain highly limited and would require further study and comparison to effects with other TUS protocols.”. Please see lines 561-562. We thank the reviewer for the supportive comments on the rest of the review.

Notice that the way the equation solves that with the solved value. If 10^pH-pKA is greater than 1, the Conjugate Base or [A-] is in higher concentration than the lewis acid [HA], alternatively an increased HA concentration means a larger denominator --> 10^pH-pKA closer to 0; Conjugate Acid is higher in concentration.

In order to obtain a buffer, the complete neutralization of either the acid or the base must be avoided. Thus weak acids are used (and my guess no strong bases as well) because the complete dissociation of its ions would prevent the suspension of the pH.

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Why is it tending so much towards yes (before becoming uncertain)? Maybe the model tends to favor positive responses, as mentioned earlier being the case for another model. But I hypothesized there to be relation hallucinations with the yes and no flipped compared to the Figure. Since the plot is averaged, are those cases so rare that they do not get represented after averaging anymore?

Why does it become uncertain only once the last layers are reached? They call it sharp change in Figure 8b.

Should be "previous", right?

That assumes that the token that has high probability in the intermediate layer (e.g. yes in Figure 7) is the correct answer.

XII Regio

this needs to be verified.

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how?

This needs to be double-checked as well.

This amount isn’t correct. We need to find a way to get the exact or a near-exact figure for that.

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eLife Assessment

This important study addresses a topic that is frequently discussed in the literature but is under-assessed, namely correlations among genome size, repeat content, and pathogenicity in fungi. Contrary to previous assertions, the authors found that repeat content is not associated with pathogenicity. Rather, pathogenic lifestyle was found to be better explained by the number of protein-coding genes, with other genomic features associated with insect association status. The results are considered solid, although there remain concerns about potential biases stemming from the underlying data quality of the analyzed genomes.

Reviewer #1 (Public review):

Summary:

The manuscript "Lifestyles shape genome size and gene content in fungal pathogens" by Fijarczyk et al. presents a comprehensive analyses of a large dataset of fungal genomes to investigate what genomic features correlate with pathogenicity and insect associations. The authors focus on a single class of fungi, due to the diversity of life styles and availability of genomes. They analyze a set of 12 genomic features for correlations with either pathogenicity or insect association and find that, contrary to previous assertions, repeat content does not associate with pathogenicity. They discover that the number of protein coding genes, including total size of non-repetitive DNA does correlate with pathogenicity. However, unique features are associated to insect associations. This work represents an important contribution to the attempts to understand what features of genomic architecture impact the evolution of pathogenicity in fungi.

Strengths:

The statistical methods appear to be properly employed and analyses thoroughly conducted. The size of the dataset is impressive and likely makes the conclusions robust. The manuscript is well written and the information, while dense, is generally presented in a clear manner.

Weaknesses:

My main concerns all involve the genomic data, how they were annotated, and the biases this could impart to the downstream analyses. The three main features I'm concerned with are sequencing technology, gene annotation, and repeat annotation. The authors have done an excellent investigation into these issues, but these show concerning trends, and my concerns are not as assuaged as the authors.

The collection of genomes is diverse and includes assemblies generated from multiple sequencing technologies including both short- and long-read technologies. From the number of scaffolds its clear that the quality of the assemblies varies dramatically, even within categories of long- and short-read. This is going to impact many of the values important for this study, as the authors show.

I have considerable worries that the gene annotation methods could impart biases that significantly effect the main conclusions. Only 5 reference training sets were used for the Sordariomycetes and these are unequally distributed across the phylogeny. Augusts obviously performed less than ideally, as the authors observe in their extended analysis. While the authors are not concerned about phylogenetic distance from the training species, due to prevailing trends, I am not as convinced. In figure S12, the Augustus features appear to have considerably more variation in values for the H2 set and possible the microascales. It is unclear how this would effect the conclusions in this study.

Unfortunately, the genomes available from NCBI will vary greatly in the quality of their repeat masking. While some will have been masked using custom libraries generated with software like Repeatmodeler, others will probably have been masked with public databases like repbase. As public databases are again biased towards certain species (Fusarium is well represented in repbase for example), this could have significant impacts on estimating repeat content. Additionally, even custom libraries can be problematic as some software (like RepeatModeler) will included multicopy host genes leading to bona fide genes being masked if proper filtering is not employed. A more consistent repeat masking pipeline would add to the robustness of the conclusions. The authors show that there is a significant bias in their set.

To a lesser degree I wonder what impact the use of representative genomes for a species has on the analyses. Some species vary greatly in genome size, repeat content and architecture among strains. I understand that it is difficult to address in this type of analysis, but it could be discussed.

Reviewer #2 (Public review):

Summary:

In this paper, the authors report on the genomic correlates of the transition to the pathogenic lifestyle in Sordariomycetes. The pathogenic lifestyle was found to be better explained by the number of genes, and in particular effectors and tRNAs, but this was modulated by the type of interacting host (insect or not insect) and the ability to be vectored by insects.

Strengths:

The main strengths of this study lie in (i) the size of the dataset, and the potentially high number of lifestyle transitions in Sordariomycetes, (ii) the quality of the analyses and the quality of the presentation of the results, (iii) the importance of the authors' findings.

Weaknesses:

The weakness is a common issue in most comparative genomics studies in fungi, but it remains important and valid to highlight it. Defining lifestyles is complex because many fungi go through different lifestyles during their life cycles (for instance, symbiotic phases interspersed with saprotrophic phases). In many fungi, the lifestyle referenced in the literature is merely the sampling substrate (such as wood or dung), which does not necessarily mean that this substrate is a key part of the life cycle. The authors discuss this issue, but they do not eliminate the underlying uncertainties.

Author response:

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

Reviewer #1 (Public review):

Summary:

The manuscript "Lifestyles shape genome size and gene content in fungal pathogens" by Fijarczyk et al. presents a comprehensive analysis of a large dataset of fungal genomes to investigate what genomic features correlate with pathogenicity and insect associations. The authors focus on a single class of fungi, due to the diversity of lifestyles and availability of genomes. They analyze a set of 12 genomic features for correlations with either pathogenicity or insect association and find that, contrary to previous assertions, repeat content does not associate with pathogenicity. They discover that the number of proteincoding genes, including the total size of non-repetitive DNA does correlate with pathogenicity. However, unique features are associated with insect associations. This work represents an important contribution to the attempts to understand what features of genomic architecture impact the evolution of pathogenicity in fungi.

Strengths:

The statistical methods appear to be properly employed and analyses thoroughly conducted. The manuscript is well written and the information, while dense, is generally presented in a clear manner.

Weaknesses:

My main concerns all involve the genomic data, how they were annotated, and the biases this could impart to the downstream analyses. The three main features I'm concerned with are sequencing technology, gene annotation, and repeat annotation.

We thank the reviewer for all the comments. We are aware that the genome assemblies are of heterogeneous quality since they come from many sources. The goal of this study was to make the best use of the existing assemblies, with the assumption that noise introduced by the heterogeneity of sequencing methods should be overcome by the robustness of evolutionary trends and the breadth and number of analyzed assemblies. Therefore, at worst, we would expect a decrease in the power to detect existing trends. It is important to note that the only way to confidently remove all potential biases would be to sequence and analyze all species in the same way; this would require a complete study and is beyond the scope of the work presented here. Nevertheless some biases could affect the results in a negative way, eg. is if they affect fungal lifestyles differently. We therefore made an attempt to explore the impact of sequencing technology, gene and repeat annotation approach among genomes of different fungal lifestyles. Details are described in Supplementary Results and below. Overall, even though the assembly size and annotations conducted with Augustus can sometimes vary compared to annotations from other resources, such as JGI Mycocosm, we do not observe a bias associated with fungal lifestyles. Comparison of annotations conducted with Augustus and JGI Mycocosm dataset revealed variation in gene-related features that reflect biological differences rather than issues with annotation.  

The collection of genomes is diverse and includes assemblies generated from multiple sequencing technologies including both short- and long-read technologies. Not only has the impact of the sequencing method not been evaluated, but the technology is not even listed in Table S1. From the number of scaffolds it is clear that the quality of the assemblies varies dramatically. This is going to impact many of the values important for this study, including genome size, repeat content, and gene number.

We have now added sequencing technology in Table S1 as it was reported in NCBI. We evaluated the impact of long-read (Nanopore, PacBio, Sanger) vs short-read assemblies in Supplementary Results. In short, the proportion of different lifestyles (pathogenic vs. nonpathogenic, IA vs non-IA) were the same for short- and long-read assemblies. Indeed, longread assemblies were longer, had a higher fraction of repeats and less genes on average, but the differences between pathogenic vs. non-pathogenic (or IA vs non-IA) species were in the same direction for two sequencing technologies and in line with our results. There were some discrepancies, eg. mean intron length was longer for pathogens with long-read assemblies, but slightly shorter on average for short-read assemblies (and to lesser extent GC and pseudo tRNA count), which could explain weaker or mixed results in our study for these features.

Additionally, since some filtering was employed for small contigs, this could also bias the results.

The reason behind setting the lower contig length threshold was the fact that assemblies submitted to NCBI have varying lower-length thresholds. This is because assemblers do not output contigs above a certain length, and this threshold can be manipulated by the user. Setting a common min contig length was meant to remove this variation, knowing that any length cut-off will have a larger effect on short-read based assemblies than long-read-based assemblies. Notably, genome assemblies of corresponding species in JGI Mycocosm have a minimum contig length of 865 bp, not much lower than in our dataset. Importantly, in a response to a comment of previous reviewer, repeat content was recalculated on raw assembly lengths instead of on filtered assembly length. 

I have considerable worries that the gene annotation methods could impart biases that significantly affect the main conclusions. Only 5 reference training sets were used for the Sordariomycetes and these are unequally distributed across the phylogeny. Augusts obviously performed less than ideally, as the authors reported that it under-annotated the genomes by 10%. I suspect it will have performed worse with increasing phylogenetic distance from the reference genomes. None of the species used for training were insectassociated, except for those generated by the authors for this study. As this feature was used to split the data it could impact the results. Some major results rely explicitly on having good gene annotations, like exon length, adding to these concerns. Looking manually at Table S1 at Ophiostoma, it does seem to be a general trend that the genomes annotated with Magnaporthe grisea have shorter exons than those annotated with H294. I also wonder if many of the trends evident in Figure 5 are also the result of these biases. Clades H1 and G each contain a species used in the training and have an increase in genes for example.

We have applied 6 different reference training sets (instead of one) precisely to address the problem of increasing phylogenetic distance of annotated species. To further investigate the impact of chosen species for training, we plotted five gene features (number of genes, number of introns, intron length, exon length, fraction of genes with introns) as a function of   branch length distance from the species (or genus) used as a training set for annotation. We don’t see systematic biases across different training sets. However,  trends are very clear for clades annotated with fusarium. This set of species includes Hypocreales and Microascales, which is indeed unfortunate since Microascales is an IA group and at the same time the most distant from the fusarium genus in this set. To clarify if this trend is related to annotation bias or a biological trend, we compared gene annotations with those of Mycocosm, between Hypocreales Fusarium species, Hypocreales non-Fusarium species, and Microascales, and we observe exactly the same trends in all gene features. 

Similarly, among species that were annotated with magnaporthe_grisea, Ophiostomatales (another IA group) are among the most distant from the training set species. Here, however, another order, Diaporthales, is similarly distant, yet the two orders display different feature ranges. In terms of exon length, top 2 species in this training set include Ophiostoma, and they reach similar exon length as the Ophiostoma species annotated using H294 as a training set. In summary, it is possible that the choice of annotation species has some effect on feature values; however, in this dataset, these biases are likely mitigated by biological differences among lifestyles and clades. 

Unfortunately, the genomes available from NCBI will vary greatly in the quality of their repeat masking. While some will have been masked using custom libraries generated with software like Repeatmodeler, others will probably have been masked with public databases like repbase. As public databases are again biased towards certain species (Fusarium is well represented in repbase for example), this could have significant impacts on estimating repeat content. Additionally, even custom libraries can be problematic as some software (like RepeatModeler) will include multicopy host genes leading to bona fide genes being masked if proper filtering is not employed. A more consistent repeat masking pipeline would add to the robustness of the conclusions.

We have searched for the same species in JGI Mycocosm and were able to retrieve 58 genome assemblies with matching species, with 19 of them belonging to the same strain as in our dataset. Overall we found no differences in genome assembly length. Interestingly, repeat content was slightly higher for NCBI genome assemblies compared to JGI Mycocosm assemblies, perhaps due to masking of host multicopy genes, as the reviewer mentioned. By comparing pathogenic and non-pathogenic species for the same 19 strains, we observe that JGI Mycocosm annotates fewer repeats in pathogenic species than Augustus annotations (but trends are similar when taking into account 58 matching species). Given a small number of samples, it is hard to draw any strong conclusions; however, the differences that we see are in favor of our general results showing no (or negative) correlation of repeat content with pathogenicity. 

To a lesser degree, I wonder what impact the use of representative genomes for a species has on the analyses. Some species vary greatly in genome size, repeat content, and architecture among strains. I understand that it is difficult to address in this type of analysis, but it could be discussed.

In our case the use of protein sequences could underestimate divergence between closely related strains from the same species. We also excluded strains of the same species to avoid overrepresentation of closely related strains with similar lifestyle traits. We agree that some changes in the genome architecture can occur very rapidly, even at the species level, though analyzing emergence of eg. pathogenicity at the population level would require a slightly different approach which accounts for population-level processes. 

