This application represents a revised version of a competing renewal of a project focusing on the role of the Nf1 gene product neurofibromin during cardiac development. In humans, NF1 is mutated in patient with von Recklinghausen Neurofibromatosis, a disease characterized by benign and malignant tumors of neural crest origin. Nf1 knockout mice display enlarged endocardial cushions and succumb during mid-gestation with evidence of cardiovascular impairment. The cardiac defects include pulmonic stenosis, double outlet right ventricle and thinned myocardium, defects that are seen in other mouse and chick models of cardiac neural crest-related congenital heart disease. However, our recent studies demonstrate that neural crest migration and patterning in the heart is normal in Nf1-deficient embryos. Furthermore, inactivation of Nf1 in neural crest (using three distinct Cre mice) does not reproduce embryonic lethality or the cardiac phenotype of null embryos. Rather, these mice are born and display hyperplasia of numerous neural crest-derived structures including peripheral ganglia and the adrenal gland. Surprisingly, inactivation of Nf1 in endothelial cells using Tie2-Cre recapitulates the embryonic cardiac defects seen in Nf1 null embryos. These results establish a novel and unexpected role for neurofibromin in endothelium. Moreover, we have observed elevated levels of activated MAPK in endothelium of mutant embryos consistent with a role for Nf1 in down-regulating Ras activity. We also demonstrate enhanced nuclear localization of NFATc1 (a factor known to be required for endocardial cushion formation) in Nf1-null endocardium. Here, we propose to test the hypothesis that Nf1 deficiency in endothelial cells results in activation of Ras. We will examine downstream effectors of Ras signaling in endothelium. We will also examine the mechanism by which Ras activation modulates subsequent nuclear localization of NFATc1 and potentially Smads, and we will test the hypothesis that nuclear localization of NFATc1 is required for enhanced epithelial-mesenchymal transformation in the endocardial cushions of Nf1-deficient mice using both genetic and biochemical approaches. We will examine the function of Nf1 in adult endothelial cells and in adult and embryonic smooth muscle with the use if inducible tissue-specific Cre mice.
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