SUMMARY Blood vessels carry oxygen and nutrients and are vital to organismic viability and continued homeostasis. Angiogenesis, or the formation of new blood vessels from pre-existing ones, is the predominant developmental process by which blood vessel network density is regulated. During angiogenic development, endothelial cells create a hollow cavity called a lumen, providing a continuous conduit for blood to reach distant tissues. The mechanisms underpinning the morphodynamic changes in endothelial architecture and signaling leading to vascular lumen formation, or tubulogenesis, are incompletely understood. In this proposal we will investigate a protein called synaptotagmin-like protein 2 (sytl2) that we believe is responsible for defining the luminal surface by directing protein transport to the apical membrane during blood vessel development. Our preliminary data suggests that sytl2 defines the apical membrane and tethers Rab GTPase proteins for delivery of vesicular cargo, such as podocalyxin. In aim 1, we will characterize the role of sytl2a during vascular lumen formation in developing zebrafish embryos using a combination of live-imaging and CRISPR-based mutant generation. In aim 2, we will comprehensively demonstrate that sylt2 works in combination with the GTPase Rab35 to deliver podocalyxin to the apical plasma membrane during lumenogenesis in vitro. In aim 3, we will further characterized how sytl2a interacts with Rab35 to deliver Podocalyxin using generation of new zebrafish reporter lines and compound mutants in vivo. How blood vessel lumen formation is regulated is still a major question in the field, this proposal will provide novel insight into critical mechanisms orchestrating this process.