PONE-D-21-28036Retinoid-X Receptor Agonists Increase Thyroid Hormone Competence in Lower Jaw Remodeling of Pre-Metamorphic Xenopus laevis tadpolesPLOS ONE

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Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

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Reviewer #1: Yes

Reviewer #2: Yes

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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: Thyroid hormone (TH) signaling plays important roles during vertebrate development and it’s prone to interference from endocrine disruptors. The manuscript entitled “Retinoid-X Receptor Agonists Increase Thyroid Hormone Competence in Lower Jaw Remodeling of Pre-Metamorphic Xenopus laevis tadpoles” reports two retinoid-X receptor agonists, pharmacologic bexarotene (Bex) and environmental tributyltin (TBT), potentiated TH-induced responses in both one-week-old tadpoles that had limited TH-competence and prometamorphic tadpoles at stage NF54 that had full TH-competence. The authors used TH-induced lower jaw remodeling in Xenopus laevis as a model to investigate if Bex and TBT could potentiate TH-induced response in morphology, cellular proliferation and gene expression, respectively, and found that both Bex and TBT increased TH-induced changes of lower jaw angling, cellular proliferation in Meckel’s cartilage, and certain gene expression such as mmp11, mmp13, and runx2, etc. in one-week-old tadpoles. Consistent with these observations, they also showed that UVI 3003, a retinoid-X receptor antagonist, produced opposite phenotypes morphologically and at gene expression level. These phenotypes were partially reproduced in prometamorphic tadpoles at stage NF54. They concluded that the retinoid-X receptor agonists could potentiate TH-induced tissue remodeling in Xenopus laevis, a natural TH-dependent phenomena during frog metamorphosis, though the retinoid-X receptor agonists themselves alone didn’t induce significant morphological or molecular gene expression changes. The manuscript was well-organized and well-written and its publication would benefit readers in the related research field. Below are some issues for consideration.

1. The data showed that both Bex and TBT increased TH-induced cell proliferation in Meckel’s cartilage but UVI didn’t cause change in cell proliferation, though it slightly decreased aurkb expression in the Meckel’s cartilage tissue (Fig. 2). Did the author normalize the counts of proliferating cells to anything, such as the area of the Meckel’s cartilage tissues or total cells in the tissues? Apparently, UVI inhibited the TH-induced lower jaw remodeling, therefore the head of tadpoles treated with TH and UVI looked more like the control tadpoles treated with vehicle only, which had larger heads (Fig. 1). Did they also have larger Meckel’s cartilage tissues? If so, normalization is necessary.

2. TH-induced endogenous thibz expression was not significantly increased from either Bex or TBT treatment in combination with TH (Fig.3b) in one-week-old tadpoles, however, the authors evaluated transgenic firefly luciferase gene expression (Luc) under the control of a thibz promoter and exhibited that TBT increased TH-induced Luc activities in lower jaws of NF54 tadpoles (Fig. 4b). Both the gene expression data and the luciferase activity data were generated from lower jaws, it would be useful to explain the discrepancy of the data? Was the endogenous thibz expression also enhanced under the same treatment in the transgenic animals? What was the rationale for performing the transgenic studies here?

3. In Fig. 2, the combinatory Bex or TBT treatment increased TH-induced cellular proliferation in Meckel’s cartilage, but UVI didn’t caused changes in TH-induced cellular proliferation, assayed by immunostaining of phosphorylated Ser10 of H3 (Fig. g-i). Interestingly, the expression of aurkb gene, which encodes the Aurora kinase B responsible for phosphorylating Ser10 of H3, didn’t change from such combinatory Bex or TBT treatment with TH. Any explanation for the increase in phosphorylated Ser10 of H3 without any change in aurkb? Is the Aurora kinase B the only kinase for phosphorylating Ser10 of H3?

4. Some typos to be corrected: “Sequences for the primers…..given S1 Table.” (page 9, line 198) to be “Sequences for the primers…..given in S1 Table.”; “Interestingly, RXR agonist s and …..” (page 18, line 430) to be “Interestingly, RXR agonists and …..”; “Meckels cartilage” (page 6, line 127, and other places) to be “Meckel’s cartilage”.

