All-Trans Retinoic Acid-Responsive LGR6 Is Transiently Expressed during Myogenic Differentiation and Is Required for Myoblast Differentiation and Fusion
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsIn this manuscript, Kitakaze and colleagues investigate functions of LGR6 in differentiation of C2C12 myoblasts to myotubes. They show that LGR6 expression increases transiently upon the induction of myogenic differentiation by switching culture conditions. They provide evidence that LGR6 regulates differentiation and fusion of C2C12 cells, LGR6 expression is regulated by retinoic acid signaling, LGR6 is degraded in a Znrf3-dependent and ubiquitin-independent manner, and LGR6 activates Wnt canonical signaling activity. Overall, the experiments are conducted appropriately, and the results look convincing. The manuscript is written succinctly and is easy to follow. It would have been more comprehensive if the authors investigated the effect of Wnt3a/RSPO2 on C2C12 differentiation and fusion and how it changes by LGR6 knockdown, but this might be too much to ask for this journal.
However, one key information that is missing in this manuscript is whether the expression of LGR6 stays low beyond 24 hours after the induction of C2C12 differentiation or it comes up again later during C2C12 differentiation process. The effect of LGR6 knockdown on C2C12 differentiation and fusion (tested 72 hours after switching media) might reflect LGR6 expression and function beyond the first 24 hours, rather than the mechanism proposed by the authors that LGR6 functions early in the differentiation process. This information needs to be provided before publication of this manuscript.
Minor comments
– While the authors concisely provide their method, it is hard to know when samples were collected in each experiment. It would be helpful to specify in the figure legends how many hours after induction these samples were collected. For example, when was LGR6 expression tested in Figure 1B? 24 hours after induction? 3-6 hours? 3 days, as shown in Figure 1C? This information is critical to know siRNA efficiency in this study. I think it is essential to test LGR6 siRNA efficiency 3-6 hours after induction, since the authors investigate LGR6 functions during this time period. Supplementary Figure S1 partially address this point, but LGR6 mRNA levels need to be also tested.
– What are the alphabetical labels in some of the graphs (Fig 1A, 1G, 3A–D, etc)? They are presumably statistical details, but the information cannot be found anywhere in the manuscript.
– Pictures in Figure 1C and 3F need to be bigger and have better resolution. Current images do not represent corresponding quantifications.
– What happens to MyHC expression when LGR6 is overexpressed? How do differentiation and fusion change?
– While the authors investigate MyoD expression in some of the experiments, they never mention it. MyoD expression is increased by LGR6 overexpression in Figure 2A, what do the authors think about this?
– What happens to LGR6 mRNA expression when both RAR antagonists are added together? Would this be toxic for the cells?
Author Response
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Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors of the manuscript demonstrated that Lgr6 mRNA expression transiently increased 3 h after the induction of myogenic differentiation in C2C12 cells. They found that the loss of LGR6 decreased myomaker, myomerger, and myogenin mRNAs during differentiation, and decreased the differentiation and fusion indices. Thus, authors thought LGR6 promotes myogenic differentiation.
Subsequently, the authors demonstrated: 2.3 ATRA Is Required for Lgr6 mRNA Expression during Myogenic Differentiation;2.4 LGR6 Expression Is Downregulated by the Ubiquitin–Proteasome System; and 2.5 LGR6 Activates Wnt/β-Catenin Signaling. Each of the three parts did a large number of experiments, especially 2.3 and 2.4, but there was no association between these three parts, they are independent of each other. This is a big flaw for good scientific articles. We prefer to see them closely connected and finally get a very rigorous and convincing conclusion.
It is hoped that the authors can supplement some experiments to further clarify the mechanism of LGR 6 in regulating myogenic differentiation.
Some suggestions:
1. In this manuscript, the authors tried to prove whether or how LGR6 regulates myogenic differentiation in C2C12 cells. But the evidence in the manuscript is not so insufficient. For example, the expression analysis of the Lgr6 or genes encoding myogenic regulatory factors (such as myogenin, myomaker, and myomerger) only detected the mRNA level, but not protein expression.
2. In Fig 1F, the loss of LGR6 decreased the expression levels of myogenin, myomaker, and myomerger mRNAs but not of myoD mRNA in C2C12 at 24 h after differentiation. However, in Fig 2B, the same results were obtained by overexpression of exogenous Lgr6. This is contradictory, and the conclusion that LGR promotes myogenic differentiation could not obtained if based on the results in Fig 2B. The authors should explain why the expression of these myokines decreases in the presence of high Lgr6 mRNA expression levels.
3. The current manuscript lacks mechanistic research and mostly consists of confirmatory experiments. For instance, in Result 2.3, the manuscript experimentally validated that ATRA was required for Lgr6 mRNA expression during myogenic differentiation, and that RARα and RARγ were involved in ATRA-responsive Lgr6 mRNA expression. However, it did not further elucidate how ATRA, RARα, and RARγ specifically promote the expression of mRNA. Moreover, the roles of these genes in myogenic differentiation are not detailed thoroughly. Similar issues exist in almost every results of the study.
Comments on the Quality of English Language
The language and writing are not coherent enough, and readability needs to be improved.
Author Response
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Author Response File: 
Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors substantially improved their manuscript. However, it appears that Figure 1 has not been fully updated in their revised manuscript (I don't find new Figure 1A). Please carefully update the manuscript before publication.
Regarding the alphabetical letters in some of the graphs, it would be clearer to state something like "columns with different letters are significantly different at p < 0.05, while columns sharing the same letters are not significantly different" as described in one of the MDPI papers mentioned in the authors' response (https://www.mdpi.com/2072-6643/15/9/2032).
Author Response
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Author Response File: Author Response.pdf
