PROJECT SUMMARY Endothelial heterogeneity is a defining characteristic of the mature circulatory system. Endothelial cells that line capillary beds or other small caliber vessels have distinct phenotypes and molecular signatures that relate to their various functions in different anatomical locations. In larger vessels, there are clear molecular and phenotypic differences between arterial and venous endothelial cells. In all of these cases, endothelial differentiation is essential for normal physiological function of the circulatory system. Importantly, endothelial heterogeneity can have a major influence on the site and severity of vascular disease. Thus, a better understanding of how endothelial cell types are determined is highly relevant. For the past 15 years, we have used the zebrafish as a model system to investigate basic mechanisms of vascular morphogenesis and patterning during embryonic development. Our efforts have revealed new insights into how blood vessels are formed and underscore the importance of endothelial differentiation in this process. Importantly, we have found that endothelial cell differentiation is a primary step that is essential for blood vessel formation and assembly. However, the developmental origins of endothelial identities and the signaling pathways that drive differentiation are largely unknown. In the studies proposed here, we will apply a number of traditional developmental biology approaches coupled with cutting-edge molecular techniques to define the hierarchy of endothelial ontogeny during embryonic development. Through Cre/lox lineage tracing we will identify where and when endothelial cell types are established. In parallel, we will apply single cell RNA sequencing on endothelial progenitors at multiple developmental stages to identify transcriptome signatures that define endothelial subtypes. At the same time, efforts to identify enhancer elements flanking subtype-specific genes will contribute to our knowledge of transcriptional regulatory pathways and upstream signals that drive differentiation. Finally, we will continue to investigate the link between endothelial differentiation and vascular morphogenesis through functional interrogation of subtype specific genes using knockout zebrafish models generated through genome editing. Together, our efforts will define the developmental endothelial hierarchy and allow us to identify essential signaling pathways responsible for endothelial heterogeneity.