Mechanisms of Delta-like 4 Endocytosis and Notch Activation During Blood Vessel Development - SUMMARY Endothelial cells (ECs) are the cell type responsible for the bulk of embryonic blood vessel formation, eventually leading to an estimated 100,000 miles of vasculature by adulthood. During development, new blood vessels emerge from pre-existing vasculature, a process termed angiogenesis. Notch signaling is fundamental to angiogenesis and adult blood vessel homeostasis. In the absence of Notch, blood vessels demonstrate a chronic sprouting phenotype marked by unchecked proliferation and overgrowth; this evidence and others overwhelmingly show Notch is required for blood vessel maturation and stability. When ligand-bound, the Notch intracellular domain is cleaved and translocates to the nucleus, acting as a transcription factor. Delta-like ligand 4 (DLL4) is a potent Notch ligand that binds to the extracellular domain of Notch. To elicit Notch activation the ectodomain of Notch requires a pulling force by DLL4 to expose a S2 cleavage site. Once exposed, the Notch extracellular domain is cleaved, allowing for release of the Notch intracellular domain (NICD) stimulating signaling activation. Very little is known about the post-transcriptional regulation of DLL4, and even less is understood about the mechanisms by which DLL4 exerts a sustained pulling force to activate Notch-mediated lateral inhibition. Our preliminary data describes, for the first time, how DLL4 endocytosis and anchoring to the actin cytoskeleton generates the mechanical force required to expose the S2 site of Notch. Specifically, this proposal will focus on two largely uncharacterized proteins we believe are central to force- generating DLL4 endocytosis, Eps15 homology domain binding protein 1 (EHBP1) and EH domain containing protein 2 (EHD2). Our broad hypothesis is that EHBP1 anchors EHD2 to f-actin filaments, aiding in the transendocytosis of DLL4 bound to Notch. In Aim1 we will detail how both EHBP1 and EHD2 work in combination to facilitate DLL4 endocytosis, and in doing so, mediate Notch signaling. In Aim2, we will comprehensively demonstrate that EHBP1 and EDH2 works in combination to regulate DLL4 endocytosis and downstream Notch activity during zebrafish blood vessel morphogenesis using live-imaging and CRISPR- based mutant generation. Overall, this proposal will answer several critically important questions pertaining to blood vessel development as well as provide a powerful training opportunity for undergraduate scholars.
