PONE-D-23-06337Metamorphic gene regulation programs in Xenopus tropicalis tadpole brainPLOS ONE

Dear Dr. Denver,

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2. To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) methods of anesthesia and/or analgesia, and (3) efforts to alleviate suffering.

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“Dr. Yun-Bo Shi kindly provided the anti-TR serum. This research was supported by NSF grant IOS 1456115 to RJD.”

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Additional Editor Comments:

Dear Robert J. Denvers,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, I feel that it is suitable for publication with some clarification and proofreading. Therefore, my decision is "Minor Revision."

First, I would like to apologize to the authors for the length of time the review process has taken and thank them for their patience.

I also want to give a brief explanation for the long delay:

After receiving the reports from two reviewers, I asked for the opinion of a third reviewer, as the first reviews resulted in a completely opposite opinions from the 2 reviewers, one being very positive and the other calling for rejection of the article. In this situation, I sought another independent opinion so that I could make my decision in full transparency with regard to these evaluations.

In their comments, two of the reviewers are very positive and agree that the work is of good quality and of interest. They underline the relevance of the data presented and feel that this is an important work.

The question of whether the events that occur during natural metamorphosis and induced metamorphosis are comparable is clearly an important issue that deserves to be addressed and debated.

Both reviewers stress this point and I would like to join them in saying that this central question in the article is a very interesting one and deserves to be opened to the scientific community.

However, if the reviewers are globally enthusiastic, the work raises some questions and comments as pointed out in some of the comments of reviewers 1 and 3, which deserves to be reconsidered and improved. There is therefore a need to revise the manuscript substantially before publication.

In conclusion, I ask the authors to give the most complete answer to the reviewers, trying as much as possible to provide arguments to answer their different questions, especially regarding the comments of reviewer 1. In addition, there is some proofreadings that must be corrected.

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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: Yes

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: 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

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

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Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: 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 proposed by Raj, Sifuentes, Kyono and Denver proposes to address an important question of thyroid hormones action in brain during metamorphosis, in the amphibian model Xenopus tropicalis.

By combining RNASeq and ChIPseq data, the authors aim to compare transcriptional variations during natural metamorphosis versus following TH treatment.

Even if the quality of the writing is certain, the manuscript is far from reaching publication quality.

Several subsections of the results section are redundant with other parts of the manuscript. The results description is very concise. Too concise.

For example, the comparison of NF50 vs NF66 stages (lines 291-293) reveals > 4 k DE genes. This represents > 20% of the total number of genes. In fact, this is so high that ANOVA (and more generally parametric models of variance) fail and should not be used. This increases the level of false positives and negatives. If the comparison was indeed NF50 vs NF66, this is a mistake. But if the list of 4k DE genes is the union of two comparisons (untreated vs T3 treatment NF50, and unterated vs T3 treated NF66), this is a different story. This would raise additional questions, but it is at least technologically sound.

This is more than an important technical point, and unfortunately, it is not addressed at all in the manuscript.

- The experimental quality of the data set produced sounds reasonable, but the authors fail to provide additional supporting experimental to help the reader estimate its quality. Not that the work hasn't been done correctly, but ChIP-Seq is notoriously difficult to run, and there are numerous (and unavoidable) caveats at the technical and analysis levels. As the manuscript stands, there are very little data to help the reader estimate how far the data the support the conclusions reached by the authors. This is particularly true for the ChIPSeq, which are always very noisy and where peak height is close to background level.

- Even if this type of comparisons is popular, the comparison of the ChipSeq peaks with the DE genesis highly speculative. The simple fact that a peak is located in a gene (DE or not) is not evidence of direct regulation. The discussion about this point is long, and they rightfully cite the seminal work of Fullwood et al. So why do the authors label this section "identification of direct thyroid hormone target genes using TR Chrip-seq" ? Can we really say this, without additional experimental data (at least 3C or 4C) ?

Minor points

- The tool CLUSTRX looks interesting, and the visual output quite telling. Its source should be referenced.

Reviewer #2: The study by Raj and colleagues is the first genome-wide analysis of gene expression changes for any tadpole tissue spanning the start to end of natural metamorphosis. Theirs is also the first study to directly compare these changes in spontaneous metamorphosis with those induced precociously by TH, a commonly-used approach.

