Epigenetic and physiological alterations in zebrafish subjected to hypergravity

Marcela Salazar; Silvia Joly; Guillem Anglada-Escudé; Laia Ribas

doi:10.1371/journal.pone.0300310

Peer Review History

Original SubmissionOctober 16, 2023
21 Nov 2023 Decision Letter - Hiroshi Kaji, Editor PONE-D-23-33854Epigenetic and physiological alterations in zebrafish subjected to hypergravityPLOS ONE Dear Dr. Ribas, 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. ============================== The reviewers raised several concerns in this paper, which should be addressed in the revised version. ============================== Please submit your revised manuscript by Jan 05 2024 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. 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[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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No Reviewer #2: No ****** 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: No ****** 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: No ****** 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: This manuscript addresses the effects of hypergravity on developmental, behavioral, gene expression, and epigenetic changes in zebrafish. The authors showed significant hypermethylation of the genome and downregulation (not significant) of gene expression levels of three genes under hypergravity for 5 days. This study is important for understanding the effects of hypergravity in DNA methylation level, but needs to be modified before publication in Plos One. 1. Figure 4 and Table 1 show the identification of three locomotor aspects. It could be more easily understood if video data or other means were used to demonstrate these typical behaviors. 2. Figure 5 shows various phenotype under hypergravity. Each is assumed to have different conditions, such as tissue inflammation and damage. Did authors perform the histological analysis? And how did the authors evaluate gene expression and genomic methylation in zebrafish exhibiting these various phenotypes? 3. The authors attempt to show in only one figure (Figure 6) the overmethylation of the zebrafish genome under 5 days of hypergravity. The results of the comprehensive analysis should be evaluated from various angles. 4. Figure 7 describes that there was a decrease in the expression of three genes, but it was not significant. Were significant decreases in gene expression also seen in other genes? 5. Are there any specific sites of DNA methylation under hypergravity condition? 6. Is "Hipergravity" misspelled in figure 6? Reviewer #2: In this study, Salazar et al. studied the effects of hypergravity on development, behavior, gene expression, and epigenetic alterations in zebrafish. The authors revealed that hypergravity decreased survival, but not hatching rate at 2 days post fertilization. Hypergravity induced physiological and morphological changes including posture, movement frequency, and swimming behavior. Moreover, hypergravity increased DNA methylation and tended to decrease epigenetic-related genes, such as dnmt1, dnmt3, and tet1, without statistical significance in the zebrafish larvae. However, there are several issues with the manuscript. The details are attached below. Major points 1. Please provide a rationale for used 3 g hypergravity condition in the present study. 2. The detailed methods for analysis of qPCR should be added in Methods section. Did the authors calculated data using ΔΔCt methods? Which endogenous control was used? 3. Although Table 2 showed primer sequence for ef1α and rpl13a, the authors did not show those quantitative data. 4. Fig. 6: According to figure legend, data were expressed as boxplot with thick line indicating median. However, figure 6 were not expressed boxplot. Please correct figure 6 and its figure legend. 5. Statistical analysis should be correct. Although the authors revealed that Shapiro-Wilk test was used to check for normality in Methods section, normality was evaluated with a Kolmogorov-Smirnov test in Fig. 3. The authors used student t-test for assessment of differences in Fig. 6, but there is no description regarding t-test in Methods section. It is not clear which statistical was used in Fig. 7. ****** 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: Yes: Masahiro Chatani 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. 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Revision 1
14 Feb 2024 Author Response Reviewer #1: This manuscript addresses the effects of hypergravity on developmental, behavioral, gene expression, and epigenetic changes in zebrafish. The authors showed significant hypermethylation of the genome and downregulation (not significant) of gene expression levels of three genes under hypergravity for 5 days. This study is important for understanding the effects of hypergravity in DNA methylation level, but needs to be modified before publication in Plos One. 1. Figure 4 and Table 1 show the identification of three locomotor aspects. It could be more easily understood if video data or other means were used to demonstrate these typical behaviors. Thanks, for the comment. We have added new support information creating Dataset 1 (https://drive.google.com/drive/folders/1PNgmMw1a-Nh0kWXqsyCIH3FOIeCAuOJe?hl=es) which are the videos exhibiting every morphological characteristic observed in both the control and hypergravity groups of zebrafish larvae. 2. Figure 5 shows various phenotypes under hypergravity. Each is assumed to have different conditions, such as tissue inflammation and damage. Did the authors perform the histological analysis? And how did the authors evaluate gene expression and genomic methylation in zebrafish exhibiting these various phenotypes? Thanks for pointing this out. Unfortunately, histological analysis was not performed because we used the whole larvae for the DNA and RNA extractions, and therefore no more tissues were available. Gene expression and methylation analysis were randomly conducted within the groups; 15 samples were ethologically evaluated from each experiment, and 10 of these were randomly selected for the aforementioned studies. We decided not to select any specific phenotype as this could bias the result. However, after evaluating our raw data from the experiment, we have created the following table with the selected phenotypes of those larvae used for molecular analysis. Teratologies Control Hypergravity Normal body shape 12 0 Overall body deformation 1 6 Tail anomalies 1 0 Body curvature 1 9 3. The authors attempt to show in only one figure (Figure 6) the over methylation of the zebrafish genome under 5 days of hypergravity. The results of the comprehensive analysis should be evaluated from various angles. The reviewer is right with the suggestion. In the current form of the study, we aimed to explore, for the first time, the epigenetic changes that hypergravity could trigger in zebrafish larvae. As the findings are promising, future studies in our lab will be addressed towards a better comprehension of the epigenome modifications from various angles. We are currently conducting RNA-seq from a similar experiment hoping that they will untangle molecular events by gene expression modifications. 