Retinoic acid signaling is critical during the totipotency window in early mammalian development

Abstract

Totipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

Cell division- and DNA replication-free reprogramming of somatic nuclei for embryonic transcription

“…ZFP281 and RAR binding motifs were found only in C3H-specific DEGs ( Figures 4 F and S4 A). RAR has been shown to affect transcriptional reprogramming, possibly through opening of inaccessible sites in somatic chromatin such as Pou5f1 and Utf1 loci ( Miyamoto et al., 2018 ), and RA activity in early embryos is required for embryonic gene activation ( Iturbide et al., 2021 ). ZFP281, a zinc finger TF, maintains the pluripotency network in ES cells through the direct activation of Nanog and other pluripotency-related genes by binding to promoters with OCT4 and SOX2 ( Kim et al., 2008 ; Wang et al., 2008 ).…”

Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

“…Notably, cluster 3 showed higher retinoid X receptors alpha (Rxra) gene expression (log 2 (FoldChange) = 1.41) (Figure 7A), which is a critical receptor that controls retinoic acid activation. Recent studies have showed that Rxra expression is upregulated in 2CLCs, and the high expression of retinoic acid can reprogram mESCs to the 2CLC state [28,29]. In the predicted pathway regulation analyses by the IPA, several low expression genes in cluster 3 revealed differentiation-related genes including Fgf2 and Vegfa, where both are differentiation factors of ESCs [16,30].…”

Cross-activation of the FGF, TGF-β and WNT pathways constrains BMP4-mediated induction of the Totipotent state in mouse embryonic stem cells

“…Taken together with these observations, our study demonstrates that BMP4 signaling plays an essential role in establishing the Totipotent state. A recent study found that retinoic acid can also induce a 2C-like Totipotent state in ESCs ( 27 ). Although retinoic acid is not used to culture mouse ESCs as it induces their differentiation towards neuroectoderm lineage ( 28 ), it may act in conjunction with BMP4 signaling to induce the Totipotent state.…”

“…The transcription factor Gata2 induces the Totipotent state by inducing MERVL expression (19). The Totipotent state can also be enhanced in cultured ESCs in various ways: over-expression of Duxf3 and Nelfa genes and miR-344 microRNA; altering chromatin states through knockout of Lsd1 and Hdac inhibitors; altering metabolic states through sodium acetate and α-ketoglutarate; treating with a combination of small-molecule inhibitors and retinoic acid (21)(22)(23)(24)(25)(26)(27). Pluripotent cells are marked by the expression of Oct4, Sox2, Klf4, Nanog, Rex1 and other transcription factors known to form a selfreinforcing gene regulatory network (28,29).…”