Zebrafish scube1 and scube2 cooperate in promoting Vegfa signaling during embryonic vascularization.

AIMS The secreted and membrane-anchored SCUBE (signal peptide-CUB-EGF domain-containing proteins) gene family composed of 3 members was originally identified from endothelial cells (ECs). We recently showed that membrane SCUBE2 binds vascular endothelial growth factor A (VEGFA) and acts as a co-receptor for VEGF receptor 2 (VEGFR2) to modulate EC migration, proliferation and tube formation during postnatal and tumor angiogenesis. However, whether these SCUBE genes cooperate in modulating VEGF signaling during embryonic vascular development remains unknown. METHODS AND RESULTS To further dissect the genetic interactions of these scube genes, transcription activator-like effector nuclease-mediated genome editing was used to generate knockout (KO) alleles of each scube gene. No overt vascular phenotypes were seen in any single scube KO mutants because of compensation by other scube genes during zebrafish development. However, scube1 and scube2 double KO (DKO) severely impaired EC filopodia extensions, migration, and proliferation, thus disrupting proper vascular lumen formation during vasculogenesis and angiogenesis as well as development of the organ-specific intestinal vasculature. Further genetic, biochemical, and molecular analyses revealed that Scube1 and Scube2 might act cooperatively at the cell-surface receptor level to facilitate Vegfa signaling during zebrafish embryonic vascularization. CONCLUSIONS We showed for the first time that cooperation between scube1 and scube2 is critical for proper regulation of angiogenic cell behaviors and formation of functional vessels during zebrafish embryonic development. TRANSLATIONAL PERSPECTIVE Our studies indicate that targeting SCUBE1 and/or SCUBE2 on modulating VEGF signaling might provide potential therapeutic treatments or VEGF-mediated proliferative pathological vascular diseases.