ECM-mediated positional cues are able to induce pattern, but not new positional information, during axolotl limb regeneration

Warren A. Vieira; Shira Goren; Catherine D. McCusker

doi:10.1371/journal.pone.0248051

Peer Review History

Original SubmissionDecember 22, 2020
2 Feb 2021 Decision Letter - Michael Schubert, Editor PONE-D-20-40259 ECM-mediated positional cues are able to induce pattern, but not new positional information, during axolotl limb regeneration PLOS ONE Dear Dr. McCusker, 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 comprehensively addresses the points raised during the review process. Please submit your revised manuscript by Mar 19 2021 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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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 We look forward to receiving your revised manuscript. Kind regards, Michael Schubert Academic Editor PLOS ONE 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 [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: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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: The authors expand upon prior work (Pham et al 2015) and a hypothesis (Vieira and McCusker 2019) to test the hypothesis that extracellular matrix materials, specifically the heparan sulfate proteoglycans (HSPGs), play a role in the upper tier of decisions that pattern limbs. They reject the hypothesis. The data and interpretation is thought-provoking and worthy of publication, but requires a little more careful exposition and qualification. Of the three component parts of the study, I query the first. The Accessory Limb Model (ALM) was an interesting choice to test the hypothesis using matrix denuded of cells as graft material. Herein lies a flaw - there is no assurance that matrix retains the incremental compositional or constitutional differences once transplanted. Unlike transplants with both matrix and cells, matrix alone does not elicit anything other than a very rudimentary hypomorphic structure. Indeed, it would be hard to state with conviction that a single cartilaginous nodule represents epimorphic versus tissue regeneration or even some sort of unusual scar cicatrix. To be definitive, the study would have needed to show that matrix retains its HSPG characteristics well beyond would healing and into blastema patterning stages. That said, I think the data is indicative, even highly suggestive, but not definitive. The loss of posterior coding in hypomorph blastema used for ALM grafts may merely reflect the gradual loss of posterior ECM features post-implantation: weeks (8 weeks of hypomorphic - ie; unusual - growth) are eons in inflamed tissues that are undergoing radical matrix (re)modelling and proliferation. I am not convinced that the mature ALM graft tissue retains or even reflects testable posterior origins that much later. You'd need mass spec, or better still some sort of immunohistochemistry to be sure that posterior attributes remain static. That said, I think these experiments have a place in the paper, but interpretation needs to be more carefully discussed and qualified. The manuscript would benefit from some editing to shorten sentences, economize on verbiage, and to correct minor grammatical errors: it was strangely difficult to understand the flow of experimental design regarding the ALM portion, and I am not sure why. Perhaps this is a feature of my COVID isolation-induced hazy state of mind...? Perhaps a flow chart encompassing all facets of the approach would help clarify? (ant/posterior source amputation result, transplant source/engraftment site process and result - the diagram in fig 2 helped, but didn't quite get me there)? Trivial small editorial stuff: p 6, - 2nd sentence from bottom - the semi-colon should be a comma p 15 under 4.2 - "Downstream cues function... ... and is communicated" should read ARE communicated (since the subject of the sentence is "cues" p 17 under 5.2.1, third line down "gentle agitate" should read gentle agitation. Reviewer #2: Author´s utilized the ALM-protocol, to generate ECM-induced structures and to show that HSPGs play a role as a molecular signal which downstream communicates information for the proper patterning during axolotl limb regeneration. Experiments are technically sound and the conclusions are coherent with the findings. I havo no major critics on the content of the manuscript. However I believe that putting more effort in the graphic work presented in the main figures, as well as the design and presentation of the figures will improve the manuscript. Besides this point, I think the manuscript is well written and has the merits to be published in PlosOne. ****** 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 Reviewer #2: 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. https://doi.org/10.1371/journal.pone.0248051.r001
Revision 1
12 Feb 2021 Author Response Response to Reviewers We would like to begin by thanking the editor and reviewers for their time and input into the review of our manuscript. The valuable feedback provided has helped to strengthen the quality of document. We have worked through the comments provided by the reviewers and made the appropriate changes to the text and figures. Below we will address each of the comments specifically (our responses provided as bolded text), detailing how the manuscript was modified to resolve the raised concerns. Reviewer #1 Comments The authors expand upon prior work (Pham et al 2015) and a hypothesis (Vieira and McCusker 2019) to test the hypothesis that extracellular matrix materials, specifically the heparan sulfate proteoglycans (HSPGs), play a role in the upper tier of decisions that pattern limbs. They reject the hypothesis. The data and interpretation is thought-provoking and worthy of publication, but requires a little more careful exposition and qualification. Of the three component parts of the study, I query the first. The Accessory Limb Model (ALM) was an interesting choice to test the hypothesis using matrix denuded of cells as graft material. Herein lies a flaw - there is no assurance that matrix retains the incremental compositional or constitutional differences once transplanted. Unlike transplants with both matrix and cells, matrix alone does not elicit anything other than a very rudimentary hypomorphic structure. Indeed, it would be hard to state with conviction that a single cartilaginous nodule represents epimorphic versus tissue regeneration or even some sort of unusual scar cicatrix. To be definitive, the study would have needed to show that matrix retains its HSPG characteristics well beyond would healing and into blastema patterning stages. That said, I think the data is indicative, even highly suggestive, but not definitive. The loss of posterior coding in hypomorph blastema used for ALM grafts may merely reflect the gradual loss of posterior ECM features post-implantation: weeks (8 weeks of hypomorphic - ie; unusual - growth) are eons in inflamed tissues that are undergoing radical matrix (re)modelling and proliferation. I am not convinced that