Reviewer #2 (Public review):

Summary:

In this paper, the authors report on the genomic correlates of the transition to the pathogenic lifestyle in Sordariomycetes. The pathogenic lifestyle was found to be better explained by the number of genes, and in particular effectors and tRNAs, but this was modulated by the type of interacting host (insect or not insect) and the ability to be vectored by insects.

Strengths:

The main strength of this study lies in the size of the dataset, and the potentially high number of lifestyle transitions in Sordariomycetes.

Weaknesses:

The main strength of the study is not the clarity of the conclusions.

(1) This is due firstly to the presentation of the hypotheses. The introduction is poorly structured and contradictory in some places. It is also incomplete since, for example, fungusinsect associations are not mentioned in the introduction even though they are explicitly considered in the analyses.

We thank the reviewer for pointing this out. We strived to address all comments and suggestions of the reviewer to clarify the message and remove the contradictions. We also added information about why we included insect-association trait in our analysis. 

(2) The lack of clarity also stems from certain biases that are challenging to control in microbial comparative genomics. Indeed, defining lifestyles is complicated because many fungi exhibit different lifestyles throughout their life cycles (for instance, symbiotic phases interspersed with saprotrophic phases). In numerous fungi, the lifestyle referenced in the literature is merely the sampling substrate (such as wood or dung), which doesn't mean that this substrate is a crucial aspect of the life cycle. This issue is discussed by the authors, but they do not eliminate the underlying uncertainties.

We agree with the reviewer that lack of certainty in the lifestyle or range of possible lifestyles of studied species is a weakness in this analysis. We are limited by the information available in the literature. We hope that our study will increase interest in collecting such data in the future.

Reviewer #3 (Public review):

Summary:

This important study combines comparative genomics with other validation methods to identify the factors that mediate genome size evolution in Sordariomycetes fungi and their relationship with lifestyle. The study provides insights into genome architecture traits in this Ascomycete group, finding that, rather than transposons, the size of their genomes is often influenced by gene gain and loss. With an excellent dataset and robust statistical support, this work contributes valuable insights into genome size evolution in Sordariomycetes, a topic of interest to both the biological and bioinformatics communities.

Strengths:

This study is complete and well-structured.

Bioinformatics analysis is always backed by good sampling and statistical methods. Also, the graphic part is intuitive and complementary to the text.

Weaknesses:

The work is great in general, I just had issues with the Figure 1B interpretation.

I struggled a bit to find the correspondence between this sentence: "Most genomic features were correlated with genome size and with each other, with the strongest positive correlation observed between the size of the assembly excluding repeats and the number of genes (Figure 1B)." and the Figure 1B. Perhaps highlighting the key p values in the figure could help.

We thank the reviewer for pointing out this sentence. Perhaps the misunderstanding comes from the fact that in this sentence one variable is missing. The correct version should be “Most genomic features were correlated with genome size and with each other, with the strongest positive correlation observed between the genome size, the genome size excluding repeats and the number of genes (Figure 1B)”. Also, the variable names now correspond better to those shown on the figure.

Reviewer #1 (Recommendations for the authors):

The authors have clearly done a lot of good work, and I think this study is worthwhile. I understand that my concerns about the underlying data could necessitate rerunning the entire analysis with better gene models, but there may be another option. JGI has a fairly standard pipeline for gene and repeat annotation. Their gene predictions are based on RNA data from the sequenced strain and should be quite good in general. One could either compare the annotations from this manuscript to those in mycocosm for genomes that are identical and see if there are systematic biases, or rerun some analyses on a subset of genomes from mycocosm. Indeed, it's possible that the large dataset used here compensates for the above concerns, but without some attempt to evaluate these issues, it's difficult to have confidence in the results.

We very appreciate the positive reception of our manuscript. Following the reviewer’s comments we have investigated gene annotations in comparison with those of JGI Mycocosm, even though only 58 species were matching and only 19 of them were from the same strain. This dataset is not representative of the Sordariomycetes diversity (most species come from one clade), therefore will not reflect the results we obtained in this study. To note, the reason for not choosing JGI Mycocosm in the first place, was the poor representation of the insect-associated species, which we found key in this study. In general, we found that assembly lengths were nearly identical, number of genes was higher, and the repeat content was lower for the JGI Mycocosm dataset. When comparing different lifestyles (in particular pathogens vs. non-pathogens), we found the same differences for our and JGI Mycocosm annotations, with one exception being the repeat content. In the small subset (19 same-strain assemblies), our dataset showed the same level of repeats between the two lifestyles, whereas JGI Mycocosm showed lower repeat content for pathogens (but notably for all 58 species, the trend was same for our and JGI Mycocosm annotations). None of these observations are in conflict with our results where we find no or negative association of repeat content with pathogens. 

The figures are very information-dense. While I accept that this is somewhat of a necessity for presenting this type of study, if the authors could summarize the important information in easier-to-interpret plots, that could help improve readability.

We put a lot of effort into showing these complicated results in as approachable manner as possible. Given that other reviewers find them intuitive we decided to keep most of them as they are. To add more clarification, we added one supplementary figure showing distributions of genomic traits across lifestyles. Moreover, in Figure 5, a phylogenetic tree was added with position of selected clades, as well as a scatterplot showing distributions of mean values for genome size and number of genes for those clades. If the reviewer has any specific suggestions on what to improve and in which figure, we’re happy to consider it. 

Reviewer #2 (Recommendations for the authors):

I have no major comments on the analyses, which have already been extensively revised. My major criticism is the presentation of the background, which is very insufficient to understand the importance or relevance of the results presented fully.

Lines are not numbered, unfortunately, which will not help the reading of my review.

(1) The introduction could better present the background and hypotheses:

(a) After reading the introduction, I still didn't have a clear understanding of the specific 'genome features' the study focuses on. The introduction fails to clearly outline the current knowledge about the genetic basis of the pathogenic lifestyle: What is known, what remains unknown, what constitutes a correlation, and what has been demonstrated? This lack of clarity makes reading difficult.

We thank the reviewer for pointing this out. We have now included in the introduction a list of genomic traits we focus on. We also tried to be more precise about demonstrated pathogenic traits and other correlated traits in the introduction. 

(b) Page 3. « Various features of the genome have been implicated in the evolution of the pathogenic lifestyle. » The cited studies did not genuinely link genome features to lifestyle, so the authors can't use « implicated in » - correlation does not imply causation.

This sentence also somehow contradicts the one at the end of the paragraph: « we still have limited knowledge of which genomic features are specific to pathogenic lifestyle

We thank the reviewer for this comment. We added a phrase “correlated with or implicated in” and changed the last sentence of the paragraph into “Yet we still have limited knowledge of how important and frequent different genomic processes are in the evolution of pathogenicity across phylogenetically distinct groups of fungi and whether we can use genomic signatures left by some of these processes as predictors of pathogenic state.”.

(c) Page 3: « Fungal pathogen genomes, and in particular fungal plant pathogen genomes have been often linked to large sizes with expansions of TEs, and a unique presence of a compartmentalized genome with fast and slow evolving regions or chromosomes » Do the authors really need to say « often »? Do they really know how often?

We removed “often”.

(d) Such accessory genomic compartments were shown to facilitate the fast evolution of effectors (Dong, Raffaele, and Kamoun 2015) ». The cited paper doesn't « show » that genomic compartments facilitate the fast evolution of effectors. It's just an observation that there might be a correlation. It's an opinion piece, not a research manuscript.

We changed the sentence to “Such accessory genomic compartments could facilitate the fast evolution of effectors”.

(e) even though such architecture can facilitate pathogen evolution, it is currently recognized more as a side effect of a species evolutionary history rather than a pathogenicity related trait ». This sentence somehow contradicts the following one: « Such accessory genomic compartments were shown to facilitate the fast evolution of effectors".

Here we wanted to point out that even though accessory genome compartments and TE expansions can facilitate pathogen evolution the origin of such architecture is not linked to pathogenicity. We reformulated the sentence to “Even though such architecture can facilitate pathogen evolution, it is currently recognized that its origin is more likely a side effect of a species evolutionary history rather than being caused by pathogenicity”.

(f) As the number of genes is strongly correlated with fungal genome size (Stajich 2017), such expansions could be a major contributor to fungal genome size. » This sentence suggests that pathogens might have bigger genomes because they have more effectors. This is contradictory to the sentence right after « At the end of the spectrum are the endoparasites Microsporidia, which have among the smallest known fungal genomes ».

The authors state that pathogens have bigger genomes and then they take an example of a pathogen that has a minimal genome. I know it's probably because they lost genes following the transition to endoparasitism and not related to their capacity to cause disease. I just want to point out that their writing could be more precise. I invite authors to think of young scholars who are new to the field of fungal evolutionary genomics.

We thank the reviewer for prompting us to clarify the text. We rewrote this short extract as follows “Notably, not all pathogenic species experience genome or gene expansions, or show compartmentalized genome architecture. While gene family expansions are important for some pathogens, the contrary can be observed in others, such as Microsporidia. Due to transition to obligatory intracellular lifestyle these fungi show signatures of strong genome contractions and reduced gene repertoire (Katinka et al. 2001) without compromising their ability to induce disease in the host. This raises questions about universal genomic mechanisms of transition to pathogenic state.”

(g) I find it strange that the authors do not cite - and do not present the major results of two other studies that use the same type of approach and ask the same type of question in Sordariomycetes, although not focusing on pathogenicity:

Hensen et al.: https://pubmed.ncbi.nlm.nih.gov/37820761/

Shen et al.: https://pubmed.ncbi.nlm.nih.gov/33148650/

We thank the reviewer for pointing out this omission. We now added more information in the introduction to highlight the importance of the phylogenetic context in studying genome evolution as demonstrated by these studies. The following part was added to introduction:  “Other phylogenomic studies investigating a wide range of Ascomycete species, while not explicitly focusing on the neutral evolution hypothesis, have found strong phylogenetic signals in genome evolution, reflected in distinct genome characteristics (e.g., genome size, gene number, intron number, repeat content) across lineages or families (Shen et al. 2020; Hensen et al. 2023). Variation in genome size has been shown to correlate with the activity of the repeat-induced point mutation (RIP) mechanism (Hensen et al. 2023; Badet and Croll 2025), by which repeated DNA is targeted and mutated. RIP can potentially lead to a slower rate of emergence of new genes via duplication (Galagan et al. 2003), and hinder TE proliferation limiting genome size expansion (Badet and Croll 2025). Variation in genome dynamics across lineages has also been suggested to result from environmental context and lifestyle strategies (Shen et al. 2020), with Saccharomycotina yeast fungi showing reductive genome evolution and Pezizomycotina filamentous fungi exhibiting frequent gene family expansions. Given the strong impact of phylogenetic membership,  demographic history (Ne) and host-specific adaptations of pathogens on their genomes, we reasoned that further examination of genomic sequences in groups of species with various lifestyles can generate predictions regarding the architecture of pathogenic genomes.”

(h) Genome defense mechanisms against repeated elements, such as RIP, are not mentioned while they could have a major impact on genome size (Hensen et al cited above; Badet and Croll https://www.biorxiv.org/content/10.1101/2025.01.10.632494v1.full).

This citation is added in the text above.

(i) Should the reader assume that the genome features to be examined are those mentioned in the first paragraph or those in the penultimate one?

In the last paragraph of the introduction we included the complete list of investigated genomic traits.

(j) The insect-associated lifestyle is mentioned only in the research questions on page 4, but not earlier in the introduction. Why should we care about insect-associated fungi?

We apologize for this omission. We added a sentence explaining how neutral evolution hypotheses can explain patterns of genome evolution in endoparasites and species with specialized vectors (traits present in insect-associated species) and added a sentence in the last paragraph that this is the reason why we have selected this trait for analysis.  

(2) Why use concatenation to infer phylogeny?

(a) Kapli et al. https://pubmed.ncbi.nlm.nih.gov/32424311/ « Analyses of both simulated and empirical data suggest that full likelihood methods are superior to the approximate coalescent methods and to concatenation »

(b) It also seems that a homogeneous model was used, and not a partitioned model, while the latter are more powerful. Why?

We thank the reviewer for the comment. When we were reconstructing the phylogenetic tree  we were not aware of the publication and we followed common practices from literature for phylogenetic tree reconstruction even though currently they are not regarded as most optimal. In fact, in the first round of submission, we have included both concatenation as well as a multispecies coalescent method based on 1000 busco sequences and a concatenation method with different partitions for 250 busco sequences. All three methods produced similar topologies. Since the results were concordant, we chose to omit these analyses from the manuscript to streamline the presentation and focus on the most important results.

(3) Other comments:

Is there a table listing lifestyles?

Yes, lifestyles (pathogenicity and insect-association) are listed in Supplementary Table S1. 

(4) Summary:

(a) seemingly similar pathogens »: meaning unclear; on what basis are they similar? why « seemingly »?

We removed “seemingly” from the sentence.

(b) Page 4: what's the difference between genome feature and genome trait?

There is no difference. We apologize for the confusion. We changed “feature” to “trait” whenever it refers to the specific 13 genomic traits analyzed in this study.

(c) Page 22: Braker, not Breaker

corrected

What do the authors mean when they write that genes were predicted with Augustus and Braker? Do they mean that the two sets of gene models were combined? Gene counts are based on Augustus (P24): why not Braker?

