Peptide growth factors, such as members of the Transforming Growth Factor beta (TGFbeta) family, play central roles in morphogenesis and organogenesis. In this proposal, we will test the hypotheses that the Type I, Type II, and Type III TGFbeta receptors are developmentally regulated during cardiogenesis and play a functional role in the TGFbeta-induced epithelial-mesenchymal transformation that occurs in the atrioventricular (AV) cushion of the heart, a key event in cardiogenesis. To test these hypotheses we have cloned the Type II and Type III receptors and will clone the Type I receptor in order to characterize receptor expression by Northern analysis, RNAse protection assay, in situ hybridization, and immunohistochemistry to determine if the receptor mRNA or protein levels are regulated during cardiogenesis. In an in vitro model of AV cushion transformation, we will use antisense oligonucleotides specific for each receptor type to determine whether the loss of a specific receptor type affects the ability of either exogenously added TGFbeta or AV cushion myocardium to induce transformation. We will also develop dominant negative mutants for the Type II TGFbeta receptor. These mutants will be tested in a well-known model system, the mink lung cell. We will subclone cDNAs encoding dominant negative mutants into avian retroviruses and infect in vitro the AV cushion to score for the effect of these forms of the receptor on epithelial-mesenchymal transformation. We will use retroviral vectors to infect these same regions in ovo during embryogenesis and score the resulting phenotype. In addition, antisense constructs for the Type II receptor will be subcloned into retroviral vectors, tested for the ability to decrease mRNA and receptor protein in cultured cardiocytes, and used in studies of AV cushion transformation both in vitro and in ovo. The results of dominant negative and antisense experiments for the Type II receptor will be compared directly. Since growth factors such as TGFbeta not only contribute to embryogenesis, but also play a role in tissue repair and wound healing, an understanding of TGFbeta receptor function and regulation is essential to both determining the molecular basis of congenital cardiac abnormalities and cardiac function in the adult. Therefore, the information obtained from the proposed experiments will increase our understanding of basic cellular mechanisms of differentiation and transformation and may help to explain both inherited and acquired human cardiac abnormalities.
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