The vascular endothelial growth factor-A (VEGF-A, or VEOF) and its receptors play critical roles in controlling blood vessel growth and function, such as embryonic vascularization and tumor angiogenesis. VEGF receptor-i (FIt-i) and VEGF receptor-2 (Flk-1) are both transmembrane tyrosine kinases, but their signaling mechanisms in the context of developing embryos are unclear. While a null inactivation of the fit-i gene led to severe vascular defects and early embryonic lethality, a limited disruption of its kinase domain did not lead to vascular defects. Hiratsuka et al. proposed that Fit-i was a mere VEGF binding protein whose role was to limit VEGF accessibility to FIk-1, and that in the fit-i null mutants, it was an increase in Flk-1 signaling that led to the vascular defects. However, null mutant phenotypes of fit-i and fik-1 indicate that Fit-i and Fik-1 do not play opposite roles. The primary defect in fit-1-/- null mutants is the excessive hemangioblast commitment, but in flk-1 null mutants, the initial commitment to hemangioblasts still occurs. To address these inconsistencies, we have proposed four specific aims. In particular, the level of Fik-1 signaling will be manipulated to see if an up-regulation of the Fik-1 activity will result in a fit-i-/- -like mutant phenotype, and if a down-regulation of the Flk-1 activity in fit-1-/- background will minimize flt-1-/- phenotype. Modulation of Flk-1 activity will be achieved in several ways: by breeding fit-i knockout mice with mice of reduced VEGF activity and by knock-in of flk-1 cDNAs encoding a constitutively activated FIk-1 mutant and FIk-1 mutants with reduced kinase activities. The requirement for Fit-i kinase signaling will be more thoroughly examined by a complete deletion of the entire genomic sequence encoding for the Fit-i kinase domain. These experiments will provide a definitive answer to the question whether Fit-i is an active signaling molecule or a passive VEGF binding protein. These experiments may also provide us with extended opportunities to address several important issues of VEGF receptor mediated signaling, in particular the role of Flk-1 signaling in regulating endothelial cell proliferation and/or survival in vivo. Together, the above experiments will facilitate us to achieve our long term goal in understanding the mechanisms of VEGF-mediated siarialino in vascular development.
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