Environmental oxygen is crucial for mammalian life. Low oxygen levels –namely hypoxia— occur naturally in the developing embryo and cells adapt to it. Nonetheless, the role of oxygen as a factor influencing stem cell behavior and developmental trajectories is not well understood. In this study, I dissected the effects of acute and prolonged hypoxia in embryonic and extraembryonic stem cells as well as the functional impact on lineage choices and differentiation potential. I show that, while stem cells maintain their cellular identity in hypoxia, low oxygen levels promote a cell type-specific and temporal transcriptional response. Specifically, in embryonic stem (ES) cells, hypoxia selectively induces a transcriptional early primitive streak signature with induction of mesendoderm marker genes, such as Wnt3, T and Eomes, without inducing spontaneous differentiation. Mechanistically, I show that HIF1a activation in normoxia recapitulates the induction of developmental genes as those observed in hypoxia. Additionally, low oxygen levels also alter the epigenetic landscape of ES cells, leading to global DNA demethylation and bivalent chromatin modification rewiring. Last, using a 3D gastruloid differentiation model and in combination with scRNA-seq, I show that hypoxia-induced WNT pathway enables symmetry breaking, polarization and axial elongation in the absence of exogenous WNT activation. When combined with exogenous WNT activation, hypoxia enhances lineage representation by enriching the cell types that are otherwise absent or underrepresented in normoxic gastruloids, such as notochord and gut endoderm, respectively. Moreover, hypoxia provides morphological cues to gut endodermal cells which self-organize in tubular structures reminiscent of the embryonic gut tube. Taken together, these findings reveal the impact of hypoxia on stem cell behavior and during gastrulation where it modulates morphogenesis and cellular composition in 3D gastrulation models. Hence, my investigation provides a direct link between microenvironmental factors and stem cell functions and strongly supports the use of physiologically relevant oxygen levels in models of embryo development.
