DOI: 10.1038/s41593-025-01937-y

Resource: RRID:BDSC_5428

Curator: @scibot

SciCrunch record: RRID:BDSC_5428

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: RRID:BDSC_7374

Curator: @scibot

SciCrunch record: RRID:BDSC_7374

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: RRID:BDSC_35512

Curator: @scibot

SciCrunch record: RRID:BDSC_35512

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: RRID:BDSC_58362

Curator: @scibot

SciCrunch record: RRID:BDSC_58362

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: RRID:BDSC_29042

Curator: @scibot

SciCrunch record: RRID:BDSC_29042

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: RRID:BDSC_64826

Curator: @scibot

SciCrunch record: RRID:BDSC_64826

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: RRID:BDSC_35836

Curator: @scibot

SciCrunch record: RRID:BDSC_35836

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: RRID:BDSC_6480

Curator: @scibot

SciCrunch record: RRID:BDSC_6480

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: RRID:BDSC_7077

Curator: @scibot

SciCrunch record: RRID:BDSC_7077

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: (Jackson ImmunoResearch Labs Cat# 705-545-003, RRID:AB_2340428)

Curator: @scibot

SciCrunch record: RRID:AB_2340428

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: (Jackson ImmunoResearch Labs Cat# 711-165-152, RRID:AB_2307443)

Curator: @scibot

SciCrunch record: RRID:AB_2307443

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: RRID:Addgene_66105

Curator: @scibot

SciCrunch record: RRID:Addgene_66105

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: (Jackson ImmunoResearch Labs Cat# 711-475-152, RRID:AB_2340616)

Curator: @scibot

SciCrunch record: RRID:AB_2340616

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: (Jackson ImmunoResearch Labs Cat# 703-545-155, RRID:AB_2340375)

Curator: @scibot

SciCrunch record: RRID:AB_2340375

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: (Jackson ImmunoResearch Labs Cat# 712-605-153, RRID:AB_2340694)

Curator: @scibot

SciCrunch record: RRID:AB_2340694

What is this?

DOI: 10.1038/s41593-025-01937-y

Resource: (Jackson ImmunoResearch Labs Cat# 715-165-151, RRID:AB_2315777)

Curator: @scibot

SciCrunch record: RRID:AB_2315777

What is this?

DOI: 10.1038/s41467-025-59490-y

Resource: University of Pittsburgh Cryo-electron Microscopy Core Facility (RRID:SCR_025216)

Curator: @scibot

SciCrunch record: RRID:SCR_025216

What is this?

DOI: 10.1038/s41467-025-59490-y

Resource: RRID:Addgene_119816

Curator: @olekpark

SciCrunch record: RRID:Addgene_119816

What is this?

DOI: 10.1038/s41467-025-59458-y

Resource: RRID:Addgene_206159

Curator: @scibot

SciCrunch record: RRID:Addgene_206159

What is this?

DOI: 10.1038/s41467-025-59218-y

Resource: (MGI Cat# 2670020,RRID:MGI:2670020)

Curator: @scibot

SciCrunch record: RRID:MGI:2670020

What is this?

DOI: 10.1038/s41467-025-59153-y

Resource: Connectivity Toolbox (RRID:SCR_009550)

Curator: @scibot

SciCrunch record: RRID:SCR_009550

What is this?

DOI: 10.1038/s41467-025-59153-y

Resource: ICBM 152 Nonlinear atlases version 2009 (RRID:SCR_008796)

Curator: @scibot

SciCrunch record: RRID:SCR_008796

What is this?

DOI: 10.1038/s41467-025-59153-y

Resource: Analysis of Functional NeuroImages (RRID:SCR_005927)

Curator: @scibot

SciCrunch record: RRID:SCR_005927

What is this?

DOI: 10.1038/s41467-025-59153-y

Resource: Mindboggle (RRID:SCR_002438)

Curator: @scibot

SciCrunch record: RRID:SCR_002438

What is this?

DOI: 10.1038/s41467-025-59153-y

Resource: FreeSurfer (RRID:SCR_001847)

Curator: @scibot

SciCrunch record: RRID:SCR_001847

What is this?

DOI: 10.1038/s41467-025-59153-y

Resource: FMRIPREP (RRID:SCR_016216)

Curator: @scibot

SciCrunch record: RRID:SCR_016216

What is this?

DOI: 10.1038/s41467-025-59153-y

Resource: ANTS - Advanced Normalization ToolS (RRID:SCR_004757)

Curator: @scibot

SciCrunch record: RRID:SCR_004757

What is this?

DOI: 10.1038/s41467-025-59153-y

Resource: Nipype (RRID:SCR_002502)

Curator: @scibot

SciCrunch record: RRID:SCR_002502

What is this?

DOI: 10.1038/s41467-025-59153-y

Resource: FSL (RRID:SCR_002823)

Curator: @scibot

SciCrunch record: RRID:SCR_002823

What is this?

DOI: 10.1038/s41467-025-59129-y

Resource: RRID:Addgene_12260

Curator: @scibot

SciCrunch record: RRID:Addgene_12260

What is this?

DOI: 10.1038/s41467-025-59129-y

Resource: RRID:Addgene_52961

Curator: @scibot

SciCrunch record: RRID:Addgene_52961

What is this?

DOI: 10.1038/s41467-025-59129-y

Resource: RRID:Addgene_107169

Curator: @olekpark

SciCrunch record: RRID:Addgene_107169

What is this?

DOI: 10.1038/s41467-025-59010-y

Resource: RRID:Addgene_139554

Curator: @scibot

SciCrunch record: RRID:Addgene_139554

What is this?

DOI: 10.1038/s41467-025-59010-y

Resource: RRID:Addgene_21189

Curator: @scibot

SciCrunch record: RRID:Addgene_21189

What is this?

DOI: 10.1038/s41467-025-58985-y

Resource: RRID:Addgene_135584

Curator: @scibot

SciCrunch record: RRID:Addgene_135584

What is this?

DOI: 10.1038/s41467-025-58753-y

Resource: RRID:Addgene_52961

Curator: @scibot

SciCrunch record: RRID:Addgene_52961

What is this?

DOI: 10.1038/s41467-025-58753-y

Resource: RRID:Addgene_48138

Curator: @scibot

SciCrunch record: RRID:Addgene_48138

What is this?

DOI: 10.1038/s41467-025-58753-y

Resource: RRID:Addgene_8454

Curator: @scibot

SciCrunch record: RRID:Addgene_8454

What is this?

DOI: 10.1038/s41467-025-58737-y

Resource: RRID:Addgene_112867

Curator: @scibot

SciCrunch record: RRID:Addgene_112867

What is this?

DOI: 10.1038/s41467-025-58737-y

Resource: RRID:Addgene_104963

Curator: @scibot

SciCrunch record: RRID:Addgene_104963

What is this?

DOI: 10.1038/s41467-025-58737-y

Resource: RRID:BDSC_36303

Curator: @scibot

SciCrunch record: RRID:BDSC_36303

What is this?

DOI: 10.1038/s41467-025-58737-y

Resource: RRID:Addgene_32396

Curator: @scibot

SciCrunch record: RRID:Addgene_32396

What is this?

DOI: 10.1038/s12276-025-01435-y

Resource: RRID:Addgene_26966

Curator: @scibot

SciCrunch record: RRID:Addgene_26966

What is this?

DOI: 10.1038/s12276-025-01435-y

Resource: RRID:Addgene_12260

Curator: @olekpark

SciCrunch record: RRID:Addgene_12260

What is this?

DOI: 10.1038/s12276-025-01435-y

Resource: RRID:Addgene_12259

Curator: @olekpark

SciCrunch record: RRID:Addgene_12259

What is this?

DOI: 10.1016/j.xcrm.2025.102126

Resource: (RRID:CVCL_3481)

Curator: @scibot

SciCrunch record: RRID:CVCL_3481

What is this?

DOI: 10.1016/j.str.2025.04.012

Resource: (Jackson ImmunoResearch Labs Cat# 715-165-150, RRID:AB_2340813)

Curator: @scibot

SciCrunch record: RRID:AB_2340813

What is this?

Mình vừa có một chuyến đi trong ngày tuyệt vời đến Ninh Thuận với tour Đồng Cừu - Vịnh Vĩnh Hy - Hang Rái - Vườn Nho của Nha Trang Travel và muốn chia sẻ nhanh trải nghiệm này. Nếu bạn không có nhiều thời gian nhưng vẫn muốn khám phá những nét đặc sắc nhất của vùng đất này, đây thực sự là một lựa chọn rất đáng cân nhắc.

Hành trình bắt đầu với Đồng Cừu Suối Tiên, một không gian xanh mát và yên bình. Những chú cừu trắng ở đây rất thân thiện, tha hồ cho bạn chụp những bức ảnh đáng yêu. Tiếp đó, chúng mình di chuyển đến Vịnh Vĩnh Hy. Cung đường biển ở đây đẹp ngoạn mục, với làn nước trong xanh và những dãy núi hùng vĩ ôm trọn lấy vịnh.

Sau bữa trưa với các món hải sản địa phương khá ngon miệng, điểm đến tiếp theo là Hang Rái. Nơi đây thực sự là một kỳ quan thiên nhiên với bãi san hô cổ hóa thạch và những khối đá có hình thù độc đáo. Khung cảnh vừa kỳ vĩ vừa có chút huyền ảo. Cuối cùng, đoàn ghé thăm Vườn Nho Ninh Thuận, được đi dạo dưới những giàn nho trĩu quả, thưởng thức nho tươi và tìm hiểu về quy trình trồng nho.

Về dịch vụ, xe đưa đón của Nha Trang Travel rất thoải mái và đúng giờ. Anh hướng dẫn viên nhiệt tình, cung cấp nhiều thông tin thú vị suốt chuyến đi. Với lịch trình được sắp xếp hợp lý, tham quan được nhiều điểm đẹp và dịch vụ chu đáo, mình thấy tour này mang lại giá trị rất tốt.

Tóm lại, nếu bạn muốn một chuyến đi nhanh gọn, tiện lợi và đầy ắp trải nghiệm đáng nhớ tại Ninh Thuận, mình hoàn toàn giới thiệu tour này của Nha Trang Travel. Chắc chắn bạn sẽ có một ngày thật vui và ý nghĩa.

DOI: 10.1016/j.scr.2025.103728

Resource: (Abcam Cat# ab124964, RRID:AB_11129103)

Curator: @scibot

SciCrunch record: RRID:AB_11129103

What is this?

DOI: 10.1016/j.isci.2025.112508

Resource: (Thermo Fisher Scientific Cat# 13-4170-82, RRID:AB_657506)

Curator: @scibot

SciCrunch record: RRID:AB_657506

What is this?

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __ Summary In this work, the authors present a careful study of the lattice of the indirect flight muscle (IFM) in Drosophila using data from a morphometric analysis. To this end, an automated tool is developed for precise, high-throughput measurements of sarcomere length and myofibril width, and various microscopy techniques are used to assess sub-sarcomeric structures. These methods are applied to analyze sarcomere structure at multiple stages in the process of myofibrillogenesis. In addition, the authors present various factors and experimental methods that may affect the accurate measurement of IFM structures. Although the comprehensive structural study is appreciated, there are major issues with the presentation/scope of the work that need to be addressed: Major Comments 1. The main weakness of the paper is in its claim of presenting a model of the sarcomere. Indeed, the paper reports a structural study that is drawn onto a 3D schematic. There is no myofibrillogenesis model that would provide insights into mechanisms. Therefore, the use of the word model is grossly overstated.

In biology, the term “model” is used in various contexts, but it generally refers to a simplified representation of a biological system, a structure or a process. Accordingly, we consider “model” the most fitting phrase for what we present in Figure 4 (Figure 7 in the revised manuscript). These are not arbitrary 3D schematics; they are scaled representations in which the length, the number and the relative three-dimensional arrangement of thin and thick filaments are based on measurements. These measurements are primarily based on our own data (presented in the main text and provided in the supplementary materials), as published data were either lacking or inconsistent. Moreover, we would like to highlight that we do not claim to present a conceptual or mechanistic model of myofibrillogenesis, but we do present structural reconstructions or models for four developmental time points. Therefore, we disagree with the remark that “the use of the word model is grossly overstated”, as our wording fully corresponds to the common sense.

In general, the major focus and contribution of the work is unclear. How does the comprehensive nature of the measurements contribute to existing literature?

We significantly revised the text to highlight the main points more firmly, and added an additional section to help non-specialist readers to better understand our aims and findings.

Figure labels are often rather confusing - for example it is unclear why there is a B, B', B' etc instead of B,C,D, etc.

The figure labels have been revised in accordance with the reviewer’s recommendation.

Some comments in the text are not clearly tied to the figures. For example, in lines 108-109, are the authors referring to the shadow along the edges of the myofibril when saying they are not clearly defined (Figure 1C)?

The lines refer to the fact that identifying the boundary of an “object” in a fluorescence microscopy image is inherently challenging - even under ideal conditions where the object’s image is not affected by nearby signals or background noise. To improve clarity, we revised this section and now it reads: The other key parameter - myofibril diameter - is typically measured using phalloidin staining. However, accurately delineating their boundaries in micrographs is difficult - even under optimal conditions (high signal‑to‑noise ratio, no overlapping fibers, etc.; Fig. 1C). This limitation arises from the fundamental nature of light microscopy as the image produced is a blurred version of the actual structure, due to convolution with the microscope’s point spread function.

In line 116, it is unclear what "surrounding structures" the authors are referring to if the myofibrils are isolated.

We revised the text for clarity. It now states: Once isolated, myofibrils lie flat on the coverslip, aligning with the focal plane of the objective lens. This orientation allows for high-resolution, undistorted imaging and accurate two-dimensional measurements, free from interference by neighboring biological structures (e.g.: other myofibrils).

In lines 141-142, there is no reference of data to back up the claim of validation.

We addressed this mistake by including a reference to Fig. S1E (Fig. S1D in the revised manuscript).

In line 170, the authors mention the mef2-Gal4/+ strain as a Gal4 driver line but do not clearly state how this strain is different from the wildtypes or how this impacts their results.

Mef2-Gal4 is a muscle-specific Gal4 driver, often used in Drosophila muscle studies. It is a convention between Drosophila geneticists that presence of a transgene (i.e. Mef2-Gal4) changes the genetic background, and although it does not necessariliy cause any phenotypic effect, it is clearly distinguished from the wild type situation, and whenever relevant, Mef2-Gal4/+ is the preferred choice (if not the correct choice) as a control instead of wild type. As clear from our data, presence of the Mef2-Gal4 driver line does not affect the length or width of IFM sarcomeres as compared to wild type.

In lines 182-185, the authors discuss the effects of tissue embedding on morphometrics. Were factors such as animal sex, age, fiber type, etc. conserved in these experiments? If not, any differences in results may be confounding.

We fully agree with the reviewer that when testing the effect of a single variable, all other variables should remain constant. This is actually one of the main points emphasized in the results section. Additionally, this information is already provided in the Source Data files for each panel.

In lines 199-201, the authors discuss results of myofibril diameter using different preparation methods, yet no data is cited to support the claims. In line 220, the phrase "6 independent experiments" is unclear. Is each independent experiment performed using a different animal? Furthermore, are 6 experiments performed for each time point?

We substantially revised the relevant paragraphs and ensured that the corresponding data (Figure 2A in the revised manuscript) is cited each time when it is discussed. We conducted six independent experiments at each time point. This is consistently indicated in the figures and can be verified in the SourceData files (specifically, Fig3SourceData in this case). To clarify what we mean by "independent experiments," we added the following sentence to the Methods section: Experiments were considered independent when specimens came from different parental crosses, and each experiment included approximately six animals to capture individual variability.

In line 254, the authors refer to "number of sarcomeres". It must be clearly stated if this refers to sarcomeres per myofibril, image area, etc.

It is now clearly stated as: "number of sarcomeres per myofibril".

In line 274, the authors refer to "myofilament number". It must be clearly stated if this refers to myofilaments per myofibril, image area, etc.

We counted the number of myofilaments in developing myofibrils, and this is now clearly stated in the text and in the legend of Figure 3 (Figure 4 in the revised manuscript).

In line 299, the authors mention that thin filaments measured less than 560 nm in length, yet no data is cited to support this.

The previously missing reference to Figure 4 (Figure 7 in the revised manuscript) has now been added in addition to the revised Supplementary Figure 5.

In the "Quantifying sarcomere growth dynamics" section of the summary (starting from line 402) the authors introduce data that would be more naturally placed in the results and discussion section.

As suggested by the reviewer, we incorporated the key aspects of sarcomere growth dynamics into the Results and Discussion section.

In lines 422-423, it is not mentioned what the controls are for.

This was already explained in the main text between lines 167 and 173.

In the caption of Figure 1C, it is not mentioned what the red dashed lines in the microscope images represent.

The caption has been updated to include the following clarification: The red dashed lines border the ROI used for generating the intensity profiles.

In the caption of Figure 1D, the difference between the lighter and darker grey points is not mentioned.

This was already explained in each relevant figure legend. In this specific case, it is stated between lines 850 and 852: “Light gray dots represent individual measurements of sarcomere length and myofibril diameter, while the larger dots indicate the mean values from independent experiments.”

In line 849, the stated p-value (0.003) does not match that mentioned in the figure (0.0003).

We thank the reviewer for noticing this small mistake; correction was made to display the accurate p-value of 0.0003 at both places.

In line 874, it is not clear what an "independent experiment" refers to (different animal, etc.?).

We refer the reviewer to point 9, where this question has already been addressed.

Figure 2A is hard to read. Using different colored dots for different time points might help.

As suggested by the reviewer, we generated a plot with the individual points color-coded by time.

The significant figures presented in Figure 4 give a completely inaccurate representation of the variability of the measurements achieved with these techniques.

Certainly, each measured parameter exhibits inherent biological and technical variability. We have made all the raw data available to the reader through the SourceData files, and this variability is also evident in Figures 1, 2, 3, Supplementary Figure 1, 3, and 5 (Figure 1, 2, 3, 4, 6, and Supplementary Figure 1 in the revised manuscript). Also we have included an additional plot (Supplementary Figure 5 in the revised manuscript) that presents the calculated thin and thick filament lengths and their uncertainty. However, in Figure 4 (Figure 7 in the revised manuscript), our goal was to present an easily understandable visual representation of the sarcomeric structures for each time point, based on the averages of the relevant measurements.

In line 877, it should be mentioned that the number of filaments is counted per myofibril. The y-axes in the figure should also be adjusted to clarify this.

As suggested by the reviewer, both the figure legend and the plot have been updated to clearly indicate that the filament count refers to the number per myofibril.

In line 883, it is not clear what an "independent experiment" refers to (different animal, etc.?).

We refer the reviewer to point 9, where this question has already been addressed.

The statement of sample sizes in all figures is a little confusing.

Following general guidelines, we used SuperPlots to effectively present the data, as nicely demonstrated in the JCB viewpoint article by Lord et al., 2020 (PMID: 32346721). Individual measurements are shown as pooled data points, allowing readers to appreciate the spread, distribution and number of measurements. Overlaid on these pooled dot plots are the mean values from each independent experiment, with error bars representing variability between independent experiments. Sample sizes are provided for both individual measurements and independent experiments. This is now clearly explained in the Materials and Methods section, and we corrected the legends to improve clarity (“n” indicates the number of independent experiments/individual measurements).

In lines 1007-1008, the authors imply that the lattice model is needed for calculation of myofilament length. However, from the equations and previous data, it seems that this can be estimated using the confocal and dSTORM images.

As the reviewer correctly noted, myofilament length can be estimated using measurements from confocal and dSTORM images, following the equations provided. However, constructing even a simplified model requires multiple constraints to be defined and applied in a specific order. In practice, one must first determine the number and arrangement of myofilaments in a cross-sectional view of an “average sarcomere” before attempting to build a longitudinal model, where length calculations become relevant. This is now clarified in the text.

A more specific discussion of future directions is needed to put this paper in context. For example: Can anything from the overall process be used to better understand sarcomere dynamics in larger animals/humans? Can this be applied to disease modelling?

To address these questions, we have added a section titled STUDY LIMITATIONS, which states: “Our study is focused on describing the growth of IFM sarcomeres during myofibrillogenesis at the level of individual myofilaments. Additionally, we developed a user-friendly software tool for precise sarcomere size measurements and demonstrate that these measurements are sensitive to varying conditions. Whereas, this tool can be used successfully on whole muscle fiber preparations as well, our pipeline was intentionally optimized for individual IFM myofibrils ensuring higher measurement precision in our hands than other type of preparations. Thus, we predict that future work will be required to extend it to sarcomeres from other muscle tissues or species. Nevertheless, our study exemplifies a workflow how to measure sarcomere dimensions precisely. With some variations, it should be possible to adopt it for other muscles, including vertebrate and human striated muscles. To facilitate this and to enhance the accessibility and usability of this dataset, we welcome any feedback and suggestions from researchers in the field.”

One of the major claims of the paper is that there is a measurable variability with sex and other parameters. However, this data is never clearly summarized, presented (except for supplement), or discussed for its implications.

We followed the suggestion of the reviewer, and we moved this supplementary data into a main figure, and thoroughly revised the corresponding paragraphs to present and discuss the findings more clearly.

Minor Comments: 1. Lines 60-65 seem to break the flow of the introduction. As the authors discuss existing methods in literature for IFM analysis in the previous couple sentences, the following sentences should clearly state the limitations of existing methods/current gap in literature and a general idea of what the current work is contributing.

We agree with this remark, and we substantially revised the Introduction to clearly define the existing gap in the literature and to articulate how our work addresses this gap.

In line 104, the acronym for ZASPs is not spelled out.

The acronym has now been spelled out for clarity.

**Referee Cross-commenting**

I agree as well.

