Addendum to: Nature Structural & Molecular Biology https://doi.org/10.1038/s41594-021-00590-w, published online 27 May 2021.
In our manuscript, we investigated the potential role of retinoic acid (RA) signaling in the conversion of mouse embryonic stem cells to 2-cell-like cells and in early mouse embryos. It has since been brought to our attention that further scholarly discussion of our findings in early mouse embryos in relation to other previously published work and genetic loss-of-function experiments would benefit a wider readership.
In particular, in the field of early development, there is often misunderstanding in the way that data from genetic knock-outs are interpreted when the products of the genes under study are present, either in mRNA or in protein form, in the maternal germline. The relevant functions cannot be examined from heterozygous mothers as the maternal contribution, in most cases, masks their early zygotic functions1. For loss-of-function studies to be meaningful in the early pre-implantation period, a genetic depletion in the mature oocyte is necessary. This is not only due to maternal contribution of mRNA but also proteins and other biomolecules inherited from the oocyte, which provides the embryo with mRNA until at least the 2-cell stage in mice, the time at which zygotic genome activation occurs2,3,4,5,6. Genetic analyses of enzymes responsible for RA synthesis show that loss of function for the enzymes does not prevent development past the 2-cell stage but leads to embryonic or postnatal lethality7,8,9,10,11,12. Because these mice do not reach adulthood, any potential role for these enzymes in zygotes and 2-cell stage embryos through maternal deposition of their mRNA or protein in the oocyte cannot be addressed in these mouse models.
Considering this, it is important to appreciate that, so far, all genetic knock-outs targeting RA-producing enzymes and their receptors are zygotic knock-outs. Therefore, a limitation of our study, and that of previous genetic loss-of-function works, is that, until now, none has been performed to specifically deplete members of this pathway in the maternal germline. Indeed, it is notable that mRNAs for all four enzymes synthesizing RA are present in oocytes and zygotes as maternal mRNA13,14. Interestingly, though, recent technological advances have made it possible to assess the presence of RA in oocytes and early embryos in mice. While biochemical approaches such as HPLC are challenging in pre-implantation embryos and oocytes, due to the limited amount of material they provide, recent work has indeed provided evidence of the presence of RA in germinal-vesicle and metaphase II stage oocytes as well as mouse zygotes15.
Collectively, these considerations point towards the need for future work in this area to fully comprehend the molecular regulation and the specific molecules involved in the generation and function of RA signaling in the mature maternal germline and embryos following fertilization.
References
Staller, M. V. et al. Depleting gene activities in early Drosophila embryos with the “maternal-Gal4-shRNA” system. Genetics 193, 51–61 (2013).
de Vries, W. N. et al. Expression of Cre recombinase in mouse oocytes: a means to study maternal effect genes. Genesis 26, 110–112 (2000).
Erhardt, S. et al. Consequences of the depletion of zygotic and embryonic enhancer of zeste 2 during preimplantation mouse development. Development 130, 4235–4248 (2003).
Schultz, R. M. The molecular foundations of the maternal to zygotic transition in the preimplantation embryo. Hum. Reprod. Update 8, 323–331 (2002).
Burton, A. & Torres-Padilla, M. E. Chromatin dynamics in the regulation of cell fate allocation during early embryogenesis. Nat. Rev. Mol. Cell Biol. 15, 723–734 (2014).
Schultz, R. M., Stein, P. & Svoboda, P. The oocyte-to-embryo transition in mouse: past, present, and future. Biol. Reprod. 99, 160–174 (2018).
Sandell, L. L. et al. RDH10 is essential for synthesis of embryonic retinoic acid and is required for limb, craniofacial, and organ development. Genes Dev. 21, 1113–1124 (2007).
Rhinn, M., Schuhbaur, B., Niederreither, K. & Dolle, P. Involvement of retinol dehydrogenase 10 in embryonic patterning and rescue of its loss of function by maternal retinaldehyde treatment. Proc. Natl Acad. Sci. USA 108, 16687–16692 (2011).
Molotkov, A., Molotkova, N. & Duester, G. Retinoic acid guides eye morphogenetic movements via paracrine signaling but is unnecessary for retinal dorsoventral patterning. Development 133, 1901–1910 (2006).
Molotkova, N., Molotkov, A. & Duester, G. Role of retinoic acid during forebrain development begins late when Raldh3 generates retinoic acid in the ventral subventricular zone. Dev. Biol. 303, 601–610 (2007).
Mic, F. A., Haselbeck, R. J., Cuenca, A. E. & Duester, G. Novel retinoic acid generating activities in the neural tube and heart identified by conditional rescue of Raldh2 null mutant mice. Development 129, 2271–2282 (2002).
Dupe, V. et al. A newborn lethal defect due to inactivation of retinaldehyde dehydrogenase type 3 is prevented by maternal retinoic acid treatment. Proc. Natl Acad. Sci. USA 100, 14036–14041 (2003).
Deng, Q., Ramskold, D., Reinius, B. & Sandberg, R. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. Science 343, 193–196 (2014).
Park, S. J., Shirahige, K., Ohsugi, M. & Nakai, K. DBTMEE: a database of transcriptome in mouse early embryos. Nucleic Acids Res. 43, D771–D776 (2015).
Gao, R. et al. Defining a TFAP2C-centered transcription factor network during murine peri-implantation. Dev. Cell 59, 1146–1158 (2024).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Iturbide, A., Ruiz Tejada Segura, M.L., Noll, C. et al. Addendum: Retinoic acid signaling is critical during the totipotency window in early mammalian development. Nat Struct Mol Biol 32, 2128–2129 (2025). https://doi.org/10.1038/s41594-025-01661-y
Published:
Version of record:
Issue date:
DOI: https://doi.org/10.1038/s41594-025-01661-y