the mature ALM graft tissue retains or even reflects testable posterior origins that much later. You'd need mass spec, or better still some sort of immunohistochemistry to be sure that posterior attributes remain static. That said, I think these experiments have a place in the paper, but interpretation needs to be more carefully discussed and qualified. Response: We thank the reviewer for this feedback. These comments helped us pinpoint places in the text that needed clarification, which we have worked to correct in the revised manuscript. The premise of our study was to determine if ECM was sufficient, on its own, to elicit the generation of new positional identities in a regeneration competent environment. Our experimental design (using amputation, grafting and transcription-based assays) was to test whether posterior ECM, when grafted into anterior innervated wound sites, was sufficient to elicit a cellular growth response that could generate new, non-anterior, positional identities and, in turn, the formation of ectopic limb structures. The aim of our experiments was not to determine whether posterior ECM could retain posterior positional information for prolonged periods, when grafted into new anatomical locations. Rather, we were testing its ability to elicit the formation of new positional information from the host cells. Based on the patterning hierarchy model, new positional information is generated early on in the regenerative process and, therefore, ECM-associated positional information cues, if present, would not require long term stability in this context. We hope that out modifications to the text and figures will help to clarify this to the reader. The manuscript would benefit from some editing to shorten sentences, economize on verbiage, and to correct minor grammatical errors: it was strangely difficult to understand the flow of experimental design regarding the ALM portion, and I am not sure why. Perhaps this is a feature of my COVID isolation-induced hazy state of mind...? Perhaps a flow chart encompassing all facets of the approach would help clarify? (ant/posterior source amputation result, transplant source/engraftment site process and result - the diagram in fig 2 helped, but didn't quite get me there)? Response: We thank Reviewer #1 for this feedback. We apologize for the difficulty in the understanding associated with our experimental design. To remedy these raised concerns, we first worked through the manuscript to economize the text. Next, we correct any grammatical errors present. All changes have been tracked and presented in the ‘Revised Manuscript with Track Changes’ document. Lastly, to improve the understanding of our experimental design, we redesigned figures 1 and 2 (both associated with ALM experiments). For figure 1, we included an experimental outline (new panel A). This describes each step of the experiment. This panel also includes two potential outcomes for the experiment - either the structures regenerate or not. We also explain the implications of each outcome in the legend of the figure. If the structures regenerate, then ECM was sufficient to generate new, non-anterior positional information in the innervated would sites. If the structures do not regenerate, then ECM is insufficient to elicit the formation of new positional information. For figure 2, we redesigned panel A. The experimental outline now includes two potential outcomes for the experiment - either regenerative responses are elicited in both anterior and posterior host sites, or only in the posterior sites. We also explain the implications of each outcome in the legend of the figure. If ectopic pattern is generated in both anterior and posterior host sites, then the donor tissue comprised of anterior and non-anterior positional identities. Therefore, the grafted ECM was sufficient to generate new, non-anterior positional information in the original innervated would sites. If ectopic pattern is only generated in the posterior host sites, then the donor tissue comprised only of anterior identities. In this situation, ECM was insufficient to elicit the generation of new positional information in the original wound sites. Trivial small editorial stuff: p 6, - 2nd sentence from bottom - the semi-colon should be a comma p 15 under 4.2 - "Downstream cues function... ... and is communicated" should read ARE communicated (since the subject of the sentence is "cues" p 17 under 5.2.1, third line down "gentle agitate" should read gentle agitation. Response: These have all been corrected. Reviewer #2 Comments Author´s utilized the ALM-protocol, to generate ECM-induced structures and to show that HSPGs play a role as a molecular signal which downstream communicates information for the proper patterning during axolotl limb regeneration. Experiments are technically sound and the conclusions are coherent with the findings. I havo no major critics on the content of the manuscript. However I believe that putting more effort in the graphic work presented in the main figures, as well as the design and presentation of the figures will improve the manuscript. Besides this point, I think the manuscript is well written and has the merits to be published in PlosOne. Response: We thank Reviewer #2 for this feedback. We have made several changes to the figures to help improve the understanding associated with them and our study. The redesigned figures, and corresponding changes to the legends, have been highlighted in the revised manuscript. Attachments Attachment Submitted filename: Response to Reviewers_PLoS One_ECM.docx https://doi.org/10.1371/journal.pone.0248051.r002
19 Feb 2021 Decision Letter - Michael Schubert, Editor ECM-mediated positional cues are able to induce pattern, but not new positional information, during axolotl limb regeneration PONE-D-20-40259R1 Dear Dr. McCusker, 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. 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, Michael Schubert Academic Editor PLOS ONE 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 #2: All comments have been addressed ******** 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 #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? 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 #2: 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 #2: Yes ****** 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 #2: I am satisfied with the new version of the manuscript, the new version of the figures. In my opinion this manuscript is ready for publication in Plos One. ****** 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. 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 #2: No https://doi.org/10.1371/journal.pone.0248051.r003
Formally Accepted
23 Feb 2021 Acceptance Letter - Michael Schubert, Editor PONE-D-20-40259R1 ECM-mediated positional cues are able to induce pattern, but not new positional information, during axolotl limb regeneration Dear Dr. McCusker: 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. Michael Schubert Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248051.r004

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