We only meant here that gene annotation was performed using Braker pipeline, which uses a particular version of Augustus. We corrected the sentence.

(d) Figure 2B and 2C:

'Undetermined sign' or 'Positive/Negative' would be better than « YES » or it's just impossible to understand the figure without reading the legend.

We changed “YES” to “UNDETERMINED SIGN” as suggested by the reviewer.

This sentence captures the essence of the transactional model by showing how communication is a collaborative, continuous process where meaning is co-created rather than simply transmitted

this sentence underscores how personal and cultural differences shape interpretation, showing that effective communication depends on understanding others’ perspectives and shared experiences

This highlights how external or internal distractions can disrupt understanding, reminding readers that effective communication depends not only on clear messages but also on managing barriers that alter meaning

self awareness occurs after communication failure

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eLife Assessment

This valuable study uses a sophisticated array of techniques to investigate the mechanisms through which the chordotonal receptors in the locust ear (Müller's organ) sense auditory signals. Ultrastructural reconstruction of the sensory organ provides convincing evidence of the organization of the scolopidial structure that wraps the sensory neuron cilium. However, the recordings of sound-evoked motion and electrophysiological activity from the chordotonal sensory neurons provide incomplete evidence for the proposed axial stretch model of mechanotransduction.

Reviewer #1 (Public review):

Chaiyasitdhi et al. set out to investigate the detailed ultrastructure of the scolopidia in the locust Müller's organ, the geometry of the forces delivered to these scolopidia during natural stimulation, and the direction of forces that are most effective at eliciting transduction currents. To study the ultrastructure, they used the FIB-SEM technique, to study the geometry of natural stimulation, they used OCT vibrometry and high-speed light microscopy, and to study transduction currents, they used patch clamp physiology.

Strengths:

I believe that the ultrastructural description of the locust scolopidium is excellent and the first of its kind in any insect system. In particular, the finding of the bend in the dendritic cilium and the position of the ciliary dilation are interesting, and it would be interesting to see whether these are common features within the huge diversity of insect chordotonal organs.

I believe the use of OCT to measure organ movements is a significant strength of this paper; however, using ex vivo preparations undermines any conclusions drawn about the system's in vivo mechanics.

The choice of Group III scolopidia is also good. Research on the mechanics of locust tympana has shown that travelling waves are formed on the tympanum and waves of different frequencies show highest amplitudes at different positions on the tympanum, and therefore also on different groups of scolopidia within the Müller's organ (Windmill et al, 2005; 2008, and Malkin et al, 2013). The lowest frequency modal waves (F0) observed by Windmill et al 2008 were at about 4.4 kHz, which are slightly higher than the ~3 kHz frequencies studied in this paper but do show large deflections where these group III scolopidia attach at the styliform body (Windmill et al, 2005).

This should be mentioned in the paper since the electrophysiology justification to use group III neurons is less convincing, given that Jacobs et al 1999 clearly point out that group III neurons are very variable and some of them are tuned much higher to 10 kHz, and others even higher to 20-30 kHz.

Weaknesses:

Specifically, it is understandable that the authors decided to use excised ears for the light microscopy, where Müller's organ would not be accessible in situ. However, it is very likely that excision will change the system's mechanics, especially since any tension or support to Müller's organ will be ablated. OCT enables in vivo measurements in fully undissected systems (Mhatre et al, Biorxiv, 2021) or in systems with minimal dissection where the mechanics have not been compromised (Vavakou et al, 2021). The choice to entirely dissect out the membrane is difficult to understand here.

My main concern with this paper, however, is the use of light microscopy very close to the Nyquist limit to study scolopidial motion, and the fact that the OCT data contradict and do not match the light microscopy data.

The light microscopy data is collected at ~8 kHz, and hence the Nyquist limit is ~4 kHz. It is possible to measure frequencies reliably this close to the limit, but the amplitude of motion is quite likely to be underestimated, given that the technique only provides 2 sample points per cycle at 4 kHz and approximately 2.66 sample points at 3 kHz. At that temporal resolution, the samples are much more likely to miss the peak of the wave than not, and therefore, amplitudes will be misestimated. A much more reasonable sample rate for amplitude estimation is generally about 10 samples per cycle. I do not believe the data from the microscopy is reliable for what the authors wish to use them for.

Using the light microscopy data, the authors claim that the strains experienced by the group III scolopidia at 3 kHz are greater along the AP axis than the ML axis (Figure 4). However, this is contradicted by the OCT data, which show very low strain along the AP axis (black traces) at and around 3 kHz (Figure 3c and extended data Figure 2f) and show some movement along the ML axis (red traces, same figures). The phase at low amplitudes of motion cannot be considered very reliable either, and hence phase variations at these frequencies in the OCT cannot be considered reliable indicators of AP motion; hence, I'm unclear whether the vector difference in the OCT is a reliable indicator of movement.

The OCT data are significantly more reliable as they are acquired at an appropriate sampling rate of 90 kHz. The authors do not mention what microphone they use to monitor or calibrate their sound field and phase measurements in OCT, but I presume this was done since it is the norm. Thus, the OCT data show that the movement within the Müller's organ is complex, probably traces an ellipse at some frequencies as observed in bushcrickets (Vavkou et al, 2021) and also thought to be the case in tree crickets based on the known attachment points of the TO (Mhatre et al, 2021). The OCT data shows relatively low AP motion at frequencies near 3 kHz, and higher ML motion, which contradicts the less reliable light microscopy data. Given that the locust membrane shows peaks in motion at ~4.5 kHz, ~11 kHz, and also at ~20 kHz (Windmill et al, 2008), I am surprised that the authors limited their OCT experiments and analyses to 5 kHz.

In summary for this section, I am not convinced of the conclusion drawn by the authors that group III scolopidia receive significantly higher stimulation along the AP axis in their native configuration, if indeed they were studied in the appropriate force regime (altered due to excision).

In the scolopidial patch clamp data, the authors study transduction currents in response to steady state stimulation along the AP axis and the ML axis. The responses to steady state and periodic forces may well be different, and the authors do not offer us a way to clearly relate the two and therefore, to interpret the data.

In addition, both stimulation types, along the AP axis and the ML, elicit clear transduction responses. Stimulation along the AP axis might be slightly higher, but there is over 40% variation around the mean in one case (pull: 26.22 {plus minus} 10.99 pA) and close to 80% variation in the other (push: 10.96 {plus minus} 8.59 pA). These data are indeed from a very high displacement range (2000 nm), which is very high compared to the native displacement levels, which are in the 1-10 nm range.

The factor change from sample to sample is not reported, and is small even overall. The statistical analyses of these data are not clearly reported, and I don't see the results of the overall ANOVA in the results section. I also find the dip in the reported transduction currents between 10 and 100 nm quite odd (Figure 5 j-m) and would like to know what the authors' interpretation of this behaviour is. It seems to me that those currents increase continuously linearly after ~50-100 nm and that the data below that range are in the noise. Thus, the transduction currents observed at the relevant displacement range (1-10 nm) may not actually be reliable. How were these small displacements achieved, and how closely were the actual levels monitored? Is it possible to reliably deliver 1-10 nm displacements using a micromanipulator?

What is clear, despite the difficulty in interpreting this data, is that both AP and ML stimulation evoke transduction currents, and their relative differences are small. Additionally, in Müller's organ itself, in the excised organ, the scolopidia are stimulated along both axes. Thus, in my opinion, it is not possible to say that axial stretch along the cilium is 'the key mechanical input that activates mechano-electrical transduction'.

Reviewer #2 (Public review):

Summary of strengths and weaknesses:

Using several techniques-FIB-SEM, OCT, high-speed light microscopy, and electrophysiology-Chaiyasitdhi et al. provide evidence that chordotonal receptors in the locust ear (Müller's organ) sense the stretch of the scolapale cell, primarily of its cilium. Careful measurements certainly show cell stretch, albeit with some inconsistencies regarding best frequencies and amplitudes. The weakest argument concerns the electrophysiological recordings, because the authors do not show directly that the stimulus stretches the cells. If this latter point can be clarified, then our confidence that ciliary stretch is the proximal stimulus for mechanotransduction will be increased. This conclusion will not come as a surprise for workers in the field, as the chordotonal organ is known as a stretch-receptor organ (e.g., Wikipedia). But it is a useful contribution to the field and allows the authors to suggest transduction mechanisms whereby ciliary stretch is transduced into channel opening.

Reviewer #3 (Public review):

Summary:

The paper 'A stretching mechanism evokes mechano-electrical transduction in auditory chordotonal neurons' by Chaiyasitdhi et al. presents a study that aims to address the mechanical model for scolopidia in Schistocerca gregaria Müller's organ, the basic mechanosensory units in insect chordotonal organs. The authors combine high-resolution ultrastructural analysis (FIB-SEM), sound-evoked motion tracking (OCT and high-speed light microscopy), and electrophysiological recordings of transduction currents during direct mechanical stimulation of individual scolopidia. They conclude that axial stretching along the ciliary axis is an adequate mechanical stimulus for activating mechanotransduction channels.

Strengths/Highlights:

(1) The 3D FIB-SEM reconstruction provides high resolution of scolopidial architecture, including the newly described "scolopale lid" and the full extent of the cilium.

(2) High-speed microscopy clearly demonstrates axial stretch as the dominant motion component in the auditory receptors, which confirms a long-standing question of what the actual motion of a stretch receptor is upon auditory stimulation.

(3) Patch-clamp recordings directly link mechanical stretch to transduction currents, a major advance over previous indirect models.

Weaknesses/Limitations:

(1) The text is conceptually unclear or written in an unclear manner in some places, for example, when using the proposed model to explain the sensitivity of Nanchung-Inactive in the discussion.

(2) The proposed mechanistic models (direct-stretch, stretch-compression, stretch-deformation, stretch-tilt) are compelling but remain speculative without direct molecular or biophysical validation. For example, examining whether the organ is pre-stretched and identifying the mechanical components of cells (tissues), such as the extracellular matrix and cytoskeleton, would help establish the mechanical model and strengthen the conclusion.

(3) To some extent, the weaknesses of the paper are part of its strengths and vice versa. For example, the direct push/pull and up/down stimulations are a great experimental advance to approach an answer to the question of how the underlying cellular components are deformed and how the underlying ion channels are forced. However, as the authors clearly state, neither of their stimulations can limit all forces to only one direction, and both orthogonal forces evoke responses in the neurons. The question of which of the two orthogonal forces 'causes' the response cannot be answered with these experiments and has not been answered by this manuscript. But the study has brought the field a considerable step closer to answering the question. The answer, however, might be that both longitudinal ('stretch') and perpendicular ('compression') forces act together to open the ion channels and that both dendritic extension via stretch and bending can provide forces for ion channel gating. The current paper has identified major components (longitudinal stretch components) for the neurons they analysed, but these will surely have been chosen according to their accessibility, and as such, the variety of mechanical responses in Müller's organ might be greater. In light of these considerations, the authors might acknowledge such uncertainties more clearly in their paper. The paper is an impressive methodological progress and breakthrough, but it simply does not "demonstrate that axial stretch along the cilium is the adequate stimulus or the key mechanical input that activates mechano-electrical transduction" as the authors write at the start of their discussion. They do show that axial stretch dominates for the neurons they looked at, which is important information. The same applies to the end of the discussion: The authors write, "This relative motion within the organ then drives an axial stretch of the scolopidium, which in turn evokes the mechano-electrical transduction current." Reading the manuscript, the certainty and display of confidence are not substantiated by the data provided. But they are also not necessary. The study has paved the road to answer these questions. Instead, the authors are encouraged to make suggestions on how the remaining uncertainties could be removed (and what experiments or model might be used).

Author response:

Reviewer #1 (Public review):

Chaiyasitdhi et al. set out to investigate the detailed ultrastructure of the scolopidia in the locust Müller's organ, the geometry of the forces delivered to these scolopidia during natural stimulation, and the direction of forces that are most effective at eliciting transduction currents. To study the ultrastructure, they used the FIB-SEM technique, to study the geometry of natural stimulation, they used OCT vibrometry and high-speed light microscopy, and to study transduction currents, they used patch clamp physiology.

Strengths:

I believe that the ultrastructural description of the locust scolopidium is excellent and the first of its kind in any insect system. In particular, the finding of the bend in the dendritic cilium and the position of the ciliary dilation are interesting, and it would be interesting to see whether these are common features within the huge diversity of insect chordotonal organs.

Thank you very much for your comments. We indeed plan to extend and continue our approach to exploit and understand diverse chordotonal organs in insects and crustaceans.

I believe the use of OCT to measure organ movements is a significant strength of this paper; however, using ex vivo preparations undermines any conclusions drawn about the system's in vivo mechanics.

Having re-read the manuscript, we failed to explicitly describe our ex vivo preparation of Müller’s organ including key references that detail the largely retained physiological function of Müller’s organ. We have now revised this detail in the method section:

“We used an excised locust ear preparation for all experiments, following a previously described dissection protocol [9]. In short, the tympanum, with Muller’s organ attached was left intact suspended between the cuticular rim. The cuticular rim of the tympanum was fixed into a hole in a preparation dish that allowed Muller’s organ to be submerged with extracellular saline, whilst the outside of the tympanum was dry and could be stimulated with airborne sound. This ex vivo preparation of Muller’s organ retained frequency tuning (Warren & Matheson, 2018), similar electrophysiological function as freshly dissected Muller’s organs (Hill, 1983a, 1983b; Michelsen, 1968: frequency discrimination in the locust ear by means of four groups of receptor cells), and amplitude coding (Warren & Matheson, 2018). Since Müller’s organ is backed by an air-filled trachea in vivo, the addition of saline solution in the ex vivo preparation decreased its displacements ~100 fold due to a dampening effect (Warren et al., 2020).”

