Endothelial tip/stalk cell selection requires BMP9-induced β_IV-spectrin expression during sprouting angiogenesis

β_IV-Spectrin is a membrane cytoskeletal protein with specialized roles in the nervous system and heart. Recent evidence also indicates a fundamental role for β_IV-spectrin in angiogenesis as its endothelial-specific gene deletion in mice enhances embryonic lethality due to hypervascularization and hemorrhagic defects. During early vascular sprouting, β_IVspectrin is believed to inhibit tip cell sprouting in favor of the stalk cell phenotype by mediating VEGFR2 internalization and degradation. Despite these essential roles, mechanisms governing β_IV-spectrin expression remain unknown. Here we identify bone morphogenetic protein 9 (BMP9) as a major inducer of β_IV-spectrin gene expression in the vascular system. We show that BMP9 signals through the ALK1/Smad1 pathway to induce β_IV-spectrin expression, which then recruits CaMKII to the cell membrane to induce phosphorylation-dependent VEGFR2 turnover. Although BMP9 signaling promotes stalk cell behavior through activation of hallmark stalk cell genes ID-1/3 and Hes-1 and Notch signaling cross-talk, we find that β_IV-spectrin acts upstream of these pathways as loss of β_IV-spectrin in neonate mice leads to retinal hypervascularization due to excessive VEGFR2 levels, increased tip cell populations, and strong Notch inhibition irrespective of BMP9 treatment. These findings demonstrate β_IV-spectrin as a BMP9 gene target critical for tip/stalk cell selection during nascent vessel sprouting.