Reviewer #1 (Significance (Required)):

In summary, this paper provides a multi-scale characterization of Drosophila flight muscle sarcomere structure under a variety of conditions, which is potentially a significant contribution for the field. However, the paper scope is overstated in that it does not provide an actual sarcomere model. Further, there are multiple issues with data presentation that impact the readability of the manuscript.

Although it is somewhat unclear what would be “an actual sarcomere model” for the reviewer, but we cannot accept that we made on overstatement by using the word “model”, because one of the main outcomes of our work are indeed the myofilament level sarcomere models depicted in Figure 4 (Figure 7 in the revised manuscript). As said above, we do not claim that these would be molecular models, or mechanistic models or developmental models, but it makes absolutely nonsense (even in common terms!) that our scaled graphical representations (based on a wealth of measurements) should not be or cannot be called models.

As to the comment with data presentation, we thank the reviewer for the numerous suggestions, and we substantially revised the manuscript to increase clarity and overall readability.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __ Summary: In this manuscript titled "A myofilament lattice model of Drosophila flight muscle sarcomeres based on multiscale morphometric analysis during development," Görög et al. perform a detailed analysis of morphological parameters of the indirect flight muscle (IFM) of D. melanogaster. The authors start by illustrating the range of measurements reported in the literature for mature IFM sarcomere length and width, showing a need to revisit and determine a standardized measurement. They develop a new Python-based tool, IMA, to analyze sarcomere lengths from confocal micrographs of isolated myofibrils stained with phalloidin and a z-disc marker. Using this tool, they demonstrate that sample preparation (especially mounting medium), as well as fiber type, sex, and age influence sarcomere measurements. Combining IMA, TEM, and STORM data, they measure sarcomere parameters across development, providing a comprehensive and up-to-date set of "standardized" sarcomere measurements. Using these data, they generate a model integrating all of the parameters to model sarcomeres at four discrete timepoints of development, recapitulating key phases of sarcomere formation and growth.

Major comments: Line 200 & 901 - Figure S1B - The authors make a strong statement about the use of liquid versus hardening media, and it is clear from the image provided in Figure S1 that there is a difference in the apparent sarcomere width. The identity of the "liquid media" versus the "hardening media" should be clearly identified in the Results, in addition to the legend for Figure S1. The authors show that "glycerol-based solutions" increase sarcomere width, but the Materials only list 90% glycerol and PBS. However, a frequently used liquid mounting media is Vectashield. Based on the literature, measurements in liquid Vectashield show diameters significantly less than 2.2 microns observed here with presumably 90% glycerol or PBS. Can the authors qualify this statement, or provide data that all forms of liquid mounting media cause this effect? Does this also apply to hemi-thorax and sectioned preparations, or just isolated myofibrils?

We used a PBS-based solution containing 90% glycerol as our liquid medium, as now stated in the main text. In response to the reviewer’s suggestion, we also tested a non-hardening version of Vectashield (H-1000). Myofibrils in Vectashield were significantly thicker than those in ProLong Gold but still thinner than those in the 90% glycerol–PBS solution, shown in Figure 2B. The mechanisms that could potentially explain these observations have been described in several studies (Miller et al., 2008; Tanner et al., 2011, 2012). Briefly, IFM is a densely packed macromolecular assembly. Upon removal of the cell membrane, myofibrillar proteins attract water, leading to overhydration of the myofilament lattice. This increases the spacing between filaments, resulting in an expansion of overall myofibril diameter. The extent of hydration depends on the osmolarity of the surrounding medium, as the system eventually reaches osmotic equilibrium. While both liquid media induced significant swelling, the observed differences likely reflect variations in their osmotic properties. In contrast, dehydration - an essential step in electron microscopy sample preparation - reduces the spacing between filaments, making myofibrils appear thinner. This explains why EM micrographs consistently show significantly smaller myofibril diameters (Chakravorty et al., 2017).

        Hardening media such as ProLong Gold introduce additional artifacts: during polymerization, these media shrink, exerting compressive forces on the tissue (Jonkman et al., 2020). We therefore propose that isolated myofibrils first expand due to overhydration in the dissection solution, and are then compressed back toward their *in vivo* dimensions during incubation in ProLong Gold. The average *in vivo* diameter of IFM myofibrils can be estimated without direct measurements, as it is determined by two key factors: (i) the number of myofilaments, which has been quantified in EM cross-sections in several studies (Fernandes & Schöck, 2014; Shwartz et al., 2016; Chakravorty et al., 2017) including our own, and (ii) the spacing between filaments, which can be measured by X-ray diffraction even in live *Drosophila* or under various experimental conditions (Irving & Maughan, 2000; Miller et al., 2008; Tanner et al., 2011, 2012). Our findings suggest that the effects of lattice overhydration and media-induced shrinkage are most pronounced in isolated myofibrils. In larger tissue preparations, the inter-myofibrillar space likely acts as a mechanical and osmotic buffer, reducing the extent of such distortions

Can the authors comment on whether the length of fixation or fixation buffer solution, in addition to the mounting medium, make a difference on sarcomere length and diameter measurements? This is another source of variation in published protocols.

The effect of fixation time on sarcomere morphometrics in whole-mount IFM preparations has been previously demonstrated by DeAguero et al. (2019), as briefly noted in our manuscript. To extend these findings, we performed a comparison using isolated myofibrils, assessing morphometric parameters after fixation for 10, 20 (standard) and 60 minutes. We found no difference between the 10- and 20-minute fixation conditions; however, fixation for 60 minutes resulted in significantly increased myofibril diameter (and these data are now shown in Supplementary Figure 1C). A comparable increase in thickness was also observed when using a glutaraldehyde-based fixative. These results suggest that more extensively fixed myofibrils may better resist the compressive forces exerted by hardening media.

Line 237-238. The authors conclude that premyofibrils are much thinner than previously measured. The use of Airyscan to more accurately measure myofibril width at this timepoint is a good contribution, as indeed diffraction and light scatter likely contribute to increased width measured in light microscopy images. I also wonder, though, how well the IMP software performs in measuring width at 36h APF, given how irregular the isolated myofibrils at this stage look (wide z-lines but thinner and weaker H and I bands as shown in Fig. 2B)?

The reviewer is correct that measurements during the early stages of myofibrillogenesis require additional effort. However, in addition to its automatic mode, IMA can also operate in semi-automatic or manual modes, ensuring complete control over the measurements. Myofibril width is determined from the phalloidin channel at the Z-line (as described in the software’s User Guide and Supplementary Figure 2), where it is at its thickest.

Also, how much of the difference in sarcomere width arises due to effects of "stripping" components off of the sarcomere at the earliest timepoint (for example alpha-actinin or Zasp proteins)?

A comparison between isolated myofibrils and those from microdissected muscles (Supplementary Figure 3B, Figure 3C in the revised manuscript) shows that the isolation process does not alter the morphometric measurements of sarcomeres. Moreover, the measured myofibril width aligns well with what we expect based on the number of myofilaments observed in TEM cross-sections of myofibrils at 36 hours APF (Figure 3A, now Figure 4A in the revised manuscript), supporting the consistency of our model.

Myofibrils at early timepoints do contain more than 4-12 sarcomeres in a line (they extend the full length of the myofiber), so it is possible they are breaking due to the detergent and mechanical disruption induced by the isolation method.

The reviewer is correct - myofibrils likely span the full length of the myofiber from the onset of myofibrillogenesis. However, during the isolation of individual myofibrils, they often break, and even mature myofibrils typically fragment into pieces of about 300 µm in length (illustrated in Figure 1E, now Figure 2A in the revised manuscript). Importantly, our measurements show that this fragmentation does not affect the assessed sarcomere length or width (as shown in Supplementary Figure 3B, now Figure 3C in the revised manuscript).

Line 312 - What does "stable association" mean in this context? The authors mention early timepoints lack stable association of alpha-Actinin or Zasp52, and they reference Fig. S4C, but this figure only shows 72h and 24 AE, not 36h and 48 h APF. Previous reports have seen localization of both alpha-Actinin and Zasp52, so presumably the detergent or mechanical isolation is stripping these components off of the isolated myofibrils up until 72h.

In agreement with previous reports, we also detected both α-Actinin (as shown in former Supplementary Figure 3B, now Figure 3C) and Zasp52 in microdissected IFM starting from 36 hours APF. However, these markers were largely absent from the isolated myofibrils of young pupae (36 to 60 hours APF). By 60 hours APF, strong α-Actinin and Zasp52 staining became evident in isolated myofibrils, whereas dTitin epitopes were clearly detectable from the earliest time point examined. This indicates that some proteins, such as α-Actinin and Zasp52, can be lost during the isolation process, whereas others like dTitin are retained and this differential sensitivity appears to depend on developmental stage. A likely explanation is that α-Actinin and Zasp52 are recruited early to Z-bodies but are only fully incorporated as more mature Z-disks form between 48 and 60 hours APF. This incomplete incorporation at the earlier stages could account for their loss during the isolation process. This interpretation is supported by our morphological analysis of the Z-discs, as shown in the dSTORM dataset (former Figure 3B, B’’, now Figure 4C, E) and in longitudinal TEM sections (former Supplementary Figure 5B, now in Figure 6B). Because α-Actinin and Zasp52 are not detected in isolated myofibrils at 36 and 48 hours APF, they are not included in Figure S4C (Figure 5C in the revised manuscript). This is explained in the updated figure legend.

This same type of issue comes up again in Lines 325-334, where the authors talk about 3E8 and MAC147. They state that 3E8 signal significantly declines in later stages and that MAC147 is not suitable to label myofibrils in young pupae, but they only show data from 72 APF and 24 AE (which looks to have decent staining for both 3E8 and MAC147). A clearer explanation here would be helpful.

To put it simply: we used one myosin antibody to label the A-band in the IFM of 36h APF and 48h APF animals, and a different antibody for the 72h APF and 24h AE stages. In more detail: Myosin 3E8 is a monoclonal antibody targeting the myosin heavy chain and labels the entire length of mature thick filaments except for the bare zone (former Supplementary Figure 4D, now in Figure 5D), suggesting its epitope is near the head domain. As a result, we expect a uniform A-band staining - excluding the bare zone - which is exactly what we observe in the IFM of young pupae (36h APF and 48h APF; formerly Figure 3B, now Figure 4C in the revised manuscript). However, at 72h APF and 24h AE, Myosin 3E8 produces a different staining pattern: two narrow stripes flanking the bare zone and two broader, more diffuse stripes near the A/I band junction (former Supplementary Figure 4D, now Figure 5D). This change is likely due to restricted antigen accessibility at these later developmental stages - a common issue in the densely packed IFM - making this antibody unsuitable for reliably measuring thick filament length in these stages.

MAC147 is another monoclonal antibody against Mhc that recognizes an epitope near the head domain. However, it only works reliably in more mature myofibrils (72h APF and 24h AE; formerly Figure 3B, now Figure 4C in the revised manuscript), likely due to its specificity for a particular Mhc isoform. This is why we do not include images from earlier developmental stages using this antibody. We added a revised, concise explanation in the main text for general readers, and provided a more detailed description for specialist readers in the legend of Supplementary Figure 4D (updated as Figure 5D in the revised manuscript).

Figure 3B. The authors show the H, Z, and I lengths in B', B', and B' and discuss these lengths in the text (lines 305-320). It would also be nice to actually have the plots showing the measured/calculated lengths for thin and thick filaments. These are mentioned in the results, but I cannot find the plots in the figures and there is no panel reference.

A summary table of the measured and calculated parameters is provided in Fig4SourceData (Fig7Source Data in the revised manuscript). However, following the reviewer’s suggestion, we also generated an additional plot (Supplementary Figure 5 in the revised manuscript) that displays the calculated thin and thick filament lengths.

Line 400. Does the model in Figure 4 actually have molecular resolution as the authors claim? From these views, thick and thin filaments appear to be represented by cylindrical objects. Localization of specific molecules would require further modeling with individual proteins. Or do the authors mean localization from STORM imaging relative to the ends of the thick and/or thin filaments? The model itself is a useful contribution, but based on Figure 4, resolution of individual molecules is not evident.

The reviewer is correct; and we fully agree that we do not present a molecular model of sarcomeres in this study - nor do we claim to. Instead we present a myofilament level model. Nevertheless, the scaled myofilament lattice model we introduce could serve as a geometric constraint when constructing supramolecular models of sarcomeres. As the reviewer rightly notes, implementing such an approach would require additional effort.

The main Results section of the text is condensed into 4 figures. However, I found myself flipping back and forth between the main figures and the supplement continuously, especially parts of Supplemental Figures 1, 3, 4, and 5. With such large amounts of detail in the Results relying on the supplement, it may be worth considering reorganizing the main and supplemental figures, and having 7 main figures, to include important panels that are currently in the supplement (esp. Fig S1B, S1C, S1D, S3B, S4, S5).

We found it a very useful suggestion, and we substantially reorganized the figures in the revised manuscript according to the recommendations of the reviewer.

Minor comments: On the plots in Fig. S1B, D, and F, it is hard to see the color of the dots because the red error bars are on top of them. Can the other distribution dots be tinted the correct color or the x-axis labels be added, so it is clear which dataset is which?

We significantly enlarged the dots to enhance visual clarity.

Line 142 needs a reference to Figure S1, Panel E, which shows the accuracy and precision measurements.

The requested panel reference has now been included in the revised manuscript.

Lines 198 - is this range from the above publications? Needs to be clearly cited.

The range has indeed been estimated using measurements from the aforementioned publications, and this point is now further clarified in the revised text.

Figure S3B is confusing - why do the blow-ups overlap both the top (presumably microdissected) and the bottom (presumably isolated) images? The identity of microdissected images should be labeled, as they are hard to see underneath of the blown-up images and the identity of individual image planes wasn't immediately obvious.

We refined the panel structure of Figure S3B (Figure 3C in the revised manuscript) to enhance clarity as the reviewer suggested.

Line 298. By "misaligned," do the authors mean the pointed ends are not uniformly anchored in the z-disc, leading to the wide z-disc measurements? At this early stage, I'm not sure "misaligned" is the right word - perhaps "were not yet aligned in register at the z-disc" or something similar.

We revised the text for clarity. It now reads: At 36 hours APF, thin filaments had not yet aligned in perfect register at the Z-disc, with most measuring less than 560 nm in length - and exhibiting considerable variability.

Figure S6 - spelling mistake in label of panel A, "sarcomer" should be "sarcomere"

The typo is corrected.

Line 487. Spelling "Zaps52" should be "Zasp52"

The typo is corrected.

Line 887. Spelling "Myofilement" should be "Myofilament"

The typo is corrected.

Line 946-947. In the legend for Supp. Fig. 3., the authors should specify which published datasets on sarcomere length are shown in the figure by including the references in the legend. Presumably the "isolated individual myofibrils" are the blue "this study" lines, leaving the "microdissected muscles" as the magenta "previous reports" on the figure. Without the reference, it is not clear if these are microdissected, isolated myofibrils, hemi-thorax sections, cryosections, or another preparation method for the "previous reports" data.

The references have now been added to both the figure and its legend.

**Referee Cross-commenting**

I agree with the comments from the other reviewers. Many of the major themes are consistent across the reviews, including regarding the model, preparation methods, and the software tool.

Reviewer #2 (Significance (Required)):

Strengths: This manuscript is an important contribution to the field of sarcomere development. The authors use modern technologies to revisit variation in morphometric measurements in the literature, and they identify parameters that influence this variation. Notably, sex-specific differences, DLM versus DVM measurements, and mouting media are potential contributors to the variability. Combining TEM and STORM with a confocal timecourse of isolated myofibrils, they refine previously published values of sarcomere length and width, and add more comprehensive data for filament length, number and spacing. This highly accurate timecourse demonstrates continual growth of sarcomeres after 48 h APF, and correct some inconsistencies from previous large-scale timecourse datasets. These data are very valuable to the field, especially Drosophila muscle biologists, and will serve as a comparative resource for future studies. Weaknesses: At early timepoints, loss of sarcomere components through mechanical or detergent-mediated artifacts may influence the authors' measurements. In addition, isolating myofibrils is not always the most ideal approach, as it loses information on myofiber structure as well as organization and structure of the myofibrils in vivo.

We believe that the control experiments we presented here adequately demonstrate that sarcomere measurements are not affected by the myofibril isolation process at early timepoints (Figure 3C). Nevertheless, we certainly agree with the reviewer that isolated myofibrils alone cannot capture the entire complexity of muscle tissues, and additional approaches should also be applied in complex projects. Yet, we are confident that our approach offers the most reliable and efficient method for precise morphometric analysis of the sarcomeres, and although alone it is very unlikely to be sufficient to address all questions of a muscle development project, it can still be applied as a very useful and robust tool.

The point regarding liquid versus hardening mounting media is valuable, but remains to be tested and validated with the diverse liquid and hardening media used by other labs.

Whereas it would not be feasible for us to test all possible liquid and hardening media used by others in all possible conditions, we tested the effect of Vectashield (the most commonly used liquid media) according to the suggestion of the reviewer, and the results are now included in the manuscript. We think that this is a valuable extension of the list of the materials and conditions we tested, although we need to point out that our primary goal was not necessarily to test as many conditions as possible (because the number of those conditions is virtually endless), rather to raise awareness among colleagues that these variables can significantly impact the data obtained and affect their comparability.

The IMA software seems to be designed specifically for analysis of isolated myofibrils, and it is unclear if it would work for other types of IFM preparations.

As stated in the manuscript, IMA is a specialized tool designed for the analysis of individual myofibrils. While it can also process other types of IFM preparations in semi-automatic or manual modes, we believe these approaches compromise both efficiency and accuracy. This is further clarified in the revised manuscript.

A last point is that TEM and STORM may not be available on a regular basis to many labs, hindering wide implementation of the approach used in this manuscript to generate very accurate and detailed measurements of sarcomere morphometrics.

Regarding the availability of TEM and STORM, we acknowledge that these techniques are not universally accessible. However, that is exactly one major value of our work that our open-source software tool now allows researchers to generate valuable data using only a confocal microscope in combination with our published datasets.

Audience: Scientists who study sarcomerogenesis or Drosophila muscle biology.

My expertise: I study muscle development in the Drosophila model.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __ Summary: This manuscripts presents a computational tool to quantify sarcomere length and myofibril width of the Drosophila indirect flight muscles, including developmental samples. This tool was applied to confocal and STORM super-resolution images of isolated myofibrils from adult and developing flight muscles. Thick filament numbers per myofibril were counted during development of flight muscles. A myofilament model of developing flight muscle myofibrils is presented that remains speculative for the early developmental stages.

Major comments: 1. The title of the manuscript appears unclear. What is a lattice model? Lattice is an ordered array. The filament array parameters for mature flight muscles was aready measured. It appears that the authors speculate how this order might be generated during sarcomere assembly, which is not studied in this manuscript as it is limited to periodic arrays after 36h APF.

As the reviewer correctly points out, a lattice refers to an ordered array - in the case of IFM sarcomeres, this includes both thin and thick filaments. Therefore, the phrase "myofilament lattice model of Drosophila flight muscle sarcomeres" specifically describes a model representing the spatial organization of these filament arrays within the sarcomere. To provide additional clarity for readers, we have revised the title to include more context. It now reads: Developmental Remodeling of Drosophila Flight Muscle Sarcomeres: A Scaled Myofilament Lattice Model Based on Multiscale Morphometrics

To create a model of these arrays, three essential pieces of information are required:

1) The length of the filaments,

2) The number of filaments, and

3) The relative position of the filaments.

While some direct measurements are available in the literature, and others can be used to calculate the necessary values, available data is often contradictory or simply different from each other (as described in our ms) making them unsuitable for constructing scaled models of the myofilament arrays. In contrast to that, here we present a comprehensive and consistent set of measurements that enabled us to build models not only of mature sarcomeres but also of sarcomeres at three other significant developmental time points.

Regarding the mention of "sarcomere assembly" in line 37, we intended it to refer to the growth of the sarcomeres, not their initial formation. We do not speculate about sarcomere assembly anywhere in the text. In fact, we have clearly stated multiple times that our focus is on the growth of the IFM myofilament array during myofibrillogenesis. Nevertheless, to avoid confusion, we revised the phrase in line 37 to "sarcomere growth".

The authors review the flight muscle sarcomere length literature and conclude it is variable because of imprecise measurements. Likely this is partially true, however, more importantly is that the sarcomere length and width changes during isolation methods of the myofibrils, as well as by various embedding methods, as the authors show here as well in Figure 1B-E.

We dedicated two sections of the Results - “An automated method to accurately measure sarcomeric parameters” and “IFM sarcomere morphometrics are affected by sex, age, fiber type, and sample preparation” - to exploring potential sources of variability in published IFM sarcomere measurements. Based on these analyses, we conclude that such variability stems from both measurement imprecision and biological or technical factors, including sex, age, fiber type and, of foremost, sample preparation. Because it is difficult to quantify the relative impact of each variable across published studies, we have refrained from speculations about the relative contribution of the different factors in the revised manuscript.

Hence, I find the strongly claims the authors make here surprising, while they are isolating the myofibrils. Hence, these myofibrils are ruptured at the ends, relaxed or contracted, depending on buffer choice and passive tension is released. On page 8, the authors correctly state that the embedding medium causes shrinkage of the myofibrils. While isolation is state of the art for electron microscopy techniques, other methods including sectioning or even whole mount preparation have been developed for high resolution microscopy of IFMs that avoid these artifacts. Unfortunately, this manuscript only uses isolated myofibrils that were fixed and then mechanically dissociated by pipetting. This method likely induces variations as seen by the large spread of sarcomere length reported in Figure 1C (2.8-3.9µm?) and even bigger spreads for myofibril widths. Are these also seen in tissue without dissections? Unfortunately, no comparision to intact flight muscles are reported with the here presented quantification tool. The sarcomere length spread in the developmental samples is even larger.

