The chromatin-remodeling enzyme CHD3 plays a role in embryonic viability but is dispensable for early vascular development

Jun Xie; Siqi Gao; Christopher Schafer; Sarah Colijn; Vijay Muthukumar; Courtney T. Griffin

doi:10.1371/journal.pone.0235799

Peer Review History

Original SubmissionApril 25, 2020
27 May 2020 Decision Letter - Edward E Schmidt, Editor PONE-D-20-12024 The chromatin-remodeling enzyme CHD3 plays a role in embryonic viability but is dispensable for early vascular development PLOS ONE Dear Dr. Griffin, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, I feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, I invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Please ensure to include the data requested by the Reviewer, as it is not acceptable to have critical supporting evidence referred to as "data not shown". Also, the speculative statements pointed out by the Reviewer need to either be supported with data or removed from the manuscript. Please submit your revised manuscript by Jul 11 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols I will look forward to receiving your revised manuscript. Kind regards, Edward E Schmidt Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. We note that you have included the phrase “data not shown” in your manuscript. Unfortunately, this does not meet our data sharing requirements. PLOS does not permit references to inaccessible data. We require that authors provide all relevant data within the paper, Supporting Information files, or in an acceptable, public repository. Please add a citation to support this phrase or upload the data that corresponds with these findings to a stable repository (such as Figshare or Dryad) and provide and URLs, DOIs, or accession numbers that may be used to access these data. Or, if the data are not a core part of the research being presented in your study, we ask that you remove the phrase that refers to these data. 3. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. 4. We noted in your submission details that a portion of your manuscript may have been presented or published elsewhere: 'The Chd3-flox allele we generated for this manuscript is briefly described in our collaborator's manuscript that is currently under consideration for publication (de Castro et al; Biorxiv; 2019). We do not consider this a dual publication, because generation of the Chd3-flox allele is not described in detail in the de Castro study, and the Cre-recombinase line used in that study is different than the ones used for this study. However, we cite this preprint in our discussion and therefore have uploaded it for reviewer consideration.' Please clarify whether this publication was peer-reviewed and formally published. If this work was previously peer-reviewed and published, in the cover letter please provide the reason that this work does not constitute dual publication and should be included in the current manuscript. [Note: HTML markup is below. Please do not edit.] 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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 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 ****** 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 ****** 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: Review of PONE-D-20-12024, “The Chromatin-remodeling enzyme CHD3 plays a role in embryonic viability but is dispensable for early vascular development.” In this manuscript, J. Xie and colleagues generated a conditional (floxed) allele of murine CHD3, a component of the NuRD chromatin-remodeling complex. Subsequent analysis of Tie2- and Sox2-Cre-mediated deletion of the Chd3-floxed allele revealed no apparent role for CHD3 in embryonic vascular development, as was previously reported for CHD4. Interestingly, a potential requirement for CHD3 activity was suggested by Chd3-knockout mice. Global deletion of CHD3 resulted in partial lethality of mice during gestation, based on observed versus expected Chd3Δ/Δ offspring at weaning. Western blot analyses of tissue-specific expression of CHD3 revealed that CHD3 is primarily expressed in adult brain and gonads. Based on these findings, the authors conclude that CHD3 may play a role in embryonic neurodevelopment. Comments: 1. Although the authors showed that efficient, Cre-mediated deletion of the Chd3-flox allele and lack of CHD3 protein in adult Chd3Δ/Δ brain tissue, validation of the Chd3-flox allele would be augmented by addition of the Southern blot analysis of targeted ES cells and PCR-based analyses of Cre-mediated recombination. The authors list these analyses as “data not shown.” In my opinion, this data should be included in Figure 1. 2. In the discussion section, the authors suggest that “temporal neuronal-specific deletion of Chd3 could further clarify its roles in embryonic and postnatal brain development…,” although no evidence was presented in the manuscript that CHD3 is actually expressed in developing neuronal tissue. Evidence of embryonic, tissue- and developmental stage-specific CHD3 expression would be useful in guiding future studies using Chd3-flox mice, and should be included in this manuscript. ****** 6. 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. If you choose “no”, your identity will remain anonymous but your review may still be made public. 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 [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. Attachments Attachment Submitted filename: Review of PONE-D-20-12024 5-19-2020.pdf https://doi.org/10.1371/journal.pone.0235799.r001
