PONE-D-25-16398Loss of Shroom3 in the developing mouse myocardium does not impact heart developmentPLOS ONE

Dear Dr. Carleton,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Specifically, the reviewers have significant concerns about the small dataset used and suggest to refocus the manuscript on the novel findings obtained with a robust dataset using an established model. Further, they also ask to add updated citations on human heart defects in SHROOM3.

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Please submit your revised manuscript by May 31 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: SHROOM3 expression has been shown in the developing heart where it has been shown to play an important role in heart development, although the cell type responsible for this effect is not known and is the focus of the study.

The authors generated a floxed Shroom3 mouse line to test loss of Shroom3 specifically in myocardial cells. The overall conclusion is that loss of Shroom3 expression in these cells is not responsible for defects in cardiac development associated with full body loss of Shroom3.

Specific findings:

• Shroom expression starting at E10.5 in developing mice.

• Expression can be detected even earlier within myocardial cells but not in endocardial cells.

• Expression of Shroom fades after birth in the heart overall but remains within the myocardium and within the left atrium.

• The authors show a significant decrease in cardiac cross-sectional area in full body Shroom3 loss but this loss is not recapitulated within the mice where Shroom3 is only lost in the myocardial cells. Similar effects are seen when examining Shroom3 associated heart defects and ventricular thinning.

Overall, the manuscript demonstrates the reported findings. I did have a few minor points that should be taken within the context of trying to make the data more accessible to readers with less background in physiology or tissue development.

Minor points:

In the quantification of compact layer thickness in Figure8C and 8F, the effect is clear between wt and heterozygous Shroom3 GT hearts but this reviewer would appreciate seeing the data points used for these graphs so that I can appreciate how representative the data in the rest of the figure is.

Additionally, is there another measurement that is not expected to change as a result of Shroom3 expression that could be quantified as a negative control?

In a related note, for measurements of heart wall thickness, is there a way to normalize these values to overall heart size or dimension so that we can be sure that the effect is not due to overall differences in heart size.

For Fig9C, as you are measuring the cross-sectional area for a large number of cells, a violin-plot would allow us to see the distribution of the data. Once again, I have no doubt as to the effect being shown, but I’d love to see if the effect was uniform for all cells or just a subset of the population (presumably a large subset of the population). This is more of a concern in Figure15 however.

For figure13, I appreciate that the point is to measure the thickness of both left and right ventricular walls in either wt of myocardial shroom loss mice but the placement of the lines used for measurements does not appear to reflect the actual thickness of the wall in 13B. The difference is dramatic and would change the conclusions for the figures. Since this is not my specific area of expertise, this reviewer would appreciate more explanation for how the boundaries are chosen (especially for the inside boundary). Once again, data points on the bar graphs would also help a reader appreciate the data with more nuance.

Reviewer #2: The manuscript by Carleton et al describe an important role for a novel candidate in human disease called SHROOM3. SHROOM3 has been implicated in human disease including primarily kidney defects, and more recently, cardiac defects. A manuscript from 2020 describes heart defects due to SHROOM3 loss of function in embryos. Carleton et al. have expanded these findings, describing a more detailed expression pattern, additional cardiac defects, including valve defects, and pathology in adult cardiomyocytes, with smaller cardiomyocytes persisting to adulthood. These findings are important to our understanding of cardiac development and relevant to human disease. In terms of placing this novel data in the literature, the manuscript describes a role for SHROOM3 in patients with heterotaxy, however a brief literature review reveals two more recent studies, that have shown a role for SHROOM3 in human cardiac disease, including patients with CHD (PMID: 36011280, PMID: 39202774) and upregulation of SHROOM3 after myocardial infarction (PMID: 39086770). The findings of Carleton et al. may have direct relevance to these studies, given their findings of left ventricle thinning and valve defects. Further their findings in adult mice, in terms of the expression pattern and smaller cardiomyocyte area, are relevant to upregulation of SHROOM3 post myocardial infarction. The study needs to highlight these more recent findings and describe their data in the full context of available literature surrounding SHROOM3. Carleton et al. also describe, for the first time, a SHROOM3 conditional loss of function mouse-line. However, this dataset is much smaller. Carleton et al describe that loss of function in the myocardium does not lead to heart defects. These findings may be important for mechanism, and the authors highlight that other cell lines like neural crest are likely contributors to cardiac defects. This finding is important data in narrowing SHROOM3‘s lineage-specific role. However, the conclusions are based on the evaluation of a small number of embryos. This small number is likely not sufficient given the partial penetrance of the described heart defects, and especially to draw primary conclusions for the manuscript. Carleton et al need to better highlight what has been previously reported in SHROOM3 loss of function mice, and the novel findings added here (which are important.) The manuscript would be stronger with enlarging this small dataset or refocusing onto their important findings in a more robust dataset using an established model. Below are comments which, if addressed, would make the manuscript suitable for publication.