The major issue raised in this paragraph is the use of isolated myofibril versus intact flight muscle preparations. The reviewer claims that the latter might be superior because the isolated myofibrils are ruptured at their ends. Clearly, the intact IFMs cannot be imaged in vivo by light microscopy because the adult fly cuticle is opaque. To visualize these muscles, one must open the thorax, but neither microdissection nor sectioning preserves them perfectly, even the cleanest longitudinal cuts sever some myofibrils, and dissection itself can damage the tissue. Although published images often show only the most pristine regions, the practice of selective cropping cannot be taken as a scientific argument. Here, by comparing sarcomere lengths measured in isolated myofibrils with those from whole-mount longitudinal DLM sections and microdissected IFM myofibers, we demonstrate that isolation does not alter sarcomere length (Figure 1E, now Figure 2A in the revised manuscript). As to myofibril width, it is determined by two parameters: the number of myofilaments and the spacing between them. In vivo filament spacing has been measured directly, and filament counts can be obtained from EM cross-sections of DLM fibers. Combining these values gives an expected in vivo myofibril diameter. While isolated myofibrils measure thinner than those in whole-mount or microdissected samples (Figure 1E, now Figure 2A in the revised manuscript), their diameter closely matches this in vivo estimate (see manuscript, lines 187–198). Therefore, we conclude that isolated myofibrils (even if it seems counterintuitive for this reviewer) are superior for sarcomere measurements than whole-mount preparations - and that is why we primarily rely on them here.

Despite that, we certainly recognize that isolated myofibrils cannot recapitulate every aspect of an IFM fiber, and the need for whole-mount preparations during our IFM studies is not questioned by us.

        In addition to this general answer to the issues raised in the above paragraph of the reviewer, we would like to specifically reflect for some of the remarks:

„Unfortunately, this manuscript only uses isolated myofibrils that were fixed and then mechanically dissociated by pipetting.”

This is a false statement that “this manuscript only uses isolated myofibrils” as we used different preparation methods for initial comparisons (see Figure 1E, now Figure 2A in the revised manuscript). Additionally, unlike the reviewer assumed, the myofibrils were first dissociated and then fixed, and not vice versa (as described in the Materials and Methods section).

„This method likely induces variations as seen by the large spread of sarcomere length reported in Figure 1C (2.8-3.9µm?) and even bigger spreads for myofibril widths. Are these also seen in tissue without dissections?”

This remark makes absolutely no sense, as we do not report sarcomere length values in Figure 1C at all. By assuming that the reviewer meant to refer to Figure 1B, it still remains a misunderstanding or a false statement, because that panel refers to the variations found in published data (not in our current data), and this is clearly explained both in the figure legend and the main text. Regardless of that, the stated spread does not appear unusual. In the article by Spletter et al. (2018), the authors report a similar spread (2.576–3.542 µm) for sarcomere length in mature IFM using whole-mount DLM cross-sections. As to the second question here, we do observe a comparable spread in other preparations as well (see Figure 1E, now Figure 2A in the revised manuscript), which is again the opposite conclusion as compared to the (clearly false) assumption of the reviewer.

„Unfortunately, no comparision to intact flight muscles are reported with the here presented quantification tool. „

This is also a false statement; as we do report comparison to whole mount cross sections which we belive the reviewer considers „intact” in Figure 1E (Figure 2A in the revised manuscript).

„The sarcomere length spread in the developmental samples is even larger.”

The spread is not larger at all than in previous reports, as clearly shown in Supplementary Figure 3A.

The authors suggest that there are sex differences in sarcomere length and pupal development duration. This is potentially interesting, unfortunately they then use mixed sex samples to analyse sarcomeres during flight muscle development.

In the revised manuscript, we now provide a more detailed description of a subtle post-eclosion difference in IFM sarcomere metrics between male and female Drosophila. We attribute this variation to the well-established observation that female pupae develop slightly faster than males, a property that may last till shortly after eclosion. Confirming this experimentally would require considerable effort with limited scientific benefit. Nonetheless, the subtle nature of this sex-linked variation reinforced our decision to include IFM sarcomeres from both male and female flies in our comprehensive developmental analysis.

The IMA software tool lacks critical assessment of its performance compared to other tools and the validation presented is too limited. IMA seems to generate systematic errors, based on Fig S1E, as it does not report the ground truth. These have to be discussed and compared to available tools. The principles of fitting used in IMA seem well adapted to IFM myofibrils in low noise conditions, but may not be usable in other situations. This should be assessed and discussed.

IMA is a specialized software tool developed to address a specific need, notably, to accurately and efficiently measure sarcomere length and myofibril diameter in individual IFM myofibril images labeled with both phalloidin and Z-disc markers. For our purposes, it remains the most suitable and reliable option, and we are confident that IMA outperforms all other available tools. To demonstrate this, we have included a table comparing the few alternatives (MyofibrilJ, SarcGraph, and sarcApp) capable of both measurements, which further supports our conclusion. Given IMA's focused application, extensive validation under artificially low signal-to-noise conditions is unnecessary. While IMA may introduce minor systematic errors (~0.01 µm for sarcomere length and ~0.03 µm for myofibril diameter), these are negligible errors relative to the limitations of the simulated ground truth data used for benchmarking. This point is now addressed in the manuscript.

It is claimed that validation was achieved on simulated IFM images: do the authors rather mean simulated isolated IFM myofibril images? This is not quite the same in terms of algorithm complexity and this should be corrected if this is the case.

Indeed, we used simulated individual IFM myofibril images, where both phalloidin labeling and Z-disc labeling are present. This is clearly shown in Supplementary Figure 1A, and stated in the text when first introduced: „we generated artificial images of IFM myofibrils with known dimensions, simulating the image formation process”

The authors need to revise their comparison to other tools. It is incomplete and seemingly incorrect. It should be clearly stated that IMA is limited to isolated myofibrils, which is a far easier segmentation task than what other tools can do, such as sarcApp (Neininger-Castro et al. 2023, PMID: 37921850). Defining the acronym would be valuable in that sense. The claim line 129-130 "none can adequately measure myofibril diameter from regular side view images" is unclear. What do the authors refer to as "side view images"? Sarc-Graph from Zhao et al 2021, PMID: 34613960, and sarcApp from Neininger-Castro et al. 2023 provide sarcomere width, in conditions that are very similar to what IMA does, e.g. on xy images based on the documentation provided on github. A performance comparison with these tools would be valuable. Does installation and use of IMA require computational skills?

Motivated by the reviewer’s comments, we revised the section introducing IMA. However, we chose not to include an extensive comparison with other software tools, as this would divert the manuscript’s focus without impacting the main conclusions. Instead, we added a summary table highlighting the key requirements for analyzing IFM sarcomere morphometrics from Z-stacks of phalloidin- and Z-line-labeled individual myofibrils and compared the available tools accordingly. In our experience, most software tools are developed to address very specific problems, even those marketed as general-purpose solutions. Consequently, applying them beyond their intended scope often results in reduced efficiency and suboptimal performance. Although sarcApp was initially available as a free tool, one of its dependencies (PySimpleGUI 5) has since adopted a commercial license model. Using a trial version of PySimpleGUI 5, we evaluated sarcApp on our dataset. The software is limited to single-plane image input, hence raw image stacks must be preprocessed into a suitable format, which is a time consuming step. Furthermore, implementation requires basic programming proficiency, as parameter adjustments must be performed directly within the source code to accommodate dataset-specific configurations. Once appropriately configured, sarcApp reliably quantifies both sarcomere length and myofibril width with accuracy comparable to that of IMA. However, it lacks built-in diagnostic feedback or visualization tools to facilitate measurement verification or troubleshooting during batch processing. SarcGraph also supports only single-plane image inputs and requires prior image preprocessing. Additionally, images must be loaded manually one by one, which further reduces processing efficiency. Parameter optimization relies on direct code modification through a trial-and-error process, demanding a certain level of programming proficiency. Even with these adjustments, the software frequently introduces artifacts - such as Z-line splitting - when applied to our dataset. Even when segmentation is successful, sarcomere length is often overestimated, whereas myofibril diameter is consistently underestimated. As compared to these issues, IMA was designed for ease of use and does not require any programming experience to install or operate. It can automatically handle raw microscopic image formats without the need for preprocessing. Segmentation is fully automated, with no requirement for parameter tuning. The tool provides visual feedback during both the segmentation and fitting steps, allowing users to confidently assess and validate the results. IMA produces accurate and precise measurements of sarcomere length and diameter. Batch processing is enabled by default, significantly improving efficiency when analyzing multiple images. Finally, unlike the reviewer stated, IMA is not limited to isolated myofibrils. It is optimized for isolated myofibrils (i.e. full performance is achieved on these samples), but it can also work on whole-mount preparations in semi-automatic and manual mode, which still allow precise measurements (with some reduction in processing efficiency).

As to the minor comments, the acronym IMA was already defined in lines 541 and 917–918 of the original submission, as well as on the software’s GitHub page. Additionally, we replaced the phrase "side view images" with "longitudinal myofibril projections" to improve clarity.

How do the authors know that the bright phallodin signal visible that the Z-disc at 36h and 48h APF is due to actin filament overlap, as suggested? An alternative solution are more short actin filaments at the early Z-discs.

It is widely accepted that the bright phalloidin signal at the Z-line in mature sarcomeres reflects actin filament overlap (e.g., Littlefield and Fowler, 2002; PMID: 11964243). Accordingly, in slightly stretched myofibrils, this bright signal diminishes, and in more significantly stretched myofibrils, a small gap appears (e.g., Kulke et al., 2001; PMID: 11535621). The width of this bright phalloidin signal corresponds to the electron-dense band seen in longitudinal EM sections (Figure 3B and Supplementary Figure 5B, now Figure 4B and Figure 6B in the revised manuscript) and matches the actin filament overlap observed in Z-disc cryo-EM reconstructions from other species (Yeganeh et al., 2023; Rusu et al., 2017), where individual thin filaments can be resolved. By extension, we interpret the bright phalloidin signals at the Z-discs observed at 36 h and 48 h APF as arising from similar actin filament overlaps, given their comparable width to the electron-dense Z-bodies described both in our study (Supplemantary Figure 5B, now Figure 6B in the revised manuscript) and by Reedy and Beall (1993). While we cannot fully rule out the reviewer’s alternative interpretation, for the time being it remains a bold speculation without supporting evidence, and therefore we prefer to stay with the conventional view.

The authors seem to doubt their own interpretation that actin filaments shrink when reading line 304 and following. This is obviously critical for the "model" presented.

Unlike the reviewer implies, we certainly do not doubt our own interpretation, but to avoid confusion we revised the corresponding paragraph in the manuscript and provided more details on our explanation, and we also provide a brief overview of it here. Between 36 h and 48 h APF we observe a pronounced structural transition in the IFM sarcomeres. In EM cross-sections, the previously irregular myofilament lattice becomes organized into a regular hexagonal pattern (Figure 3A, now Figure 4A in the revised manuscript) with filament spacing typical of mature myofibrils (Supplementary Figure 5A, now Figure 6A in the revised manuscript). In longitudinal EM sections, the elongated, amorphous Z-bodies condense along the myofibril axis to form well-defined, adult-like Z-discs (Supplementary Figure 5B, now Figure 6B in the revised manuscript). Similarly, dSTORM imaging shows that the Z-disc associated D-Titin epitopes become more compact and organized during this period (Supplementary Figure 4E, now Figure 5E in the revised manuscript). The edges of the thick filament arrays also become more sharply defined, and the appearance of a distinct bare zone indicates the establishment of a regular register (Figure 3B, now Figure 4B in the revised manuscript). By assuming that a similar reorganization occurs within the thin filament array, the apparent length of the thin filament array would decrease—not due to shortening of individual filaments, rather due to improved alignment. Although we cannot directly resolve single thin filaments, this reorganization offers the most plausible explanation for the observed change.

Minor comments: 1. Figure S1B is not called out in the text.

The reviewer might have missed this, but in fact, it is explicitly called out in line 181.

Fig. 1: Please state whenever images are simulations?

We appreciate the reviewer’s observation that the simulated IFM myofibril images are indistinguishable from the real ones, as this confirms the adequacy of these images for testing our software tool. However, this is already clearly indicated: Figure 1B features simulated images, as noted in the figure legend (line 824), and Supplementary Figure 1A similarly shows simulated images, as stated both in the legend (line 886) and in the figure.

Fig. 2: Length-width correlation - please provide individual points color-coded by time point?

As suggested by the reviewer, we generated a plot with the individual points color-coded by time.

"newly eclosed males and females, we observed that males have slightly shorter sarcomeres and narrower myofibrils". Please provide a statistical test supporting the difference.

In the revised manuscript, we compared sarcomere length and myofibril width between males and females from 0 to 96 hours AE using a two-way ANOVA with Sidak’s multiple comparisons test. We expanded our description of these observations in the main text, and details of the statistical analysis are now included in the revised figure legend (Figure 1E). Briefly, newly eclosed males showed slightly shorter sarcomeres than females - a consistent but non-significant trend (p = 0.9846) - which resolved by 12 h AE, with sarcomere lengths remaining similar thereafter (p = 0.1533; Figure 1E). In contrast, myofibril width was significantly narrower in the newly eclosed males (p = 0.0374), but this difference disappeared between 24 and 48 h AE as myofibrils expanded in diameter during post-eclosion development (p

Were statistical tests performed using animals as sample numbers? Please clarify in the images what are animal and what are sarcomere numbers.

Following standard guidelines, statistical tests were performed using the means of independent experiments, as noted in the figure legends. For each experiment, we used approximately 6 animals, and this information is now included in the Materials and Methods section.

mef2-Gal4 should be spelled Mef2-GAL4 according to Flybase.

This has been corrected in the revised text and figures.

Are the images shown in Figure 2B representative? 96h AE appears thicker than 24h AE but the graph reports no difference.

We aimed to show representative images, however, in the case of 96h APF we may have selected a wrong example. We now changed the image for a more appropriate one.

The authors only found Zasp52 and alpha-Actinin at the Z-discs from 72h APF onwards, which is different to what others have reported.

Similarly to former reports, we detected both α-Actinin (see Supplementary Figure 3B, now Figure 3C in the revised manuscript) and Zasp52 in microdissected IFMs as early as 36 hours APF. However, these markers were largely absent in isolated myofibrils from the early pupal stages (36–60 hours APF). By 60 hours APF, strong α-Actinin and Zasp52 signals were clearly visible in isolated myofibrils (the closest timepoint captured by dSTORM is 72h APF). As discussed in the manuscript, a likely explanation is that α-Actinin and Zasp52 are recruited to developing Z-bodies early on but are only fully incorporated into mature Z-discs between 48 and 60 hours APF. Their incomplete integration at earlier stages may lead to their loss during the isolation procedure.

Thick filament length during development has also been estimated by Orfanos and Sparrow, which should be cited (PMID: 23178940)

Contrary to the reviewer’s claim, the article 'Myosin isoform switching during assembly of the Drosophila flight muscle thick filament lattice' does not provide any measurements or estimates of thick filament length; it only includes a schematic illustration where the length of the thick filaments is not based on empirical data.

**Referee Cross-commenting**

I also agree with my colleagues comments, which are largely consistent.

Reviewer #3 (Significance (Required)):

This paper introduces a tool to measure sarcomere length. Easy to use tools that do this as well already exist. The tool can also measure sarcomere width, which it claims as unique point, which is not the case, see above comment.

We are aware that other tools exist to measure sarcomere parameters (and we did not claim the opposite in our ms), nevertheless, we need to emphasize that based on our comparisons, IMA is superior to all three alternatives. Three software tools could, in principle, be used to measure both sarcomere length and myofibril diameter: MyofibrilJ, SarcGraph, and sarcApp. However, two of them - MyofibrilJ and SarcGraph - consistently under- or overestimate these values. The only tool capable of performing these measurements reliably, sarcApp, is no longer freely available, it requires programming expertise, and it does not support raw image file formats, making it difficult to use in practice (see above comments for more details). In contrast, IMA is user-friendly and does not require any programming expertise to install or operate. It can automatically process raw microscopic image formats without the need for preprocessing. Segmentation is fully automated, and no parameter tuning is necessary. The tool offers visual feedback on both the segmentation and fitting processes, enabling users to validate results with confidence. IMA delivers accurate and precise measurements of sarcomere length and diameter. Additionally, batch processing is enabled by default, significantly enhancing workflow efficiency.

This manuscript shows that depending on the isolation and embedding media sarcomere and myofibrils width changes and hence artifacts can be introduced. While this is not suprising, it has not been well controlled in a number of previous publications.

Furthermore, this paper measures sarcomere length and width during flight muscle development and consolidates what was already known from previous publications. Sarcomeres are added until 48 h APF, then they grow in diameter. Despite strong claims in the text, I do not see any significant novel findings how sarcomeres grow in length or width or any significant deviations from what has been published before. This is even documented in the supplementary graphs by comparing to published data. It is close to identical.

The overall process has been quantitatively described in four previous studies (Reedy and Beall, 1993, Orfanos et al., 2015, Spletter et al., 2018, Nikonova et al., 2024). While there is general agreement on the pattern of sarcomere development, significant discrepancies exist among these datasets; differences that become particularly problematic when attempting to build structural models. More specifically: Reedy and Beall (1993) report substantially shorter sarcomeres compared to all other datasets, including ours. This discrepancy likely stems from two factors: (i) their use of longitudinal EM sections, where sample preparation is known to cause considerable tissue shrinkage; and (ii) the maintenance of their flies at 23 °C, a temperature that clearly delays development relative to the more commonly used 25 °C. Interestingly, Spletter et al. (2018) and Nikonova et al. (2024) conducted their experiments at 27 °C, which also deviates from standard conditions and may complicate comparisons. Orfanos et al. (2015) suggested that mature sarcomere length is reached by approximately 88 hours after puparium formation (APF). In contrast, our measurements show that sarcomeres continue to elongate beyond this point, reaching mature length between 12 and 24 hours post-eclosion. All four earlier studies report a mature sarcomere length around 3.2-3.3 µm, only slightly longer than the ~3.2 µm length of thick filaments (Katzemich et al., 2012; Gasek et al., 2016). This would imply an I-band length below ~100 nm, which is an implausibly short distance. In contrast, our data, along with several recent studies (González-Morales et al., 2019; Deng et al., 2021; Dhanyasi et al., 2020; DeAguero et al., 2019), support a mature sarcomere length of approximately 3.45 µm, placing the length of the I-band at around 250 nm. This estimate is more consistent with high-resolution structural observations from longitudinal EM sections and fluorescent nanoscopy (Szikora et al., 2020; Schueder et al., 2023). Although Reedy and Beall (1993) provide limited data on myofibril diameter during myofibrillogenesis, a more detailed quantitative analysis is presented by Spletter et al. (2018) and by Nikonova et al. (2024). Interestingly, Spletter et al. report two separate datasets - one based on longitudinal sections and another on cross-sections of DLM fibers. While the measurements are consistent during early pupal stages, they diverge significantly in mature IFMs (1.116 ± 0.1025 µm vs. 1.428 ± 0.0995 µm), a discrepancy that is not addressed in their publication. Nikonova et al. (2024) report even narrower myofibril widths (0.9887 ± 0.1273 µm). Moreover, the reported diameters of early myofibrils in all three datasets are nearly twice as large as those reported by Reedy and Beall (1993) and in our own measurements, directly contradicting the reviewer's claim that the values are “close to identical.” Finally, our data clearly demonstrate that both the length and diameter of IFM sarcomeres reach a plateau in young adults, which is a key developmental feature not examined in previous studies.

In summary, we did not and we do not intend to claim that our conclusions are novel as to the general mechanisms of myofibril and sarcomere growth. Rather, our contribution lies in providing a high-precision, robust analysis of the growth process using a state-of-the-art toolkit, resulting in a comprehensive description that aligns with structural data obtained from TEM and dSTORM. We therefore believe that expert readers will recognize numerous valuable aspects of our approaches that will advance research in the field.

Counting the total number of thick filaments during myofibril development is nice, however, this also has been done (REEDY, M. C. & BEALL, C. 1993, PMID: 8253277). In this old study, the authors reported the amount of filament across one myofibril. How does this compare to the new data here counting all filaments? Unfortunatley, this is not discussed.

Indeed, the study by Reedy and Beall (1993) was primarily based on longitudinal DLM sections, which were used to estimate myofibril width and count the number of thick filaments on this lateral view images (e.g., ~15 thick filaments wide at 75 hours APF), but total thick filament numbers were not provided. While such data could theoretically be used to estimate the number of myofilaments per myofibril, these estimations would depend on the unverified assumption that the section includes the full width of the myofibril. Additionally, the study did not provide standard deviations or the number of measurements, limiting the interpretability and reproducibility of their findings. These points highlight the need for a more rigorous and quantitative approach. For these reasons, we chose to quantify myofilament number using cross-sections, providing more accurate and reliable assessments.

Besides the difference between the lateral versus cross sections, a direct comparison of our studies is further complicated by differences in the developmental time points and experimental conditions used. Reedy and Beall (1993) reports data from pupae aged 42, 60, 75 and 100 hours, as well as from adults, whereas we present data from 36, 48, and 72 hours APF, and from 24 hours after eclosion, which corresponds to approximately 124 hours APF. Moreover, their experiments were carried out at 23 °C, a temperature that somewhat slows down pupal development and results in adult eclosion at around 112 hours APF, as stated in their study. In contrast, our experiments were carried out at the more commonly used 25 °C, where adults typically emerge around 100 hours APF.

Collectively, these differences prevented meaningful comparisons between the two datasets, and therefore we preferred to avoid lengthy discussions on this issue.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #1

Evidence, reproducibility and clarity

Summary

In this work, the authors present a careful study of the lattice of the indirect flight muscle (IFM) in Drosophila using data from a morphometric analysis. To this end, an automated tool is developed for precise, high-throughput measurements of sarcomere length and myofibril width, and various microscopy techniques are used to assess sub-sarcomeric structures. These methods are applied to analyze sarcomere structure at multiple stages in the process of myofibrillogenesis. In addition, the authors present various factors and experimental methods that may affect the accurate measurement of IFM structures. Although the comprehensive structural study is appreciated, there are major issues with the presentation/scope of the work that need to be addressed:

Major Comments

1. The main weakness of the paper is in its claim of presenting a model of the sarcomere. Indeed, the paper reports a structural study that is drawn onto a 3D schematic. There is no myofibrillogenesis model that would provide insights into mechanisms. Therefore, the use of the word model is grossly overstated.

2. In general, the major focus and contribution of the work is unclear. How does the comprehensive nature of the measurements contribute to existing literature?

3. Figure labels are often rather confusing - for example it is unclear why there is a B, B', B' etc instead of B,C,D, etc.

4. Some comments in the text are not clearly tied to the figures. For example, in lines 108-109, are the authors referring to the shadow along the edges of the myofibril when saying they are not clearly defined (Figure 1C)?

5. In line 116, it is unclear what "surrounding structures" the authors are referring to if the myofibrils are isolated.

6. In lines 141-142, there is no reference of data to back up the claim of validation.

7. In line 170, the authors mention the mef2-Gal4/+ strain as a Gal4 driver line but do not clearly state how this strain is different from the wildtypes or how this impacts their results.

8. In lines 182-185, the authors discuss the effects of tissue embedding on morphometrics. Were factors such as animal sex, age, fiber type, etc. conserved in these experiments? If not, any differences in results may be confounding.

9. In lines 199-201, the authors discuss results of myofibril diameter using different preparation methods, yet no data is cited to support the claims. In line 220, the phrase "6 independent experiments" is unclear. Is each independent experiment performed using a different animal? Furthermore, are 6 experiments performed for each time point?

10. In line 254, the authors refer to "number of sarcomeres". It must be clearly stated if this refers to sarcomeres per myofibril, image area, etc.

11. In line 274, the authors refer to "myofilament number". It must be clearly stated if this refers to myofilaments per myofibril, image area, etc.

12. In line 299, the authors mention that thin filaments measured less than 560 nm in length, yet no data is cited to support this.

13. In the "Quantifying sarcomere growth dynamics" section of the summary (starting from line 402) the authors introduce data that would be more naturally placed in the results and discussion section.

14. In lines 422-423, it is not mentioned what the controls are for.

15. In the caption of Figure 1C, it is not mentioned what the red dashed lines in the microscope images represent.

16. In the caption of Figure 1D, the difference between the lighter and darker grey points is not mentioned.

17. In line 849, the stated p-value (0.003) does not match that mentioned in the figure (0.0003).

18. In line 874, it is not clear what an "independent experiment" refers to (different animal, etc.?).

19. Figure 2A is hard to read. Using different colored dots for different time points might help.

20. The significant figures presented in Figure 4 give a completely inaccurate representation of the variability of the measurements achieved with these techniques.

21. In line 877, it should be mentioned that the number of filaments is counted per myofibril. The y-axes in the figure should also be adjusted to clarify this.

22. In line 883, it is not clear what an "independent experiment" refers to (different animal, etc.?).

23. The statement of sample sizes in all figures is a little confusing.

24. In lines 1007-1008, the authors imply that the lattice model is needed for calculation of myofilament length. However, from the equations and previous data, it seems that this can be estimated using the confocal and dSTORM images.

25. A more specific discussion of future directions is needed to put this paper in context. For example: Can anything from the overall process be used to better understand sarcomere dynamics in larger animals/humans? Can this be applied to disease modelling?

26. One of the major claims of the paper is that there is a measurable variability with sex and other parameters. However, this data is never clearly summarized, presented (except for supplement), or discussed for its implications.

Minor Comments

1. Lines 60-65 seem to break the flow of the introduction. As the authors discuss existing methods in literature for IFM analysis in the previous couple sentences, the following sentences should clearly state the limitations of existing methods/current gap in literature and a general idea of what the current work is contributing.

2. In line 104, the acronym for ZASPs is not spelled out.

Referee Cross-commenting

I agree as well.

Significance

In summary, this paper provides a multi-scale characterization of Drosophila flight muscle sarcomere structure under a variety of conditions, which is potentially a significant contribution for the field. However, the paper scope is overstated in that it does not provide an actual sarcomere model. Further, there are multiple issues with data presentation that impact the readability of the manuscript.

DOI: 10.1242/jeb.191106

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1136/jitc-2024-010966

Resource: RRID:Addgene_12259

Curator: @olekpark

SciCrunch record: RRID:Addgene_12259

What is this?

DOI: 10.1038/s42003-025-08083-y

Resource: None

Curator: @olekpark

SciCrunch record: RRID:Addgene_149415

What is this?

DOI: 10.1038/s41467-025-59377-y

Resource: RRID:Addgene_35614

Curator: @olekpark

SciCrunch record: RRID:Addgene_35614

What is this?

DOI: 10.1038/s41467-025-59377-y

Resource: RRID:Addgene_119942

Curator: @olekpark

SciCrunch record: RRID:Addgene_119942

What is this?

DOI: 10.1038/s41419-025-07693-y

Resource: RRID:Addgene_18782

Curator: @olekpark

SciCrunch record: RRID:Addgene_18782

What is this?

DOI: 10.1038/s41419-025-07685-y

Resource: None

Curator: @olekpark

SciCrunch record: RRID:Addgene_73055

What is this?

DOI: 10.1038/s41419-025-07685-y

Resource: None

Curator: @olekpark

SciCrunch record: RRID:Addgene_73056

What is this?

DOI: 10.1038/s41392-025-02221-y

Resource: RRID:Addgene_8454

Curator: @olekpark

SciCrunch record: RRID:Addgene_8454

What is this?

DOI: 10.1038/s41392-025-02221-y

Resource: RRID:Addgene_12260

Curator: @olekpark

SciCrunch record: RRID:Addgene_12260

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:Addgene_19514

Curator: @olekpark

SciCrunch record: RRID:Addgene_19514

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:Addgene_22487

Curator: @olekpark

SciCrunch record: RRID:Addgene_22487

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:BDSC_458

Curator: @maulamb

SciCrunch record: RRID:BDSC_458

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:BDSC_8530

Curator: @maulamb

SciCrunch record: RRID:BDSC_8530

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:BDSC_33623

Curator: @maulamb

SciCrunch record: RRID:BDSC_33623

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:BDSC_58469

Curator: @scibot

SciCrunch record: RRID:BDSC_58469

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:BDSC_33773

Curator: @scibot

SciCrunch record: RRID:BDSC_33773

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:BDSC_66101

Curator: @scibot

SciCrunch record: RRID:BDSC_66101

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:BDSC_5846

Curator: @scibot

SciCrunch record: RRID:BDSC_5846

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:BDSC_67976

Curator: @scibot

SciCrunch record: RRID:BDSC_67976

What is this?

DOI: 10.1007/s00018-025-05711-y

Resource: RRID:BDSC_9146

Curator: @scibot

SciCrunch record: RRID:BDSC_9146

What is this?

DOCUMENT DE BRIEFING - AUDITION DE FRANÇOIS BAYROU SUR LES VIOLENCES SCOLAIRES, NOTAMMENT À NOTRE-DAME DE BÉTHARRAM

Date : [Insérer la date de l'audition si disponible, sinon indiquer "Non spécifié"] Sujet : Audition de François Bayrou devant la commission d'enquête sur les violences scolaires, en particulier à Notre-Dame de Bétharram. Source : Extraits de la retranscription de l'audition. Intervenant Principal : François Bayrou (Premier Ministre) Rapporteurs : Violette Spilbou, Paul Vanier Présidente : [Nom de la présidente non précisé dans les extraits]

1. Synthèse Exécutive

François Bayrou, auditionné sous serment, a abordé les accusations portées à son encontre concernant sa connaissance et sa gestion des violences, notamment sexuelles, au sein de l'établissement Notre-Dame de Bétharram.

Il a vivement contesté les allégations selon lesquelles il aurait protégé des pédocriminels ou minimisé la gravité des faits.

Il a insisté sur l'importance de son audition pour les victimes, qu'il considère comme le cœur du sujet, tout en dénonçant une instrumentalisation politique de l'affaire visant à l'abattre et à déstabiliser le gouvernement.

Bayrou a fourni une chronologie précise de ses liens avec Bétharram (en tant que parent d'élève de 1987 à 2002) et de son action en tant que Ministre de l'Éducation Nationale (1993-1997) face aux alertes reçues, notamment un rapport d'inspection de 1996 qu'il a commandé.

Il a longuement débattu avec les rapporteurs, en particulier sur les variations perçues dans ses déclarations antérieures et sur l'existence et le contenu d'une conversation avec le juge Mirand en 1998.

Il a également défendu son approche face à la violence, y compris un incident personnel controversé.

2. Points Clés et Thèmes Principaux

• La Protection des Victimes et le "Continent Caché" des Violences : Bayrou place les victimes au centre de sa préoccupation. Il décrit les violences, en particulier sexuelles, comme un "continent caché" qui émerge enfin, notant que cela ne se limite pas aux établissements scolaires mais touche de nombreux domaines (associations, sport, famille, etc.). Il exprime sa reconnaissance envers ceux qui ont "permis de dévoiler ce qui devait l'être".
• "pour moi le premier mot qui me vient quand je pense à cette audition c'est enfin... elle est très importante pour les garçons et les filles qui ont été victimes de violence et particulièrement de violence sexuelle depuis des décennies que ce soit à Betaram ou comme nous le découvrons tous les jours en beaucoup d'autres établissements scolaires et en beaucoup d'autres institutions associatives sportives dans le monde du spectacle en famille hélas c'est un j'ai employé cette expression un continent caché qui apparaît qui surgit"
• "ce sont celles-là les victimes qui m'intéressent qui trop souvent se sont tu parce qu'elles ont honte parce qu'elles n'osent pas parce qu'elles ne veulent pas faire de peine à leurs proches"
• Accusations Personnelles et Instrumentalisation Politique : Bayrou réfute vigoureusement les accusations selon lesquelles il aurait protégé des pédocriminels ou ignoré les alertes. Il perçoit l'affaire comme une "manœuvre", une "instrumentalisation" politique visant à "abattre ce gouvernement abattre le suivant et le suivant encore", utilisant le "scandale" comme arme, notamment via les réseaux sociaux.
• "Je n'ai jamais été informé de quoi que ce soit de violence ou de violence à forcerie sexuelle jamais" (déclaration contestée par les rapporteurs mais que Bayrou maintient dans son principe)
• "ce n'est pas parce que j'exprime cette reconnaissance que je n'identifie pas les manœuvres l'instrument l'instrumentalisation de tout cela en reprenant une phrase d'un des inspirateurs de certains d'entre vous abattre ce gouvernement abattre le suivant et le suivant encore et l'arme qui est utilisée c'est l'arme du scandale"
• "vous m'interrogez vous en montant à la tribune pour m'accuser d'avoir protégé des péd criminels"
• Lien avec Notre-Dame de Bétharram : Bayrou détaille son unique lien avec l'établissement, en tant que parent d'élèves de 1987 à 2002. Il réfute avoir été membre d'un organe de gouvernance. Il mentionne avoir été désigné représentant du conseil régional au conseil d'administration en 1985 mais affirme n'y avoir "jamais siégé". Il indique n'être entré dans l'établissement que lors d'événements ponctuels (inauguration d'un gymnase, réparation de la chapelle, inondation).
• "notre fille aînée est entrée à Betaram en première en 1987 il y a presque 40 ans et notre dernier fils a quitté cet établissement en 2002 il y a presque un quart de siècle voilà exactement mon lien avec Betaram"
• "vous m'avez demandé si j'avais été membre des organes de direction de Betaram jamais"
• "j'ai été désigné sans jamais siéger"
• Connaissance des Faits et Variation des Déclarations : Le débat central porte sur le moment où Bayrou a eu connaissance des violences. Il maintient initialement n'avoir été informé que "par la presse". Face aux questions insistantes des rapporteurs sur les variations de ses déclarations entre le 11 et le 18 février, il finit par reconnaître avoir évoqué les accusations de viol visant le père Caricar avec le juge Mirand, mais affirme que cette conversation n'a pas apporté d'informations nouvelles qui n'étaient pas déjà dans la presse. Il accuse les rapporteurs de "méthode malveillante" et de vouloir "tirer la réalité pour nourrir un procès en scandale".
• "je maintiens que les seules informations que j'ai eu étaient celles qui étaient dans le journal je n'en ai pas eu d'autres"
• "je n'ai jamais entendu parler de violence sexuelle avant que le journal La République des Pyrénées lesclair des Pyrénées et Sud-Ouest fassent mention de ces violences sexuelles on doit être le 29 mai de l'année 2016 de l'année 2000 hein 98 de l'année 1998 vous avez raison 29 mai"
• "j'ai pu parler avec le juge Christian Mirand de ces accusations de viol qui visent le père Caricalcal donc de violence sexuelle sans doute oui" (cité par le rapporteur, confirmé indirectement par Bayrou dans la suite)
• "je regrette je ne me laisserai pas entraîner par vous ma version n'a pas varié" (affirmation contestée par les rapporteurs)
• Le Rapport d'Inspection de 1996 : Face aux alertes de violences physiques en 1995, Bayrou, alors Ministre de l'Éducation Nationale, a commandé un rapport d'inspection en avril 1996. Il défend cette action comme une "vraie vérification", notant que l'inspecteur a entendu une vingtaine de personnes. Il rappelle que ce rapport concluait que l'établissement était "sage, objectif et favorable". Il a redécouvert ce rapport récemment via une publication dans la presse, n'en ayant pas conservé de trace. Il affirme avoir demandé un "suivi" et cite un courrier du directeur de l'établissement daté de novembre 1996 indiquant que les conclusions ont été exécutées (licenciement d'un surveillant, suppression des élèves surveillants).
• "il n'est pas vrai qu'il y a eu des articles de presse ou en tout cas je les ai jamais vu sur le jugement que vous évoquez en 1993 jamais moi j'ai jamais vu ça" (concernant les violences physiques mentionnées par les rapporteurs)
• "du au le 9 ou le 10 avril je demande une inspection de l'établissement"
• "il a entendu 20 personnes... je trouve moi que c'est une vraie vérification"
• "ce rapport d'inspection qui n'existait chez personne s'est retrouvé il a été publié par le Figaro et ce rapport d'inspection en effet on va en parler donne toutes les garanties et les... il est sage objectif et favorable à l'établissement"
• "je demande un suivi au recteur au-delà du rapport"
• "Je viens de licencier même si cela risque d'avoir des retombées le surveillant qui avait une certaine conception de la discipline" (citant le courrier du directeur)
• La Conversation avec le Juge Mirand (1998) et l'Accusation d'Intervention : Bayrou confirme avoir eu une conversation avec le juge Mirand (un ami proche, beau-frère d'une victime tragique de son village) en 1998, après que les articles de presse ont révélé les accusations de viol contre le père Caricar. Il maintient que Mirand n'a pas violé le secret de l'instruction et qu'il n'a rien appris de nouveau. Il réfute catégoriquement l'accusation d'être intervenu dans l'affaire, notamment en lien avec la libération du père Caricar. Il cite le témoignage sous serment de l'ex-Ministre de la Justice Elisabeth Guigou et un document écrit du procureur général de l'époque pour prouver que l'intervention était celle du numéro 2 de la Direction des Affaires Criminelles et des Grâces, Laurent Lemel, et non la sienne. Il accuse le gendarme Hontan et Matrassou d'affabuler ou de se tromper, et les rapporteurs de "manipulation".
• "je connais très bien les deux gendarmes Matrasou et en qui j'ai toujours toute confiance et s'il dit cela on a dû arriver" (cité par le rapporteur concernant la déclaration de Mirand, partiellement corroborant Hontan selon le rapporteur)
• "le gendarme entend soit il ment soit il affabule" (concernant la déclaration de Hontan)
• "celui qui est intervenu c'est le numéro 2 de la direction des affaires criminelles et des grâces or Laurent Lemel est encore en vie... il est assez facile de lui poser la question : est-ce que c'est moi qui suis intervenu auprès de lui ou quelqu'un d'autre"
• "je sous serment et pour moi un serment c'est pas rien j'affirme que je ne suis pas intervenu et que ceux qui m'en ont accusé ont conduit une manipulation"
• La Libération du Père Caricar : Bayrou revient sur la libération du père Caricar en 1998, notant que la chambre d'accusation a justifié cette décision notamment par le fait que l'instruction n'avait pas avancé pendant un an. Il s'étonne de l'absence d'actes d'instruction pendant cette période, mais réfute l'idée que sa libération ou son envoi en Italie soient dus à des pressions politiques, jugeant cette idée "délirante" dans le monde judiciaire. Il cite un courrier de l'avocat de la partie civile, Maître Blazi, "profondément choqué" par cette suggestion de pressions.
• "Il est libéré parce que la chambre de l'instruction dit en réalité il s'est présenté de lui-même et le maintenir en détention n'apportera rien à la recherche de la vérité"
• "pourquoi est-ce que la longueur du délai d'exécution de cette commission rogatoire ne peut justifier que soit maintenu à l'encontre de Pierre Siv Caricard des mesures restrictives de liberté qui ne sont absolument pas indispensables au bon déroulement de l'instruction" (citant l'ordonnance de la chambre de l'instruction)
• "je suis profondément choqué... que l'on puisse laisser entendre que des pressions auraient été exercées sur la chambre de l'instruction concernant la libération du père Caricar" (citant le courrier de Maître Blazi)
• Le Financement de Bétharram par le Conseil Général : Interrogé sur les subventions facultatives versées par le conseil général qu'il présidait après l'affaire Caricar, Bayrou répond qu'il n'y a jamais eu de "financement particulier" pour Bétharram, mais un règlement général pour tous les établissements privés du département. Il mentionne une participation exceptionnelle pour le remplacement de bâtiments préfabriqués dangereux, conforme à la loi Falloux.
• "il n'y a jamais eu de financement particulier pour Betaram il y a un règlement comme dans tous les départements"
• "Betaram a reconstruit en dur au lieu des préfabriqués pailleront point et je crois pour quelque chose comme le conseil général a dû donner quelque chose comme 50000 € une participation à la sécurité des enfants conformément à la loi fallou"
• La Déclaration de Madame Gulung : Bayrou conteste avec force le témoignage sous serment de Madame Gulung, enseignante, selon laquelle le père Vaillant lui aurait dit en 1996 "vous êtes là pour venger mon ami Caricar". Il affirme que cela est "pas possible" car le père Caricar n'intervient dans l'affaire qu'en 1998 et serait parti à Rome des années avant 1996. Il qualifie son témoignage d'"affabulation sous serment".
• "vous n'avez pas compris que vous êtes là pour venger mon ami Caricar c'est ce qu'elle a déclaré sousement devant vous" (cité par le rapporteur)
• "je répète Caricar est parti à Rome selon les interprétations entre 91 et 93 donc des années avant ces événements et elle dit que le père vaillant lui dit c'est pour venger Caricar"
• "je dis que l'affirmation qu'elle a faite sous serment devant vous est une affirmation qui ne peut pas tenir qui ne peut pas être acceptée et donc je dis que cette affirmation est une affabulation souserment"
• Le Contact entre son Cabinet et le Juge Mirand : Questionné sur l'appel de son conseiller, ancien procureur de Pau, au juge Mirand pour parler du secret de l'instruction, Bayrou confirme qu'il est "tout à fait possible et légitime" qu'un tel échange ait eu lieu entre personnes qui se connaissent, surtout face aux "déclarations" qui l'accusaient de manquer au secret de l'instruction. Il assume la responsabilité de ses collaborateurs.
• "je connais le juge Mirande que mon conseiller le connaît il a été procureur à peau pendant des années et que tous les journaux nous expliquaient que vous disiez qu'il y avait eu rupture du secret de l'instruction"
• "il est tout à fait possible et légitime que entre personnes qui se connaissent... il y a pas excommunication parce que quelqu'un dit à quelqu'un d'autre que on lui raconte que il a manqué au secret de l'instruction"
• "j'affirme je dis que mes collaborateurs sont sous ma responsabilité et j'ai pas l'intention de dire qu'ils font des trucs sans que je le sache"
• La Vision de la Violence Éducative et l'Incident de Strasbourg : Bayrou est interrogé sur l'incident de 2002 où il a donné une "tape" à un enfant. Il replace l'événement dans un contexte tendu (lapidation d'une mairie par des militants islamistes suite à son interdiction du voile à l'école) et justifie son geste comme une "tape de père de famille" face au vol de son portefeuille par l'enfant. Il maintient que "ce n'est pas de la violence" et qu'il soutient la lutte contre les violences éducatives ordinaires. Il affirme sa vision éducative basée sur le "langage", l'"esprit critique" et la "sécurité affective".
• "il est vrai qu'à Strasbourg en 2002 dans un moment extrêmement tendu... j'ai le réflexe quand je suis dans tout le temps de de vérifier si mon stylo mon portefeuille est à sa place... et en passant la main j'ai trouvé la main d'un petit garçon qui était en train de sortir mon portefeuille de ma poche et je lui ai donné une tape pas une claque"
• "pour moi ça n'est pas de la violence"
• "c'était un geste éducatif"
• "ma vision éducative c'est que ce qui permet d'accéder à un enfant c'est de lui parler je pense que la clé la plus importante c'est le langage"
• Omerta et Dysfonctionnements de l'État : Le thème de l'omerta est débattu. Bayrou réfute l'idée d'une omerta locale organisée dans sa région. Il reconnaît en revanche des dysfonctionnements systémiques dans la transmission d'informations entre administrations (Justice, Éducation) et le principe du "pas de vague" dans les institutions. Il propose de changer de méthode en créant un "mini commando de responsables" pour agir directement face aux problèmes.
• "il n'est pas vrai que chez nous y ait une omerta c'est pas vrai"
• "si le ministère de la justice avait informé et que le ministère de l'éducation avait provoqué des inspections par exemple peut-être ça aurait été différent mais c'est incommunicable chacun est dans son tuyau d'orge la justice parle pas en dépit de la circulaire que j'avais prise"
• "des grandes administrations comme ça elles vivent selon le principe du pas de vague chef d'établissement il dit pas de vague parce que l'inspecteur d'académie préfère qu'il ait pas de vague et l'inspecteur d'académie dit au recteur pas de vague et le recteur les recteurs il disent au ministre ça se passe très bien c'est comme ça"
• Propositions pour l'Avenir : En conclusion, Bayrou évoque la nécessité de garantir que chaque victime soit écoutée et que les signalements soient mieux recueillis. Il propose de s'inspirer de la loi allemande du 8 avril créant une autorité indépendante couvrant l'école, la culture et le sport, ainsi qu'un conseil scientifique et un conseil des victimes. Il mentionne le travail en cours de la Haute Commissaire à l'Enfance et de la ministre chargée de l'enfance placée.
• "comment pouvons-nous garantir aujourd'hui dans chaque établissement que chaque victime chaque famille chaque élève sera écouté et entendu ?" (Question posée par la rapporteur, reformulée par Bayrou comme un objectif)
• "j'ai proposé que regarde si on ne pouvait pas transplanter en France la loi qui a été votée le 8 avril en Allemagne... qui met en place une autorité qui touche tous les secteurs à la fois"
• "une autorité et deux conseils un conseil scientifique et un conseil des victimes"
• "j'ai nommé quelqu'un pour qui j'ai une très grande estime Sarah Elie au commissaire à l'enfance et elle est précisément en train de travailler... je pense qu'on est oui en en situation d'apporter des réponses qui ne s'enferment pas uniquement dans le cadre strict scolaire"

