Characterization of the in vivo function of Neuropilin1 during development

Neuropilin1 (Npn1) is a transmembrane receptor that is critical for development of both the nervous and vascular systems. It is a ligand for both the chemorepulsive Semaphorin3s and for vascular endothelial growth factor (VEGF), which is a protein critical for proper development, particularly for angiogenesis. Npn1 knockout mice die during early development due to cardiovascular abnormalities, and mice lacking Npn1 in endothelial cells (ECs) die perinatally with similar cardiovascular deficits. Because of the known importance of VEGF in cardiovascular development, it had been thought that the VEGF-Npn1 interaction was responsible for the premature death seen in Npn1 mutants. We identified one amino acid residue (D320) in the b1 domain of Npn1 that is necessary for VEGF-Npn1 binding. By mutating this site, we eliminated VEGF-Npn1 binding in vitro. We then made a knock-in mouse containing the D320K mutation, thus creating a mouse with no VEGF-Npn1 binding (Npn1VEGF-mouse). Surprisingly, the Npn1VEGF-mutant mouse does not have the premature death or vascular phenotype seen in the Npn1-null. In particular, it does not recapitulate the decreased vascular density or decreased endothelial cell number seen in the Npn1 null. This indicates that the vascular phenotype seen in the Npn1 null is not a result of VEGF-Npn1 binding, and instead implicates that the phenotype is the result of the interaction of Npn1 with its VEGF co-receptor VEGFR2.