PONE-D-22-00900

MYL9 deficiency is neonatal lethal in mice due to abnormalities in the lung and the muscularis propria of the bladder and intestine

PLOS ONE

Dear Dr. Chong,

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Academic Editor

PLOS ONE

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Reviewers' comments:

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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: Partly

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: No

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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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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: Huang et al., established a line of Myl9-/- mice and characterized the phenotypes briefly. It would be interesting to explore the role of Myl9 beyond smooth muscle contraction in this animal model. However, I feel a little confusing about the logic of this paper. The aim of this paper is try to explore the in vivo physiological roles of Myl9 such as hematopoietic stem cells as author mentioned in abstract and introduction. But, the authors did not measure the corresponding functions in MYL9-/-

mice. Particularly, the hematopoietic process of the mutant mice was not described or discussed. If do not have this data, it seems there is no new finding of this report.

Major

1. P16 Line 348: the authors claim “from our study, we now know that MYL9 is highly expressed in the smooth muscle of the bladder but not expressed by skeletal muscle”. It is not true, it has been well known that MYL9 is specifically expressed in smooth muscle.

2. P15: the authors discuss too much about the overlapping phenotypes of MYL9-deficient mice and

other mutant mice as well as gene-mutated patients, because this paper did not provide any data about the interaction of these proteins.

3. Lymphocytes did not express MYL9 as claimed in this report, how about the function in MYL9-deficient lymphocytes.

Minors

1. P3 last line: have role should be have a role

2. P4 line 72: “.. is activity transcribed” means “..actively transcribed”?

Reviewer #2: The manuscript "MYL9 deficiency is neonatal lethal in mice due to abnormalities in the lung and the muscularis propria of the bladder and intestine" by Huang et al, is a promising start to understanding the nuances of different myosin isoforms. They deftly produced a Myl9 deficient mouse that also expresses LacZ in its place, thus revealing where Myl9 has been abrogated. There are several issues needing addressing before this manuscript should be considered for publication that are listed below:

1. Quantification of knockout

There is a severe lack of quantitative information about the knockout of Myl9 in their mice. They mention that genotyping was performed to show the increased LacZ in the place of exons 3 and 4 but do not attach a gel showing that this is indeed the case. This is a minor issue that can be added to supplementary data but should be attached. Even further, the authors could add supplemental sequencing data from the creation of the mouse line ordered from KOMP to increase confidence in successful insertion and removal of active Myl9 expression and genotype.

Next, considering that the authors' construct removes exons 3 & 4 of the Myl9 gene and replace it with LacZ, this presents an opportunity for quantification. While acknowledging that Myl9 shares extremely high sequence similarity with Myl12a Myl12b, designing primers within/across exons 3 & 4 of Myl9 would be ideal for qPCR quantification of Myl9 mRNA. By choosing primers containing distinct sequences within Myl9 you would likely show a decrease in Myl9 transcripts, even if there is 100% overlap of primers with Myl12a & Myl12b (as there does not appear to be any functional redundancy or compensation). Additionally, by quantifying LacZ mRNA, you could show that the loss of wild-type Myl9 has happened and that LacZ expression has taken it's place.

2. Protein Expression of MYL9

Fully understanding that Myl9 and Myl12a and Myl12b have incredibly high sequence homology, I agree that detecting Myl9 separate from Myl12a and Myl12b is extremely difficult. However, there are indeed MYL9 antibodies commercially available that could be utilized. Similar to my qPCR suggestion, the antibody might not be able to distinguish MYL9, MYL12A and MYL12B but it would show a reduction in expression within the high expressing smooth muscle cells/tissue through immunohistochemistry or western blotting. While the authors are correct that sequence similarity makes it very hard to distinguish these proteins, they do not present any data to support this other than amino acid sequence similarity. The authors could state that there was a reduction of regulatory light chains (RLC) instead of MYL9 itself. When this is combined with qPCR data showing a loss of wild-type Myl9 it would solid evidence of protein loss. Despite sequence homology, I believe data concerning protein level difference of MYL9 or RLC in general is necessary.

3. Additional FACS

The authors use FACS to isolate TCR-beta+ cells to test levels of beta-galactosidase and find a lack of expression, which goes against previous data showing that it is expressed. I feel that this needs to be resolved further with other, more general hematopoietic cell markers such as CD45 or CD34 and then isolating either RNA/protein from these cells and quantifying Myl9/LacZ expression. It is a striking conundrum that the beta-galactosidase is only showing up in smooth muscle but not other known cellular sources. This undercuts their contention that knockout is indeed globally accomplished and is in direct contrast to previous publications. At the very least, further exposition as to why this contrast is occurring is needed.

4. Poor Image Quality

The cryosection images throughout the manuscript must be of better resolution. By mentioning that hypertrophy is occurring in both the bladder and intestinal muscle layers, the authors have presented another opportunity for quantification that is not present. With clearer images, the authors will be able to show how much hypertrophy is present in both qualitative and quantitative forms. Within the intestinal images, it is unclear what level of hypertrophy is present and without a clearer image and quantification, it will remain unclear.

5. Minor English Editing

There are a few incorrectly used (abnormal instead of abdominal on pg. 9 of submission) and missing words in the manuscript but this is of minor concern.

With these additions and corrections, I believe the manuscript will be of higher quality and complete for submission.

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Reviewer #1: No

Reviewer #2: Yes: Brian G Jorgensen

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PONE-D-22-00900R1MYL9 deficiency is neonatal lethal in mice due to abnormalities in the lung and the muscularis propria of the bladder and intestinePLOS ONE

Dear Dr. Chong,

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 by the Reviewer 1.

Please submit your revised manuscript by Jul 27 2022 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:

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• 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,

Seungil Ro, PhD

Academic Editor

PLOS ONE

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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: All comments have been addressed

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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: Yes

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

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: Yes

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: Yes

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: The authors clarified the purpose of this study is investigating the physiological function of Mly9 because the physiological function of Mly9 remains unclear. The result shows the importance of Myl9 in smooth muscle. It has been documented that Myl9 serves as the regulatory light chain (RLC) of smooth muscle myosin and initiates smooth muscle contraction when it is phosphorylated by Mylk. The function of Myl9 is clear at least in smooth muscle. Aso, there are reports showing that tissue specific or global deletion of Myl9 causes abolished smooth muscle contraction and hence the dilation phenotypes of hollow organs. The question is what is the new finding of this study?

Reviewer #2: Huang et al, have properly addressed each issue that I raised in my initial review and gone above and beyond to resolve the questions and concerns that were initially expressed. The addition of the use of the Myl9/Myl12a/Myl12b antibodies, with LacZ protein data, adds a strong component to the paper allowing for direct visualization of clear loss of the protein in the smooth muscle layer. Furthermore, the completion of more robust FACS information via the addition of other hematopoietic markers to solidify their conclusions on isolated cells. The combination of the molecular and gross anatomical data that Huang et al have responded to my comments with was thoughtful, well-crafted, and much more complete for publication at this time.

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Reviewer #1: No

Reviewer #2: Yes: Brian Jorgensen

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MYL9 deficiency is neonatal lethal in mice due to abnormalities in the lung and the muscularis propria of the bladder and intestine

PONE-D-22-00900R2

Dear Dr. Chong,

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.

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Seungil Ro, PhD

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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

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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: Yes

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

Reviewer #1: Yes

**********

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: Yes

**********

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: 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: I have no more comments and recommend this paper acceptable.

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Reviewer #1: No

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