Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail

Satoshi Ishishita; Shumpei Kitahara; Mayuko Takahashi; Sakura Iwasaki; Shoji Tatsumoto; Izumi Hara; Yoshiki Kaneko; Keiji Kinoshita; Katsushi Yamaguchi; Akihito Harada; Yasushige Ohmori; Yasuyuki Ohkawa; Yasuhiro Go; Shuji Shigenobu; Yoichi Matsuda; Takayuki Suzuki

doi:10.1371/journal.pone.0265008

Peer Review History

Original SubmissionOctober 4, 2021
30 Dec 2021 Decision Letter - Karthikeyan Adhimoolam, Editor PONE-D-21-31931Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quailPLOS ONE Dear Dr. Suzuki, 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. ACADEMIC EDITOR: Minor revision. Please submit your revised manuscript by Feb 10 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. 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If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. 4. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [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 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: In this manuscript entitled “Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail”, the author has mainly demonstrated a causative gene of white eggshells in Japanese quail by the approach of mRNA sequencing, ddRAD sequencing, whole-genome resequencing. In Summary, the author in this manuscript have clearly demonstrated the transcriptomic profiling to uncover the genes responsible for white eggshells. The manuscript can be accepted without any further revision. Reviewer #2: The manuscript entitled “Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail”. The authors analysed the cause of low PPIX production in a classical Japanese quail mutant exhibiting white eggshells by comparing its gene expression in the uterus with that of the wild type using transcriptome analysis and performed genetic linkage mapping to identify the causative genomic region of the white eggshell phenotype. It also suggests that uterus-specific transcriptional regulation of heme-biosynthesis-related genes is an evolutionarily acquired mechanism of eggshell pigment production in Japanese quail. Based on our findings, we discuss the molecular basis of PPIX production in the uterus of Japanese quail. The manuscript comprises all the necessary elements of scientific novelty. The experimental designing and execution of the study were appreciable. I recommend this manuscript for the reconsideration for publication in PlosOne after incorporating minor changes given in below. COMMENTS FOR THE AUTHOR: Authors must concentrate on the formatting, and use of symbols, etc., throughout the manuscript. Framework figure is required. It will be useful to the readers for better understanding of the studied issue. In materials and method section: authors should explain why each item of methodology was done. Proper formatting needed in the materials and methods, results, discussion and conclusion section. Introduction was comprehensive to the studied issue. Real Time-PCR’s raw data in the supplementary for scrutiny. In conclusion section authors should provide some future prospectus related to present study. ****** 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.0265008.r001
Revision 1
15 Feb 2022 Author Response Response to Reviewer #2 We wish to express our appreciation to the Reviewer for his or her insightful comments, which have helped us significantly improve the paper. 1) “Authors must concentrate on the formatting, and use of symbols, etc., throughout the manuscript.”, “Proper formatting needed in the materials and methods, results, discussion and conclusion section.” Response: We have revised the formatting properly for readability. The descriptions in the Materials and methods, results, and discussion sections have been arranged. Please see the revised manuscript with track changes for details. Main revisions are as follows. Lines 140. “The average number of mapped read pairs is also shown in S1 Data.”, which was placed in the result section in the original manuscript, has been moved to the Materials and Methods section, for readability. Lines 250-256. “For the synthesis of RNA� paired-like homeodomain 3 (PITX3))”, which was placed at the end of this paragraph, has been moved next to the first sentence of the paragraph for readability. Line 469. The line break position has been changed. In the original manuscript, it was placed before “The 172 kb candidate region harbored�” (line 443 in the revised manuscript). Lines 566-591. Discussion about the candidate gene and the hypothetical model has been moved from the 4th and 5th paragraphs in the original manuscript to the 3rd and 4th paragraphs in the revised manuscript. Lines 603-608. The discussion about in situ hybridization analysis has been moved from the 3rd paragraph to the 5th paragraph. Consequently, the numbering of reference has been changed as follows. #53 in the original manuscript > #55 in the revised manuscript #54 > #53 #55 > #54 The reference list has been changed along with these alterations. The result of biochemical assay and the hypothetical model of the genetic cascade in the original Fig 6 have been divided into Fig 6 and Fig 7 in the revised manuscript, for readability. We have defined “uterus-specific genes” and “the candidate region” as follows for readability. Lines 106-110. “To identify the differentially expressed genes (DEGs) between the wild-type and mutant uteri and detect the genes that are specifically upregulated in the uterus (uterus-specific genes) compared to the other six organs (heart, liver, intestine, kidneys, lungs, and brain) and two tissues (muscle and isthmus), we performed mRNA sequencing of the uteri and isthmuses from the wild-type and mutant quail.” Line 193-195. “sequence variants that were found in the candidate causative region (candidate region) were annotated using SnpEff v4.3t [33].” In the author contribution section (at the end of the manuscript), the short names of several authors have been changed, as follows. Lines 917-923. Ishishita > Is Iwasaki > Iw Ohkawa > Ohk Ohmori > Ohm We have arranged or added several subtitles in Materials and Methods and Result sections for readability, as follows. Line 105. “mRNA sequencing” has been change to “mRNA sequencing and differential gene expression analysis”. Line 152-153. “Double-digest restriction site-associated DNA (ddRAD) sequencing and association test” has been changed to “Construction of a reference family, double-digest restriction site-associated DNA (ddRAD) sequencing, and association test”. Line 232. “Transmission electron microscopy” has been changed to “Transmission electron microscopy (TEM)”. Line 286. “Biochemical assay for the PPIX-forming ability of the uterus” has been changed to “Biochemical assay to evaluate the PPIX-forming ability of the uterus”. Line 323-324. “Fig 1. The white eggshell phenotype” has been changed to “Fig 1. White eggshell phenotype and low accumulation of brown pigments in the uterine mucosa of the mutant quail.” for scientific accuracy. Line 342-343. “Characterization of the mutant uterus by gene expression analysis” has been changed to “Characterization of the mutant uterus by gene expression analysis and identification of uterus-specific genes”. Line 356-357. “Fig 2. Genes that are specifically upregulated in the wild-type uterus and downregulated in the mutant uterus.” has been changed to “Fig 2. Identification of genes that are specifically upregulated in the wild-type uterus and downregulated in the mutant uterus.”