3. Principales Contradictions et Points de Tension

• Variation des Déclarations sur la Connaissance des Faits : Les rapporteurs ont insisté sur les changements perçus dans les déclarations de Bayrou entre le 11 et le 18 février concernant sa connaissance des violences physiques et sexuelles. Bayrou a contesté l'idée d'une variation, mais ses propres propos ont évolué de "Je n'ai jamais été informé de quoi que ce soit" à la reconnaissance d'avoir évoqué les accusations de viol avec le juge Mirand.
• Intervention dans l'Affaire Judiciaire : Malgré les preuves documentaires et les témoignages cités par les rapporteurs suggérant une intervention du procureur général suite à une démarche "de monsieur Bayrou", Bayrou a catégoriquement nié toute intervention personnelle, affirmant qu'il s'agissait de Laurent Lemel. La confrontation des témoignages sous serment du gendarme Hontan et du juge Mirand a ajouté à la confusion sur l'origine de cette information.
• La Crédibilité du Témoignage de Madame Gulung : Bayrou a attaqué frontalement la crédibilité du témoignage sous serment de Madame Gulung, le qualifiant d'"affabulation", ce qui a suscité la réaction des rapporteurs qui en avaient fait une "lanceuse d'alerte" crédible.

4. Faits Importants et Idées Clés à Retenir

• François Bayrou a été parent d'élèves à Bétharram de 1987 à 2002.
• En tant que Ministre de l'Éducation Nationale, il a commandé un rapport d'inspection sur Bétharram en avril 1996 suite à des alertes de violences physiques.
• Ce rapport de 1996 a conclu que l'établissement était "sage, objectif et favorable".
• Bayrou affirme avoir demandé un suivi et que le directeur de l'établissement a indiqué avoir mis en œuvre les conclusions (licenciement d'un surveillant, suppression des élèves surveillants).
• Bayrou a eu une conversation avec le juge Mirand en 1998 concernant les accusations de viol contre le père Caricar, mais affirme que cette conversation n'a pas révélé d'informations qui n'étaient pas déjà publiques.
• Il réfute catégoriquement toute intervention personnelle dans l'affaire judiciaire du père Caricar et cite des preuves pour étayer sa position.
• Il conteste la crédibilité du témoignage de Madame Gulung.
• Il reconnaît des dysfonctionnements systémiques dans la transmission d'informations entre administrations et le phénomène du "pas de vague".
• Il propose la création d'une autorité indépendante et de conseils scientifiques et des victimes pour lutter contre les violences sur les enfants dans différents domaines.
• Il défend son incident de 2002 à Strasbourg comme une "tape éducative de père de famille" et non comme de la violence.

The Web Content Accessibility Guidelines:

Operable – since I cannot select images, the navigation and interaction is good, it is understandable, basic but works in a way that anyone of different ages can identify and browse the colors of the web page.

il y aurait donc une diversification des publics en particulier une meilleure inclusion des publics moins connaissants? il serait pertinent de citer ici une ou deux références

Sobre la necesidad de diferentes clasificaciones para diferentes propósitos. Argumentan que los psicólogos y neurocientíficos tienen objetivos diversos y que una sola clasificación no puede servir para todos ellos.

How about x or y?

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Manuscript number: RC-2025-02953

Corresponding author(s): Andreas, Villunger

1. General Statements [optional]

*We would like to thank the reviewers for their constructive input and overall support. We appreciate to provide a provisional revision plan, as outlined here, and are happy to engage in additional communication with journal editors via video call, in case further clarifications are needed. *

2. Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

Reviewer #1

__Evidence, reproducibility and clarity __

Summary: This manuscript by Leone et al describes the role of the PIDDosome in cardiomyocytes. Using a series of whole body and cardiomyocyte specific knockouts, the authors show that the PIDDosome maintains correct ploidy in these cells. It achieves this through inducing cell cycle arrest in cardiomyocytes in a p53 dependent manner. Despite this effect on ploidy, PIDDosome-deficient hearts show no structural or functional defects. Statistics and rigor appear to be adequate.

We thank this referee for taking the time to evaluate our work and their valuable comments. We assume that this reviewer by mistake indicates that the phenomenon we describe, depends on p53. As outlined in the abstract and throughout the manuscript, the effect is independent of p53, but may additionally still involve p21, acting along or parallel to the PIDDosome.

Major comments: 1. Figure 1 uses fluorescent intensity of a nuclear stain to determine ploidy per nucleus and they further separate the results into mononucleated, binucleated or multinucleated cells. It is hard to know how to interpret these results without further information or controls. Is there a good positive control that can be used to help to show whether this assay is quantitative? The differences are larger with the Raidd and caspase-2 knockouts than with the Pidd knockouts but this is not addressed.

*We appreciate this concern. Regarding a "good positive control" we can say that we follow state-of the art in the cardiomyocyte field and studies by the Evans (PMID: 36622904), Kuhn (PMID: 32109383), Bergmann (PMID: 26544945) and Patterson labs (PMID: 28783163, 36912240) all use the identical approach to discriminate 2n from 4n nuclei in microscopy images at the cellular level. The fact that the majority of rodent CM nuclei is indeed diploid (PMID: 31175264, 31585517 and 32078450) and a large number of nuclei has been evaluated to assess their mean fluorescence intensity (MFI) reduces the risk of a systematic bias in our analysis. Moreover, we have used an orthogonal approach that is indeed quantitative to define DNA content, i.e,. flow-cytometry based evaluation of DNA content in isolated CM nuclei (Fig. 1C). We hence are confident our assays are quantitative. *

Regarding the fact that loss of Pidd1 causes a more saddle phenotype, we can offer to discuss this in light of the fact that Pidd1 has additional functions, outside the PIDDosome (PMID: 35343572), and that we made similar observations when analyzing ploidy in hepatocytes (PMID: *31983631). Given the fact that all components of the PIDDosome show a similar phenotype, and that this phenotype is mimicked by loss of the protein that connects PIDD1 and centrosomes, ANKRD26 (Fig. 4a), we are confident that this biological variation in our analysis is not affecting our conclusions. *

On line 459 the authors state that the increase in polyploidy in PIDDosome knockouts occurs in adult hood but this is not directly tested. In fact, in the next section the polyploidy is assessed in early postnatal development. This statement should be explained or removed.

We see that we have made an unclear statement here. In fact, we first noted increases in ploidy in adult heart and then define the time window in development when this happens. This sentence will be rephrased.

In Figure 4. The authors obtained RNAseq data for P1, P7 and P14 but only show the differences with and without caspase-2 at P7. Given that the differences in ploidy are more significant at P14 (Fig 3D), all the comparisons should be shown along with analysis of whether the same genes/gene families are altered in the absence of caspase-2.

The reason why we focus on postnatal day 7 (P7) is that data from Alkass et al (PMID: 26544945) and other labs (PMID: 31175264 ) document that on this day the initial wave of binucleation peaks. Hence, we hypothesized that the PIDDosome must be active in most CM, which aligns well with the increased mRNA levels of all of its components (Figure 3). Interestingly, it seems that its action is tightly regulated, as mRNA of PIDDosome components drop on P10, suggesting PIDDosome shut-down or downregulation. Similar findings have been noted in the liver (PMID: *31983631). Alkass and colleagues also show that very few CMs enter another round of DNA synthesis between P7 and P14, and hence possible transcriptome changes in the absence of the PIDDosome will be strongly diluted. *

Please note that on P1, there is no difference between genotypes to be expected as all CM are mononucleated diploids and cytokinesis competent, as previously demonstrated (PMID: *26544945). Moreover, PIDDosome expression levels are extremely low (Fig. 3A). As such, no difference between genotypes are expected on P1. In addition, on P14 the ploidy phenotype observed in PIDDosome knockout mice reaches the maximum and ploidy increases are comparable to adult tissue. Thus, at this time the trigger for PIDDosome activation (cytokinesis failure) is no longer observed as the majority of CMs are post-mitotic, (PMID: 26247711). As such the impact of PIDDosome activation on the P14 transcriptome is most likely negligible. However, if desired, we can expand our bioinformatics analysis summarizing findings made related to DEGs over time in wt animals by comparing genotypes also on day 1 and day 14. In light of the above, analysis between genotypes on P7 holds still appears as the one most meaningful. *

Some validation of the RNAseq and/or proteomics results would be an important addition to this study

We agree with this notion and propose to validate key candidates related to cardiomyocyte proliferation and polyploidization, some of which we found to be differentially expressed at the mRNA level on day 7in the RNAseq data (e.g., p21, Foxm1, Kif18a, Lin37 and others)

Regarding the proteomics results, we face the challenge that we can only try to confirm if candidate proteins are likely caspase substrates in silico using DeepCleave*, and potentially pick one or two candidates linked to CM differentiation for further analysis in vitro and in heterologous cell based assays (e.g. 293T cells), as no bona-fide ventricular cardiomyocyte cell lines exist. Primary postnatal CMs are extremely difficult to transfect, nor they proliferate without drug-treatment, or fail cytokinesis ex vivo. *

Figure 4D: the authors make the conclusion that p21 is downstream of PIDD (et p53 independent). However, this is not supported by the data because the increase in 4N cells/decrease in 2N cells, although statistically significant, is nowhere near that of caspase-2 KO and caspase-2/p21 KO. Statistics should also compare p32KO with c2KO. In the absence of any other data, the more likely conclusion is that p21 is not involved.

*We agree that the findings related to the impact seen upon loss of p21 suggest that it is not the only effector involved in ploidy control and it may not even be an effector engaged by caspase-2, as C2/p21 DKO mice have an even higher ploidy increase, albeit not statistically significant. However, it is important to highlight that p21 (Cdkn1a) was found to be downregulated in our transcriptomic analysis suggesting an involvement in the caspace2-cascade. We are happy to highlight this when presenting the results and in the discussion. *

*We assume that this referee refers to p73 KO data that should be compared to Casp2 KO data (could be read as p73 or p53, but the latter we compare side by side with Casp2 in Fig. 4 already). As p73 KO mice were not found to be viable beyond day 7 (our attempt to find animals on day 10 failed, in line with published literature (PMID: 24500610, 10716451)), we can only offer to compare this data set to the data presented in Figure 3C, where we have analyzed ploidy increases on day 7 from wt and PIDDosome mutant mice. This re-analysis will show that only Caspase-2 mutant mice display a significant ploidy increase on P7, when compared to wt or p73 mutant animals, while no difference are noted between wt and p73 mutant mice (to be included in new Suppl. Fig. 3C) *

Minor comments: Suggest moving Figure 4A to Figure 3 as it seems to fit better there based on the citation of this figure in the text

*We can see some benefit in this recommendation and included panel 4A now in an updated version of Figure 3. *

Recommend enhancing the brightness of microscopy images in Figure 1E and 2D

We will try to improve image quality, may have been due to PDF conversion

Significance

This study provides interesting information for the role of the PIDDosome in protecting from polyploidy and adds to the body of work by this same group studying this pathway in the liver.

The main weakness in terms of significance is the lack of a phenotype in the hearts of these animals. Therefore, it is clear that ploidy (or at least PIDDosome dependent ploidy) has minimal impact on cardiac development.

We respectfully disagree with the comment that the lack of impact on cardiac function constitutes a weakness of our findings. Several studies on ploidy control in the liver (PMID 34228992) but importantly also heart (PMID: 36622904) have failed to document a clear impact of increased ploidy on organ function. This does not infer insignificance, but maybe rather that the context where this becomes relevant has not been identified. We are happy expand on this in our discussion

The authors mention that they have not tried giving these mice an myocardial infarct (MI) or inducing any other type of cardiac damage. Although it is understood that these experiments are likely outside of the scope of the present study, without this information the impact of this study is moderate. I recommend expanding the discussion to provide a more in-depth possible rationale as to why ploidy perturbations do not lead to structural changes like in the liver.

Despite this, the insights to the pathway itself are interesting to investigators in the caspase-2 field if a little underdeveloped, especially concerning the role of p21.

My expertise is in cell death and caspase biology (especially caspase-2). I have sufficient expertise to evaluate all parts of this paper.

*As mentioned above, we will amend our conclusions on p21, in light of potential findings made when validating DEG candidates, as stated above. *

*We hope that the changes and amendments proposed here will be satisfactory to this referee to recommend publication of a revised manuscript. *

Reviewer #2

__Evidence, reproducibility and clarity: __

__Summary: __

In this study, the authors investigated the role of the PIDDosome during cardiomyocyte polyploidization. PIDDosome is a multi-protein complex activating the endopeptidase Caspase-2, and shown to be involved in eliminating cells with extra centrosomes or in response to genotoxic stress (Burigotto & Fava, 2021, Sladky and Villunger, 2020). In both cases, the PIDDosome is recruited in a ANKRD26-dependent manner at the centrosomes leading to p53 stabilization and cell death (Burigotto & Fava, 2021; Evans et al., 2020; Burigotto et al., 2021).

Here, by studying mouse cardiomyocyte differentiation, the authors showed that PIDDosome is imposing ploidy restriction during cardiomyocyte differentiation. Importantly, in contrast to a previous report in the liver (Sladky et al., 2020), they showed that PIDDosome acts in a p53-independent manner in cardiomyocytes. Indeed, they suggested that PIDDosome controls ploidy in cardiomyocytes through p21 activation.

We want to thank this reviewer for the time taken to evaluate our work and provide critical feedback that will help to improve our revised manuscript.

__Major comments: __

In general the conclusions of the authors are well supported by the experiments. However, I would suggest the following experiments/analysis to strengthen the paper:

The authors should improve the Figure 1 to help the readers who are not familiar with cardiomyocyte polyploidization. For instance, I would suggest to add a scheme to summarize cardiomyocyte polyploidization (in terms of nuclear size, mono vs multi and so on).

We agree that a visual summary of the postnatal timing of CM polyploidization will be helpful for the generalist not familiar with the topic and have added a scheme, adapted from a study by Alkass et al. (PMID: *26544945), who elegantly defined the timing of this process during postnatal mice life (now Fig. 1A). *

Based on the images they presented in 1B, the authors should also measure the nuclear area or volume in the different conditions in which components of the PIDDosome were depleted. Indeed, these two parameters should be easier to conceptualize for the readers (instead of the fluorescence nuclear intensity). This could help to understand if the nuclear size is maintained between the different conditions and if this is comparable between mono, bi or multinucleated cardiomyocytes.

We have acquired this data and it can be used to provide additional information on nuclear area and/or volume. We propose to focus on re-analyzing data from wt, Casp2 and XMLC2CRE/Casp2f/f mice. The additional information can be included in Figures 1 & 2, respectively.

• In Figure 2A, the authors presented cross section of heart from animals showing that PIDDosome depletion has no effect on heart size. This is a surprising result since cardiomyocytes have higher ploidy levels and this could have an effect on their function. Since the importance of this observation, the authors should present a quantification of the heart size in the different conditions shown in Figure 2A.

We agree with this comment. We can measure the heart vs. body weight ratio or tibia length in adult Casp2-/- vs. WT (3 month old) in order to indirectly evaluate possible increases in CM size linked to increased ploidy.

Also, the percentage of cardiomyocytes presenting higher levels of ploidy seems quite low. The authors should discuss this point. In particular because this could explain the absence of consequences on heart size and function at steady state.

We agree with this conclusion and will expand on this in our discussion. It is important to note that as opposed to findings made in liver (PMID: *31983631), genetic manipulation of ploidy regulators such as E2f7/8 (PMID: 36622904), only led to modest changes in CM ploidy, suggesting that either a small band-width compatible with normal heart function exists, or that additional mechanisms exist that take control when these thresholds set by the PIDDosome or E2f7/8 are exceeded. These mechanisms could involve Cyclin G (PMID: 20360255), or TNNI3K (PMID: 31589606). Importantly, a recent publication has shown that overexpression of Plk1(T210D) and Ect2 from birth causes increased heart weight coupled with a minor decrease in CM size. These mice undergo to premature death (PMID: 39912233) suggesting that CM polyploidization is a tight regulated process regulated by several independent mechanisms during heart development. *

In Figure 2D, the authors measured the cardiomyocyte cross-sectional area and concluded that removing PIDDosome components have no effect on cardiomyocyte cell size. Since it has been shown that ploidy increase is normally associated with an increase in cell area, the authors should measure cell area of cardiomyocytes analyzed in Figure 1B. It could be then interesting to establish a correlation with nuclear area and the mono, bi or multinucleated status. This will strengthen the results showing that ploidy increases without affecting cell area.

Indeed, studies in PIDDosome deficient livers suggest that tissue is containing fewer but bigger cells (PMID: *31983631). As opposed to the liver the percentage of cardiomyocytes presenting higher levels of ploidy is relatively low. Thus, a possible increase in CM size in PIDDosome deficient mice may be masked in heart cross-sections. In order to better correlate the ploidy with cell size, we propose to reanalyze our microscopy images used to extract the data displayed in Fig. 1D. We may run into the problem though that the number of cells acquired may become limiting to achieve sufficient statistical power. In this case we could pool data from different PIDDosome mutant CM to increase statistical power. Again, we propose to initially prioritize wt vs. Casp2 vs. XMLC2/Casp2f/f mice. In addition, we can offer to quantify heart to body weight ratio or tibia length as an additional read-out (see answer to a previous reviewer comment). *

The authors should discuss the fact that PIDDosome depletion lead only to a mild increase in ploidy levels (4N) in a small percentage of cardiomyocyte. If the PIDDosome is controlling ploidy, one could expect that removing it should lead to a drastic increase in the ploidy levels. Is PIDDosome depletion leading to cell death in some cardiomyocyte? The authors should discuss this point in the discussion or if relevant show a staining with an apoptosis marker. Is another mechanism compensating to prevent higher ploidy levels in cardiomyocytes?

These are valid thoughts, some of which we contemplated before. In part, we have addressed them in our response to Reviewer#1, above, discussing similar findings made in E2f7/8 deficient hearts (PMID: 36622904), or Cyclin G overexpressing hearts (PMID: 20360255), where also only modest changes in ploidy were achieved. Together these observations are suggesting alternative control mechanism able to act, or limited tolerance towards larger shifts in ploidy, incompatible with proper cell function and survival. Towards this end, we can offer to test if we find increased signs of cell death in PIDDosome mutant hearts by TUNEL staining of histological sections. Of note, we did not find evidence for such a phenomenon in the liver (PMID: 31983631).

Even if the authors presented RNAseq data suggesting that the PIDDosome is activated during cardiomyocyte differentiation, they should clearly demonstrate this point to strengthen the message of the paper. Indeed, the conclusions are based on the absence of PIDDosome components triggering higher ploidy in cardiomyocytes. However, we don't know whether (and when) the PIDDosome is activated during cardiomyocyte differentiation to control their ploidy levels. I would suggest to analyze PIDDosome activation markers by immunofluorescence in *cardiomyocytes at different developmental stages. *

*We agree with this referee that direct proof of PIDDosome activation would be helpful and that we only infer back from loss of function phenotypes when and where the PIDDosome becomes activated. However, several technical issues prevent us from collecting more direct evidence of PIDDosome activation in the developing heart. 1) Polyploidization in heart CM appears to happen gradually in CM from day 3 on with a peak at day 7 (PMID: 26544945). Hence, this is not a synchronous process, where we could pinpoint simultaneous activation of the PIDDosome in all cells at the same time, which would facilitate biochemical analysis, e.g., by western blotting for signs of Caspase-2 activation (i.e. the loss of its pro-form, PMID: 28130345). 2) Our most reliable readout, MDM2 cleavage by caspase-2 giving rise to specific fragments detectable in western, is not applicable to mouse tissue, as the antibody we use only detects human MDM2 (PMID: 28130345) and no other MDM2 Ab we tested gave satisfactory results. Independent of that, 3) we do not see involvement of p53 in CM ploidy control (arguing against a role of MDM2). *

*As such, we can only offer to look at extra centrosome clustering in postnatal binucleated CM (as also suggested further below), as a putative trigger for PIDDosome activation. However, this has been published by the first author of this study before (PMID 31301302). Given that we have made the significant effort to time resolve the increase in ploidy in postnatal mice (please note that several hearts needed to be pooled for each time point, analyzed in multiple biological replicates), we think that our conclusions are well-justified based on the genetic data provided. *

Concerning the methods, the authors must add the references for each product they used and not only the origin. When relevant, the RRID should be indicated. Without this information the method and the data cannot be reproduced.