And in the last section of the introduction:

“Here, we combined FIB-SEM to resolve the 3D ultrastructure of a scolopidium, OCT and high-speed microscopy to examine sound-evoked motion at both the organ and individual scolopidium levels, and direct mechanical stimulation of the scolopale cap, where the ciliary tip is anchored, whilst simultaneously recording transduction currents. Here, Muller’s organ and the tympanum was excised from the locust for physiological experiments. This ex vivo preparation of Muller’s organ retained frequency tuning, amplitude coding and electrophysiological function. This preparation also permitted the enzymatic isolation of individual scolopidia whilst recording transduction currents (Warren & Matheson, 2018).”  

To further clarify physiological differences between the in vivo and ex vivo operation of the tympanum and Müller’s organ, we will perform an additional experiment for the revised manuscript by quantifying the changes in the sound-evoked tonotopic travelling wave of the tympanum using Laser Doppler Vibrometry (LDV). This result will be added to the Supplementary Text.

The choice of Group III scolopidia is also good. Research on the mechanics of locust tympana has shown that travelling waves are formed on the tympanum and waves of different frequencies show highest amplitudes at different positions on the tympanum, and therefore also on different groups of scolopidia within the Müller's organ (Windmill et al, 2005; 2008, and Malkin et al, 2013). The lowest frequency modal waves (F0) observed by Windmill et al 2008 were at about 4.4 kHz, which are slightly higher than the ~3 kHz frequencies studied in this paper but do show large deflections where these group III scolopidia attach at the styliform body (Windmill et al, 2005).

Thank you very much. We accept that the frequencies studied in this manuscript were lower than the lowest modal wave observed by Windmill et al., 2008. Other authors, according to Jacobs et al. 1999, found broad tuning form 3.4-3.74 kHz (Michelson et al., 1971) and 2-3.5 kHz (Halex et al., 1988). We settled on tuning previously measured for Group-III neurons in the same kind of preparation as in this manuscript, which was broadly around 3 kHz (Warren & Matheson, 2018).

This should be mentioned in the paper since the electrophysiology justification to use group III neurons is less convincing, given that Jacobs et al 1999 clearly point out that group III neurons are very variable and some of them are tuned much higher to 10 kHz, and others even higher to 20-30 kHz.

Looking at Fig. 7 from Jacobs et al., 1999, we indeed see that the four Group-III neurons recorded in this study are broadly tuned to 3-4 kHz. Often these tuning curves have threshold dips at higher frequencies at least 20 dB higher. We settled on the most sensitive frequency that we previously measured, and which also overlaps the most sensitive frequencies from several other studies.

Weaknesses:

Specifically, it is understandable that the authors decided to use excised ears for the light microscopy, where Müller's organ would not be accessible in situ. However, it is very likely that excision will change the system's mechanics, especially since any tension or support to Müller's organ will be ablated.

We completely understand this criticism. We have now added descriptions in the methodology and introduction (as detailed previously). In short, the tympanum was left intact suspended on the cuticle. Müller’s organ retains all (measured) physiological properties: frequency tuning, amplitude coding and electrophysiological function. To further investigate whether this excised preparation is a representative of the in vivo conditions, we plan to measure tympanal mechanics, such as the travelling wave, as part of the revisions.

OCT enables in vivo measurements in fully undissected systems (Mhatre et al, Biorxiv, 2021) or in systems with minimal dissection where the mechanics have not been compromised (Vavakou et al, 2021). The choice to entirely dissect out the membrane is difficult to understand here.

The pioneering OCT works by Mhatre et al, Biorxiv, 2021 and Vavakou et al, 2021 set the new standard of in vivo measurements in the field. We also totally agree with Reviewer#1’s view that OCT is best performed on in vivo Müller’s organ and we tried OCT imaging of Müller’s organ for several months in vivo. Although the OCT penetrates the tympanum the OCT beam does not penetrate the tracheal air sac that surrounds Müller’s organ and therefore OCT cannot be used in vivo. Please also see previous comment with regards to the intact physiological operation of Muller’s organ in the ex vivo preparation.

My main concern with this paper, however, is the use of light microscopy very close to the Nyquist limit to study scolopidial motion, and the fact that the OCT data contradict and do not match the light microscopy data. The light microscopy data is collected at ~8 kHz, and hence the Nyquist limit is ~4 kHz. It is possible to measure frequencies reliably this close to the limit, but the amplitude of motion is quite likely to be underestimated, given that the technique only provides 2 sample points per cycle at 4 kHz and approximately 2.66 sample points at 3 kHz. At that temporal resolution, the samples are much more likely to miss the peak of the wave than not, and therefore, amplitudes will be mis-estimated. A much more reasonable sample rate for amplitude estimation is generally about 10 samples per cycle. I do not believe the data from the microscopy is reliable for what the authors wish to use them for.

We understand your concern that the study of sound-evoked motion of the scolopidium using light microscopy was done near the Nyquist limit (with our average sampling rate at 8.6 ± 0.3 kHz and the Nyquist limit at 4.3 kHz). We also agree with your comment that amplitude of the motion could be underestimated at frequencies closer to the limit. However, we find that this systematic error does not change the key observation from our direct light microscopy observation that axial stretch of the scolopidium occurs around 3 kHz.

To address this concern, we plan to study the scolopidial motion within Group 1 auditory neurons, which are tuned to lower frequencies (0.5-1.5 kHz). This new set of data will allow us to obtain more data points per cycle (up to ~8.6 data points at 1 kHz). We will consider adding this result into the revised Fig. 4 or its extended data.

Regarding increasing the sampling rate, we did try to achieve higher sampling rate (> 10 kHz), however, there is a technical limitation of our camera and a trade-off between other key parameters, such as the size of the region of interest (ROI) and magnification. To increase the sampling rate, we will have to reduce the magnification or the ROI and in turn lose the spatial resolution required for quantification of the scolopidial motion or the ROI does not cover the whole scolopidial motion. The sampling rate at 8.6 ± 0.3 kHz was the best we could achieve.

Using the light microscopy data, the authors claim that the strains experienced by the group III scolopidia at 3 kHz are greater along the AP axis than the ML axis (Figure 4). However, this is contradicted by the OCT data, which show very low strain along the AP axis (black traces) at and around 3 kHz (Figure 3c and extended data Figure 2f) and show some movement along the ML axis (red traces, same figures). The phase at low amplitudes of motion cannot be considered very reliable either, and hence phase variations at these frequencies in the OCT cannot be considered reliable indicators of AP motion; hence, I'm unclear whether the vector difference in the OCT is a reliable indicator of movement.

This is our fault for not clearly explaining the orientation of the light microscopy measurement, which then leads to the reviewer’s concern about contradiction between OCT and light microscopy. Our OCT measurements was done along the Antero-Posterior (AP) and Mesio-Lateral axes (ML), while the axial stretch of the scolopidium occurs along the Dorso-Ventral (DV) axis. We recognise that the anatomical references in this manuscript can be confusing, and we tried to show the orientation of the scolopidium relative to Müller’s organ in Fig. 3b. To further clarify the orientation of our observations, we will add anatomical references in Fig. 4a and Fig. 5a. in the revised manuscript.

As stated in our result section (Line 165-167)

“Notably, we could not resolve the Group-III scolopidia along the ventro-dorsal axis—which runs parallel to the dendrite—as the OCT beam was obstructed by either the cuticle or the elevated process”

We did try to perform OCT measurement along the VD axis, but we could not resolve the scolopidial region along the scolopidial or ciliary axes because the OCT beam could not go through the thick cuticle at the edge of the tympanic membrane and the elevated process. For this reason, it is impossible for us to find an agreement or rule out any contradiction between the OCT and light microscopy since they are measuring motion along different axes. We plan to address this accessibility issue in a separate work using OCT measurements in combination with mirrors.

The OCT data are significantly more reliable as they are acquired at an appropriate sampling rate of 90 kHz. The authors do not mention what microphone they use to monitor or calibrate their sound field and phase measurements in OCT, but I presume this was done since it is the norm.

We use a condenser microphone (MK301, Microtech) and measuring amplifier (type 2610, Brüle & Kjær) for calibration. The calibration microphone was also calibrated beforehand using  a sound calibrator type 4231 from B&K.

Thus, the OCT data show that the movement within the Müller's organ is complex, probably traces an ellipse at some frequencies as observed in bushcrickets (Vavkou et al, 2021) and also thought to be the case in tree crickets based on the known attachment points of the tympanal organ (Mhatre et al, 2021). The OCT data shows relatively low AP motion at frequencies near 3 kHz, and higher ML motion, which contradicts the less reliable light microscopy data. Given that the locust membrane shows peaks in motion at ~4.5 kHz, ~11 kHz, and also at ~20 kHz (Windmill et al, 2008), I am surprised that the authors limited their OCT experiments and analyses to 5 kHz.

We found that immediately above 5 kHz the displacements reduced to undetectable magnitudes. We accept that there may be other modes of vibration at higher frequencies >10 kHz (based on Jacobs et al., 1999) that we could have detected with OCT. However, we focused our analysis on Group-III neurons at the best frequency and frequencies that we could cross-compere between our high-speed imaging system and OCT.

In summary for this section, I am not convinced of the conclusion drawn by the authors that group III scolopidia receive significantly higher stimulation along the AP axis in their native configuration, if indeed they were studied in the appropriate force regime (altered due to excision).

Again, we accept our faults for not clearly displaying the anatomical references of the scolopidial and ciliary axes in Fig. 4 and Fig. 5. We also did not clearly describe in detail that our ex vivo preparation largely retains its physiological properties. We will address the errors of our measurement near Nyquist and provide additional information from Group 1 scolopidia where we could achieve higher data points per cycle.

In the scolopidial patch clamp data, the authors study transduction currents in response to steady state stimulation along the AP axis and the ML axis. The responses to steady state and periodic forces may well be different, and the authors do not offer us a way to clearly relate the two and therefore, to interpret the data.

We will revise the Fig. 5a to clarify that the push-pull were done along the Dorso-Ventral (DV) axis and the push-pull were done along the Antero-Posterior (AP) axis. We do agree that steady-state and periodic forces may well be very different. However, valuable insight can be gained from mechanical systems when displaced outside of their normal physiological frequency (e.g. the transformative work on vertebrate hair bundle mechanics, Howard & Hudspeth, 1988). For the same reason, we believe artificial stimulation of the scolopidium gives us new and crucial information to understand scolopidial mechanics. Our main finding that stretch is the dominant stimulus should still, or at least provide strong support, that stretch is the dominant stimulus in periodical motion.

In addition, both stimulation types, along the AP axis and the ML, elicit clear transduction responses. Stimulation along the AP axis might be slightly higher, but there is over 40% variation around the mean in one case (pull: 26.22 {plus minus} 10.99 pA) and close to 80% variation in the other (push: 10.96 {plus minus} 8.59 pA). These data are indeed from a very high displacement range (2000 nm), which is very high compared to the native displacement levels, which are in the 1-10 nm range.

In this experiment, we wished to establish the upper limits (and plateau region) of displacement-transduction current response. However, even at 2000 nm we still did not see a plateau. Therefore, we believe that the strain on the scolopidium is still in the operating range even though our displacement is not. This discrepancy can be explained because the base of the scolopidium is not fixed. Therefore, the displacement imposed in our experiment is not equivalent to the strain on the cilium but a combination of pulling and stretching along the length of the dendrite. The force, however, remains along that particular axis, supporting our main finding.

Another important consideration is that the cilium is surrounded by the scolopale wall. It is assumed that the scolopale wall is far stiffer than the ciliary and will therefore limit the amount of ciliary strain.

The factor change from sample to sample is not reported and is small even overall. The statistical analyses of these data are not clearly reported, and I don't see the results of the overall ANOVA in the results section.

We reported the statistical analyses in the Fig. 5 Source Data. We will now add tables displaying these statistics in the supplementary text of the revised manuscript.

I also find the dip in the reported transduction currents between 10 and 100 nm quite odd (Figure 5 j-m) and would like to know what the authors' interpretation of this behaviour is. It seems to me that those currents increase continuously linearly after ~50-100 nm and that the data below that range are in the noise. Thus, the transduction currents observed at the relevant displacement range (1-10 nm) may not actually be reliable. How were these small displacements achieved, and how closely were the actual levels monitored? Is it possible to reliably deliver 1-10 nm displacements using a micromanipulator?

One interpretation is that the cilium has both sensitive and insensitive mechanically gated ion channels. A finding that is also supported by Effertz et al., 2012. We will add a sentence in the discussion highlighting this interpretation. We will also provide our calibration of displacement vs voltage delivered to the piezo in the Supplementary Text.

What is clear, despite the difficulty in interpreting this data, is that both AP and ML stimulation evoke transduction currents, and their relative differences are small. Additionally, in Müller's organ itself, in the excised organ, the scolopidia are stimulated along both axes. Thus, in my opinion, it is not possible to say that axial stretch along the cilium is 'the key mechanical input that activates mechano-electrical transduction'.