Reviewer #2: In this paper Dr Mengeling and co-authors, analyze the effects of treating NF48 or NF54 X. laevis tadpoles with T3 in the presence or not of RXR agonists (bexarotene or tributylin) or agonists UVI3003. They focus specifically on lower jaw morphology and on the effects on cell proliferation and on the expression of selected genes on lower jaw. They conclude that RXR agonists increase TH competence in lower jaw remodeling of X.l. tadpoles.

The conclusion reached of the paper is not innovative, they showed essentially the same conclusion in their 2018 paper in ‘Endocrinology’ “: RXR Ligands Modulate Thyroid Hormone Signaling Competence in Young Xenopus laevis Tadpoles.

Here they focus more specifically on what they call the “angle of Meckels cartilage” which they measure on pictures of whole tadpoles heads. This measure is very superficial, the authors should have analyzed, at a minimum, tadpole cartilage skeletons and described the effects on all skeletal components as, for example, in : Baltzinger M, et al. Dev Dyn. 2005 Dec;234(4):858-67.or Vieux-Rochas M, et al. Birth Defects Res B Dev Reprod Toxicol. 2010. A description of the phenotype is needed e.g. is there any unilateral or bilateral fusion between the Quadrate, the Ceratohyal (Ch) and/or Meclels Cartilage? Can you relate this to specific effects described in the (vast) literature on craniofacial development? Do different treatments result in different phenotypes? No considerations are made on the mechanisms of of action of TRs or RARs, RXR…on craniofacial morphogenesis, on endodermal or epidermal signals to neural crest cells, on CNCCs migration etc . Actually, no considerations at all is made on the potential mechanism, all the vast literature on receptor involvement in the mechanisms of craniofacial morphogenesis is completely ignored.

As this paper does not really provide any novel information and considering the superficiality of the analysis and discussion I am not suggesting the paper to be published.

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Retinoid-X Receptor Agonists Increase Thyroid Hormone Competence in Lower Jaw Remodeling of Pre-Metamorphic Xenopus laevis tadpoles

PONE-D-21-28036R1

Dear Dr. Mengeling,

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.

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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

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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: 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

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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

**********

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: Thyroid hormone (TH) signaling plays important roles during vertebrate development and it’s prone to interference from endocrine disruptors. The manuscript entitled “Retinoid-X Receptor Agonists Increase Thyroid Hormone Competence in Lower Jaw Remodeling of Pre-Metamorphic Xenopus laevis tadpoles” reports two retinoid-X receptor agonists, pharmacologic bexarotene (Bex) and environmental tributyltin (TBT), potentiated TH-induced responses in both one-week-old tadpoles that had limited TH-competence and prometamorphic tadpoles at stage NF54 that had full TH-competence. The authors used TH-induced lower jaw remodeling in Xenopus laevis as a model to investigate if Bex and TBT could potentiate TH-induced response in morphology, cellular proliferation and gene expression, respectively, and found that both Bex and TBT increased TH-induced changes of lower jaw angling, cellular proliferation in Meckel’s cartilage, and certain gene expression such as mmp11, mmp13, and runx2, etc. in one-week-old tadpoles. Consistent with these observations, they also showed that UVI 3003, a retinoid-X receptor antagonist, produced opposite phenotypes morphologically and at gene expression level. These phenotypes were partially reproduced in prometamorphic tadpoles at stage NF54. They concluded that the retinoid-X receptor agonists could potentiate TH-induced tissue remodeling in Xenopus laevis, a natural TH-dependent phenomena during frog metamorphosis, though the retinoid-X receptor agonists themselves alone didn’t induce significant morphological or molecular gene expression changes. The manuscript was well-organized and well-written and its publication would benefit readers in the related research field.

The changes and responses are satisfactory in the revised version.

Reviewer #2: The inclusion of the analysis of tadpole cartilage skeletons has greatly improved the quality of the paper.

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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