We will update this information where relevant to reproduce our results

Minor comments:

In general, the text and the figures are clear. Nevertheless, I would suggest the following changes:

• Figures 1B, 2B and 2C: the y-axis must start at 0.

We will adopt axes accordingly

Figure 4A: The authors should stain centrosomes in cardiomyocytes. This should strengthen the conclusion taken by the authors based on the results obtained in mice depleted for ANKRD26. Indeed, for the moment they are insufficient to conclude about the role of the centrosomes. The authors should show that centrosomes cluster in cardiomyocytes (a condition necessary for PIDDosome activation in polyploid cells) and if possible that component of the PIDDosome are recruited here.

*This point is well taken and addressed in part above. Clustering of extra centrosomes has been documented and published by the first author of this study in rat polyploid cardiomyocytes (PIMID; cited). We can offer to show clustering of centrosomes in mouse CM isolated from day 7 hearts, but while PIDD1 can be detected well in MEF, we repeatedly failed to stain fro PIDD1 in primary CMs. *

Figure 4F: I would suggest to modify the working model to emphasize more the differences between WT and PIDDosome KO.

We will aim to improve this cartoon/graphical abstract

The prior studies are referenced appropriately.

Reviewer #2 (Significance (Required)):

How polyploid cells control their ploidy levels during differentiation remains poorly understood. The data presented here represent thus an advance concerning this question. The actual model concerning PIDDosome activation relies on the presence of extra centrosomes that drives the ANKDR26-dependent recruitment of the PIDDosome. Then, Caspase 2 is activated leading to a p53-p21 dependent cell cycle arrest (Burigotto & Fava, 2021, Sladky and Villunger, 2020; Janssens & Tinel, 2012; Evans et al., 2020; Burigotto et al., 2021). In this study, the authors showed that similar pathway takes place during cardiomyocyte differentiation to control ploidy levels. These data are reminiscent of previous work showing PIDDosome involvement during hepatocyte polyploidization (Sladky et al. 2020). Together, these data highlight the prominent role of the PIDDosome complex in controlling ploidy levels in physiological context. Importantly, this study identified that the classical p53-dependent cell cycle arrest described after PIDDosome activation is not involved here. Instead, the data established that independently of p53, p21 contribute to control cardiomyocyte ploidy. In consequence, this study extends the initial pathway associated with PIDDosome activation and suggest that other mechanisms could take place to restrain cell proliferation upon PIDDosome activation. Overall, this makes this paper significant and of interest for the following fields: polyploidy, heart/cardiomyocyte development and PIDDosome.

My field of expertise includes polyploidy, cell cycle and genetic instability.

We thank this reviewer for the time taken and the positive feedback provided.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

N/A

4. Description of analyses that authors prefer not to carry out

Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

• *As outlined above, limited tools are available to validate putative caspase-2 substrates, identified in proteomics analysis, in an impactful manner. *
• *Also, as discussed above, we deem myocardial infarction experiments in mice as unsuitable to improve our work, as with all likely-hood, they will yield negative results. *

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary:

In this study, the authors investigated the role of the PIDDosome during cardiomyocyte polyploidization. PIDDosome is a multi-protein complex activating the endopeptidase Caspase-2, and shown to be involved in eliminating cells with extra centrosomes or in response to genotoxic stress (Burigotto & Fava, 2021, Sladky and Villunger, 2020). In both cases, the PIDDosome is recruited in a ANKRD26-dependent manner at the centrosomes leading to p53 stabilization and cell death (Burigotto & Fava, 2021; Evans et al., 2020; Burigotto et al., 2021).

Here, by studying mouse cardiomyocyte differentiation, the authors showed that PIDDosome is imposing ploidy restriction during cardiomyocyte differentiation. Importantly, in contrast to a previous report in the liver (Sladky et al., 2020), they showed that PIDDosome acts in a p53-independent manner in cardiomyocytes. Indeed, they suggested that PIDDosome controls ploidy in cardiomyocytes through p21 activation.

Major comments:

In general the conclusions of the authors are well supported by the experiments. However, I would suggest the following experiments/analysis to strengthen the paper:

• The authors should improve the Figure 1 to help the readers who are not familiar with cardiomyocyte polyploidization. For instance, I would suggest to add a scheme to summarize cardiomyocyte polyploidization (in terms of nuclear size, mono vs multi and so on). Based on the images they presented in 1B, the authors should also measure the nuclear area or volume in the different conditions in which components of the PIDDosome were depleted. Indeed, these two parameters should be easier to conceptualize for the readers (instead of the fluorescence nuclear intensity). This could help to understand if the nuclear size is maintained between the different conditions and if this is comparable between mono, bi or multinucleated cardiomyocytes.
• In Figure 2A, the authors presented cross section of heart from animals showing that PIDDosome depletion has no effect on heart size. This is a surprising results since cardiomyocytes have higher ploidy levels and this could have an effect on their function. Since the importance of this observation, the authors should present a quantification of the heart size in the different conditions shown in Figure 2A. Also, the percentage of cardiomyocytes presenting higher levels of ploidy seems quiet low. The authors should discuss this point. In particular because this could explain the absence of consequences on heart size and function at steady state.
• In Figure 2D, the authors measured the cardiomyocyte cross-sectional area and concluded that removing PIDDosome components have no effect on cardiomyocyte cell size. Since it has been shown that ploidy increase is normally associated with an increase in cell area, the authors should measure cell area of cardiomyocytes analyzed in Figure 1B. It could be then interesting to establish a correlation with nuclear area and the mono, bi or multinucleated status. This will strengthen the results showing that ploidy increases without affecting cell area.
• The authors should discuss the fact that PIDDosome depletion lead only to a mild increase in ploidy levels (4N) in a small percentage of cardiomyocyte. If the PIDDosome is controlling ploidy, one could expect that removing it should lead to a drastic increase in the ploidy levels. Is PIDDosome depletion leading to cell death in some cardiomyocyte? The authors should discuss this point in the discussion or if relevant show a staining with an apoptosis marker. Is another mechanism compensating to prevent higher ploidy levels in cardiomyocytes?
• Even if the authors presented RNAseq data suggesting that the PIDDosome is activated during cardiomyocyte differentiation, they should clearly demonstrate this point to strengthen the message of the paper. Indeed, the conclusions are based on the absence of PIDDosome components triggering higher ploidy in cardiomyocytes. However, we don't know whether (and when) the PIDDosome is activated during cardiomyocyte differentiation to control their ploidy levels. I would suggest to analyze PIDDosome activation markers by immunofluorescence in cardiomyocytes at different developmental stages.

Concerning the methods, the authors must add the references for each product they used and not only the origin. When relevant, the RRID should be indicated. Without this information the method and the data cannot be reproduced.

The statistics are well indicated in the figures and in the figure legends.

Minor comments:

In general, the text and the figures are clear. Nevertheless, I would suggest the following changes:

• Figures 1B, 2B and 2C: the y-axis must start at 0.
• Figure 4A: The authors should stain centrosomes in cardiomyocytes. This should strengthen the conclusion taken by the authors based on the results obtained in mice depleted for ANKRD26. Indeed, for the moment they are insufficient to conclude about the role of the centrosomes. The authors should show that centrosomes cluster in cardiomyocytes (a condition necessary for PIDDosome activation in polyploid cells) and if possible that component of the PIDDosome are recruited here.
• Figure 4F: I would suggest to modify the working model to emphasize more the differences between WT and PIDDosome KO.

The prior studies are referenced appropriately.

Significance

How polyploid cells control their ploidy levels during differentiation remains poorly understood. The data presented here represent thus an advance concerning this question.

The actual model concerning PIDDosome activation relies on the presence of extra centrosomes that drives the ANKDR26-dependent recruitment of the PIDDosome. Then, Caspase 2 is activated leading to a p53-p21 dependent cell cycle arrest (Burigotto & Fava, 2021, Sladky and Villunger, 2020; Janssens & Tinel, 2012; Evans et al., 2020; Burigotto et al., 2021). In this study, the authors showed that similar pathway takes place during cardiomyocyte differentiation to control ploidy levels. These data are reminiscent of previous work showing PIDDosome involvement during hepatocyte polyploidization (Sladky et al. 2020). Together, these data highlight the prominent role of the PIDDosome complex in controlling ploidy levels in physiological context.

Importantly, this study identified that the classical p53-dependent cell cycle arrest described after PIDDosome activation is not involved here. Instead, the data established that independently of p53, p21 contribute to control cardiomyocyte ploidy. In consequence, this study extend the initial pathway associated with PIDDosome activation and suggest that other mechanisms could take place to restrain cell proliferation upon PIDDosome activation.

Overall, this makes this paper significant and of interest for the following fields: polyploidy, heart/cardiomyocyte development and PIDDosome.

My field of expertise includes polyploidy, cell cycle and genetic instability.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

Reply to the reviewers

Manuscript number: RC-2025-02953

Corresponding author(s): Andreas, Villunger

[The “revision plan” should delineate the revisions that authors intend to carry out in response to the points raised by the referees. It also provides the authors with the opportunity to explain their view of the paper and of the referee reports.

• *

The document is important for the editors of affiliate journals when they make a first decision on the transferred manuscript. It will also be useful to readers of the reprint and help them to obtain a balanced view of the paper.

• *

If you wish to submit a full revision, please use our "Full Revision" template. It is important to use the appropriate template to clearly inform the editors of your intentions.]

1. General Statements [optional]

*We would like to thank the reviewers for their constructive input and overall support. We appreciate to provide a provisional revision plan, as outlined here, and are happy to engage in additional communication with journal editors via video call, in case further clarifications are needed. *

2. Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

• *

Reviewer #1

__Evidence, reproducibility and clarity __

Summary: This manuscript by Leone et al describes the role of the PIDDosome in cardiomyocytes. Using a series of whole body and cardiomyocyte specific knockouts, the authors show that the PIDDosome maintains correct ploidy in these cells. It achieves this through inducing cell cycle arrest in cardiomyocytes in a p53 dependent manner. Despite this effect on ploidy, PIDDosome-deficient hearts show no structural or functional defects. Statistics and rigor appear to be adequate.

We thank this referee for taking the time to evaluate our work and their valuable comments. We assume that this reviewer by mistake indicates that the phenomenon we describe, depends on p53. As outlined in the abstract and throughout the manuscript, the effect is independent of p53, but may additionally still involve p21, acting along or parallel to the PIDDosome.

Major comments: 1. Figure 1 uses fluorescent intensity of a nuclear stain to determine ploidy per nucleus and they further separate the results into mononucleated, binucleated or multinucleated cells. It is hard to know how to interpret these results without further information or controls. Is there a good positive control that can be used to help to show whether this assay is quantitative? The differences are larger with the Raidd and caspase-2 knockouts than with the Pidd knockouts but this is not addressed.

*We appreciate this concern. Regarding a “good positive control” we can say that we follow state-of the art in the cardiomyocyte field and studies by the Evans (PMID: 36622904), Kuhn (PMID: 32109383), Bergmann (PMID: 26544945) and Patterson labs (PMID: 28783163, 36912240) all use the identical approach to discriminate 2n from 4n nuclei in microscopy images at the cellular level. The fact that the majority of rodent CM nuclei is indeed diploid (PMID: 31175264, 31585517 and 32078450) and a large number of nuclei has been evaluated to assess their mean fluorescence intensity (MFI) reduces the risk of a systematic bias in our analysis. Moreover, we have used an orthogonal approach that is indeed quantitative to define DNA content, i.e,. flow-cytometry based evaluation of DNA content in isolated CM nuclei (Fig. 1C). We hence are confident our assays are quantitative. *

Regarding the fact that loss of Pidd1 causes a more saddle phenotype, we can offer to discuss this in light of the fact that Pidd1 has additional functions, outside the PIDDosome (PMID: 35343572), and that we made similar observations when analyzing ploidy in hepatocytes (PMID: *31983631). Given the fact that all components of the PIDDosome show a similar phenotype, and that this phenotype is mimicked by loss of the protein that connects PIDD1 and centrosomes, ANKRD26 (Fig. 4a), we are confident that this biological variation in our analysis is not affecting our conclusions. *

On line 459 the authors state that the increase in polyploidy in PIDDosome knockouts occurs in adult hood but this is not directly tested. In fact, in the next section the polyploidy is assessed in early postnatal development. This statement should be explained or removed.

We see that we have made an unclear statement here. In fact, we first noted increases in ploidy in adult heart and then define the time window in development when this happens. This sentence will be rephrased.

In Figure 4. The authors obtained RNAseq data for P1, P7 and P14 but only show the differences with and without caspase-2 at P7. Given that the differences in ploidy are more significant at P14 (Fig 3D), all the comparisons should be shown along with analysis of whether the same genes/gene families are altered in the absence of caspase-2.

The reason why we focus on postnatal day 7 (P7) is that data from Alkass et al (PMID: 26544945) and other labs (PMID: 31175264 ) document that on this day the initial wave of binucleation peaks. Hence, we hypothesized that the PIDDosome must be active in most CM, which aligns well with the increased mRNA levels of all of its components (Figure 3). Interestingly, it seems that its action is tightly regulated, as mRNA of PIDDosome components drop on P10, suggesting PIDDosome shut-down or downregulation. Similar findings have been noted in the liver (PMID: *31983631). Alkass and colleagues also show that very few CMs enter another round of DNA synthesis between P7 and P14, and hence possible transcriptome changes in the absence of the PIDDosome will be strongly diluted. *

Please note that on P1, there is no difference between genotypes to be expected as all CM are mononucleated diploids and cytokinesis competent, as previously demonstrated (PMID: *26544945). Moreover, PIDDosome expression levels are extremely low (Fig. 3A). As such, no difference between genotypes are expected on P1. In addition, on P14 the ploidy phenotype observed in PIDDosome knockout mice reaches the maximum and ploidy increases are comparable to adult tissue. Thus, at this time the trigger for PIDDosome activation (cytokinesis failure) is no longer observed as the majority of CMs are post-mitotic, (PMID: 26247711). As such the impact of PIDDosome activation on the P14 transcriptome is most likely negligible. However, if desired, we can expand our bioinformatics analysis summarizing findings made related to DEGs over time in wt animals by comparing genotypes also on day 1 and day 14. In light of the above, analysis between genotypes on P7 holds still appears as the one most meaningful. *

Some validation of the RNAseq and/or proteomics results would be an important addition to this study

We agree with this notion and propose to validate key candidates related to cardiomyocyte proliferation and polyploidization, some of which we found to be differentially expressed at the mRNA level on day 7in the RNAseq data (e.g., p21, Foxm1, Kif18a, Lin37 and others)

Regarding the proteomics results, we face the challenge that we can only try to confirm if candidate proteins are likely caspase substrates in silico using DeepCleave*, and potentially pick one or two candidates linked to CM differentiation for further analysis in vitro and in heterologous cell based assays (e.g. 293T cells), as no bona-fide ventricular cardiomyocyte cell lines exist. Primary postnatal CMs are extremely difficult to transfect, nor they proliferate without drug-treatment, or fail cytokinesis ex vivo. *

Figure 4D: the authors make the conclusion that p21 is downstream of PIDD (et p53 independent). However, this is not supported by the data because the increase in 4N cells/decrease in 2N cells, although statistically significant, is nowhere near that of caspase-2 KO and caspase-2/p21 KO. Statistics should also compare p32KO with c2KO. In the absence of any other data, the more likely conclusion is that p21 is not involved.

*We agree that the findings related to the impact seen upon loss of p21 suggest that it is not the only effector involved in ploidy control and it may not even be an effector engaged by caspase-2, as C2/p21 DKO mice have an even higher ploidy increase, albeit not statistically significant. However, it is important to highlight that p21 (Cdkn1a) was found to be downregulated in our transcriptomic analysis suggesting an involvement in the caspace2-cascade. We are happy to highlight this when presenting the results and in the discussion. *

*We assume that this referee refers to p73 KO data that should be compared to Casp2 KO data (could be read as p73 or p53, but the latter we compare side by side with Casp2 in Fig. 4 already). As p73 KO mice were not found to be viable beyond day 7 (our attempt to find animals on day 10 failed, in line with published literature (PMID: 24500610, 10716451)), we can only offer to compare this data set to the data presented in Figure 3C, where we have analyzed ploidy increases on day 7 from wt and PIDDosome mutant mice. This re-analysis will show that only Caspase-2 mutant mice display a significant ploidy increase on P7, when compared to wt or p73 mutant animals, while no difference are noted between wt and p73 mutant mice (to be included in new Suppl. Fig. 3C) *

Minor comments: Suggest moving Figure 4A to Figure 3 as it seems to fit better there based on the citation of this figure in the text

*We can see some benefit in this recommendation and included panel 4A now in an updated version of Figure 3. *

Recommend enhancing the brightness of microscopy images in Figure 1E and 2D

We will try to improve image quality, may have been due to PDF conversion

Significance

This study provides interesting information for the role of the PIDDosome in protecting from polyploidy and adds to the body of work by this same group studying this pathway in the liver.

The main weakness in terms of significance is the lack of a phenotype in the hearts of these animals. Therefore, it is clear that ploidy (or at least PIDDosome dependent ploidy) has minimal impact on cardiac development.

We respectfully disagree with the comment that the lack of impact on cardiac function constitutes a weakness of our findings. Several studies on ploidy control in the liver (PMID 34228992) but importantly also heart (PMID: 36622904) have failed to document a clear impact of increased ploidy on organ function. This does not infer insignificance, but maybe rather that the context where this becomes relevant has not been identified. We are happy expand on this in our discussion

• *

The authors mention that they have not tried giving these mice an myocardial infarct (MI) or inducing any other type of cardiac damage. Although it is understood that these experiments are likely outside of the scope of the present study, without this information the impact of this study is moderate. I recommend expanding the discussion to provide a more in-depth possible rationale as to why ploidy perturbations do not lead to structural changes like in the liver.

Despite this, the insights to the pathway itself are interesting to investigators in the caspase-2 field if a little underdeveloped, especially concerning the role of p21.

My expertise is in cell death and caspase biology (especially caspase-2). I have sufficient expertise to evaluate all parts of this paper.

*As mentioned above, we will amend our conclusions on p21, in light of potential findings made when validating DEG candidates, as stated above. *

*We hope that the changes and amendments proposed here will be satisfactory to this referee to recommend publication of a revised manuscript. *

• *

Reviewer #2

__Evidence, reproducibility and clarity: __

__Summary: __

In this study, the authors investigated the role of the PIDDosome during cardiomyocyte polyploidization. PIDDosome is a multi-protein complex activating the endopeptidase Caspase-2, and shown to be involved in eliminating cells with extra centrosomes or in response to genotoxic stress (Burigotto & Fava, 2021, Sladky and Villunger, 2020). In both cases, the PIDDosome is recruited in a ANKRD26-dependent manner at the centrosomes leading to p53 stabilization and cell death (Burigotto & Fava, 2021; Evans et al., 2020; Burigotto et al., 2021).

Here, by studying mouse cardiomyocyte differentiation, the authors showed that PIDDosome is imposing ploidy restriction during cardiomyocyte differentiation. Importantly, in contrast to a previous report in the liver (Sladky et al., 2020), they showed that PIDDosome acts in a p53-independent manner in cardiomyocytes. Indeed, they suggested that PIDDosome controls ploidy in cardiomyocytes through p21 activation.

We want to thank this reviewer for the time taken to evaluate our work and provide critical feedback that will help to improve our revised manuscript.

__Major comments: __

In general the conclusions of the authors are well supported by the experiments. However, I would suggest the following experiments/analysis to strengthen the paper:

The authors should improve the Figure 1 to help the readers who are not familiar with cardiomyocyte polyploidization. For instance, I would suggest to add a scheme to summarize cardiomyocyte polyploidization (in terms of nuclear size, mono vs multi and so on).

We agree that a visual summary of the postnatal timing of CM polyploidization will be helpful for the generalist not familiar with the topic and have added a scheme, adapted from a study by Alkass et al. (PMID: *26544945), who elegantly defined the timing of this process during postnatal mice life (now Fig. 1A). *

Based on the images they presented in 1B, the authors should also measure the nuclear area or volume in the different conditions in which components of the PIDDosome were depleted. Indeed, these two parameters should be easier to conceptualize for the readers (instead of the fluorescence nuclear intensity). This could help to understand if the nuclear size is maintained between the different conditions and if this is comparable between mono, bi or multinucleated cardiomyocytes.

We have acquired this data and it can be used to provide additional information on nuclear area and/or volume. We propose to focus on re-analyzing data from wt, Casp2 and XMLC2CRE/Casp2f/f mice. The additional information can be included in Figures 1 & 2, respectively.

• In Figure 2A, the authors presented cross section of heart from animals showing that PIDDosome depletion has no effect on heart size. This is a surprising result since cardiomyocytes have higher ploidy levels and this could have an effect on their function. Since the importance of this observation, the authors should present a quantification of the heart size in the different conditions shown in Figure 2A.

