Genetic and chemical analysis of vascular development in zebrafish.

摘要

The development of the vascular system requires cooperative contributions of multiple signaling and metabolic pathways and transcription factors. To investigate the molecular mechanisms regulating blood vessel formation, I analyzed the regulatory elements of the kdrl gene and conducted genetic and chemical screens. From these approaches, I discovered new molecules and pathways controlling vascular development in zebrafish embryos. The kdrl gene is essential for vascular development, however, the regulatory mechanisms of kdrl gene expression are not fully understood. By biochemical and genetic approaches, I showed that FoxH1, a transcription factor mediating TGFβ signaling, directly binds to the kdrl upstream regulatory region and functions as a negative modulator of kdrl expression in a Smad2-dependent manner. To examine vascular development in vivo, we used the kdrl promoter to generate a transgenic fish, Tg(kdrl:GFP)la116. Using this animal model, I performed genetic and chemical screens to identify genes and pathways important for vascular development. From the forward genetic screen, I found that embryos deficient in the sizzled and mind bomb genes exhibit defects in vascular development. Phenotypic analysis revealed that sizzled mutant embryos develop an expanded caudal vein due to the upregulation of BMP signaling and mind bomb mutants develop a defective axial vessel and caudal vein because of reduced Notch signaling. Finally, from the chemical screen, I discovered a novel small molecule that can affect arteriovenous angiogenesis differently. The development of intersegmental vessels (ISVs) has been well characterized and used as in vivo model for arterial angiogenesis. In this study, I characterized the development of caudal vein plexus (CVP) and established it as a new in vivo model for venous angiogenesis. I performed a chemical screen using the ISVs and CVP as indicators for arterial and venous angiogenesis and discovered a small molecule, aplexone, which inhibits venous angiogenesis preferentially. Microarray analysis and molecular and cellular approaches revealed that aplexone reduces protein geranylgeranylation by affecting the HMGCR pathway, and results in reduced endothelial cell migration leading to the inhibition of angiogenesis. Overall, my study has provided a new in vivo model system and expanded our understanding of the molecular mechanisms underlying vascular development in zebrafish.