PONE-D-21-17584

CD9 tetraspanins convey robustness to CXCR4b signalling during collective cell migration

PLOS ONE

Dear Dr. Roehl,

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Academic Editor

PLOS ONE

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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: No

Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

Reviewer #2: Yes

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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: No

Reviewer #2: Yes

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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

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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 manuscript contains plenty of errors and most of the figures do not show all images/panels described in the text. At this stage, the manuscript is not competent for publication and should be prepared again with more attention.

Reviewer #2: The paper by Marsay et al revisits the role of the tetraspanin CD9 in the primordium. A previous microarray analysis study in 2010 had shown that CD9 is expressed in the primordium and that that CD9 morpholino injected embryos have delayed migration and fewer neuromasts. This study examines expression of cd9b and the closely related cd9a with whole mount in situ hybridization, confirming expression of these cd9 paralogues in the primordium. It recapitulates previous results showing that cd9 knockdown delays migration and reduces the number of neuromasts. It goes on to show that a TALEN generated mutant, predicted to have loss of CD9b function, does not recapitulate the morphant phenotype and though migration is delayed the number of neuromasts is not altered. The authors go on to determine if the difference between the morphant and mutant phenotype is related to the redundant role of cd9a by generating a CRISPR based mutant expected to have of function. However, the phenotype of the double mutant is not significantly different from that of cd9b alone. Finally, the authors explore the potential role of CD9 in chemokine dependent migration in the primordium by showing that a subcritical knock down of cxcr4b or cxcl12a results in slowing of migration only in the background of a double mutant loss of cd9b and cd9a.

Overall, though the study documents their results effectively, the observations only add modestly to what was previously known about cd9b function. Beyond showing that, unlike morphants, mutants do not have a reduction in the number of neuromasts, they also show that the difference between cd9b morphant and mutant phenotypes cannot be accounted for by redundant function of cd9a.

The authors demonstrate a reduction in primordium migration specifically in double cd9b/cd9a mutants but not wild-type embryos following partial knock down of cxcr4b or cxcl12a. They interpret this to suggest cd9 functions to make cxcr4b function more robust. While strictly speaking it may be correct to say the function is less “robust”, the analysis is minimal and it is remains unclear what the role of cd9 in chemokine might be or whether the exaggeration of deficits seen specifically in the double mutants necessarily result from a role of cd9 in determining efficacy of chemokine signaling. The deficits seen in cd9 double mutants are not typical of those seen when chemokine signaling alone is compromised as it is not even clear whether primordium migration is slower or simply delayed in its initiation. A deficit in chemokine signaling alone might be expected to slow migration and reduce spacing between deposited neuromasts, however, this is not what is seen in the mutants. Furthermore, as collective migration of the primordium is not just dependent on chemokine signaling but also, at a minimum, on Fgf signaling, it is not clear if the observed deficits in migration seen specifically in cd9 mutants might also be seen when Fgf signaling dependent migration is compromised. The authors should test if compromised migration, observed in cd9 double mutants in the background of compromised cxcr4b/cxcl12a function, is also seen when migration is slowed with subcritical doses of an Fgf signaling inhibitor. Are synergistic effects on migration only seen following manipulation of chemokine dependent migration and not following subcritical interference with Fgf-dependent migration? The literature suggests quite a broad range of potential roles for Cd9 and it would be premature to conclude that changes following loss of cd9b function demonstrated in this study are necessarily related to a role for CD9 in facilitating chemokine signaling.

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Reviewer #1: No

Reviewer #2: No

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PONE-D-21-17584R1Augmentation of chemokine signalling by CD9 tetraspanins facilitates collective cell migrationPLOS ONE

Dear Dr. Roehl,

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.

As you will see from the reviewers' comments below, both reviewers agree that the manuscript has improved with the revisions made. On the other hand, they both still feel that title and parts of the abstract overstate the findings made in this study and request to rephrase your wording. I agree with both that the experimental evidence provided is not sufficient to directly link Cd9 function to expression or activity of Cxcr4b. Other interpretations of your findings, as outlined by reviewer 2, are possible. Both reviewers suggest alternative titles and text changes. I recommend that you consider their proposed changes. In your response, please also address the comment raised by reviewer 2 on the movies of LLP migration, whether a delayed start of the LLP is possible in mutants. In your response letter, please also describe the contributions of the newly added co-authors.

Please submit your revised manuscript by Dec 18 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.

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: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Christoph Winkler, Dr.

Academic Editor

PLOS ONE

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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.