Revision 1
15 Jun 2020 Author Response Editorial/Journal Requests: 1. Ensure that the manuscript meets PLOS ONE’s style requirements: We have read the requirements carefully and have reformatted the manuscript and figures accordingly. 2. “Data not shown” cannot be used in the manuscript: The phrase “data not shown” was originally located at the end of our first “results” paragraph and referred to Southern blotting of ES cells to validate homologous recombination of the Chd3-flox targeting vector prior to blastocyst injection. This Southern blotting was performed by Cyagen Biosciences, with whom we contracted to make our Chd3-flox mice. Cyagen never sent us the Southern blot images, so we cannot include them and have therefore removed the phrase “data not shown” from the manuscript and indicated that Southern blotting was performed by Cyagen. In the same sentence, “Data not shown” also referred to PCR genotyping validation of Chd3-excision events in our mice carrying a Cre recombinase. We have now included these genotyping data in Fig 1E. 3. Show all original uncropped and unadjusted images underlying all blot or gel results: We have now provided all original blots and gels as Supporting Information. Please note that in collecting our original blots for Figure 4B (female mouse tissues blotted for CHD3 and GAPDH), we were unhappy with the aesthetics of the background bands on the GAPDH blot. Therefore, we re-ran the same lysates and re-probed them for CHD3 and GAPDH. The new data recapitulate the original data very closely but give a better exposure for CHD3 and a cleaner blot for GAPDH. Therefore, we have substituted these new blots for the original ones that we had submitted for Figure 4B (see uploaded "Response to Reviewer" document, which includes a figure showing the side-by-side original and updated blots). 4. Dual publication issue: As we reported upon submission, the Chd3-flox allele we generated for this manuscript is briefly described in our collaborator’s manuscript that was reviewed by Development in April. Our collaborator (Dr. Pezza) is still revising the manuscript for resubmission to Development. Since it (1) has not yet been re-reviewed and accepted, (2) does not describe the generation of the Chd3-flox allele in detail, and (3) does not use the same Cre-recombinase lines used in this study, we do not consider this a dual publication. However, we have cited the preprint of that study in our discussion (de Castro et al; bioRxiv; 2019). Reviewer Comments: 1. “Although the authors showed that efficient, Cre-mediated deletion of the Chd3-flox allele and lack of CHD3 protein in adult Chd3��/� brain tissue, validation of the Chd3-flox allele would be augmented by addition of the Southern blot analysis of targeted ES cells and PCR-based analyses of Cre-mediated recombination. The authors list these as “data not shown.” In my opinion, this data should be included in Figure 1.” We appreciated this suggestion and refer the Reviewer to our response to the Editor (see #2 under “Editor/Journal Requests” above and new Fig 1E). 2. “In the discussion section, the authors suggest that “temporal neuronal-specific deletion of Chd3 could further clarify its roles in embryonic and postnatal brain development…,” although no evidence was presented in the manuscript that CHD3 is actually expressed in developing neuronal tissue. Evidence of embryonic, tissue- and developmental stage-specific CHD3 expression would be useful in guiding future studies using Chd3-flox mice, and should be included in this manuscript.” In response to this request, we have revised our Discussion (2nd to last paragraph) to cite a published report that CHD3 is expressed in embryonic mouse brain cortices as early as E15.5 (Nitarska et al, 2016, Cell Reports, 17, 1683-1698). This study also shows that electroporation of CHD3 shRNA into developing cortices compromises neuronal migration and laminar identity during development. We feel that this important citation fulfills the Reviewer’s request in providing evidence for CHD3 expression in developing neuronal tissue and in demonstrating that CHD3 is functionally important for embryonic brain development. https://doi.org/10.1371/journal.pone.0235799.r002
23 Jun 2020 Decision Letter - Edward E Schmidt, Editor The chromatin-remodeling enzyme CHD3 plays a role in embryonic viability but is dispensable for early vascular development PONE-D-20-12024R1 Dear Dr. Griffin, I am 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. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Edward E Schmidt Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0235799.r003
Formally Accepted
26 Jun 2020 Acceptance Letter - Edward E Schmidt, Editor PONE-D-20-12024R1 The chromatin-remodeling enzyme CHD3 plays a role in embryonic viability but is dispensable for early vascular development Dear Dr. Griffin: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Edward E Schmidt Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0235799.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .