Intermediate-filament Nestin and group B1 SOX transcription factors (SOX1/2/3) are often employed as markers for neural primordium, suggesting their regulatory link. We have identified adjacent and essential SOX and POU factor binding sites in the Nestin neural enhancer. The 30-bp sequence of the enhancer including these sites (Nes30) showed a nervous system-specific and SOX-POU-dependent enhancer activity in multimeric forms in transfection assays and was utilized in assessing the specificity of the synergism; combinations of either group B1 or group C SOX (SOX11) with class III POU proved effective. In embryonic day 13.5 mouse spinal cord, Nestin was expressed in the cells with nuclei in the ventricular and subventricular zones. SOX1/2/3 expression was confined to the nuclei of the ventricular zone; SOX11 localized to the nuclei of both subventricular (high-level expression) and intermediate (low-level expression) zones. Class III POU (Brn2) was expressed at high levels, localizing to the nucleus in the ventricular and subventricular zones; moderate expression was observed in the intermediate zone, distributed in the cytoplasm. These data support the model that synergic interactions between group B1/C SOX and class III POU within the nucleus determine Nestin expression. Evidence also suggests that such interactions are involved in the regulation of neural primordial cells.Specific gene regulation for the determination of neural primordial cells is of high interest for its possible contribution to stem cell biology, where neural tissues are of primary importance. The majority of the neural primordial cells present in the central nervous system (CNS) from embryonic to adult stages (2, 47) have been documented to express the intermediate filament protein Nestin (11,28,41). Given that transcriptional regulation determining a cell type is largely reflected by the regulation of genes specific to the cell type, we focused on the regulation of the Nestin gene to better understand gene regulation that is determinative for the neural primordial state.Previous studies of rat and human Nestin genes indicated that Nestin expression in the CNS, at least during embryonic stages, is largely regulated by a nervous system-specific enhancer located in the 3Ј half of the second intron (21,29,58,61). After an analysis of a 257-bp-long Nestin enhancer in the rat, Josephson et al. (21) identified putative binding sites for POU, AP2, and a hormone-responsive element; further assessment of their involvement in Nestin enhancer activity was performed using transgenic mouse embryos. Mutational alterations of the downstream POU factor sites, bound by class III POU factors in vitro, largely eliminated the activity of the enhancer; mutations of other sites altered the domain of the CNS in which the enhancer showed activity (21). These previous results indicated that the activation of the Nestin neural enhancer is fundamentally dependent on the binding of a POU factor to the downstream site and that other nuclear factors may cooperate in est...

Embryonic stem cells can be incorporated into the developing embryo and its germ line, but, when cultured alone, their ability to generate embryonic structures is restricted. They can interact with trophoblast stem cells to generate structures that break symmetry and specify mesoderm, but their development is limited as the epithelial-mesenchymal transition of gastrulation cannot occur. Here, we describe a system that allows assembly of mouse embryonic, trophoblast and extra-embryonic endoderm stem cells into structures that acquire the embryo's architecture with all distinct embryonic and extra-embryonic compartments. Strikingly, such embryo-like structures develop to undertake the epithelial-mesenchymal transition, leading to mesoderm and then definitive endoderm specification. Spatial transcriptomic analyses demonstrate that these morphological transformations are underpinned by gene expression patterns characteristic of gastrulating embryos. This demonstrates the remarkable ability of three stem cell types to self-assemble in vitro into gastrulating embryo-like structures undertaking spatio-temporal events of the gastrulating mammalian embryo.

SUMMARY Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions, but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs, and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.