Discovery of a pre-vein progenitor that requires VEGF/ERK inhibition to complete vein differentiation

Despite substantial insight into mechanisms underlying arterial blood vessel development, multiple aspects of vein development remain elusive, including vein-determining extracellular signals and cell-fate trajectories1-10. One might expect arteries and veins to arise simultaneously during development, as both are needed for a functional circulatory system. Nevertheless, arteries often precede veins in vivo, as exemplified by the first intraembryonic blood vessels11-17. Here we present a model of vein differentiation that answers longstanding questions in the field. By reconstituting human vein endothelial cell (EC) differentiation from mesoderm in vitro, we discovered that vein development unfolds in two steps driven by opposing signals. First, VEGF is necessary to differentiate mesoderm into "pre-vein" ECs--a newly-defined intermediate state--and to endow endothelial identity. Second, once cells have acquired pre-vein EC identity, VEGF/ERK inhibition is necessary to specify vein ECs. Pre-vein ECs co-expressed certain arterial (SOX17) and venous (APLNR) markers and harbored poised chromatin at future venous genes. However, VEGF/ERK inhibition was necessary to activate poised venous genes (e.g., NR2F2), and for pre-vein ECs to complete venous differentiation. Intersectional lineage tracing supported a pre-vein intermediate step in vivo: early Sox17+ Aplnr+ ECs also formed veins in mouse embryos. We leveraged this developmental knowledge for disease modeling by differentiating human pluripotent stem cells into artery and vein ECs, and comparing their responses to Ebola and Andes viruses under biosafety-level-4 containment. Artery and vein ECs responded divergently to the same virus, thus revealing that developmentally specified cell identity impacts viral infection. Taken together, we propose a two-step model for vein development wherein VEGF first differentiates mesoderm into pre-vein ECs, but subsequent VEGF/ERK inhibition generates vein ECs. VEGF activation is thought to be broadly essential for vascular development6,18, and thus our discovery that VEGF/ERK inhibition specifies vein identity has potential implications for understanding current therapies that either activate or inhibit VEGF signaling19,20.