A complex set of developmental events transform early primordia within endocardial cushions into the atrioventricular (AV) valves and septa of the mature heart. Endocardial cushions are composed of an inner endocardial layer, and outer myocardial layer, and an acellular extracellular matrix rich layer separating these two layers. A subset of endocardial cells within cushions transform into mesenchymal cells that migrate into the extracellular matrix and differentiate into mature valve and septum tissue. This cell transformation process coupled with asymmetrical cell proliferation within endocardial cushions remodels simple early endocardial cushions into elongated valves. Investigation into molecular mechanisms involved in valve and septum morphogenesis centered on a family of growth factors, called Wnts, due to previous reports showing involvement of this growth factor family in similar developmental processes. Expression analysis of Wnt growth factors, Wnt receptors, and soluble Wnt inhibitors was performed in search of Wnt signaling components with restricted expression in AV endocardial cushions. Initial RT-PCR analysis demonstrated expression of several Wnts (Wnt-5a, Wnt-6, Wnt-9a, Wnt-10a and Wnt-11), Wnt receptors (Fz-1, Fz-3, Fz-4, and Fz-6) and the soluble Wnt antagonist Frzb in early endocardial cushions. Spatial and temporal expression analysis revealed restricted endocardial cushion expression of Wnt-9a and Frzb. Wnt-9a is expressed in the endocardial cell layer while Frzb is expressed in the endocardium and transformed mesenchyme of AV endocardial cushions. Overexpression of Wnt-9a in endocardial cushions increases cell proliferation resulting in enlarged hypercellular endocardial cushions. Overexpression of a truncated mutant form of Wnt-9a, which acts in a dominant negative manner, increases programmed cell death in endocardial cushions. Overexpression of Wnt-9a also induces beta-catenin responsive transcription in AV canals consistent with Wnt-9a signaling in a canonical Wnt/beta-catenin pathway. Wnt-9a induced increases in cell proliferation are inhibited by overexpression of Frzb. Spatial expression patterns suggest that Frzb delineates a zone of Wnt-9a induced cell proliferation near the endocardial cell layer to promote endocardial cushion outgrowth and remodeling into mature valve leaflets.
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