We agree with this comment. We can measure the heart vs. body weight ratio or tibia length in adult Casp2-/- vs. WT (3 month old) in order to indirectly evaluate possible increases in CM size linked to increased ploidy.

Also, the percentage of cardiomyocytes presenting higher levels of ploidy seems quite low. The authors should discuss this point. In particular because this could explain the absence of consequences on heart size and function at steady state.

We agree with this conclusion and will expand on this in our discussion. It is important to note that as opposed to findings made in liver (PMID: *31983631), genetic manipulation of ploidy regulators such as E2f7/8 (PMID: 36622904), only led to modest changes in CM ploidy, suggesting that either a small band-width compatible with normal heart function exists, or that additional mechanisms exist that take control when these thresholds set by the PIDDosome or E2f7/8 are exceeded. These mechanisms could involve Cyclin G (PMID: 20360255), or TNNI3K (PMID: 31589606). Importantly, a recent publication has shown that overexpression of Plk1(T210D) and Ect2 from birth causes increased heart weight coupled with a minor decrease in CM size. These mice undergo to premature death (PMID: 39912233) suggesting that CM polyploidization is a tight regulated process regulated by several independent mechanisms during heart development. *

• *

In Figure 2D, the authors measured the cardiomyocyte cross-sectional area and concluded that removing PIDDosome components have no effect on cardiomyocyte cell size. Since it has been shown that ploidy increase is normally associated with an increase in cell area, the authors should measure cell area of cardiomyocytes analyzed in Figure 1B. It could be then interesting to establish a correlation with nuclear area and the mono, bi or multinucleated status. This will strengthen the results showing that ploidy increases without affecting cell area.

Indeed, studies in PIDDosome deficient livers suggest that tissue is containing fewer but bigger cells (PMID: *31983631). As opposed to the liver the percentage of cardiomyocytes presenting higher levels of ploidy is relatively low. Thus, a possible increase in CM size in PIDDosome deficient mice may be masked in heart cross-sections. In order to better correlate the ploidy with cell size, we propose to reanalyze our microscopy images used to extract the data displayed in Fig. 1D. We may run into the problem though that the number of cells acquired may become limiting to achieve sufficient statistical power. In this case we could pool data from different PIDDosome mutant CM to increase statistical power. Again, we propose to initially prioritize wt vs. Casp2 vs. XMLC2/Casp2f/f mice. In addition, we can offer to quantify heart to body weight ratio or tibia length as an additional read-out (see answer to a previous reviewer comment). *

The authors should discuss the fact that PIDDosome depletion lead only to a mild increase in ploidy levels (4N) in a small percentage of cardiomyocyte. If the PIDDosome is controlling ploidy, one could expect that removing it should lead to a drastic increase in the ploidy levels. Is PIDDosome depletion leading to cell death in some cardiomyocyte? The authors should discuss this point in the discussion or if relevant show a staining with an apoptosis marker. Is another mechanism compensating to prevent higher ploidy levels in cardiomyocytes?

These are valid thoughts, some of which we contemplated before. In part, we have addressed them in our response to Reviewer#1, above, discussing similar findings made in E2f7/8 deficient hearts (PMID: 36622904), or Cyclin G overexpressing hearts (PMID: 20360255), where also only modest changes in ploidy were achieved. Together these observations are suggesting alternative control mechanism able to act, or limited tolerance towards larger shifts in ploidy, incompatible with proper cell function and survival. Towards this end, we can offer to test if we find increased signs of cell death in PIDDosome mutant hearts by TUNEL staining of histological sections. Of note, we did not find evidence for such a phenomenon in the liver (PMID: 31983631).

Even if the authors presented RNAseq data suggesting that the PIDDosome is activated during cardiomyocyte differentiation, they should clearly demonstrate this point to strengthen the message of the paper. Indeed, the conclusions are based on the absence of PIDDosome components triggering higher ploidy in cardiomyocytes. However, we don't know whether (and when) the PIDDosome is activated during cardiomyocyte differentiation to control their ploidy levels. I would suggest to analyze PIDDosome activation markers by immunofluorescence in *cardiomyocytes at different developmental stages. *

*We agree with this referee that direct proof of PIDDosome activation would be helpful and that we only infer back from loss of function phenotypes when and where the PIDDosome becomes activated. However, several technical issues prevent us from collecting more direct evidence of PIDDosome activation in the developing heart. 1) Polyploidization in heart CM appears to happen gradually in CM from day 3 on with a peak at day 7 (PMID: 26544945). Hence, this is not a synchronous process, where we could pinpoint simultaneous activation of the PIDDosome in all cells at the same time, which would facilitate biochemical analysis, e.g., by western blotting for signs of Caspase-2 activation (i.e. the loss of its pro-form, PMID: 28130345). 2) Our most reliable readout, MDM2 cleavage by caspase-2 giving rise to specific fragments detectable in western, is not applicable to mouse tissue, as the antibody we use only detects human MDM2 (PMID: 28130345) and no other MDM2 Ab we tested gave satisfactory results. Independent of that, 3) we do not see involvement of p53 in CM ploidy control (arguing against a role of MDM2). *

*As such, we can only offer to look at extra centrosome clustering in postnatal binucleated CM (as also suggested further below), as a putative trigger for PIDDosome activation. However, this has been published by the first author of this study before (PMID 31301302). Given that we have made the significant effort to time resolve the increase in ploidy in postnatal mice (please note that several hearts needed to be pooled for each time point, analyzed in multiple biological replicates), we think that our conclusions are well-justified based on the genetic data provided. *

Concerning the methods, the authors must add the references for each product they used and not only the origin. When relevant, the RRID should be indicated. Without this information the method and the data cannot be reproduced.

We will update this information where relevant to reproduce our results

Minor comments:

In general, the text and the figures are clear. Nevertheless, I would suggest the following changes:

• Figures 1B, 2B and 2C: the y-axis must start at 0.

We will adopt axes accordingly

Figure 4A: The authors should stain centrosomes in cardiomyocytes. This should strengthen the conclusion taken by the authors based on the results obtained in mice depleted for ANKRD26. Indeed, for the moment they are insufficient to conclude about the role of the centrosomes. The authors should show that centrosomes cluster in cardiomyocytes (a condition necessary for PIDDosome activation in polyploid cells) and if possible that component of the PIDDosome are recruited here.

*This point is well taken and addressed in part above. Clustering of extra centrosomes has been documented and published by the first author of this study in rat polyploid cardiomyocytes (PIMID; cited). We can offer to show clustering of centrosomes in mouse CM isolated from day 7 hearts, but while PIDD1 can be detected well in MEF, we repeatedly failed to stain fro PIDD1 in primary CMs. *

Figure 4F: I would suggest to modify the working model to emphasize more the differences between WT and PIDDosome KO.

We will aim to improve this cartoon/graphical abstract

The prior studies are referenced appropriately.

Reviewer #2 (Significance (Required)):

How polyploid cells control their ploidy levels during differentiation remains poorly understood. The data presented here represent thus an advance concerning this question. The actual model concerning PIDDosome activation relies on the presence of extra centrosomes that drives the ANKDR26-dependent recruitment of the PIDDosome. Then, Caspase 2 is activated leading to a p53-p21 dependent cell cycle arrest (Burigotto & Fava, 2021, Sladky and Villunger, 2020; Janssens & Tinel, 2012; Evans et al., 2020; Burigotto et al., 2021). In this study, the authors showed that similar pathway takes place during cardiomyocyte differentiation to control ploidy levels. These data are reminiscent of previous work showing PIDDosome involvement during hepatocyte polyploidization (Sladky et al. 2020). Together, these data highlight the prominent role of the PIDDosome complex in controlling ploidy levels in physiological context. Importantly, this study identified that the classical p53-dependent cell cycle arrest described after PIDDosome activation is not involved here. Instead, the data established that independently of p53, p21 contribute to control cardiomyocyte ploidy. In consequence, this study extends the initial pathway associated with PIDDosome activation and suggest that other mechanisms could take place to restrain cell proliferation upon PIDDosome activation. Overall, this makes this paper significant and of interest for the following fields: polyploidy, heart/cardiomyocyte development and PIDDosome.

My field of expertise includes polyploidy, cell cycle and genetic instability.

We thank this reviewer for the time taken and the positive feedback provided.

• *

• *

3. Description of the revisions that have already been incorporated in the transferred manuscript

Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

• *

N/A

4. Description of analyses that authors prefer not to carry out

Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

• *

• *As outlined above, limited tools are available to validate putative caspase-2 substrates, identified in proteomics analysis, in an impactful manner. *

• *Also, as discussed above, we deem myocardial infarction experiments in mice as unsuitable to improve our work, as with all likely-hood, they will yield negative results. *

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

Summary:

In this study, the authors investigated the role of the PIDDosome during cardiomyocyte polyploidization. PIDDosome is a multi-protein complex activating the endopeptidase Caspase-2, and shown to be involved in eliminating cells with extra centrosomes or in response to genotoxic stress (Burigotto & Fava, 2021, Sladky and Villunger, 2020). In both cases, the PIDDosome is recruited in a ANKRD26-dependent manner at the centrosomes leading to p53 stabilization and cell death (Burigotto & Fava, 2021; Evans et al., 2020; Burigotto et al., 2021).

Here, by studying mouse cardiomyocyte differentiation, the authors showed that PIDDosome is imposing ploidy restriction during cardiomyocyte differentiation. Importantly, in contrast to a previous report in the liver (Sladky et al., 2020), they showed that PIDDosome acts in a p53-independent manner in cardiomyocytes. Indeed, they suggested that PIDDosome controls ploidy in cardiomyocytes through p21 activation.

Major comments:

In general the conclusions of the authors are well supported by the experiments. However, I would suggest the following experiments/analysis to strengthen the paper:

• The authors should improve the Figure 1 to help the readers who are not familiar with cardiomyocyte polyploidization. For instance, I would suggest to add a scheme to summarize cardiomyocyte polyploidization (in terms of nuclear size, mono vs multi and so on). Based on the images they presented in 1B, the authors should also measure the nuclear area or volume in the different conditions in which components of the PIDDosome were depleted. Indeed, these two parameters should be easier to conceptualize for the readers (instead of the fluorescence nuclear intensity). This could help to understand if the nuclear size is maintained between the different conditions and if this is comparable between mono, bi or multinucleated cardiomyocytes.
• In Figure 2A, the authors presented cross section of heart from animals showing that PIDDosome depletion has no effect on heart size. This is a surprising results since cardiomyocytes have higher ploidy levels and this could have an effect on their function. Since the importance of this observation, the authors should present a quantification of the heart size in the different conditions shown in Figure 2A. Also, the percentage of cardiomyocytes presenting higher levels of ploidy seems quiet low. The authors should discuss this point. In particular because this could explain the absence of consequences on heart size and function at steady state.
• In Figure 2D, the authors measured the cardiomyocyte cross-sectional area and concluded that removing PIDDosome components have no effect on cardiomyocyte cell size. Since it has been shown that ploidy increase is normally associated with an increase in cell area, the authors should measure cell area of cardiomyocytes analyzed in Figure 1B. It could be then interesting to establish a correlation with nuclear area and the mono, bi or multinucleated status. This will strengthen the results showing that ploidy increases without affecting cell area.
• The authors should discuss the fact that PIDDosome depletion lead only to a mild increase in ploidy levels (4N) in a small percentage of cardiomyocyte. If the PIDDosome is controlling ploidy, one could expect that removing it should lead to a drastic increase in the ploidy levels. Is PIDDosome depletion leading to cell death in some cardiomyocyte? The authors should discuss this point in the discussion or if relevant show a staining with an apoptosis marker. Is another mechanism compensating to prevent higher ploidy levels in cardiomyocytes?
• Even if the authors presented RNAseq data suggesting that the PIDDosome is activated during cardiomyocyte differentiation, they should clearly demonstrate this point to strengthen the message of the paper. Indeed, the conclusions are based on the absence of PIDDosome components triggering higher ploidy in cardiomyocytes. However, we don't know whether (and when) the PIDDosome is activated during cardiomyocyte differentiation to control their ploidy levels. I would suggest to analyze PIDDosome activation markers by immunofluorescence in cardiomyocytes at different developmental stages.

Concerning the methods, the authors must add the references for each product they used and not only the origin. When relevant, the RRID should be indicated. Without this information the method and the data cannot be reproduced.

The statistics are well indicated in the figures and in the figure legends.

Minor comments:

In general, the text and the figures are clear. Nevertheless, I would suggest the following changes:

• Figures 1B, 2B and 2C: the y-axis must start at 0.
• Figure 4A: The authors should stain centrosomes in cardiomyocytes. This should strengthen the conclusion taken by the authors based on the results obtained in mice depleted for ANKRD26. Indeed, for the moment they are insufficient to conclude about the role of the centrosomes. The authors should show that centrosomes cluster in cardiomyocytes (a condition necessary for PIDDosome activation in polyploid cells) and if possible that component of the PIDDosome are recruited here.
• Figure 4F: I would suggest to modify the working model to emphasize more the differences between WT and PIDDosome KO.

The prior studies are referenced appropriately.

Significance

How polyploid cells control their ploidy levels during differentiation remains poorly understood. The data presented here represent thus an advance concerning this question.

The actual model concerning PIDDosome activation relies on the presence of extra centrosomes that drives the ANKDR26-dependent recruitment of the PIDDosome. Then, Caspase 2 is activated leading to a p53-p21 dependent cell cycle arrest (Burigotto & Fava, 2021, Sladky and Villunger, 2020; Janssens & Tinel, 2012; Evans et al., 2020; Burigotto et al., 2021). In this study, the authors showed that similar pathway takes place during cardiomyocyte differentiation to control ploidy levels. These data are reminiscent of previous work showing PIDDosome involvement during hepatocyte polyploidization (Sladky et al. 2020). Together, these data highlight the prominent role of the PIDDosome complex in controlling ploidy levels in physiological context.

Importantly, this study identified that the classical p53-dependent cell cycle arrest described after PIDDosome activation is not involved here. Instead, the data established that independently of p53, p21 contribute to control cardiomyocyte ploidy. In consequence, this study extend the initial pathway associated with PIDDosome activation and suggest that other mechanisms could take place to restrain cell proliferation upon PIDDosome activation.

Overall, this makes this paper significant and of interest for the following fields: polyploidy, heart/cardiomyocyte development and PIDDosome.

My field of expertise includes polyploidy, cell cycle and genetic instability.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

This study investigates the potential of targeting specific regions within the RNA genome of the Porcine Epidemic Diarrhea Virus (PEDV) for antiviral drug development. The authors used SHAPE-MaP to analyze the structure of the PEDV RNA genome in infected cells. They categorized different regions of the genome based on their structural characteristics, focusing on those that might be good targets for drugs or small interfering RNAs (siRNAs).

They found that dynamic single-stranded regions can be stabilized by compounds (e.g., to form G-quadruplexes), which inhibit viral proliferation. They demonstrated this by targeting a specific G4-forming sequence with a compound called Braco-19. The authors also describe stable (structured) single-stranded regions that they used to design siRNAs showing that they effectively inhibited viral replication.

Strengths:

There are a number of strengths to highlight in this manuscript.

(1) The study uses a sophisticated technique (SHAPE-MaP) to analyze the PEDV RNA genome in situ, providing valuable insights into its structural features.

(2) The authors provide a strong rationale for targeting specific RNA structures for antiviral development.

(3) The study includes a range of experiments, including structural analysis, compound screening, siRNA design, and viral proliferation assays, to support their conclusions.

(4) Finally, the findings have potential implications for the development of new antiviral therapies against PEDV and other RNA viruses.

Overall, this interesting study highlights the importance of considering RNA structure when designing antiviral therapies and provides a compelling strategy for identifying promising RNA targets in viral genomes.

Weaknesses:

I have some concerns about the utility of the 3D analyses, the effects of their synonymous mutants on expression/proliferation, a potentially missed control for studies of mutants, and the therapeutic utility of the compound they tested vs. Gquadruplexes.

We thank the reviewer for their positive assessment and insightful comments. Below, we address each point of concern:

(1) The utility of the 3D analyses:

In the revised manuscript, we have toned down this discussion and moved Figure 3A to the supplementary materials to reduce any sense of fragmentation in the overall story. While SHAPE-MaP technology is mature and convenient to use and can indeed capture some RNA structural elements with special functions in certain case; we acknowledge that its application for 3D analyses requires further validation. We believe this approach will become more prevalent in future research.

(2) The effects of synonymous mutants on expression/proliferation:

In the PEDV genome, the PQS1 mutation site encodes lysine (AAG). Given that lysine has only two codons (AAG and AAA), the G3109A synonymous mutation represented our sole viable option. Published studies (Ding et al., 2024) confirm that neither AAG nor AAA are classified as rare or dominant codons in mammalian cells. Therefore, the observed changes in viral proliferation levels are likely to stem from alterations in RNA secondary structure rather than codon usage effects.

REFERENCES:

Ding W, Yu W, Chen Y, et al. Rare codon recoding for efficient noncanonical amino acid incorporation in mammalian cells. Science. 2024;384(6700):1134-1142. 

(3) Potentially missed control for studies of mutants:

In the revised manuscript, we have incorporated additional control experiments evaluating Braco-19's therapeutic effects on the PQS3 mutant strain (Figure 4 – figure supplement 3)：

(4) The therapeutic utility of Braco-19 vs. G-quadruplexes:

While Braco-19 is indeed a broad-spectrum G4 ligand, our data clearly show that not all PQSs in the viral genome can form G4 structures. Our findings primarily provide proof-of-concept that sequences with high G4-forming potential in viral genomes represent viable targets for antiviral therapy. Future studies could leverage SHAPEguided structural insights to design ligands with enhanced specificity for viral G4s, potentially improving therapeutic utility while minimizing off-target effects.

Reviewer #2 (Public review):

Summary:

Luo et. al. use SHAPE-MaP to find suitable RNA targets in Porcine Epidemic Diarrhoea Virus. Results show that dynamic and transient structures are good targets for small molecules, and that exposed strand regions are adequate targets for siRNA. This work is important to segment the RNA targeting.

Strengths:

This work is well done and the data supports its findings and conclusions. When possible, more than one technique was used to confirm some of the findings.

Weaknesses:

The study uses a cell line that is not porcine (not the natural target of the virus).

We thank the reviewer for their insightful comments and recognition of our study's value. The most commonly employed cell models for in vitro PEDV studies are monkey-derived Vero E6 cells and porcine PK1 cells. However, PEDV (particularly our strain) exhibits significantly lower replication efficiency in PK1 cells compared to Vero cells, and no cytopathic effects were observed in PK1 cells. In our preliminary attempts to perform SHAPE-MaP experiments using infected PK1 cells, the sequencing data showed less than 0.03% alignment to the PEDV genome, rendering subsequent analysis and downstream experiments unfeasible.

Reviewer #3 (Public review):

Summary:

This manuscript by Luo et al. applied SHAPE-Map to analyze the secondary structure of the Porcine Epidemic Diarrhoea Virus (PEDV) RNA genome in infected cells. By combining SHAPE reactivity and Shannon entropy, the study indicated that the folding of the PEDV genomic RNA was nonuniform, with the 5' and 3' untranslated regions being more compactly structured, which revealed potentially antiviral targetable RNA regions. Interestingly, the study also suggested that compounds bound to well-folded RNA structures in vitro did not necessarily exhibit antiviral activity in cells, because the binding of these compounds did not necessarily alter the functions of the well-folded RNA regions. Later in the manuscript, the authors focus on guanine-rich regions, which may form G-quadruplexes and be potential targets for small interfering RNA (siRNA). The manuscript shows the binding effect of Braco-19 (a G-quadruplex-binding ligand) to a predicted G4 region in vitro, along with the inhibition of PEDV proliferation in cells. This suggests that targeting high SHAPE-high Shannon G4 regions could be a promising approach against RNA viruses. Lastly, the manuscript identifies 73 singlestranded regions with high SHAPE and low Shannon entropy, which demonstrated high success in antiviral siRNA targeting.

Strengths:

The paper presents valuable data for the community. Additionally, the experimental design and data analysis are well documented.

Weakness:

The manuscript presents the effect of Braco-19 on PQS1, a single G4 region with high SHAPE and high Shannon entropy, to suggest that "the compound can selectively target the PQS1 of the high SHAPE-high Shannon region in cells" (lines 625-626). While the effect of Braco-19 on PQS1 is supported by strong evidence in the manuscript, the conclusion regarding the G4 region with high SHAPE and high Shannon entropy is based on a single target, PQS1.

We thank the reviewer for their positive assessment of our methodology and dataset. We propose that dynamic RNA structures in high SHAPE-high Shannon regions, when stabilized by small molecules, can serve as viable targets for antiviral therapy. Gquadruplexes represent a characteristic type of such dynamic structures that compete with local stem-loop formations in the genome. While we identified seven highly conserved PQSs in the PEDV genome, only PQS1 was located within a high SHAPEhigh Shannon region. To further validate this concept, we have supplemented the revised manuscript with Thioflavin T (ThT) fluorescence turn-on assays (Figures 3D, 3E, and Figure 3 – figure supplement 6), which provide additional evidence for the differential G4-forming capabilities of PQSs across regions with distinct structural features.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors):

Major Comments:

(1) It could be valuable for the authors to spend some more effort comparing their approach to siRNA target discovery and design to current methods for siRNA design. It would be good to highlight which components are novel, and which might offer superior performance with respect to other existing methods.

