Endothelial cell signaling pathways in cardiovascular development: Vascular/neural connections.

摘要

My doctoral dissertation involves studying the growth and development of the vasculature and centers around two projects involving novel endothelial cell signaling mechanisms. I begin by analyzing the role of the Nf1 gene in cardiovascular development. The cardiac phenotype of homozygous null Nf1 embryos is complex and the structures in the affected hearts are derived from multiple cell types. Using Cre/loxP technology, I ablated Nf1 function in myocardium, cardiac neural crest, and endothelium. Endothelial-specific Nf1 inactivation recapitulated the complete cardiac phenotype seen in null embryos. I found elevated ras/MAPK signaling and ectopic activation of NFATc1. A subset of the endothelial-specific Nf1 knockout mice survived embryogenesis and developed a leukemia resembling the type children with NF1 are predisposed to. I continued to investigate endothelial cell signaling pathways and explored the hypothesis: mechanisms that guide axons in the nervous system may have been co-opted to function independently in endothelial cells to pattern the vasculature. I identified a novel Plexin gene that we called plexinD1. In the nervous system, Plexins are receptors for the Semaphorin family of axon guidance molecules. The sequence of plexinD1 was highly similar to other Plexin family members, however its expression pattern was restricted to endothelium. To explore the in vivo role of plexinD1, I generated a mouse knockout. plexinD1-/- mice survived embryogenesis and were born, but all became cyanotic and died within 24 hours of birth and all had congenital heart defects. The phenotype was remarkably similar to sema3C-/- and to endothelial-restricted Neuropilin-1 knockouts, suggesting a possible connection in a common signaling pathway. Consistent with this, I was able to immunoprecipitate a complex containing PlexinD1 and Npn1. Moreover, in an in vitro cell-based system, this interaction enhanced Sema3C binding to Npn1. I identified a zebrafish plexinD1 ortholog. Intriguingly, inhibiting PlexinD1 function in zebrafish results in dramatic vascular patterning defects, consistent with a role for PlexinD1 in blood vessel guidance. Together, my studies show essential roles for endothelial cells in heart development and suggest that there exist similar patterning mechanisms between nerves and blood vessels.