PONE-D-21-32307Regulation of myoepithelial differentiationPLOS ONE

Dear Dr. LaFlamme,

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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: Partly

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: I Don't Know

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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: Overall the manuscript offers some novel observations about the control of differentiation of cells to a myoepithelial phenotype. The core observation is that the transcription factor Taz drives changes in several genes associated with a myoeothelial phenotype. Thus with the Pro-Mmedia Figure 6 shows Taz upregulated on outer cells of the spheroids and Figure 1 shows upregulation of aSMA and Calponion.

However as one reads the manuscript, there are various points that could have been presented with greater clarity, and other areas where additional immunohistochemistry would confirm location of the changes implied by the averaged RTPCR arising from analysis of the spheroids.

1. Readers encounter the mSG-PAC1 cell line name on page 4. This is th ekey line upon which the whole paper is based. There is no explanation offered as to what the cells are which would have helped. I ran off to the Methods to find that the only explanation was a reference. Help your readers with a brief explanation and reference in the results.

2. In the paper the changes in levels of aSMA, b4, aquaporin 5, calponoin and YAP are through RNA analysis of the spheroids. Thu sthe data re the average of the whole spheroid despite the data presented clearly implying that the myoepithelial phenotype induced by the FCS containing media predominantly occurs at the outer surface of the oral submandibular spheroids. Immunohistochemistry of these proteins on the spheroids should be supplied to show where the expression of these proteins is occurring. This is especially true for integrin beta4 which is an epithelial marker not a myoepithelial marker. All the cells in the spheroid are epithelial in origin so I would expect that all the cells express this integrin. Again, the aquaporin should be higher in the inner epithelial cells of the spheroids compared with the outer layer, so show it by IHC. As presented it is not valid to imply the changes in these genes is associated with the myoepithelial phenotype if the cells with that phenotype have not been directly analysed.

3.The data on TAZ transition to the nucleus, required for transcriptional activity, should be presented more clearly by showing images of just the TAZ staining. This would show exactly where the TAZ is within cells. The purple colour (red and blue pixels co-localised) in the overlays is not wholly clear.

4. The paper is based on an RNAseq analysis of the spheroids treated with different media. 1000s of genes changed. The manuscript has chosen to pick out a handful of genes that addresses the myoepithelial phenotype, linking them with TAZ. Where is the link to the uploaded transcriptional data for the other changed genes? Isnt it possible that there are other genes that have changed that may also have implications for the myoepithelial phenotype?

Overall there are some correlations that implicate molecular processes in myoepithelial differentiation. With additional data outlined the message would be more secure.

Reviewer #2: The authors of manuscript PONE-D-21-32307 described a 3-D culture system with mSG-PAC1 salivary gland derived cell line that was useful for identifying factors involved in differentiation into the myoepithelial lineage. Supporting data were provided by IF analysis developing murine salivary gland. The authors convincingly demonstrated that TAZ (WWTR1) is and important transcription factor for myoepithelial differentiation.

Critiques:

1) Figure 1A (particularly the PRO-A condition) would benefit from addition of nuclear staining. It kind of looks like a hazy green blob, even though I am pretty sure it is an acinus.

2) Figure 1F would be substantially improved by showing every data point that was used to derive the mean w/h ratio. In its current form it is impossible to understand how the error bars are so tight, and why the statistic looks so strong. Perhaps it really is that good, but cells in nature tend towards variation in morphometrics.

3) There is no reference in the text to Figure 5C.

4) Statements about the subcellular localization of TAZ in developing glands in vivo (Figure 5) or in the mSG-PAC1 model (Figure 6) were not clearly supported by the microscopy. It was difficult to see supposed examples of localization at the cell-cell junction vs cytoplasm vs nucleus. e.g. Cytoplasmic vs Nuclear quantification would help, as would higher resolution images.

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Reviewer #1: No

Reviewer #2: No

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Regulation of myoepithelial differentiation

PONE-D-21-32307R1

Dear Dr. LaFlamme,

Congratulations!!! 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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Kind regards,

Rajeev Samant

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

The revised manuscript has addressed Reviewers comments. The edited/revised manuscript is much improved.

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 #2: All comments have been addressed

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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 #2: Yes

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

Reviewer #2: I Don't Know

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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

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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 #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 #2: (No Response)

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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 #2: No