A strong aspect of the current study is that the main conclusions derived from this genome-wide analysis are supported by findings from previous smaller-scale studies. For example, one of the more interesting findings of the study was that, like previous smaller-scale studies, most DEGs in tadpole brain during spontaneous metamorphosis or after T3 treatment did not have TR peaks. The authors put forth several quite plausible explanations for these observations. Another very interesting observation also supported by previous smaller-scale studies was that two thirds of the genes that changed their mRNA levels during spontaneous metamorphosis were not modulated by exogenous T3 in premetamorphic tadpole brain. The authors present several plausible technical and biological explanations for these findings, and conclude that “experiments conducted with exogenous T3 may not be accurate representations of normal developmental processes.” While it is somewhat disappointing that current methods of inducing metamorphosis with T3 and evaluating gene expression do not appear to correspond as well as might be expected with spontaneous metamorphosis, this information is in fact extremely useful to other researchers who use on this method to draw (possibly erroneous) conclusions about natural metamorphosis. One might wonder if inducing metamorphosis instead with a combination of T3 + glucocorticoid may resolve this intriguing discrepancy?

Reviewer #3: In this manuscript Raj et al., have done three complementary experiments on Xenopus metamorphosis: (i) they analysed the gene expression changes in the central nervous system at 4 different time point during spontaneous metamorphosis ; (ii) they perform a classical T3 treatment at 5nM during premetamorphosis and look the gene expression change after 16hr; (iii) last they performed a ChIP-seq experiment to detect the regions of chromatin in which the TRs are binding. These three experiments have been on the central nervous system. The main surprise come from the low number of genes that are found in the three methods: As mentioned by the authors, only half of the genes modulated by treatment of premetamorphic tadpoles with TH changed expression during metamorphosis and these represented only 33% of the genes whose mRNA levels changed during metamorphosis. Similarly, only 24% of genes whose mRNA levels changed during metamorphosis had TR ChIP-seq peaks suggesting that they are direct targets. In the Discussion the authors list all the factors that can explain these low numbers.

This is a very interesting paper, whose conclusions will probably be disliked by many people working on this field, but it is very useful because it clearly reveals the limits of our assays. Despite our fantastic technical abilities, we are still doing very crude and brutal experiments and by doing that we strongly interfere with the systems we are studying.

In addition to this very strong interest, I found the paper well written and the experiments convincing. My main problem is in fact the figures and tables that I find really out of step in terms of quality compared to the text of the paper.

Figure 1B and the relevant text lines 217-228 are a bizarre way of presenting the simple fact that the authors have analyzed 4 stages: I would rather present the developmental serie and would link the various stages to show the comparisons that have been made.

On Figure 2 and 3 I would add heat maps of the 100 or 200 of top genes and would find a way to visualize the genes that are common between spontaneous metamorphosis and TH regulated. Simply put those genes in color in each of the two heat maps.

Figure 4, 5 and 6 are quite boring images of individual genes. I would rather suggest presenting with a scheme the various pathways that are discussed (e.g. cell cycle regulation) and then, again to visualize on each of them the genes that are found by the three technics. There is honestly a strong effort of illustration to do in order to sustain the very nice findings and the great amount of novel information that is present in this paper. Remember that a good part of bioinformatic is to find ways to illustrate very complex and extensive set of information.

Also, there is in my point of view one figure missing. At a minimum we need a Vent diagram to compare the number (and %) of genes that are found associated by each of the three methods. These numbers are discussed lines 294-308 and 400-412 and in the Discussion but we need to see in one clear figure how they relate to each other. What is the number of genes that are regulated/associated in the 3 experiments? Are they TH signalling genes? Could we detect that way new genes that would be very important but have escape attention ?

Minor points

There is an inversion of Figure 5 and 6 with their legends.

Lines 241-244 and Figure 2C: there is not a single gene differentially regulated (positively or negatively) in the three stages? This is curious…

Fig 2D I would be curious to have few examples of the most interesting genes in each of the C1 tp C5 categories. What I have seen in other paper is the line of 3-4 key gene indicated in color with their names. That would be useful.

In our case (in fish) we found many genes implicated in metabolism and a major shift between glycolysis and TCA cycle at different stages during metamorphosis. I think the analysis is a bit short on this point, especially given the importance of the brain as an organ for metabolism also. The link with the appetite control and adipokynes would be super interesting to discuss.

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Reviewer #1: No

Reviewer #2: Yes: Alexander M Schreiber

Reviewer #3: No

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Metamorphic gene regulation programs in Xenopus tropicalis tadpole brain

PONE-D-23-06337R1

Dear Dr. Denver,

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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Kind regards,

Laurent Coen, Ph.D.

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

After a careful reading of the corrections made by the authors, I consider that all the points raised by the reviewers have been taken into account.

All the modifications and comments asked by the reviewers have been addressed in the revised version of the manuscript, and important changes in the text and in figs presentation have been done.

Responses to comments, rewordings of the text and changes to the figures have clarified the paper's weaknesses pointed out in its initial version, and it can therefore be accepted for publication in its revised form, without the need for further consideration by the reviewers.

Reviewers' comments: