To understand the processes of cardiac pattern formation and cellular differentiation at the molecular level, the investigators wish to study the molecular properties of Cripto, a gene that encodes a secreted growth factor-like molecule that is distantly related to epidermal growth factor (EGF). In their analysis of Cript expression during mouse embryogenesis, the investigators have discovered that Cripto is specifically expressed by early cardiac progenitors in the anterior lateral plate mesoderm. During morphogenesis of the primitive heart tube, Cripto expression becomes localized to myocardial cells of the outflow tract, and is one of the first markers for regional specification of the hearts tube. In further studies, the investigators have found that putative CRIPTO receptor are expressed by embryonic myocardial cells and by primary cardiac myocytes in culture. Because Cripto is related to a Xenopus gene that can activate fibroblast growth factor (FGF) receptors, the investigators are studying whether Cripto may also act through the FGF receptor signaling pathway, which has been previously implicated in cardiac development. Taken together, these observations suggest that Cripto may represent an important signaling factor during early cardiac differentiation and pattern formation. Therefore, the investigators will pursue four specific aims that will examine the role of Cripto in murine cardiogenesis: I) Analysis of Cripto mRNA and protein expression and of CRIPTO receptor distribution by in situ hybridization, immunohistochemistry, and CRIPTO binding, in order to provide insights into potential sites of Cripto function. II) Analysis of CRIPTO protein function by expression and purification of active recombinant CRIPTO protein, by examination of its effects on FGF receptor signaling, and by assaying its effects on primary cardiac myocytes, in order to elucidate the earliest essential biological requirements for Cripto activity. IV) Analysis of regulatory regions of the Cripto promoter by identification of sequences that direct appropriate reporter gene expression in differentiating ES cells and in transgenic mouse embryos, in order to initiate studies on the regulation of cardiac-specific gene expression.