We confirm that the scolopidia are displaced along both. We also note that displacements of the scolopidium limited to the up-down axis will also produce a strain on the scolopidium along the push-pull axis. However, we tried to disentangle this complex motion by limiting the displacements to one axis during recordings of the transduction current. We found that displacement along the scolopidial axis generated the largest transduction currents. Even though there is large variation our statistical analysis confirmed a significant difference as stated in the result section (Line 283 – 286)

“Additionally, the transduction current evoked by pull from the resting position was larger than displacement upward, 12.17 ± 5.37 pA (N = 11, n = 11) (Tukey's procedure, p = 1.75e-03, t = -3.83) or downward 7.28 ± 9.76 pA (N = 11, n = 11) (Tukey's procedure, p = 5.10e-06, t = -4.53).”

The reason for large variation is that the discrete depolarisations (random depolarisations of unknown function and a common feature of chordotonal neurons so far recorded) have a similar magnitude to the transduction current produced by the step displacements. We will highlight these discrete depolarisations in Figure 4d and mention them in the results.

Reviewer #2 (Public review):

Summary of strengths and weaknesses:

Using several techniques-FIB-SEM, OCT, high-speed light microscopy, and electrophysiology-Chaiyasitdhi et al. provide evidence that chordotonal receptors in the locust ear (Müller's organ) sense the stretch of the scolapale cell, primarily of its cilium. Careful measurements certainly show cell stretch, albeit with some inconsistencies regarding best frequencies and amplitudes.

Thank you very much for acknowledging the strength of our study. Regarding the inconsistencies between best frequencies and amplitude, we believe that this concern largely arises from our faults for not clearly displaying the anatomical references of the scolopidial and ciliary axes in Fig. 4 and Fig. 5. As previously addressed in our response to Reviewer#1, we will add the anatomical references and revised the text to clarify the orientation of our measurements.

The weakest argument concerns the electrophysiological recordings, because the authors do not show directly that the stimulus stretches the cells. If this latter point can be clarified, then our confidence that ciliary stretch is the proximal stimulus for mechanotransduction will be increased.

We agree that the displacement is not solely stretching the scolopidium. However, the force is still constrained and acting along the push-pull axis. Due to this reason, we overestimate the displacement required to open the MET channels but stand by our conclusion that stretch is the dominant stimulus. For future work, we wish to devise a technique to mechanically clamp the base of the scolopidium and measure the more physiological relevant current-strain relationship.

This conclusion will not come as a surprise for workers in the field, as the chordotonal organ is known as a stretch-receptor organ (e.g., Wikipedia). But it is a useful contribution to the field and allows the authors to suggest transduction mechanisms whereby ciliary stretch is transduced into channel opening.

One of the goals of this manuscript is to highlight the lack of direct evidence for stretch-sensitivity of chordotonal organs, as this is assumed from their structure. More importantly the acceptance of chordotonal organs, as being stretch sensitive does not address the mechanism of how organs work. For instance, one candidate for the MET channel, NompC, is shown to be sensitive to compression (Wang et al., 2021). We find that a preconceived concept of “stretch-sensitive” mechanism, without an appreciation of scolopidium mechanics, cannot explain how NompC can be opened in chordotonal organs.

P. .E. Howse wrote in his work on ‘The Fine Structure and Functional Organisation of Chordotonal Organs’ in 1968 (Symp. Zool. Soc. Lon.) No. 23

“There is, however, a common tendency to refer to chordotonal organs in which scolopidia are contained in a connective tissue strand as “stretch receptor”. This is unfortunate in two senses, for firstly the implied function may not have been proved and secondly even if the organ responds to stretch the scolopidia may not.” then he proceeded to cite a pioneering work in the chordotonal organs of the hermit crab by R.C. Taylor (Comp. Biochem. Physiol. 1966) showing that the scolopidia may experience flexing when the connective strand are stretched.

This work represents the first efforts to investigate the problematic assumption of stretch-sensitivity of scolopidia since it was first highlighted 57 years ago.

Reviewer #3 (Public review):

Summary:

The paper 'A stretching mechanism evokes mechano-electrical transduction in auditory chordotonal neurons' by Chaiyasitdhi et al. presents a study that aims to address the mechanical model for scolopidia in Schistocerca gregaria Müller's organ, the basic mechanosensory units in insect chordotonal organs. The authors combine high-resolution ultrastructural analysis (FIB-SEM), sound-evoked motion tracking (OCT and high-speed light microscopy), and electrophysiological recordings of transduction currents during direct mechanical stimulation of individual scolopidia. They conclude that axial stretching along the ciliary axis is an adequate mechanical stimulus for activating mechanotransduction channels.

Strengths/Highlights:

(1) The 3D FIB-SEM reconstruction provides high resolution of scolopidial architecture, including the newly described "scolopale lid" and the full extent of the cilium.

(2) High-speed microscopy clearly demonstrates axial stretch as the dominant motion component in the auditory receptors, which confirms a long-standing question of what the actual motion of a stretch receptor is upon auditory stimulation.

(3) Patch-clamp recordings directly link mechanical stretch to transduction currents, a major advance over previous indirect models.

Weaknesses/Limitations:

(1) The text is conceptually unclear or written in an unclear manner in some places, for example, when using the proposed model to explain the sensitivity of Nanchung-Inactive in the discussion.

We will rephrase and make clearer the context of our findings for Nanchung-Inactive mechanism of MET in the introduction and the discussion. We will also refine and simplify unclear text overall.

(2) The proposed mechanistic models (direct-stretch, stretch-compression, stretch-deformation, stretch-tilt) are compelling but remain speculative without direct molecular or biophysical validation. For example, examining whether the organ is pre-stretched and identifying the mechanical components of cells (tissues), such as the extracellular matrix and cytoskeleton, would help establish the mechanical model and strengthen the conclusion.

We agree with the speculative nature of our four proposed hypotheses. We have, however, narrowed down from at least ten previous hypotheses (Field and Matheson, 1998). These hypotheses will enable us, and hopefully the field, to test them and more rapidly advance our understanding of how scolopidia work. We will add a section in the discussion as to the best way to experimentally test these four hypotheses (e.g pushing directly onto the cap should elicit sensitive responses for the cap-compression hypothesis).

(3) To some extent, the weaknesses of the paper are part of its strengths and vice versa. For example, the direct push/pull and up/down stimulations are a great experimental advance to approach an answer to the question of how the underlying cellular components are deformed and how the underlying ion channels are forced. However, as the authors clearly state, neither of their stimulations can limit all forces to only one direction, and both orthogonal forces evoke responses in the neurons. The question of which of the two orthogonal forces 'causes' the response cannot be answered with these experiments and has not been answered by this manuscript. But the study has brought the field a considerable step closer to answering the question. The answer, however, might be that both longitudinal ('stretch') and perpendicular ('compression') forces act together to open the ion channels and that both dendritic extension via stretch and bending can provide forces for ion channel gating.

Thank you very much for your acknowledgement of our experimental advances. We agree that this study cannot identify and localise the forces on the cilium as it is enclosed in the scolopidial unit. As previously explained, we plan to address this question in our next work by improving and expanding our experimental techniques, including modelling, to study the scolopidial mechanics based on our experiments using patch-clamp recording in combination with individual and direct manipulation the scolopidium.

The current paper has identified major components (longitudinal stretch components) for the neurons they analysed, but these will surely have been chosen according to their accessibility, and as such, the variety of mechanical responses in Müller's organ might be greater. In light of these considerations, the authors might acknowledge such uncertainties more clearly in their paper.

Our high-speed and OCT imaging confirms complex multi-dimensional displacements (and presumably forces) acting on the scolopidium. We agree that our mechanical stimulation cannot recapitulate such complex motions. But for future work we wish to extend our mechanical stimulation to three axis and also to pivot on the axis of the scolopidial cap.

The paper is an impressive methodological progress and breakthrough, but it simply does not "demonstrate that axial stretch along the cilium is the adequate stimulus or the key mechanical input that activates mechano-electrical transduction" as the authors write at the start of their discussion.

We rephrase to clarity that stretching along the “scolopidial axis”, not “along the ciliary axis” is the adequate stimulus. We cannot yet verify how this translates to forces acting on the cilium, hence the four speculative hypotheses. We will re-write the discussion to make clear that we are only interpretating the forces and displacements at the level of the cilium.

They do show that axial stretch dominates for the neurons they looked at, which is important information. The same applies to the end of the discussion: The authors write, "This relative motion within the organ then drives an axial stretch of the scolopidium, which in turn evokes the mechano-electrical transduction current." Reading the manuscript, the certainty and display of confidence are not substantiated by the data provided. But they are also not necessary. The study has paved the road to answer these questions. Instead, the authors are encouraged to make suggestions on how the remaining uncertainties could be removed (and what experiments or model might be used).

We will moderate our conclusion in the discussion, but we are confident that we have experimental repeats, and the statistical test, to support our conclusion that stretching of the scolopidium provides that largest transduction current responses (although not at the level of the cilium). As mentioned previously, we will include a section in the discussion for the best way to test the hypotheses arising from this work.

eLife Assessment

This study provides new and interesting findings that SCoR2 acts as a denitrosylase to control cardioprotective metabolic reprogramming and prevent injury following ischemia/reperfusion. The compelling evidence is supported by a novel multi-omics approach, but questions remain regarding the stability and human relevance of BDH1 as well as the sufficiency of SCoR2. Overall, the work will be of interest to cardiovascular researchers and provides valuable information to the field, though some mechanistic aspects require further clarification.

Reviewer #1 (Public review):

Summary:

This study shows a novel role for SCoR2 in regulating metabolic pathways in the heart to prevent injury following ischemia/reperfusion. It combines a new multi-omics method to determine SCoR2 mediated metabolic pathways in the heart. This paper would be of interest to cardiovascular researchers working on cardioprotective strategies following ischemic injury in the heart.

Strengths:

(1) Use of SCoR2KO mice subjected to I/R injury.

(2) Identification of multiple metabolic pathways in the heart by a novel multi-omics approach.

Comments on revisions:

Authors have addressed all concerns raised in the previous round of review. Substantial modifications have been made in response to those concerns. There are no further comments.

Reviewer #2 (Public review):

Summary:

This manuscript addresses the gap in knowledge related to the cardiac function of the S-denitrosylase SNO-CoA Reductase 2 (SCoR2; product of the Akr1a1 gene). Genetic variants in SCoR2 have been linked to cardiovascular disease, yet its exact role in heart remains unclear. This paper demonstrates that mice deficient in SCoR2 show significant protection in a myocardial infarction (MI) model. SCoR2 influenced ketolytic energy production, antioxidant levels, and polyol balance through the S-nitrosylation of crucial metabolic regulators.

Strengths:

Addresses a well-defined gap in knowledge related to the cardiac function of SNO-CoA Reductase 2. Besides the in-depth case for this specific player, the manuscripts sheds more light on the links between S-nytrosylation and metabolic reprogramming in heart.

Rigorous proof of requirement through the combination of gene knockout and in vivo myocardial ischemia/reperfusion

Identification of precise Cys residue for SNO-modification of BDH1 as SCoR2 target in cardiac ketolysis

Weaknesses:

The experiments with BDH1 stability were performed in mutant 293 cells. Was there a difference in BDH1 stability in myocardial tissue or primary cardiomyocytes from SCoR2-null vs -WT mice? Same question extends to PKM2.

In the absence of tracing experiments, the cross-sectional changes in ketolysis, glycolysis or polyol intermediates presented in Figures 4 and 5 are suggestive at best. This needs to be stressed while describing and interpreting these results.

The findings from human samples with ischemic and non-ischemic cardiomyopathy do not seem immediately or linearly in line with each other and with the model proposed from the KO mice. While the correlation holds up in the non-ischemic cardiomyopathy (increased SNO-BDH1, SNO-PKM2 with decreased SCoR2 expression), how do the Authors explain the decreased SNO-BDH1 with preserved SCoR2 expression in ischemic cardiomyopathy? This seems counterintuitive as activation of ketolysis is a quite established myocardial response to the ischemic stress. It may help the overall message clarity to focus the human data part on only NICM patients.

(partially linked to the point above) an important proof that is lacking at present is the proof of sufficiency for SCoR2 in S-Nytrosylation of targets and cardiac remodeling. Does SCoR2 overexpression in heart or isolated cardiomyocytes reduce S-nitrosylation of BDH1 and other targets, undermining heart function at baseline or under stress?

Comments on revisions:

Some of my points have been addressed. However, the points related to 1) BDH1 stability effect in cardiomyocytes; 2) human relevance of SNO-BDH1; 3) SCoR2 sufficiency remain unclear. That said, this manuscript will provide useful information to the field as such.

Reviewer #3 (Public review):

Summary:

This manuscript demonstrates that mice lacking the denitrosylase enzyme SCoR2/AKR1A1 demonstrate a robust cardioprotection resulting from reprogramming of multiple metabolic pathways, revealing<br /> widespread, coordinated metabolic regulation by SCoR2.

Strengths:

The extensive experimental evidence provided the use of the knockout model

Weaknesses:

No direct evidence for the underlying mechanism.

The mouse model used is not a tissue-specific knock-out.

Author response:

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

Reviewer #1 (Public review): 

Summary: 

This study shows a novel role for SCoR2 in regulating metabolic pathways in the heart to prevent injury following ischemia/reperfusion. It combines a new multi-omics method to determine SCoR2 mediated metabolic pathways in the heart. This paper would be of interest to cardiovascular researchers working on cardioprotective strategies following ischemic injury in the heart. 