We thank the reviewer for highlighting this important point. In response, we have rewritten the relevant section in the discussion:

“Our approach uniquely integrates in situ RNA structural data (SHAPE reactivity and Shannon entropy) to prioritize siRNA targets within stable single-stranded regions (high SHAPE reactivity, low Shannon entropy), which are experimentally validated as accessible in infected cells. This represents a significant departure from traditional siRNA design methods that rely primarily on sequence conservation, thermodynamic rules (e.g., Tuschl rules), or in vitro structural predictions (Ali Zaidi et al., 2023; Qureshi et al., 2018; Tang and Khvorova, 2024),which may not accurately reflect intracellular RNA accessibility. Bowden-Reid et al. designed 39 antiviral siRNAs against various SARS-CoV-2 variants based on sequence conservation, ultimately identifying 8 highly effective sequences (Bowden-Reid et al., 2023). Notably, five of these effective sequences targeted regions that were located in high SHAPE-high Shannon regions according to SARS-CoV-2 SHAPE datasets (Supplementary Table 8) (Manfredonia et al., 2020). This independent finding aligns perfectly with our conclusions and demonstrates that SHAPE-based siRNA design outperforms sequence/structureagnostic approaches, at least in terms of significantly improving antiviral siRNA screening efficiency. Given the growing availability of SHAPE datasets for numerous viruses, we are confident that our methodology will facilitate more precise design of antiviral siRNAs.”

(2) The section targeting their discovered G4 structure with Braco-19 is interesting, particularly showing effects on viral proliferation; however, it's not clear to me how this compound could be used therapeutically against PEDV, as it is a non-selective binder of G4 structures. Their results are good support for the presence and functionality of a G4 structure in PEDV, but I don't see any strategy outlined in the manuscript on how this could be specifically targeted with Braco-19.

While Braco-19 is indeed a broad-spectrum G4 ligand, our data demonstrate that not all PQSs in the viral genome can form G4 structures under physiological conditions. Our results specifically show that Braco-19 exerts its anti-PEDV activity by targeting PQS1, which is located in a high SHAPE-high Shannon entropy region. This target specificity was further confirmed by the complete resistance of the PQS1mut strain (lacking G4-forming ability) to Braco-19 treatment in our in vitro assays. 

Additionally, previous studies have reported that during rapid viral replication, viral RNA accumulates to levels that significantly exceed host RNA concentrations. This "concentration advantage" suggests that G4 ligands like Braco-19 would preferentially bind viral G4 structures over host targets, thereby enhancing their antiviral specificity in vivo. In summary, our data provide proof-of-concept that viral genomic regions with high G4-forming potential - particularly those in high SHAPE-high Shannon entropy regions - represent promising targets for antiviral therapy.

(3) The section where they proposed 3D RNA structures based on sequence similarity feels "tacked on" and I don't see how it adds to the overall story. The authors identify a short RNA hairpin in the PEDV genome with some sequence similarity to the CPEB3 nuclease P4 hairpin. However, they don't provide any evidence that this motif functions in a similar way or that it's important for the virus's life cycle. They also don't explain how this similarity could be exploited for antiviral drug development. It's not clear whether targeting this motif would have any effect on the virus. It's interesting that these two sequences share nucleotides, but it's unlikely that they share any homology...perhaps they convergently evolved (or were captured), but the similarity could also be coincidental.

We appreciate the reviewer's insightful observation regarding this section. While our intention was to demonstrate that flexible conformations in high SHAPE-high Shannon regions could potentially be targeted, we acknowledge that extensive discussion of these motifs' functions would exceed the scope of this study, resulting in some disconnection from the main narrative. In response to this valuable feedback, we have consequentially removed it from the manuscript.

(4) The authors should consider the optimality of the synonymous mutation (G3109A) that they introduced, as G3109A could swap a rare codon for a more optimal one. Even though the protein sequence is unaffected, the translation rate (and ability to proliferate) could be very different due to altered codon optimality. Additionally, to show the inactivity of the PQS3 mutant, the Braco-19 treatment studies performed on the PQS1 mutants could be repeated with PQS3 - using this as a control for these experiments.

We appreciate the reviewer's insightful comment regarding codon optimization. In the PEDV genome, the PQS1 mutation site encodes lysine (AAG). Since lysine has only two codons (AAG and AAA), the G3109A synonymous mutation was our only viable option. Published literature (Ding et al. 2024) confirms that neither AAG nor AAA are classified as either preferred or rare codons in mammalian cells. Therefore, this substitution should have minimal direct impact on translation efficiency. Compared to nonsynonymous mutations that would alter amino acid sequences, we believe this synonymous mutation represents the optimal approach for maintaining native protein function while introducing the desired structural modification.

REFERENCES:

Ding W, Yu W, Chen Y, et al. Rare codon recoding for efficient noncanonical amino acid incorporation in mammalian cells. Science. 2024;384(6700):1134-1142.

In the revised version, we have added control experiments showing the inhibitory activity of Braco-19 against the PQS3 mutant strain (Figure 4—figure supplement 3C) and discussed it in the results section.

“Furthermore, as a control, we observed nearly identical inhibitory activity of Braco19 against both the PQS3 mutant strain (AJ1102-PQS3mut) and wild-type virus (Figure 4—figure supplement 3C), demonstrating the specificity of Braco-19's action on PQS1.”

Minor Comments:

(5) The authors' description of the Shannon Entropy could be improved. The current description makes it seem like the Shannon Entropy only provides information on base pairing, however, the Shannon entropy quantifies the uncertainty of structural states at each position and is calculated based on the probabilities of the different states (paired or unpaired) that a nucleotide can adopt.

We have revised the description of Shannon entropy in the manuscript：

"The pairing probability of each nucleotide derived from SHAPE reactivities was subsequently used to calculate Shannon entropy. Regions with high Shannon entropy may adopt alternative conformations, while those with low Shannon entropy correspond to either well-defined RNA structures or persistently single-stranded regions (MATHEWS, 2004; Siegfried et al., 2014)."

(6) The overall writing of the manuscript is very good, but there are some minor grammatical issues throughout, e.g., here are some of the ones that I caught:

a) Lines 71-3: "various types of RNA structures such as hairpin structure, RNA singlestrand, RNA pseudoknot and RNA G-quadruplex (G4)" - the examples should be plural and, rather than "hairpins" (or in addition), perhaps add "helixes" to be more generically correct(?).

We have revised the relevant description: 

"various types of RNA structures such as stem-loop structures (with double-helical stems), RNA single-strand, RNA pseudoknot and RNA G-quadruplex (G4)"

b) Lines 74-5: "Of these, RNA G4 has shown considerable promise because of the high stability and modulation by small molecules" should be "Of these, RNA G4 has shown considerable promise because of its high stability and ability for modulation by small molecules."

We have revised the sentence:

“Of these, RNA G4 has shown considerable promise because of its high stability and ability for modulation by small molecules.”

c) Line 76: "have" should be "has".

We have revised the sentence.

d) Lines 104-5 (and elsewhere): "frameshift stimulation element (FSE)" should be "frameshift stimulatory element (FSE)".

We have revised the sentence.

e) Lines 428-9: following the Manfredonia's methods" should be "following Manfredonia's method" or "following the Manfredonia method".

We have made the appropriate edit.

These edits ensure grammatical accuracy and consistency with standard scientific terminology. We appreciate the reviewer's attention to detail, which has significantly improved the clarity of our manuscript.

Reviewer #2 (Recommendations for the authors):

(1) There are some important references missing, on shape-seq from Julius Lucks.

We have added citations to the foundational work by Lucks et al. (2011, PNAS) that pioneered in vitro RNA structure probing using SHAPE-seq.

(2) Describe the acronym "SHAPE",

We have now included the full name of SHAPE:“Selective 2’-Hydroxyl Acylation and Primer Extension”.

(3) Line 81: 2"-hydroxyl-selective - the prime is incorrect.

We thank the reviewer for catching this technical error. We have corrected "2"hydroxyl" to "2'-hydroxyl".

(4) Explaining a bit better how shape reagent works would be beneficial (one sentence should suffice).

We have revised the Introduction section:

“SHAPE reagents like NAI selectively modify flexible, unpaired 2′-OH groups in RNA, and these modifications are detected as mutations during reverse transcription, enabling precise mapping of RNA secondary structures through sequencing.”

(5) Line 128: cite the paper that introduced NAI.

We have now properly cited the original publication introducing NAI（Spitale et al., 2012）.

(6) Line 243: Can you describe what the compound is?

The compound is Braco-19. This has now been included in the methods section. 

(7) Line 272: describe what 3Dpol is and the source of it.

We have supplemented the relevant information as follows:

"3Dpol (recombinant RNA-dependent RNA polymerase; Abcam, ab277617, 0.02 mg/reaction)"

(8) Figure 1 legend: For both C and D, the explanation of the G4 structure and the RISC complex should be added, otherwise, it becomes unclear why they are there.

We have revised the captions for Figure 1 as follows:

"(A) Well-folded regions (low SHAPE reactivity and low Shannon entropy; 26.40% of genome). These regions represent stably folded RNA structures with minimal conformational flexibility, likely serving as structural scaffolds or functional elements in viral replication. (B) Dynamic structured regions (low SHAPE reactivity and high Shannon entropy; 11.70% of genome). These conformationally plastic domains likely mediate regulatory switches between alternative secondary structures during infection. (C) Dynamic unpaired regions (high SHAPE reactivity and high Shannon entropy; 26.90% of genome). These regions are prone to form non-canonical nucleic acid structures (e.g., G-quadruplexes), which can be stabilized by small-molecule ligands to inhibit viral replication. (D) Persistent unpaired regions (high SHAPE reactivity and low Shannon entropy; 9.67% of genome). These regions are more accessible for siRNA binding, facilitating recruitment of Argonaute proteins and Dicer to form the RNAinduced silencing complex (RISC) for targeted cleavage."

(9) Figure S2 panel A should be in Figure 1. This is a nice picture showing the backbone of the research.

In the revised manuscript, we have reorganized Figure 1 and Figure S2 by incorporating the SHAPE-MaP workflow diagram (previously Figure S2A) into Figure 1 as panel (A): 

(10) Please add the citation to Braco-19.

We have now added the appropriate citation for Braco-19 (Gowan et al., 2002) in the revised manuscript.

(11) Figure 5 legend: could you add in parenthesis the what ds means (and call Figure S28).

We appreciate the reviewer's attention to detail. In the revised manuscript, we have clarified the abbreviations in the Figure 5 legend: ss (single-stranded targeting siRNAs); ds (dual-stranded targeting siRNAs). 

(12) Line 107: I would argue that the "stabilization of a G4" inhibited viral proliferation. And that supports the point of the paper, that a small molecule that stabilizes the G4 can be used to reduce viral replication. I suggest emphasizing this thorough the paper.

We fully concur with the reviewer's insightful perspective. In the revised manuscript, we have comprehensively strengthened the point of 'G4 stabilization' as an antiviral mechanism through the following enhancements:

(1) In the Results section: We present Thioflavin T (ThT) fluorescence assays demonstrating the G4-forming capability of PQSs in the full-length PEDV genomic RNA context:

“These findings indicate that although most PQSs can form G4 structures in vitro, PQS1—located in the high SHAPE-high Shannon entropy region—demonstrates the most robust G4-forming capability when competing with local secondary structures in the genomic context.”

(2) In the Results section: The inclusion of Braco-19 inhibition assays using PQS3 mutant virus as control provides robust evidence that Braco-19 exerts its antiviral effects specifically through PQS1 stabilization:

“Furthermore, as a control, we observed nearly identical inhibitory activity of Braco-19 against both the PQS3 mutant strain (AJ1102-PQS3mut) and wild-type virus, demonstrating the specificity of Braco-19's action on PQS1.”

(3) In the Discussion section: We have rewritten the mechanistic interpretation to emphasize: 

"Crucially, Braco-19 showed no inhibitory activity against the PQS1-mutant strain while maintaining potent activity against the PQS3-mutant strain (Figure 4E, Figure 4—figure supplement 3C). This suggests that the compound can selectively target the PQS1 of the high SHAPE-high Shannon region in cells." 

(13) For PQS1, it's suggested that it is indeed a competing and transient conformation that forms the G4. I wonder if using an extended PQS1 (perhaps what is shown in Figure 3E) and using fluorescence, and/or K+ vs Li+, and/or in-vitro SHAPE could tell us more about this dynamic structure. Thioflavin T or any other fluorescent molecule that binds to G4s could be easily used to show how the formation of G4 may happen or not. In addition, how Braco-19 could really lock the dynamic structure in-vitro as well. I think the field would benefit from a deeper investigation of it.

To address the dynamic competition between G4 and alternative RNA conformations, we performed Thioflavin T (ThT) fluorescence turn-on assay (now in Figure 3D-E and Figure 3—figure supplement 6) under physiological K⁺ conditions (100 mM), with PRRSV-G4 RNA as a positive control. This reads as:

“To validate whether SHAPE analysis could reflect the competitive conformational folding of PQSs in the PEDV genome, we performed in vitro transcription to obtain local intact structures containing PQSs within dynamic single-stranded regions and stable double-stranded regions (Table S6). Thioflavin T (ThT) fluorescence turn-on assays were conducted under physiological K⁺ conditions (100 mM), with the G4 sequence of porcine reproductive and respiratory syndrome virus (PRRSV) serving as a positive control (Control-G4)(Fang et al., 2023). The results demonstrated that for short PQSs sequences containing only G4-forming motifs (Table S7), PQS1, PQS3, PQS4, and PQS6 all induced significant ThT fluorescence enhancement (Figure 3D-E, Figure 3—figure supplement 6), confirming their ability to form G4 structures. However, in long RNA fragments encompassing PQSs and their flanking sequences, only PQS1 and PQS4 exhibited pronounced ThT fluorescence responses (Figure 3DE), whereas PQS2, PQS3, and PQS6 showed negligible signals (Figure 3E, Figure 3— figure supplement 6). Notably, the PQS1-long chain displayed the strongest fluorescence signal, while its mutant counterpart (PQS1mut-long chain) exhibited the lowest background fluorescence (Figure 3D). These findings indicate that although most PQSs can form G4 structures in vitro, PQS1—located in the high SHAPE-high Shannon entropy region—demonstrates the most robust G4-forming capability when competing with local secondary structures in the genomic context. Therefore, PQS1 was selected for further structural and functional validation.”

(14) Figure S29 is nice and informative. Consider moving it to the main text.

We appreciate the reviewer's positive assessment of Figure S29. Now we have renamed this figure as "Figure 5—Supplement 2".

DOI: 10.1186/s13041-025-01204-y

Resource: RRID:Addgene_20298

Curator: @olekpark

SciCrunch record: RRID:Addgene_20298

What is this?

DOI: 10.1186/s12935-025-03805-y

Resource: (RRID:CVCL_2091)

Curator: @dhovakimyan1

SciCrunch record: RRID:CVCL_2091

What is this?

DOI: 10.1080/19336934.2018.1549419

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1080/19336934.2018.1549419

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s44319-025-00391-y

Resource: Image-Pro Plus (RRID:SCR_007369)

Curator: @evieth

SciCrunch record: RRID:SCR_007369

What is this?

DOI: 10.1038/s42003-025-07829-y

Resource: RRID:BDSC_25685

Curator: @maulamb

SciCrunch record: RRID:BDSC_25685

What is this?

DOI: 10.1038/s42003-025-07829-y

Resource: RRID:BDSC_60317

Curator: @maulamb

SciCrunch record: RRID:BDSC_60317

What is this?

DOI: 10.1038/s42003-025-07829-y

Resource: RRID:BDSC_66806

Curator: @maulamb

SciCrunch record: RRID:BDSC_66806

What is this?

DOI: 10.1038/s42003-025-07829-y

Resource: RRID:BDSC_60312

Curator: @maulamb

SciCrunch record: RRID:BDSC_60312

What is this?

DOI: 10.1038/s42003-025-07829-y

Resource: RRID:BDSC_64277

Curator: @maulamb

SciCrunch record: RRID:BDSC_64277

What is this?

DOI: 10.1038/s42003-025-07829-y

Resource: RRID:BDSC_4539

Curator: @maulamb

SciCrunch record: RRID:BDSC_4539

What is this?

DOI: 10.1038/s41598-018-36277-4

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41467-025-59002-y

Resource: RRID:Addgene_107167

Curator: @olekpark

SciCrunch record: RRID:Addgene_107167

What is this?

DOI: 10.1038/s41467-025-58923-y

Resource: RRID:Addgene_50920

Curator: @olekpark

SciCrunch record: RRID:Addgene_50920

What is this?

DOI: 10.1038/s41467-025-56294-y

Resource: RRID:BDSC_31056

Curator: @maulamb

SciCrunch record: RRID:BDSC_31056

What is this?

DOI: 10.1038/s41467-018-07540-z

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41467-018-07540-z

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41467-018-07540-z

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41467-018-07540-z

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41467-018-07540-z

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41467-018-07540-z

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41467-018-07540-z

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41467-018-07540-z

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41467-018-07101-4

Resource: Bloomington Drosophila Stock Center (RRID:SCR_006457)

Curator: @bdscstockkeepers

SciCrunch record: RRID:SCR_006457

What is this?

DOI: 10.1038/s41420-025-02422-y

Resource: None

Curator: @olekpark

SciCrunch record: RRID:Addgene_42516

What is this?

DOI: 10.1038/s41419-025-07623-y

Resource: RRID:Addgene_125839

Curator: @olekpark

SciCrunch record: RRID:Addgene_125839

What is this?

DOI: 10.1038/s41419-025-07623-y

Resource: RRID:Addgene_19999

Curator: @olekpark

SciCrunch record: RRID:Addgene_19999

What is this?

DOI: 10.1038/s41419-025-07623-y

Resource: RRID:Addgene_19234

Curator: @olekpark

SciCrunch record: RRID:Addgene_19234

What is this?

DOI: 10.1038/s41419-025-07623-y

Resource: RRID:Addgene_52962

Curator: @olekpark

SciCrunch record: RRID:Addgene_52962

What is this?

DOI: 10.1038/s41419-025-07623-y

Resource: RRID:Addgene_48138

Curator: @olekpark

SciCrunch record: RRID:Addgene_48138

What is this?

DOI: 10.1038/s41419-025-07623-y

Resource: RRID:Addgene_12260

Curator: @olekpark

SciCrunch record: RRID:Addgene_12260

What is this?

DOI: 10.1038/s41419-025-07623-y

Resource: RRID:Addgene_12259

Curator: @olekpark

SciCrunch record: RRID:Addgene_12259

What is this?

DOI: 10.1038/s41419-018-1022-y

Resource: RRID:Addgene_13316

Curator: @areedewitt04

SciCrunch record: RRID:Addgene_13316

What is this?

DOI: 10.1038/s41419-018-1022-y

Resource: None

Curator: @areedewitt04

SciCrunch record: RRID:Addgene_23955

What is this?

DOI: 10.1016/j.celrep.2025.115631

Resource: (BioLegend Cat# 105812, RRID:AB_313221)

Curator: @scibot

SciCrunch record: RRID:AB_313221

What is this?

DOI: 10.1016/j.ccell.2025.04.003

Resource: (Abcam Cat# ab214437, RRID:AB_3096032)

Curator: @scibot

SciCrunch record: RRID:AB_3096032

What is this?

DOI: 10.1016/j.ccell.2025.04.001

Resource: (Cell Signaling Technology Cat# 9272, RRID:AB_329827)

Curator: @scibot

SciCrunch record: RRID:AB_329827

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_3083762

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: (Abcam Cat# ab226930, RRID:AB_2893433)

Curator: @scibot

SciCrunch record: RRID:AB_2893433

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: (Zen Bio Cat# 380709, RRID:AB_2924434)

Curator: @scibot

SciCrunch record: RRID:AB_2924434

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: GraphPad Prism (RRID:SCR_002798)

Curator: @scibot

SciCrunch record: RRID:SCR_002798

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_3083766

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: Jeol JEOL JEM-1400Flash Transmission Electron Microscope (RRID:SCR_020179)

Curator: @scibot

SciCrunch record: RRID:SCR_020179

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: Nikon Eclipse 80i Advanced Research Microscope (RRID:SCR_015572)

Curator: @scibot

SciCrunch record: RRID:SCR_015572

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_2939057

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: (Abcam Cat# ab104224, RRID:AB_10711040)

Curator: @scibot

SciCrunch record: RRID:AB_10711040

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: (Abbkine Cat# A23220, RRID:AB_2737289)

Curator: @scibot

SciCrunch record: RRID:AB_2737289

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_3083765

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: ImageJ (RRID:SCR_003070)

Curator: @scibot

SciCrunch record: RRID:SCR_003070

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: Nikon Eclipse E600 Fluorescence Microscope (RRID:SCR_018606)

Curator: @scibot

SciCrunch record: RRID:SCR_018606

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: (Proteintech Cat# 10494-1-AP, RRID:AB_2263076)

Curator: @scibot

SciCrunch record: RRID:AB_2263076

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: (Cell Signaling Technology Cat# 8945, RRID:AB_10860076)

Curator: @scibot

SciCrunch record: RRID:AB_10860076

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_3083764

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: (Proteintech Cat# 26593-1-AP, RRID:AB_2818996)

Curator: @scibot

SciCrunch record: RRID:AB_2818996

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: None

Curator: @scibot

SciCrunch record: RRID:AB_3081690

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: BioRad ChemiDoc Touch Imaging System (RRID:SCR_021693)

Curator: @scibot

SciCrunch record: RRID:SCR_021693

What is this?

DOI: 10.1007/s12017-025-08856-y

Resource: (Cell Signaling Technology Cat# 8242, RRID:AB_10859369)

Curator: @scibot

SciCrunch record: RRID:AB_10859369

What is this?

DOI: 10.1007/s11306-025-02254-y

Resource: RRID:Addgene_47048

Curator: @scibot

SciCrunch record: RRID:Addgene_47048

What is this?

DOI: 10.1007/s10522-025-10233-y

Resource: None

Curator: @olekpark

SciCrunch record: RRID:Addgene_67639

What is this?

I now wonder if I can build a powerful CRM on Django.

w h y???