. Line 386. “Genetic linkage mapping of the candidate region responsible for the white eggshell phenotype” has been changed to “Genetic linkage mapping of the responsible locus”. Line 404. “Fig 3. The candidate region of the white eggshell (we) phenotype is mapped to chromosome 6.” has been changed to “Fig 3. Genetic linkage mapping of the we locus.”. Line 440-441. “Identification of the causative mutation and causative gene around the we locus” has been added. Lines 494-495. “Distribution analysis of PITX3, ALAS1, and HEPHL1 transcripts in the uterine mucosa” has been added. 2) ” Framework figure is required.” Response: We have added framework of this study in S2 Fig, therefore, Line 77-78. We have mentioned “The framework of this study in shown in S2 Fig.” at the end of Introduction. Line 814. We have added a figure caption “S2 Fig. Framework of this study.”. The numbering of the remaining Supporting Figures has also been changed. 3) ”In materials and method section: authors should explain why each item of methodology was done.” Response: Lines 91-92, 106-110, 154-157, 181-183, 224, 233-235, 249-250, 277-278. We have described the objective of each item at the beginning of paragraphs. Please read the revised manuscript with track changes for details. We have also mentioned objectives of experiments in the result section at lines 343-345, 389, 442, and 496. 4) ”Real Time-PCR's raw data in the supplementary for scrutiny.” Response: As suggested, we have added raw data of Real Time PCRs as S6�S11, S14, and S15 Data. Because we have added these data, numbering of Supporting Data and has been changed and captions in pages 39�41 have been changed. 5) ”In conclusion section authors should provide some future prospectus related to present study.” Response: We have added the following sentences at the end of the conclusion. Lines 635-640. “In future studies, we should identify the causative gene and mutation responsible for white eggshells and investigate the functional roles and transcriptional mechanisms of the 11 genes that were specifically expressed in the wild-type uterus and downregulated in the mutant uterus. In addition, the specific roles of apical and LP cells in PPIX production should be further elucidated.” 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 We have corrected the manuscript style. 2. We note that you have indicated that data from this study are available upon request. PLOS only allows data to be available upon request if there are legal or ethical restrictions on sharing data publicly. For more information on unacceptable data access restrictions, please see http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. In your revised cover letter, please address the following prompts: a) If there are ethical or legal restrictions on sharing a de-identified data set, please explain them in detail (e.g., data contain potentially sensitive information, data are owned by a third-party organization, etc.) and who has imposed them (e.g., an ethics committee). Please also provide contact information for a data access committee, ethics committee, or other institutional body to which data requests may be sent. b) If there are no restrictions, please upload the minimal anonymized data set necessary to replicate your study findings as either Supporting Information files or to a stable, public repository and provide us with the relevant URLs, DOIs, or accession numbers. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. We will update your Data Availability statement on your behalf to reflect the information you provide. We have uploaded all data and procedures to replicate our study onto PLOS ONE submission system or public database. Therefore, we would like to remove the following statement “Other miscellaneous information is available from the corresponding author upon request.” 3. We note that you have stated that you will provide repository information for your data at acceptance. Should your manuscript be accepted for publication, we will hold it until you provide the relevant accession numbers or DOIs necessary to access your data. If you wish to make changes to your Data Availability statement, please describe these changes in your cover letter and we will update your Data Availability statement to reflect the information you provide. Our data deposited to public database (SRA in DDBJ) and github.com are already accessible, without restriction. 4. 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. There are no papers that have been retraced in our reference list. We have mentioned about changes to the reference in rebuttal letter. 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. We have checked all the figures using the PACE tool. Revised figures (Figs 2, 6, and 7; S2 Fig) have been corrected via PACE. 5. Others The following careless mistakes have been corrected as follows. Line 431. Fig 3E (at line 452 in the original manuscript) > Fig 3F In Fig 2C. The number of DEG (down) in the circle has been corrected from 250 (all 261 DEGs – 11 overlapping genes) to 102 (=113 downregulated genes – 11 overlapping genes). Many minor corrections in terms of grammatical accuracy have been made though professional English proofreading. See the revised manuscript with track changes for details. Attachments Attachment Submitted filename: response letter 220215.docx https://doi.org/10.1371/journal.pone.0265008.r002
22 Feb 2022 Decision Letter - Karthikeyan Adhimoolam, Editor Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail PONE-D-21-31931R1 Dear Dr. Suzuki, 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, Karthikeyan Adhimoolam 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: (No Response) Reviewer #2: Authors have addressed all my queries and now the manuscript is technically sound. Therefore, I recommend this manuscript is accepted for publication in its current form. ****** 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.0265008.r003
Formally Accepted
28 Feb 2022 Acceptance Letter - Karthikeyan Adhimoolam, Editor PONE-D-21-31931R1 Uterus-specific transcriptional regulation underlies eggshell pigment production in Japanese quail Dear Dr. Suzuki: 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. Karthikeyan Adhimoolam Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0265008.r004

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