1. SHROOM3 loss of function has been shown to result in heart defects in mouse embryos, however the current study adds novel findings. The study needs to describe what has been previously reported and what are the novel findings. The novel findings need to be highlighted. This includes: 1) Evaluation of embryos at e18.5, (versus 14.5 in the Durbin et all study, where E18.5 represent a more robust timepoint for evaluation of cardiac defects than Durbin et al.) 2) A more detailed expression pattern is described 3) A spectrum of valve defects are reported – for the first time. 4) Ventricle thinning is isolated to left ventricle 5) ventricle thinning persists to adulthood. 6) They measure cardiomyocyte area and the smaller size persists into adulthood

2. The manuscript needs to cite more recent literature PMID: 36011280, PMID: 39202774, PMID: 39086770), and place their data in context (for example, their findings related to SHROOM3 valve defects and ventricle thinning may be relevant in hypoplastic left heart syndrome patients, and SHROOM3 expression pattern post-myocardial infarction.

3. The manuscript presents a robust dataset from an established gene trap model. They then present a smaller dataset using a conditional loss of function approach. The later findings are important, given they are the first lineage-specific deletion of SHROOM3 reported. However, the dataset is much smaller than the evaluation of nearly 100 gene trap embryos. From my reading, the conclusions are based on n=8 flox/flox embryos for cardiac defects and n=3 for wall thickness. The results section needs to make more transparent how many embryos were analyzed in each group. Furthermore, 8 embryos does not seam sufficient to make a conclusion, given the partial penetrance of the cardiac defects. These numbers need to be increased, or, at least, the data needs to be described objectively without such strong conclusion.

4. The authors state their findings indicate other cell populations, like the neural crest cells, may be important for heart defects. Are neural crest cells important to cardiomyocyte size and ventricle thickness? Perhaps myocardial SHROOM3 expression is important in conjunction with other cell populations like the cardiac neural crest? If the authors have data about conditional loss of function using a Wnt1-Cre resulting in the neural crest deletion of SHROOM3, they should report it here.

5. To the best of their ability, the authors should refocus the manuscript on the novel findings obtained with a robust dataset using an established model, versus findings from a smaller number of embryos using a new model.

6. The title of the article needs to be modified to highlight the novel findings in the manuscript, obtained from a robust dataset, versus the conclusions drawn from a small cohort of embryos. Perhaps: (as stated in the first line of the conclusion), “SHROOM3 is an important contributor to mammalian heart development and postnatal heart structure.” or “The role of SHROOM3 in cardiac defects.”

7. The abstract also needs to be modified to highlight the novel findings obtained from a robust dataset versus the negative conclusions drawn from a much smaller number of embryos.

The manuscript presents important and novel findings. However, a better description of the numbers utilized in the dataset is needed. Furthermore, if possible, the numbers for the conditional loss of function embryos should be increased, and a second lineage-specific deletion should be performed. At the very least, they should describe their novel findings in the context of what was previously demonstrated in SHROOM3 loss of function embryo heart defects. The authors should also add updated citations on human heart defects in SHROOM3, properly placing the data in the literature. Overall, the authors should refocus the manuscript on the novel findings obtained with a robust dataset using an established model. These findings may have clinical relevance in heart disease given recent findings in patients.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-25-16398R1Understanding the role of Shroom3 in the developing mouse myocardiumPLOS ONE

Dear Dr. Carleton,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================There are still concerns about the presence of Shroom 3 in the outflow tract 

==============================

Please submit your revised manuscript by Sep 14 2025 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org . When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols . Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols .

We look forward to receiving your revised manuscript.

Kind regards,

Federica Limana

Academic Editor

PLOS ONE

Journal Requirements:

1. If the reviewer comments include a recommendation to cite specific previously published works, please review and evaluate these publications to determine whether they are relevant and should be cited. There is no requirement to cite these works unless the editor has indicated otherwise. 

2. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: (No Response)

Reviewer #2: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: (No Response)

Reviewer #2: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: (No Response)

Reviewer #2: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: (No Response)

Reviewer #2: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: (No Response)

Reviewer #2: The increased number of embryos analyzed, clarification of the number of embryos used in each analysis, and the revised title, abstract and discussion satisfy previous concerns.

One minor point that needs to be addressed before acceptance.

The manuscript describes the Shroom3 expression pattern, which is robust and an important contribution to the manuscript. The manuscript states: “This expression was specific to the myocardium of the atria and ventricles of the heart and was not seen in the outflow tracts or great arteries….” and then later …” Shroom3 expression was seen in the base of the outflow tract“

The staining pattern is clear and there is clearly a sharp drop-off and absence in the great arteries. However, it seems contradictory to say staining is not present in the outflow tract and then later that it is present in the base of the outflow tract. It seems more accurate to state that staining was localized to the base of the outflow tract and absent from the great arteries.

This is an important point given that cardiac neural crest cells are responsible for outflow tract, aortopulmonary, and ventricular septation, and the article hypothesizes (logically) that neural crest cells may be responsible for the septal defects seen in whole body loss of function embryos.

Clarification of this minor point would make the manuscript acceptable for publication.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean? ). If published, this will include your full peer review and any attached files.

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Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy .

Reviewer #1: No

Reviewer #2: No

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[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/ . PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org . Please note that Supporting Information files do not need this step.

Understanding the role of Shroom3 in the developing mouse myocardium

PONE-D-25-16398R2

Dear Dr. Carleton,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Federica Limana

Academic Editor

PLOS ONE

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