Mechanisms of endothelial cell differentiation during vasculogenesis

About This Grant

PROJECT SUMMARY/ ABSTRACT Blood vessels are the earliest organs formed during embryonic development, and the embryonic cardiovas- cular system is required for nutrient exchange and organogenesis. As early as mouse embryonic day (E) 7.5 in the extraembryonic yolk sac blood islands and embryos, mesodermal precursors undergo the first cell fate de- cision to form the endothelial cell (EC) lineage. ECs further differentiate into arterial, venous, and lymphatic subtypes that are necessary to build the vascular system. However, the clonal relationships and spatial origins of EC subtypes that form the vasculature are poorly defined, and the mechanisms of EC specification and dif- ferentiation in vivo remain incompletely understood, posing a barrier to vascular regenerative medicine. Etv2, an ETS-family transcription factor, is a master regulator of EC specification. Etv2 is transiently ex- pressed in mesoderm progenitors in the yolk sac and developing mouse embryo from E7.5 - E9.5. Mice lacking Etv2 fail to develop blood or vasculature, and Etv2 is responsible for endothelial and hematopoietic lineage specification. Conversely, forced expression of Etv2 induces EC reprogramming. We have carefully interro- gated mechanisms by which ETV2 promotes EC specification in mesoderm progenitors derived from human induced pluripotent stem cells (iPSCs), using scRNA-seq, scATAC-seq, CUT&RUN, and functional CRISPR screens. Novel observations included: (1) the strength or timing of ETV2 expression influenced arteriovenous EC differentiation; (2) in addition to stimulating EC specification, ETV2 also suppressed specification of other mesodermal lineages; and (3) ETV2 suppression of alternative fates required its recruitment of the transcrip- tional repressor REST. These observations lead to our central hypothesis that ETV2 drives EC specification and arteriovenous differentiation, with REST cooperating with ETV2 to restrict alternative lineages during em- bryonic vasculogenesis. To track cell state transitions and fate of Etv2-expressing cells during vasculogenesis, we will integrate cut- ting edge single cell, spatial transcriptomics, and barcoded lineage tracing approaches to track the progeny of Etv2-expressing progenitors. To focus our efforts, in Aim 1 we will study the initial steps of vessel formation in the yolk sac. We will perform spatial and clonal analysis of Etv2-lineage cells during early yolk sac vasculogen- esis and late vascular plexus remodeling. To determine the role of Rest in Etv2-directed EC specification, in Aim 2 we will determine the effect of Rest inactivation on vasculogenesis and Etv2-lineage diversification, un- cover the Rest-Etv2 transcriptional regulatory network, and identify Rest-regulated genes and TFs in Etv2-line- age progenitors required for EC specification. The work will produce a high-resolution phylogenetic tree of vas- cular development originating from individual Etv2+ progenitors and bring new insights into the molecular mechanisms that direct EC subtype differentiation, informing future efforts in vascular regeneration.