Normal heart morphogenesis requires the controlled migration of several populations of cells including endocardial cushion mesenchymal cells, epicardial cells and cells of neural crest origin. Fibulin-1 is an extracellular matrix (ECM) protein, whose expression coincides spatially and temporally with growth and migratory events occurring in the endocardial cushions and epicardium as well as other sites of epithelial- mesenchymal transformation including neural crest. Our recent findings indicate that fibulin-1 is a potent inhibitor of cells motility promoted fibronectin, including that of cardiac cushion mesenchymal cells in vitro. A novel hypothesis that will be addressed by experiments proposed in this application is that fibulin-1 functions to regulate morphogenic cell movements in the developing heart by suppressing the motility-promoting activity of fibronectin and perhaps other ECM components of the endocardial cushion, myocardium and epicardium. The first part of the study will focus on characterizing the effects of fibulin-1 on mortality of the major cell types involved with valvuloseptal morphogenesis using in vitro assays that measure motility and haptotactic migration of the endocardial, myocardial, epicardial and neural crest cells. The second part of the study will evaluate the consequence of heart morphogenesis of experimental in vivo modulation of fibulin-1 expression in the epicardium, myocardium and endocardium. This will involve embryonic heart microinjection of fibulin-1 and fibulin-1 antibodies as well as transgenic approaches designed to augment fibulin-1 expression in specific embryonic heart tissues. The transgenic approaches will include heart injection of fibulin 1-expressing adenovirus and tissue-specific expression of fibulin-1 under the control of endocardial and myocardial- specific gene promoters. The third part of the study will explore the underlying mechanisms for the suppressive effects of fibulin-1 on cell motility by identifying the cell surface receptor(s) that mediates its effect on cell motility and mapping the region(s) within fibulin 1 responsible for suppressing cell motility. Experiments proposed in this later aim will also seek to characterize the major intracellular signaling pathways that fibulin-1 modulates with emphasis on the regulation of the actomyosin complex.
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