Characterization of a member of the CEACAM protein family as a novel marker of proton pump-rich ionocytes on the zebrafish epidermis

Julien Kowalewski; Théo Paris; Catherine Gonzalez; Etienne Lelièvre; Lina Castaño Valencia; Morgan Boutrois; Camille Augier; Georges Lutfalla; Laure Yatime

doi:10.1371/journal.pone.0254533

Peer Review History

Original SubmissionApril 27, 2021
25 May 2021 Decision Letter - Sheng-Ping Lucinda Hwang, Editor PONE-D-21-13921 Characterization of a member of the CEACAM protein family as a novel marker of proton pump-rich ionocytes on the zebrafish epidermis PLOS ONE Dear Dr. YATIME, 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. Please reply each comment raised by the reviewer. In addition, Fig. 3 mentioned in # 1 and 2 of comments should be Fig. 4. Please submit your revised manuscript by Jul 09 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. 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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: Overall, this is an excellent piece of work. The expression results, including the PCR analysis for tissue distribution and temporal expression of developing stages, in situ hybridization/ immunocytochemistry, as well as the fluorescent images from the transgenic lines, are clear, well presented, and convincing. These results bring us a new marker for HR cells and expand our pieces of knowledge for ionocytes functions and their potential interactions with adjacent cells or the surrounding environment. The only weakness of this study is the lack of a bit in-depth functional analysis of ceacamz1, such as gain- or loss-of-function and external stimulation experiments (different pH or ion strength, pathogen exposure…). Although the expression data connected ceacamz1 role to the HR cell function, readers still have no idea about its exact physiological role in fish or how it involves in the ionocyte functions. On the whole, though, this is an interesting and very valuable study. I only provide a few minor comments/questions below to be considered before publication. 1. The quality of Fig. 3 (A-G) needs to be improved. I assume it’s the problem of image acquisition rather than that of ISH staining. It’s better to show a clear image covering the whole body, including the yolk sac part, where abundant ionocytes also distribute. 2. Fig3.: Did the authors performed the ISH of ceacamz1 at 18 hrs or earlier stages? Because the RT-PCR result doesn’t necessarily represent its expression in ionocytes. 3. Did the authors check the expression patterns of other putative ceacam genes? Are there any other family members abundantly expressed in gill? Since some subtypes of ionocytes share the common structural features (intercalated between keratinocyte, direct contact with external environment….), why is the ceacamz1 uniquely expressed in HR cells if its functions are related to cell adhesion or protection against external pathogens? 4. Discussion Line536-547: The authors try to analogize the zebrafish epidermis/ionocytes to the mammalian intestine epithelium/ goblet cells. However, there exists the mucous secreting cell in the gill or skin epithelium. I’m just curious why the mucous secreting in fish epidermis didn’t show the expression of ceacamz1 if I understand the “parallelism” correctly? (if not, please deifine the “parallelism”) or Is the mammalian ceacamz gene also expressed in ion-transporting cells other than the mucous-secreting goblet cells in intestine epithelium? ****** 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". 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Revision 1
24 Jun 2021 Author Response We thank the reviewer for his/her appreciation of our work and for the very constructive comments which have helped us improve our manuscript. We have taken into consideration all the concerns raised by the reviewer and added new experiments/figures to answer some of the questions. Please find below a detailed, point-by-point response to the reviewer’s comments. ________________________________________ Reviewers' comments: Reviewer #1: Overall, this is an excellent piece of work. The expression results, including the PCR analysis for tissue distribution and temporal expression of developing stages, in situ hybridization/ immunocytochemistry, as well as the fluorescent images from the transgenic lines, are clear, well presented, and convincing. These results bring us a new marker for HR cells and expand our pieces of knowledge for ionocytes functions and their potential interactions with adjacent cells or the surrounding environment. The only weakness of this study is the lack of a bit in-depth functional analysis of ceacamz1, such as gain- or loss-of-function and external stimulation experiments (different pH or ion strength, pathogen exposure…). Although the expression data connected ceacamz1 role to the HR cell function, readers still have no idea about its exact physiological role in fish or how it involves in the ionocyte functions. Answer: we thank the reviewer for his/her appreciation of our work. We agree that functional studies on the physiological role of ceacamz1, in connection with HR-cells, would give more depth to the study. This requires much more work and we could unfortunately not provide such data at this stage of the study, especially due to the work restrictions imposed by the Covid-19 pandemic which slowed down our work quite substantially. We hope to be able to answer these questions in a follow-up study. On the whole, though, this is an interesting and very valuable study. I only provide a few minor comments/questions below to be considered before publication. 1. The quality of Fig. 3 (A-G) needs to be improved. I assume it’s the problem of image acquisition rather than that of ISH staining. It’s better to show a clear image covering the whole body, including the yolk sac part, where abundant ionocytes also distribute. Answer: we now provide a new Fig.4 for ISH analysis where images covering the whole body of the embryo/larva are shown. For each developmental stage, images for 2 representative larvae hybridized with antisense RNA probe are shown, followed by a panel displaying a zoom on the region where ISH staining is most visible. As in the former Fig. 4, only 1 representative larva is shown for hybridization with the sense RNA probe, but the image included now covers the whole body of the larva. 