Strengths:

(1) Use of SCoR2KO mice subjected to I/R injury. 

(2) Identification of multiple metabolic pathways in the heart by a novel multi-omics approach.

We thank the Reviewer for the positive review of our manuscript.

Weaknesses:

(1) Use of a global SCoR2KO mice is a limitation since the effects in the heart can be a combination of global loss of SCoR2. 

(2) Lack of a cell type specific effect. 

We agree that global KOs limit the cell type-specific mechanistic conclusions that can be drawn. Global knockouts are nonetheless informative in their own right and serve to identify phenotypes worthy of further study.

Reviewer #2 (Public review):

Summary: 

This manuscript addresses the gap in knowledge related to the cardiac function of the S-denitrosylase SNOCoA Reductase 2 (SCoR2; product of the Akr1a1 gene). Genetic variants in SCoR2 have been linked to cardiovascular disease, yet their exact role in the heart remains unclear. This paper demonstrates that mice deficient in SCoR2 show significant protection in a myocardial infarction (MI) model. SCoR2 influenced ketolytic energy production, antioxidant levels, and polyol balance through the S-nitrosylation of crucial metabolic regulators. 

Strengths: 

(1) Addresses a well-defined gap in knowledge related to the cardiac function of SNO-CoA Reductase 2. Besides the in-depth case for this specific player, the manuscript sheds more light on the links between Snitrosylation and metabolic reprogramming in the heart.

(2) Rigorous proof of requirement through the combination of gene knockout and in vivo myocardial ischemia/reperfusion. 

(3) Identification of precise Cys residue for SNO-modification of BDH1 as SCoR2 target in cardiac ketolysis 

We thank the Reviewer for their kind words.

Weaknesses: 

(1) The experiments with BDH1 stability were performed in mutant 293 cells. Was there a difference in BDH1 stability in myocardial tissue or primary cardiomyocytes from SCoR2-null vs -WT mice? The same question extends to PKM2. 

We have not assessed BDH1 stability directly in cardiomyocytes. However, S-nitrosylation increased BDH1 stability in HEK293 cells, and BDH1 expression was increased in (injured) hearts of SCoR2KO mice, together with increased SNO-BDH1. 

For PKM2, there is a wealth of published evidence from us and others that S-nitrosylation does not regulate protein stability but rather inhibits tetramerization required for full activity.  

(2) In the absence of tracing experiments, the cross-sectional changes in ketolysis, glycolysis, or polyol intermediates presented in Figures 4 and 5 are suggestive at best. This needs to be stressed while describing and interpreting these results. 

We now acknowledge this limitation in the ‘Limitations’ section of the manuscript and in edits made to the text. 

(3) The findings from human samples with ischemic and non-ischemic cardiomyopathy do not seem immediately or linearly in line with each other and with the model proposed from the KO mice. While the correlation holds up in the non-ischemic cardiomyopathy (increased SNO-BDH1, SNO-PKM2 with decreased SCoR2 expression), how do the authors explain the decreased SNO-BDH1 with preserved SCoR2 expression in ischemic cardiomyopathy? This seems counterintuitive as activation of ketolysis is a quite established myocardial response to ischemic stress. It may help the overall message clarity to focus the human data part on only NICM patients. 

We find it interesting and important that SNO-BDH1 is readily detected in human heart tissue and its level is correlated to disease state. Our findings suggest conservation of this mechanism in human heart failure. However, we caution against drawing further conclusions related to NICM or ICM. Our animal model (based on a single time point) cannot faithfully recapitulate patients with chronic heart disease or differences between NICM and ICM. 

(4) This is partially linked to the point above. An important proof that is lacking at present is the proof of sufficiency for SCoR2 in S-nitrosylation of targets and cardiac remodeling. Does SCoR2 overexpression in the heart or isolated cardiomyocytes reduce S-nitrosylation of BDH1 and other targets, undermining heart function at baseline or under stress? 

The Reviewer proposes to test the effect of SCoR2 overexpression on cardioprotection. This is an interesting experiment for future study with the following caveats. First, it presupposes that native expression of SCoR2 is insufficient to control basal steady state S-nitrosylation of SNO-BDH1 and SNO-PKM2 (this does not seem to be the case). Second, overexpressed SCoR2 may be mislocalized within cells or associated with unnatural targets. Thank you.

Reviewer #3 (Public review): 

Summary: 

This manuscript demonstrates that mice lacking the denitrosylase enzyme SCoR2/AKR1A1 demonstrate a robust cardioprotection resulting from reprogramming of multiple metabolic pathways, revealing widespread, coordinated metabolic regulation by SCoR2. 

Strengths: 

(1) The extensive experimental evidence. 

(2) The use of the knockout model. 

We thank the Reviewer for identifying strengths in our work.

Weaknesses: 

(1) The connection of direct evidence for the mechanism. 

We believe we have identified a novel mechanism for cardioprotection entailing coordinate reprogramming of multiple metabolic pathways and suggesting a widescale role for SCoR2 in metabolic regulation. This is the key message we convey. While genetic dissection of individual pathways may be worthwhile, these investigations will have their own limitations. 

(2) The mouse model used is not tissue-specific. 

Please see our response to Reviewer 1, above. 

Reviewer #1 (Recommendations for the authors):

In the study, titled "The denitrosylase SCoR2 controls cardioprotective metabolic reprogramming", Grimmett ZW et al., describe a role for SNO-CoA Reductase 2 (SCoR2) in promoting cardioprotection via metabolic reprogramming in the heart after I/R injury. Authors show that loss SCoR2 coordinates multiple metabolic pathways to limit infarct size. Overall, the hypothesis is interesting, however there are some limitations as described below: 

(1) It is unclear whether SCoR2 mice are global or cardiomyocyte specific. 

We apologize for any confusion. These are global SCoR2^-/- mice. This is now stated in the Results when first identifying the strain, as well as in the Methods.  

(2) Can the authors clarify how divergent metabolic pathways such as Ketone oxidation, glycolysis, PPP and polyol metabolism work downstream of SCoR2 to impact cardioprotection in mice with I/R. 

The metabolic pathways of ketone oxidation, glycolysis, PPP and polyols appear to converge to support ischemic cardioprotection in SCoR2^-/- mice, as depicted in the model shown in Fig. 5L. Subsequent to SNO-PKM2 blockade of flux through glycolysis (detailed in this manuscript and in Zhou et al, 2019, PMID: 30487609, as well as by others), substrates of ketolysis and glycolysis are funneled into the PPP, producing the antioxidant NADPH and energy precursor phosphocreatine, which are well-known to be cardioprotective. This occurs more readily in SCoR2^-/- mice due to elevated SNO-BDH1 (detailed in this manuscript). 

Polyols, thought to be products of the PPP carbohydrate intermediates arabinose, ribulose, xylulose (among others), have recently been shown to be harmful to cardiovascular health in humans. These polyols are uniformly downregulated in SCoR2^-/- mice. We suggest this is likely the result of S-nitrosylation of SCoR2-substrate enzymes that form polyols (SCoR2/Akr1a1 is unable to directly reduce carbohydrates to their corresponding polyols). Regulation of endogenous polyol production in humans is a new concept and the mechanisms whereby these compounds increase risk of cardiac events are a subject of active investigation. This is detailed in the final paragraph of both the Results and Discussion sections, and in Fig. 5L. 

(3) The only functional outcome of SCoR2 loss in echocardiography and measurements for apoptosis. However, it would be important to determine whether the cardioprotective effect persists. It seems cardiac function was recorded 24hours post injury and whether the benefit remains till later time point such as 2 or 4 weeks is not shown. Without this time point, loss of SCoR2 only leads to an acute increment in function. 

Loss of SCoR2 reduced post-MI mortality at 4 hr; cardiac functional changes (plus troponin, LDH, and apoptosis) were studied in surviving animals at 24 hr post-MI. Cardiac response to acute injury and to chronic injury (weeks post-MI) are not the same metabolically. This is well elucidated in the literature and exemplified by the role of PKM2, which is protective in the chronic response to MI (28 days post-MI; PMID: 32078387), but implicated in injury at shorter timepoints post-MI (PMID: 33288902, 28964797). All that said, functional changes at 2-4 weeks will be important to determine in the future, as the Reviewer indicates. 

Reviewer #2 (Recommendations for the authors): 

(1) The last paragraph of the Results section should be divided into the statement related to Table S2 in the Results section, and the rest of the paragraph should be put somewhere in the Discussion. 

Thank you for this suggestion, which we have taken. 

(2) The number of mice alive/dead should be reported in the histogram in Figure 1G. 

Done.

(3) A concise Graphical Abstract will be useful to grasp the overall logic and message of the manuscript from the beginning. 

We thank you for this suggestion and have added a graphical abstract to the manuscript.

Reviewer #3 (Recommendations for the authors): 

I would suggest having more evidence on the effect of metabolic reprogramming on which cell type. The use of a global knockout is a major limitation, and probably some in vitro experiments with shRNA knockdown in endothelial cells and fibroblasts would provide more insights. 

The reviewer suggests one direction for future study. We identify a novel mechanism for cardioprotection entailing coordinate reprogramming of multiple metabolic pathways and suggesting a widescale role for SCoR2 in metabolic regulation. This is the message we wish to convey. The role of cardiomyocytes vs contributing cell types is a thoughtful direction for future study. Thank you. 

Editor's additional comment:

The editors wish to highlight a critical issue concerning the characterization of the SCoR2−/− mice employed in this study. 

In the Methods section (page 20), the manuscript states that "SCoR2+/− mice were made by Deltagen, Inc. as described previously (33)." However, reference 33 does not describe SCoR2−/− mice; instead, it refers to other genetically modified strains, including Akr1a1+/−, eNOS−/−, and PKM2−/− mice, with no mention of a SCoR2-targeted model. 

The editors fully acknowledge that the authors may be using the term "SCoR2" as a functional synonym for Akr1a1, based on its described role as a mammalian homologue of yeast SCoR. If this is the case, such equivalence should be explicitly stated in the manuscript to prevent potential confusion. Moreover, considering that the genetic deletion of Akr1a1 (i.e., SCoR2) underlies the key mechanistic findings presented, it is essential that the manuscript include a clear and comprehensive description of the generation and validation of the mouse model used. 

We therefore ask the authors to (1) clarify the nomenclature and relationship between "SCoR2" and Akr1a1, and (2) provide full details on the generation of the knockout mice, including the targeting strategy and the genotyping procedures. This information is necessary not only to ensure transparency and reproducibility but also to allow readers to fully appreciate the biological relevance of the findings.

Thank you for identifying this inconsistency. We have adjusted the manuscript text accordingly to clearly state that SCoR2 is a functional name for the product of the Akr1a1 gene and that these SCoR2^-/- mice are the same as Akr1a1^-/- mice described in Ref 33. We have augmented the Methods text to describe the generation and genotyping of these SCoR2/Akr1a1 knockout mice.

je možné něco dělat s těmi volnými předložkami na konci řádků? je možné tam dát nějakou pevnou mezeru jako ve wordu apod.?

přeformulovat větu, aby nebyl rok a číslo hned za sebou

We can analyze key data tabs ('2022 Final S1 S2', 'Targeted % Reduction S1+2', and '2022 Final S3') and categorize companies into strategic profiles like 'beginners', 'leaders', and 'influencers'. Then we choose those who are willing to work with us and have good financial viability.

Nejednoznačný

Všude změňme na Česko

Mohli bychom u všech map ještě přidat tlačítko domů, prosím?

test de commentaires

eLife Assessment

Using high-throughput small-molecule screening, this study discloses novel modulators of the mitochondrial transcription factor A (TFAM), a key regulator of mitochondrial function. Reviewers viewed the targeting of TFAM as innovative and the study's conclusions as potentially important (especially the effects on inflammation). However, the lack of evidence for a direct effect of the compounds on TFAM activity weakens the paper's key conclusion and renders the study incomplete.

Reviewer #1 (Public review):

Summary:

The authors identify small-molecule compounds modulating the stability of the mitochondrial transcription factor A (TFAM) using a high-throughput CETSA screen and subsequent secondary assays. The identified compounds increased the protein levels of TFAM without affecting its RNA levels and led to an increase in mtDNA levels. As a read-out for dose-dependent action of the identified compounds, the authors investigated cGAS-STING and ISG activation in cellular inflammation models in the presence or absence of their compounds. The addition of TFAM modulators led to a decrease in cGAS-STING/ISG activation and decreased mtDNA release. Furthermore, beneficial effects could be determined in models of mtDNA disease (rescue of ATP rates), sclerotic fibroblasts (decreased fibrosis), and regulatory T cells (decreased activation of effector T cells). The study thus proposes novel first-in-class regulators of TFAM as a therapeutic option in conditions of mitochondrial dysfunction.

Strengths:

The authors identified TFAM as a promising target in conditions of mitochondrial dysfunction, as it is a key regulator of mitochondrial function, serving both as a transcription and packaging factor of mtDNA. Importantly, TFAM is a key regulator of mtDNA copy number, and a moderate increase in TFAM/mtDNA levels has been shown to be beneficial in a number of pathological conditions. Furthermore, mtDNA release leading to activation of inflammatory responses has been linked to a variety of pathological conditions in the last decade. Thus, the identification of small molecule modulators of TFAM that have the potential to increase mtDNA copy number and decrease inflammatory signaling is of great importance. Furthermore, the authors highlight potential applications in the field of mitochondrial disease, fibrosis, and autoimmune disease.