4. Figure 7 describes that there was a decrease in the expression of three genes, but it was not significant. Were significant decreases in gene expression also seen in other genes? We did not evaluate other genes because we wanted to assess these important genes related to methylation. Based on our experience in the lab, we used them as epimarkers for gene expression studies related to methylation alterations (Ribas et al 2017 https://doi.org/10.1186/s13072-017-0168-7; Valdivieso et al 2023 DOI: 10.3390/ijms242116002). However, as previously mentioned, we are working in the lab on RNA-sequencing technologies and we hope we will be able to identify differential gene expression. 5. Are there any specific sites of DNA methylation under hypergravity conditions? For the current study on Global DNA methylation (Zymo Research), the methodology utilized unfortunately lacks the capability to discern specific methylation sites. Despite this limitation, we are actively engaged in the experimentation and evaluation of an alternative technique for our upcoming study. In particular, we are exploring the application of the MinION, a cutting-edge technology that holds promise for providing insights into the methylation patterns. This innovative approach is expected to enhance our ability to pinpoint and analyze specific methylation sites with greater precision, thereby contributing to a more comprehensive understanding of the underlying molecular mechanisms. 6. Is "Hipergravity" misspelled in figure 6? Thanks, the figure has been amended Reviewer #2: In this study, Salazar et al. studied the effects of hypergravity on development, behavior, gene expression, and epigenetic alterations in zebrafish. The authors revealed that hypergravity decreased survival, but not hatching rate at 2 days post fertilization. Hypergravity induced physiological and morphological changes including posture, movement frequency, and swimming behavior. Moreover, hypergravity increased DNA methylation and tended to decrease epigenetic-related genes, such as dnmt1, dnmt3, and tet1, without statistical significance in the zebrafish larvae. However, there are several issues with the manuscript. The details are attached below. Major points 1. Please provide a rationale for used 3 g hypergravity condition in the present study. Thanks for your comments. We performed two pilot studies before selection 3g hypergravity as the experimental conditions for our experiments. The explanation concerning the selection of 3g have been incorporated into the manuscript. 2. The detailed methods for analysis of qPCR should be added in Methods section. Did the authors calculated data using ΔΔCt methods? Which endogenous control was used? Yes, we used the ΔΔCt method with two endogenous genes, RPL13A (Ribosomal Protein L13A) and EFα (Elongation Factor α). We have already amended the text accordingly. 3. Although Table 2 showed primer sequence for ef1α and rpl13a, the authors did not show those quantitative data. These genes were the endogenous controls that were used to normalize the target genes to evaluate the expression making a qualitative study. This method allows to study of gene expression based on qualitative data all the values normalized to their endogenous genes to later compare them with their corresponding controls. Thus, these analyses are not quantitative, in compared to using a specific probe for each gene by qPCR. We hope that adding information to the text as mentioned in the previous comment makes the analysis clearer. 4. Fig. 6: According to figure legend, data were expressed as boxplot with thick line indicating median. However, figure 6 were not expressed boxplot. Please correct figure 6 and its figure legend. Thanks, the text in the legend has been amended accordingly. 5. Statistical analysis should be correct. Although the authors revealed that the Shapiro-Wilk test was used to check for normality in the Methods section, normality was evaluated with a Kolmogorov-Smirnov test in Fig. 3. The authors used a student t-test for assessment of differences in Fig. 6, but there is no description regarding t-test in the Methods section. It is not clear which statistics were used in Fig. 7. Thanks, the text has been amended accordingly. ________________________________________ Attachments Attachment Submitted filename: Response to reviewers_final_050224.docx https://doi.org/10.1371/journal.pone.0300310.r002
27 Feb 2024 Decision Letter - Pierre Denise, Editor Epigenetic and physiological alterations in zebrafish subjected to hypergravity PONE-D-23-33854R1 Dear Dr. Ribas, 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, Pierre Denise, Ph.D, M.D. Academic Editor PLOS ONE Additional Editor Comments (optional): 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 ******** 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 ****** 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 ****** 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 have fully complied with this request and have revised the text and graphic and visual data. Reviewer #2: The revised version bring additional information. The authors well addressed most of my concerns by revising the paper. ****** 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 #1: No Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0300310.r003
Formally Accepted
26 Apr 2024 Acceptance Letter - Pierre Denise, Editor PONE-D-23-33854R1 PLOS ONE Dear Dr. Ribas, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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 Pr. Pierre Denise Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0300310.r004

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