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Abstract

Vasculogenesis initiated by BMP4 was investigated using a zebrafish model. Both hematopoiesis and vasculogenesis arise from an embryonic blood-island where outer endothelial progenitors surround the inner hematopoietic precursors. We observed that transgenic zebrasfish embryos, which transiently expressed human BMP4, obtained an over-growth of blood-islands, in which whole-mount in-situ hybridizations (WISH) revealed strong and long lasting expression of zebrafish VEGF and elevated expression flk-1 (VEGF receptor 2), an endothelial cell marker in early vasculogenesis. It is well known that Smad1/5, the intracellular signaling partners of BMP4, can translocate with a co-Smad (Smad-4) from the cytoplasm to the nucleus, binding to the promoter of target genes and interacting with other transcriptional factors when BMP4 is applied to the cell. In the over-grown blood-island, the up-regulation of zebrafish smad1 expression was also demonstrated by WISH. These results suggest that BMP4 induced VEGF expression, and consequently the growth of endothelial cell in the hemangioblasts. To further dissect the signaling pathway from BMP4 to VEGF expression, we isolated a 1.2kb region of zebrafish VEGF promoter by genomic walking from the 5' end of VEGF cDNA (Genbank AF016244) and genomic library (Mobitech) screening. The biological functionality of the promoter was tested with the DsRed reporter (BD Bioscience) in zebrafish embryos. We synthesized a His₆-tagged DNA binding domain (MH1) of zebrafihs Smad1 and Smad5 in E.coli. The binding between Smad proteins and the DNA oligos with Smad binding elements (SBE) in the zebrafish VEGF promoter was demonstrated by electro-mobility shift assay (EMSA). We conclude that BMP4 not only is a morphogen and a cytokine during mesodermal development and dorsal-ventral patterning, but also functions in embryonic vasculogeneis by regulating vegf gene expression zebrafish.