Weaknesses:

The central weakness of the study is the fact that the authors propose compounds as modulators or even activators of TFAM without sufficiently proving a direct effect on TFAM itself. There are no data indicating a direct effect on TFAM activity (e.g., mtDNA transcription, replication, packaging), and it is not sufficiently ruled out that other proteins (e.g., LONP1) mediate the effect. Additionally, important information on the performed screen is not provided. Thus, the data presented is currently incomplete to support the described findings. Furthermore, the introduction and discussion are lacking key references.

Reviewer #2 (Public review):

Summary:

The present paper aims to identify small molecules that could possibly affect mitochondrial DNA (mtDNA) stability, limiting cytosolic mtDNA abundance and activation of interferon signaling. The authors developed a high-throughput screen incorporating HiBiT technology to identify possible target compounds affecting mitochondrial transcription factor A (TFAM) content, a compound known to impact mtDNA stability. Cells were subsequently exposed to target compounds to investigate the impact on TNFα-stimulated interferon signaling, a process activated by cytosolic mtDNA abundance. Compound 2, an analog of arylsulfonamide, was highlighted as a possible mitochondrial transcription factor A (TFAM)-activator, and emphasized as a small molecule that could stabilize mtDNA and prevent stress-induced interferon signaling.

Strengths:

Identifying compounds that positively affect mitochondrial biology has diverse implications. The combination of high-throughput screening and assay development to connect identified compounds with cellular interferon signalling events is a strength of the current approach, and the authors should be commended for identifying compounds that broadly impact interferon signalling. The authors have incorporated diverse measurements, including TFAM content, mtDNA content, interferon signaling, and ATP content, as well as verified the necessity of TFAM in mediating the beneficial effects of the emphasized small molecule (Compound 2).

Weaknesses:

(1) While the identified compound clearly works through TFAM, Compound 2 was identified as an arylsulfonamide, which would be expected to affect voltage-gated sodium channels (e.g. PMID: 31316182). Alterations in cellular sodium content and membrane polarization could affect metabolism to indirectly influence mtDNA and TFAM content. It remains unclear if this compound directly or indirectly affects TFAM content, especially as the authors have utilized various cancer cell lines, which could have aberrant sodium channels.

(2) TFAM is nuclear encoded - if this compound directly functions to 'activate TFAM', why/how would TFAM content increase independent of nuclear transcription?

(3) While a listed strength is the incorporation of diverse readouts, this is also a weakness, as there is a lack of consistency between approaches. For instance, data is not provided to show compound 2 increases TFAM or mtDNA content following TNFα stimulation, and extrapolating between cell lines may not be appropriate. The authors are encouraged to directly report TFAM and mtDNA for target compounds 2 and 15 to support their data reported in Figure 2. Ideally, the authors would also report for compound 1 as a control.

(4) While the authors indicate compound 11 displayed the strongest effect on ISRE activity, this appears not to be identified in Figure 1B as a compound affecting TFAM content? Can the authors identify various Compounds in Figure 1B to better highlight the relationship between compounds and TFAM content?

(5) The authors suggest Compound 2 increases cellular ATP - but they are encouraged to normalize luminescence to cellular protein and OXPHOS content to better interpret this data. Additionally, the authors are encouraged to report cellular ATP content following TNFα stimulation/stress (the key emphasis of the present data) and test compound 11, which the authors have implicated as a more sensitive compound.

The discussion is really a perspective, theorizing the diverse implications of small molecule activation of TFAM. The authors are encouraged to provide a balanced discussion, including a critical evaluation of their own work, including an acknowledgement that evidence is not provided that Compound 2 directly activates TFAM or decreases mtDNA cytosolic leakage.

eLife Assessment

This study presents a useful inventory of genes that are up- and down-regulated in the mouse small intestine (duodenum and ileum) during the first postnatal month; the data were collected and analyzed using solid and validated methodology and can be used as a starting point for additional validation of specific markers and for follow-up functional studies. Some aspects of the study were incomplete, with claims being only partially supported by the data, and it is suggested that additional validation be performed. The authors attempted to correlate gene expression changes with periods of high and low NEC susceptibility, but these correlations are speculative and not supported by functional follow-up studies. Discussion of gene expression changes with NEC susceptibility would be more appropriate to include in the Discussion section and to be tempered in the results section.

Reviewer #1 (Public review):

Summary:

In this manuscript, the authors aimed to clarify the transcriptional changes across murine postnatal small intestinal development (0 days to 1 month) in both the duodenum and ileum, a period that shows morphological similarity to 20-30 week old fetal humans. This is an especially critical stage in human intestinal development, as necrotizing enterocolitis (NEC) usually manifests during these stages.

Strengths:

The authors assessed numerous timepoints between 0 days and 1 month in the postnatal mouse duodenum and ileum using bulk RNA transcriptomics of bulk-isolated tissues. Cellular deconvolution, based on relative marker expression, was used to clarify immune cell proportions in the bulk RNA sequencing data. They confirmed some transcriptional targets found in vivo primarily in mouse via qrtPCR and immunohistochemistry, but also in human fetal tissues and isolated organoids, and are of decent quality.

Weaknesses:

The overall weakness of this study, as mentioned by the authors themselves, is that the bulk transcriptomic data generated for the study were isolated from non-fractionated bulk intestinal tissue. This makes it difficult to interpret much of this data regarding cellular fractions found across developmental time. It is difficult to rationalize the approach here, as even isolation protocols of epithelial-only or mesenchyme-only tissues for bulk RNA sequencing are well established. The authors address some of these concerns using cellular deconvolution for immune cell populations, which I think might be helpful if they expanded this analysis to other cell types (mesenchyme, endothelium, glia). However, I would assume that bulk isolations across developmental time are going to be influenced primarily by the bulk of tissue-type found at each time point - primarily epithelium. But this is also confirmed by the immune transcripts becoming more apparent later in their time series, as this system becomes more established during weaning. This study might also be strengthened by comparison with data that is publicly available for early fetal stage development in humans. Comparisons between the duodenum and ileum could be strengthened by what we already know from adult data, from both epithelial- and mesenchyme-isolated fractions. The rationale of using the postnatal mouse as a comparison to NEC is also a little unclear- perhaps some of the developmental processes are similar, however, the environments are completely different. For example, even in early postnatal mouse development, you would find microbial activity and milk.

Reviewer #2 (Public review):

Summary:

This work presents a valuable resource by generating a comprehensive bulk RNA sequencing catalogue of gene expression in the mouse duodenum and ileum during the first postnatal month. The central findings of this work are based on an analysis of this dataset. Specifically, the authors characterized molecular shifts that occur as the intestine matures from an immature to an adult-like state, investigating both temporal changes and regional differences between the proximal and distal small intestine. A key objective was to identify gene expression patterns relevant to understanding the region-specific susceptibility and resistance to necrotizing enterocolitis (NEC) observed in humans during the postnatal period. They also sought to validate key findings through complementary methods and to provide comparative context with human intestinal samples. This study will provide a solid reference dataset for the community of researchers studying postnatal gastrointestinal development and diseases that arise during these stages. However, the study lacks functional validation of the interpretations.

Strengths:

(1) The inclusion of numerous time points (day 0 through 4 weeks) and comparative analyses throughout the first postnatal month.

(2) Validation of key interpretations of RNA-seq data by other methods.

(3) Linking mouse postnatal development to human premature infant development, enhancing its clinical relevance, particularly for NEC research. The inclusion of human intestinal biopsy and organoid data for comparison further strengthens this link.

(4) The investigation covers a wide array of developmental gene categories with known significance, including epithelial differentiation markers (e.g., Vil1, Muc2, Lyz1), intestinal stem cell markers (e.g., Lgr5, Olfm4, Ascl2), mesenchymal markers (e.g., Pdgfra, Vim), Wnt signaling components (e.g., Wnt3, Wnt5a, Ctnnb1), and various immune genes (e.g., defensins, T cell, B cell, ILC, macrophage markers).

Weaknesses:

(1) The primary limitation is that there is no functional validation. The study primarily focuses on the interpretation of RNA expression. This is a common limitation of transcriptomic "atlas" studies, but the functional and mechanistic relevance of these interpretations remains to be determined.

(2) The data are derived from bulk RNA-Seq of full-thickness intestinal tissue. While this approach helps capture rare cell types and both epithelial and mesenchymal components simultaneously, it does not provide cell-type-specific gene expression profiles, which might obscure important nuances. Future investigations using single-cell sequencing would be a logical follow-up.

(3) The day 4 samples were omitted due to quality issues, which might have led to missing some dynamic changes, especially given that some ISC genes show dynamic changes around day 6.

Reviewer #3 (Public review):

Summary:

This study uses bulk mRNA sequencing to profile transcriptional changes in intestinal cells during the early postnatal period in mice - a developmental window that has received relatively little attention despite its importance. This developmental stage is particularly significant because it parallels late gestation in humans, a time when premature infants are highly vulnerable to necrotizing enterocolitis (NEC). By sampling closely spaced timepoints from birth through postnatal week four, the authors generate a resource that helps define transcriptional trajectories during this phase. Although the primary focus is on murine tissue, the authors also present limited data from human fetal intestinal biopsy samples and organoids. In addition, they discuss potential links between observed gene expression changes and factors that may contribute to NEC.

Strengths:

The close temporal sampling in mice offers a detailed view of dynamic transcriptional changes across the first four weeks after birth. The authors leverage these close timepoints to perform hierarchical clustering to define relationships between developmental stages. This is a useful approach, as it highlights when transcriptional states shift most dramatically and allows for functional predictions about classes of genes that vary over time. This high-level analysis provides an effective entry point into the dataset and will be useful for future investigations. The inclusion of human fetal intestinal samples, although limited, is especially notable given the scarcity of data from late fetal timepoints. The authors are generally careful in their presentation of results, acknowledging the limitations of their approach and avoiding over-interpretation. As they note, this dataset is intended as a foundation for their lab and others, with secondary approaches required to more fully explore the biological questions raised.

Weaknesses:

One limitation of the study is the use of bulk mRNA sequencing to draw conclusions about individual cell types. It has been documented that a few genes are exclusively expressed in single cell types. For instance, markers such as Lgr5 and Olfm4 are enriched in intestinal stem cells (ISCs), but they are also expressed at lower levels in other lineages and in differentiating cells. Using these markers as proxies for specific cell populations lowers confidence in the conclusions, particularly without complementary validation to confirm cell type-specific dynamics.

Validation of the sequencing data was itself limited, relying primarily on qPCR, which measures expression at the same modality rather than providing orthogonal support. It is unclear how the authors selected the subset of genes for validation; many key genes highlighted in the sequencing data were not assessed. Moreover, the regional differences reported in Lgr5, Olfm4, and Ascl2, appearing much higher in proximal samples than in distal ones, were not recapitulated by qPCR validation of Olfm4, and this discrepancy was not addressed. Resolving such inconsistencies will be important for interpreting the dataset.

The basis for linking particular gene sets to NEC susceptibility rests largely on their spatial restriction to the distal intestine and their temporal regulation between early (day 0-14) and later (weeks 3-4) developmental stages. While this is a reasonable approach for generating hypotheses, the correlations have limited interpretive power without experimental validation, which is not provided here. Many factors beyond NEC may drive regional and temporal differences in intestinal development.

Finally, the contribution of human fetal biopsy samples is minimal. The central figure presenting these data (Figure 4A) shows immunofluorescence for LGR5, a single stem cell marker. The staining at day 35 is not convincing, and the conclusions that can be drawn are limited to confirming the localization of LGR5-positive cells to crypts as early as 26 weeks.

Navrhuju přeformulovat:

Situace je ale složitá – mladí lidé obvykle stojí na začátku své kariéry, nemají stabilní zaměstnání ani stabilní příjmy, zároveň jejich platy (zatím) nejsou tak vysoké a neměli ještě šanci si vytvořit dostatečné úspory.

eLife Assessment

This valuable study examined the roles of the posterior parietal cortex in rats performing an auditory change-detection decision task. It provided solid evidence for two subpopulations with opposing modulation patterns during decision formation and for a correspondence between neural and behavioral measures of the short timescale used for evidence evaluation.

Joint Public Review:

In this study, the authors sought to characterize the relationship between the timescales of evidence integration in an auditory change detection task and neural activity dynamics in the rat posterior parietal cortex (PPC), an area that has been implicated in the accumulation of sensory evidence. Using the state-of-the-art Neuropixel recording techniques, they identified two subpopulations of neurons whose firing rates were positively and negatively modulated by auditory clicks. The timescale of click-related response was similar to the behaviorally measured timescale for evidence evaluation. The click-related response of positively modulated neurons also depended on when the clicks were presented, which the authors hypothesized to reflect a time-dependent gain change to implement an urgency signal. Using muscimol injections to inactivate the PPC, they showed that PPC inactivation affected the rats' choices and reaction times.

There are several strengths of this study, including:

(1) Compelling evidence for short temporal integration in behavioral and neural data for this task.

(2) Well-executed and interpretable comparisons of psychophysical reverse correlation with single-trial, click-triggered neuronal analyses to relate behavior and neural activity.

(3) Inactivation experiments to test for causality.

(4) Characterization of neural subpopulations that allows for complex relationships between a brain region and behavior.

(5) Experimental evidence for an interesting way to use sensory gain change to implement urgency signals.

