Direct visualization of renal vascular morphogenesis in Flk1 heterozygous mutant mice

Flk1, a receptor tyrosine kinase for vascular endothelial growth factor (VEGF), is the earliest known marker for endothelial precursors (angioblasts). We examined heterozygous mice in which the Flk1 gene was partially replaced by a promoter-less LacZ insert and used beta-galactosidase histochemistry to view cells transcribing Flk1. In day 10 (E10) embryos, a Flk1-positive network surrounded the metanephric blastema, and, at E11, a vessel entered the metanephros from its ventral aspect alongside the ingrowing ureteric bud. However, aortic branches did not engage embryonic kidneys at these time points. In newborns, beta-galactosidase was localized exclusively and intensely to endothelial cells of all vessels and glomeruli. In contrast, when E12 kidneys grown in organ culture for 6 days were examined, only scattered Flk1-positive cells were seen, glomeruli were unlabeled, and vessels were absent. When organ-cultured kidneys were then grafted into wild-type anterior eye chambers, numerous Flk1-positive endothelial cells in vessels and glomeruli were found, all stemming from the graft. Image analysis showed that grafts with the most abundant glomerulo- and tubulogenesis were also those with the richest expression of Flk1. We conclude that 1) kidney microvessels precede renal artery development, 2) angioblast differentiation is arrested in organ culture but released on grafting when vasculogenesis resumes, and 3) nephrogenesis and microvessel assembly are tightly coupled in vivo.