Synergistic activation of RARβ and RARγ nuclear receptors restores cell specialization during stem cell differentiation by hijacking RARα-controlled programs

Synergistic activation of RARβ and RARγ nuclear receptors in mouse stem cells restores cell specialization during neuronal differentiation by hijacking RARα-controlled programs.

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Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance. Sincerely, The authors utilized an experimental paradigm of retinoic acid-induced neural differentiation of P19 embryonic carcinoma cells and performed transcriptome, epigenetics and regulatory region analyses. Very similar methodologies were used in their previous work (Mendoza-Parra et al, Genome Res 2016), in which they dissected retinoic acid receptor subtype-specific signal pathways by means of receptor subtype-specific agonists, BMS753 for RARα, BMS641 for RARβ, and BMS961 for RARγ. Although the authors made knockout P19 cell lines for those receptors in the present study, novelty of this study appears quite limited. In addition, and more importantly, the in vivo relevance of the conclusions is not tested at all. Are the conclusions validated in embryonic brains deficient for RARα, RARβ or RARγ?
It would be interesting to test if the same gene regulatory networks found in this study play significant roles in other experimental paradigms -for example, ES cell-derived neural precursor cell differentiation or embryonic brain-derived neural stem cell differentiation.
Reviewer #2 (Comments to the Authors (Required)): The paper successfully analyses the effects of retinoic acid receptors synthetic agonist on stem cell differentiation into neural cell lineages. The experiments thoroughly investigate the effects of receptor stimulation under different conditions, including the concomitant use of receptors or selective exclusion. With that, the findings add to previous experiments in describing cell differentiation protocols important for research and perhaps future biomedical treatments. Despite the complexity of the topic, the manuscript was written in a clear and organized language, the research methods are adequate and the results are properly discussed.
Nevertheless, few minor text mistakes were detected: -Page 2, Paragraph 2, first line: "Using RAR istoype knockout lines", there is a typo. -Page 7, first paragraph: "...we generated global transcriptomes after 2, 4 and 10 days of treatment of WT P19 cells.This has been performed for samples..." These sentences need to be separated. The authors utilized an experimental paradigm of retinoic acid-induced neural differentiation of P19 embryonic carcinoma cells and performed transcriptome, epigenetics and regulatory region analyses. Very similar methodologies were used in their previous work (Mendoza-Parra et al, Genome Res 2016), in which they dissected retinoic acid receptor subtype-specific signal pathways by means of receptor subtype-specific agonists, BMS753 for RARα, BMS641 for RARβ, and BMS961 for RARγ.
We acknowledge the comments of reviewer #1 and notably the reference performed to our previous work, which is indeed the seminal effort for the presented manuscript.
Although the authors made knockout P19 cell lines for those receptors in the present study, novelty of this study appears quite limited.
We consider that the presented manuscript goes beyond the fact that we have used Rar knockout P19 lines for our readouts. In fact, there are two major aspects that differ to our previous article: In contrast to our previous article which focused on the fist 72 hours of P19 cell differentiation; i.e. addressing neurogenesis, herein we present a study covering 10 days of cell culture; hence addressing cell specialization (Neuronal subtypes + glial cells) (ii) We have revealed for the first time that the synergistic action of the RARb and Rarg agonists (BMS641 + BMS961) could lead to neuronal cell differentiation and specialization.
In this context, the use of the P19 knockouts, combined with the use of the various RAR subtype-specific agonists, allowed us to decorticate the relevant gene programs that are used during this process. Hence, our manuscript does not only describe the synergistic action of the RARb and Rarg agonists, but in addition it reveals major players that are reactivated in absence of the Rara receptor.
In addition, and more importantly, the in vivo relevance of the conclusions is not tested at all. Are the conclusions validated in embryonic brains deficient for RARα, RARβ or RARγ?
While we do agree that the in-vivo relevance would be of interest, this aspect is out of the scope of this manuscript. The main aim of our work is to understand the major programs that are activated in presence of retinoids. Notably, the fact that the pan-agonist All-trans retinoic acid (ATRA) can activate all three Rar receptors at the same time suggests the use of Rar-specific as well as Rar redundant programs; or these programs (and their cross-talks) are not fully addressed yet by the scientific community. Our work provides a piece of information by revealing the gene programs that are reactivated by the synergistic action of the Rarb and Rarg specific agonists.
It would be interesting to test if the same gene regulatory networks found in this study play significant roles in other experimental paradigms -for example, ES cell-derived neural precursor cell differentiation or embryonic brain-derived neural stem cell differentiation.
As part of the revised version of our manuscript, we have extended this study to mouse ES differentiation driven by retinoids action and we have demonstrated that (i) the combined treatment of mES cells with the Rarb and Rarg specific agonists (BMS641+BMS961) leads to neuronal cell differentiation and cell specialization (glial cells + neuron subtypes detected); (ii) the synergistic action of the Rarb and Rarg specific agonists reactivates most of the major transcription factors revealed in P19 cell differentiation and notably the Prdm8 regulome ( Figure 6).
Reviewer #2 (Comments to the Authors (Required)): The paper successfully analyses the effects of retinoic acid receptors synthetic agonist on stem cell differentiation into neural cell lineages. The experiments thoroughly investigate the effects of receptor stimulation under different conditions, including the concomitant use of receptors or selective exclusion. With that, the findings add to previous experiments in describing cell differentiation protocols important for research and perhaps future biomedical treatments. Despite the complexity of the topic, the manuscript was written in a clear and organized language, the research methods are adequate and the results are properly discussed.
We acknowledge the fact that Reviewer #2 fully grasped the spirit of this work and that he/she finds our contribution of interest, and notably our effort to provide a clear description of our studies.
Nevertheless, few minor text mistakes were detected: -Page 2, Paragraph 2, first line: "Using RAR istoype knockout lines", there is a typo. -Page 7, first paragraph: "...we generated global transcriptomes after 2, 4 and 10 days of treatment of WT P19 cells.This has been performed for samples..." These sentences need to be separated.
We apologize for these typos. We have corrected them as part of this resubmitted version.
I consider the research of extreme relevance and recommend the approval of the manuscript after minor corrections. Thank you for submitting your Research Article entitled "Combination of RARb and RARg agonists restores neuronal maturation by hijacking RARa-driven programs". It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance. Congratulations on this interesting work.

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