There are also some concerns, including:

(1) The work could be better contextualized. From a normative Bayesian perspective, the observed adaptation of timescales and gain aligns closely with optimal strategies for change detection in noisy streams: placing greater weight on recent sensory samples and lowering evidence requirements as decision urgency grows. However, the manuscript could go further in explicitly connecting the experimental findings to normative models, such as leaky accumulator or dynamic belief-updating frameworks. This would strengthen the broader impact of the work by making clear how the observed PPC dynamics instantiate computationally optimal strategies.

(2) It is unclear how the rats are performing the task, both in terms of the quality of performance (they only show hit rates, but the rats also seem to have high false alarm rates), and in terms of the underlying strategy that they seem to be using.

(3) A major conceptual weakness lies in the claim that PPC "dynamically modulates evidence evaluation in a time-adaptive manner to suit the behavioral demands of a free-response change detection task." To support this claim, it would require direct comparison of neural activity between two task demands, either in two tasks or in one task with manipulations that promote the adoption of different timescales.

(4) Some analyses of neural data are lacking or seem incomplete, without considering alternative interpretations.

(5) The muscimol inactivation results did not provide a clear interpretation about the link between PPC activity and decision performance.

Animation: carton et fils de laine entre les cartons : direction des AM en traction (normales et/ou cisaillement)

A la lecture des documents, trouver quelle est la problématique juridique majeur

La problématique est que les liens hypertexte sont soumis aux droits d'auteur. Sauf qu'il est possible d'en obtenir l'accès d'oeuvre proteger sans consentement de l'auteur. Ici c'est considerer que la communication est a l'auteur de choisir mais en soit le lien hypertexte n'est pas soumis c'est comment on la met en forme. La c'est à un large public sans restriction. Une exception à la règle pourrait être le framing.

Faire le lien avec le cours : à quelle partie du cours se rattache la thématique ?

cela renvoie aux droits d'auteur dans le cadre de la propriété intellectuelle sur les propriété littéraire et artistique.

eLife Assessment

This study presents valuable findings regardingg a rare mode of reproduction called hybridogenesis in a species pair of frogs. While parts of the study provide solid support for the claim of hybridogenesis, other parts are incomplete with certain claims being only partially supported, as alternative modes of reproduction cannot be fully ruled out.

Reviewer #1 (Public review):

Summary:

(1) Introduction Hybridogenesis involves one genome being clonally transmitted while the other is replaced by backcrossing. It results in high heterozygosity and balanced ancestry proportions in hybrids. Distinguishing it from other hybrid systems requires a combination of nuclear, mitochondrial, and population-genetic evidence. Hybridogenesis has been identified in only a few taxa (e.g., some fish, frogs, and stick insects), but no new cases have been reported in over a decade. Advancements in high-throughput sequencing now allow for the detection of high individual heterozygosity, which can indicate hybridization, but it is difficult to distinguish hybridogenesis from other similar asexual systems based solely on genome-wide data. To differentiate these systems, researchers look at several key indicators: Presence of pure-species offspring from hybrids (possible only in hybridogenesis); sex ratio (male presence in hybridogenetic systems); nuclear and mitochondrial haplotype sharing with co-distributed parental species; geographic distribution patterns, especially the lack of both parental species in hybrid populations.

(2) What the authors were trying to achieve The paper studies Quasipaa Frogs. Q. robertingeri (narrowly endemic) and Q. boulengeri (widespread), which are morphologically similar and found sympatrically in parts of China. Preliminary RAD-seq data revealed bimodal heterozygosity in Q. boulengeri samples. Some individuals had extremely high heterozygosity, consistent across loci and suggestive of F1 hybrids. These high-heterozygosity individuals had one haplotype from each species. The study investigates the high heterozygosity observed in Quasipaa frogs, particularly in individuals morphologically resembling Q. boulengeri but genetically appearing to be F1 hybrids with Q. robertingeri. The goal is to determine whether these patterns are consistent with hybridogenesis, rather than other atypical reproductive modes. The authors also suggest the hypothesis that hybridogenesis could enable range expansion of an endemic species through hybridization with a widespread relative.

(3) Methods A total of 107 individuals from 53 localities were collected for the study. This sample included 58 sexed adults-27 males and 31 females-as well as a majority of tadpoles. Of these individuals, 31 had previously determined karyotypes. DNA was extracted and sequenced. Individual heterozygosity and ancestry were estimated using bioinformatics tools. F1 hybrids were compared to one of the parental species to examine patterns of fixed heterozygous loci. Mitochondrial DNA was also extracted from sequencing data, and phylogenetic trees were constructed

(4) Results Two groups of individuals were detected based on heterozygosity: one group exhibited high heterozygosity and consisted of F1 hybrids, while the other group showed low heterozygosity, representing pure-species types. The F1 hybrids demonstrated approximately equal ancestry from Q. robertingeri and Q. boulengeri, consistently maintaining a high proportion of heterozygous loci at around 16.7%. In contrast, pure individuals had much lower heterozygosity, approximately 2.9%. F1 hybrids were found across 21 different sites, including both male and female individuals. The presence of numerous fixed heterozygous loci in F1 hybrids confirmed their hybrid origin, and these loci were absent in pure Q. boulengeri samples. F1 individuals typically carried one haplotype from each parental species. There was minimal haplotype sharing between the two pure species, but extensive sharing was observed between F1 hybrids and co-occurring pure-species individuals. In fact, F1 types shared haplotypes with local Q. boulengeri in over 90% of cases, which supports the occurrence of local backcrossing and parental contribution. In terms of mitochondrial DNA, F1 hybrids possessed mitochondrial haplotypes that clustered with Q. boulengeri and often shared these haplotypes directly. Genetic structure and phylogenetic analyses, revealed three distinct genetic clusters corresponding to F1 hybrids, Q. boulengeri, and Q. robertingeri. The F1 hybrids positioned themselves intermediate between the two pure species. Neighbor-joining trees and TreeMix analyses confirmed a strong separation between pure-species types, with F1 hybrids clustering alongside local Q. boulengeri subpopulations, indicating local formation of hybrids.

(5) Discussion In summary, the study reveals hybridogenesis (a reproductive system where hybrids clonally transmit one parental genome) in Quasipaa boulengeri and Q. robertingeri. Hybrids show high genetic heterozygosity and coexist with parental species, ruling out other reproductive modes like parthenogenesis or kleptogenesis. Evidence suggests hybridogenesis enables Q. robertingeri genomes to appear far outside their normal range, possibly aiding range expansion. Chromosomal abnormalities are linked to hybrid hybrids, supporting clonal genome transmission. The genetic divergence between parental species fits patterns seen in other hybridogenetic systems, highlighting a unique, understudied case in East Asia.

Strengths:

Overall, the authors carefully interpret their genetic data to support hybridogenesis as the reproductive mode in this system and propose that this mechanism may aid range expansion. They also appropriately acknowledge the need for further cytogenetic and ecological studies, demonstrating scientific caution. In summary, the discussion reasonably follows from the results, offering cautious interpretation where necessary.

Weaknesses:

Direct reproductive or cytological evidence is still lacking. While alternative reproductive modes are discussed and mostly ruled out logically, some require further empirical testing. The authors maintain a cautious interpretation, appropriately suggesting further research. Some outstanding questions remain.

(1) The elevated heterozygosity and presence of fixed heterozygous loci in hybrids compared to parental species strongly indicate hybridogenesis. However, alternative explanations such as repeated F1 hybridization or some form of balanced polymorphism, while less likely, are not fully excluded.

(2) The coexistence of hybrids and parental species, along with high nuclear and mitochondrial haplotype sharing between hybrids and Q. boulengeri, argues against reproductive modes like parthenogenesis, gynogenesis, or kleptogenesis. However, the assumption that hybrid sterility or multiple local hybrid origins are unlikely could be challenged if undetected local variation or cryptic reproductive strategies exist.

(3) The presence of Q. robertingeri nuclear genomes far outside their known geographic range, genetically linked to nearby populations, fits a hybridogenetic-mediated dispersal model. Although the authors dismiss human-mediated or accidental transport as explanations, these scenarios are not necessarily unlikley.

Reviewer #2 (Public review):

This study describes F1 hybrid frog lineages that use an "unusual" form of reproduction, perhaps hybridogenesis. Identifying such species is important for understanding the biodiversity of reproduction in animals, and animals that do not reproduce via "canonical" sex can be useful model systems in ecology and evolution. The conclusion of the study are based on reduced representation sequencing (RAD-seq with a de-novo assembly of loci) of 107 wild-caught individuals from 53 localities (plus 4 outgroup individuals), including 27 males, 31 females, and 49 juveniles of unknown sex. Conclusive inferences of unusual forms of reproduction typically require breeding studies and parent-offspring genotype comparisons but such information is not available (and perhaps impossible to generate) for the focal frog lineages.

(1) Conclusion 1: there are two pure species and F1 hybrids

The authors infer that there are two lineages RR and BB (corresponding to two named species), and F1 interspecific hybrids RB. This inference is based on the results presented in Figure 1 (PCA, admixture, and heterozygosity analyses) as well as analyses of fixed SNP differences between R and B. I think that this conclusion is well supported; my only comment on this part is that it would be useful to have the admixture plots & cross-validation for the 107 samples with other k values (not only k=2) as a supplemental figure. The plots in the supplemental file S1 are for the subset of 55 inds inferred to be BB only.

(2) Conclusion 2: F1 hybrids most likely reproduce via hybridogenesis

This conclusion is based on the sex ratio of hybrids and haplotype sharing between species and lineages at different, ~150 bp long loci. Parthenogenesis (including sperm-dependent parthenogenesis) is unlikely to generate males, yet sexed F1 hybrid individuals include 18 females and 10 males which prompts the exclusion of parthenogenesis in the present paper. Specific haplotype-sharing patterns are also discussed in the study and used as further support, but these arguments (and the related main and supplementary figures) are difficult to read/interpret. To clarify the arguments related to haplotype sharing and haplotype diversities, I suggest that the authors phase the R and B haplotypes from all their hybrids by using their pure (RR and BB individuals) as references. The concatenated lineage-specific haplotypes can then be used to reconstruct a single phylogenetic tree for all loci (easier to visualize and interpret that the separate haplotype networks for the loci). The authors can then draw cartoon phylogenies for what would be the expected pattern for haplotype clustering and diversity for different reproductive modes, and discuss their observed phylogenies in this regard. Similarly, the migration weights (represented in Figure 4) can then also be computed for separate haplotypes in the hybrids.

However, independently of the outcome of the phasing, it is important to note that there is no a priori reason why all F1 hybrid individuals would reproduce via the same reproductive mode. Notably, work by Barbara Mantovani and Valerio Scali on stick insects has shown that different F1 hybrid lineages involving the same parental species reproduce via hybridogenesis or parthenogenesis. I don't see how the presented data can allow excluding that some F1 hybrid frogs are parthenogenetic while others are hybridogenetic for example.

(3) Conclusion 3: Crosses between hybridogenetic RB males and hybridogenetic RB females gave rise to a new population of RR individuals outside of the RR species range (this new population would correspond to location 30 from Figure 1).

It is not entirely clear to me which data this conclusion is based on, I believe it is the combination of known species ranges for the species R (location 30 being outside of this) and the relatively low heterozygosity of RR individuals at location 30.

However, as the authors point out, the study focuses on an understudied geographic range. Isolated or rare populations of the R species may easily have been overlooked in the past, especially since the R and B species are morphologically difficult to distinguish. Furthermore, an isolated, perhaps vestigial population may also likely be inbred/feature low diversity. It seems most appropriate to discuss different (equally likely) scenarios for the RR population at location 30 rather than implying a hybridogenetic origin of RR individuals. I would also choose a title that does not directly imply this scenario but reflects the solid (not speculative) findings of the study.

Reviewer #3 (Public review):

Summary:

This work reports a new case of hybridogenetic reproduction in the frog genus Quasipaa. Only one other example of this peculiar reproductive mode is known in amphibians, and fewer than a dozen across the tree of life. Interestingly, a population of one of the parental species (Q. robertingeri) was found away from the core of its distribution, within the distribution of the hybridogens. This range expansion might have been mediated by hybridogenesis, whereby two copies of the same parental genome came together again after many generations of hybridogenesis.

Strengths:

Evidence for hybridogenesis is solid. The state of the art would be to genotype parents and offspring, but other known alternative scenarios have been considered carefully and can be ruled out convincingly. In addition, the authors are very careful in their phrasing and made sure to never overinterpret their data.

The explicit predictions under different reproductive modes (and Table 1) are a useful resource for future studies and could inspire new findings of unusual reproductive modes in other taxa.

The sampling is very impressive, with over 50 populations sampled across a very large area.

The comparison of p-distances between pairs of species involved in hybridogenesis is interesting.

Weaknesses:

The current phylogenetic reconstruction with the F1s does not enable to infer the number of origins of hybridogenesis, nor whether the population of Q. robertingeri that was found far from the core of the species' distribution indeed derives from hybridogenesis. This is because some of the signal is driven by the Q. boulengeri haplome, which is replaced every generation and therefore does not reflect the evolutionary history of the lineage.

All known reproductive modes except hybridogenesis can be excluded, but without genotyping parents and offspring, it is impossible to rule out another, yet undescribed reproductive mode.

DOI: 10.1101/2025.06.02.656652

Resource: Nipype (RRID:SCR_002502)

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Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

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