2. Fig.3: Did the authors performed the ISH of ceacamz1 at 18 hrs or earlier stages? Because the RT-PCR result doesn’t necessarily represent its expression in ionocytes. Answer: when performing ISH, we had quite substantial damage of the yolk sac at early larval stages. As a consequence, we could not properly visualize the ISH signal on the yolk region and the images recorded were of insufficient quality for publication. For this reason, we did not attempt extensively to perform ISH at earlier stages than 1 dpf and no ISH data obtained before1 dpf are shown in the manuscript. However, we do agree with the reviewer that it is important to know the distribution profile of ceacamz1 at the very beginning of its expression (i.e. sometimes between 14 and 18 hpf as suggested by our RT-PCR experiments). To answer the reviewer’s question, we have now added a figure in the supporting information (S2 Fig) where we follow the apparition of the mCherry signal of our Tg(ceacamz1:mCherry-F) reporter line at different developmental stages ranging from 10 to 24 somites (14 to 21 hpf). We recurrently observe that mCherry signal starts being clearly visible at around 16 somites stage (17 hpf) and it is localized on the yolk and yolk extension, as observed later on during development. As mRNA would appear slightly earlier, maybe 1 or 2 hours prior to CEACAMz1 protein, and as it is not detected at 14 hpf, our data suggest that almost from the beginning, of its expression, ceacamz1 localizes in the HR-ionocytes present on the ventral skin of the embryos. We have included an additional paragraph in the manuscript to describe these new findings. 3. Did the authors check the expression patterns of other putative ceacam genes? Are there any other family members abundantly expressed in gill? Since some subtypes of ionocytes share the common structural features (intercalated between keratinocyte, direct contact with external environment….), why is the ceacamz1 uniquely expressed in HR cells if its functions are related to cell adhesion or protection against external pathogens? Answer: so far, we have not checked for expression of other putative ceacam genes, mainly because there is very little data on them and their categorization in the ceacam family has not been confirmed. But of course, we would very much like to do so in the future, in a follow-up study. Almost no expression data are available on putative genes from the ceacam family in zebrafish. In fact, there are only 2 publications in the literature describing the existence of ceacam orthologues in Danio rerio, based solely on genome analysis and sequence comparisons. Data on gene expression for only one other putative ceacam gene are available and suggest that it may be expressed predominantly in the intestine (zgc:198329) but no expression has been reported in the gills and it is still not clear whether this gene belongs to the ceacam family or not. It is indeed intriguing that ceacamz1 is only expressed in HR-ionocytes if its main function resides in cell adhesion or interaction with the external environment. On the other hand, we cannot rule out that the receptor is involved in other cellular processes that are specific to HR-cells and may relate to ionoregulation or gas exchange. Furthermore, in humans, many ceacam proteins are GPI-anchored, which forces them to use a yet uncharacterized co-receptor for signal transduction. Ceacamz1 is also GPI-anchored. One explanation as to why it is only expressed in HR-cells may also be that its co-receptor is only expressed in HR-cells. We have now added a few lines in the Discussion to address this point. 4. Discussion Line536-547: The authors try to analogize the zebrafish epidermis/ionocytes to the mammalian intestine epithelium/ goblet cells. However, there exists the mucous secreting cell in the gill or skin epithelium. I’m just curious why the mucous secreting in fish epidermis didn’t show the expression of ceacamz1 if I understand the “parallelism” correctly? (if not, please define the “parallelism”) or Is the mammalian ceacam gene also expressed in ion-transporting cells other than the mucous-secreting goblet cells in intestine epithelium? Answer: in the intestine, human ceacam proteins are expressed predominantly in goblet cells and intestinal epithelial cells. So it is true that it is not a perfect parallelism in terms of cell-type expression since ceacamz1 is not expressed in mucous-secreting cells. What we meant by parallelism was rather a parallelism in terms of patho-physiological context, i.e. that ceacamz1 is expressed in an epithelium covered with a mucus layer that acts as a barrier against external invaders. In that respect, it mimicks perfectly the physiological context encountered by human ceacams. To clarify this point, we have no rephrased the corresponding paragraph in the Discussion section. Attachments Attachment Submitted filename: Response-to-reviewers-comments_Kowalewski-et-al.docx https://doi.org/10.1371/journal.pone.0254533.r002
29 Jun 2021 Decision Letter - Sheng-Ping Lucinda Hwang, Editor Characterization of a member of the CEACAM protein family as a novel marker of proton pump-rich ionocytes on the zebrafish epidermis PONE-D-21-13921R1 Dear Dr. YATIME, 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, Sheng-Ping Lucinda Hwang, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0254533.r003
Formally Accepted
1 Jul 2021 Acceptance Letter - Sheng-Ping Lucinda Hwang, Editor PONE-D-21-13921R1 Characterization of a member of the CEACAM protein family as a novel marker of proton pump-rich ionocytes on the zebrafish epidermis Dear Dr. Yatime: 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. Sheng-Ping Lucinda Hwang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0254533.r004

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