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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: (No Response)

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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: Partly

Reviewer #2: Yes

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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

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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

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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: This manuscript aimed to investigate the function of Cd9 tetraspanins in pLLP migration and neuromast formation in zebrafish. First, the authors showed that knocking down of cd9b by morpholinos delayed pLLP migration and reduced the number of deposited neuromasts along the trunk. This observation confirmed the data previously shown by Gallardo et al. To confirm the observed phenotypes the authors went on to use cd9b mutant. Unexpectedly, cd9b mutant only partly recapitulated the phenotypes seen in morphants in which the pLLP migration was transiently delayed, while the number of neuromasts was not affected in the mutant. The authors also looked into the phenotypes of cd9a mutant and cd9a/cd9b double mutants. While no obvious phenotype related to pLLP migration and neuromast formation was seen in cd9a mutant, the double mutant showed similar phenotype as of cd9b single mutant. The different phenotypes of cd9b morphants and cd9b mutant were explained that there could have compensations by other members of the tetraspanin family.

In another attempt, the authors injected morpholinos blocking Cxcr4b and Cxcl12a which previously known to interfere with the primodium migration, into cd9 double mutants. The authors showed that even the low concentrations of morpholinos, which had been known to cause no effect on pLLP migration, could induce further delay of pLLP migration in cd9 double mutants.

Overall, although the new findings in this study is not abundant, the manuscript provides certain clues for future research on the potential interplays among members of tetraspanin family, and between cd9b with Cxcl12/Cxcr4 pathway in the control of primodium migration and neuromast formation.

Minor comments:

The title “Augmentation of chemokine signalling by Cd9 tetraspanins facilitates collective cell migration” is missleading as there is no direct data showing that Cd9 enhances Cxcl12/Cxcr4 expresion or activity which in turn facilitates collective cell migration. The authors may consider a more direct title, for instance “Cd9b tetraspanin and Cxcl12a/Cxcr4b convey synergistic effect on the control of collective cell migration”.

Line 311: typo “Cxcrb” change to “Cxcr4b”

Reviewer #2: Marsay et al have resubmitted a revised version of their original manuscript “CD9 tetraspanins convey robustness to CXCR4b signaling during collective cell migration” with a new title “Augmentation of chemokine signaling by CD9 tetraspanins facilitates collective cell migration”.

A primary issue raised with the original submission was that, with limited analysis of deficits following morpholino, TALEN and/or CRISPR manipulation of cd9a and cd9b function in the zebrafish, the authors were not in position to definitively link Cd9 function to chemokine signaling. No new experiments help to establish a stronger functional relationship between cd9 and chemokine signaling in the primordium and the authors have addressed this weakness of the paper by toning down the suggested link, which is an improvement. However, both the title of the paper and some of the text still in my mind would suggest to the casual reader of the paper a stronger link than has been established.

What is not in doubt is that partial knock down of the cxcr4b in a cd9 double KO has a significant effect on retarding the position of the primordium compared to a situation, while the same subcritical dose of morpholino has no obvious effect in a wild-type background. This confirms that while the deficit in cxcr4b caused by the morpholino alone is not adequate to slow migration, an additional deficit caused by the loss of cd9 function results in delayed migration. Such observations on their own are not adequate to further conclude that the exaggerated deficits in migration resulting from the combined interference with cxcr4b or cxcl12a with cd9 necessarily result from a role of cd9 in the chemokine signaling pathway when it is known that multiple signaling systems in combination with chemokines contribute to primordium migration. The authors do raise this issue in the discussion, however the title “Augmentation of chemokine signaling by CD9 tetraspanins…” still suggests a stronger link between cd9 and chemokine signaling than the authors have evidence for in the paper and I think both the title and the abstract should be further modified to prevent unintentionally perpetuating a potentially erroneous conclusion based on limited analysis.

The title should be changed. A subtly different but perhaps less misleading title could be:

“Loss of Cd9 exaggerates deficits in collective migration caused by interference with chemokine signaling in the posterior Lateral Line primordium”

Similarly, the abstract should be edited to simply remove or edit the last line:

Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels.

Together these results provide evidence that Cd9 modulates collective cell migration of the

pLLP during zebrafish development.”

OR

Loss of both Cd9a and Cd9b sensitized embryos to reduced Cxcr4b and Cxcl12a levels.

Together these results provide evidence that Cd9 modulates collective cell migration of the

pLLP during zebrafish development. One interpretation of these observations is that Cd9 contributes to more effective chemokine signaling.

One additional issue that requires comment. The video of pLLP migration does not show any obvious deficits in migration and migration speed was never assessed or reported. Is it possible that the difference in position of primordium observed by the authors results from a delay in the start of migration. Do the authors have evidence that argues against this? If not, this possibility should also be raised in the discussion.

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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

[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.]

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Tetraspanin Cd9b and Cxcl12a/Cxcr4b have a synergistic effect on the control of collective cell migration

PONE-D-21-17584R2

Dear Dr. Roehl,

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.

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Christoph Winkler, Dr.

Academic Editor

PLOS ONE

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