Malformations of the heart occur at the rate of 7 to 10 cases/1,000 live births making congenital heart disease the most frequent form of birth defect. Despite impressive advances in diagnosis and surgical treatment, there is as yet no understanding of the molecular basis of these malformations. Many are undoubtedly the result of errors in cell migration or cell-cell interactions that occur during early heart development. A family of cell surface receptors known as the integrins has been shown to mediate cellular interactions with one another and with the extracellular matrix. Interference with the function of these receptors results in aberrant behavior of cells during early morphogenesis. We hypothesize that integrins are important determinants of early heart morphogenesis. We further postulate that they are programmatically expressed during heart development such that different groups of integrins are expressed at different times and locations during cardiac morphogenesis and that morphogenesis is at least partially dependent not only on integrin expression, but upon the expression of the right integrin in the right place and time. We will test these hypotheses using the mouse embryo. To accomplish this we will first develop cDNA probes, polyclonal antibodies and monoclonal antibodies specific for the alpha subunits of the major integrins expressed in the mouse embryo. This will be accomplished by assembling complete mouse cDNA constructs from mouse libraries, expressing them in the baculovirus system and using the purified proteins as immunogens for antibody production. Such reagents do not, for the most part, exist for mouse integrins. Second, we will monitor the program of integrin expression during early mouse development 1) by reverse transcriptase- PCR amplification of endogenous integrin mRNA. In situ hybridization will be used to detect the cellular source of the amplified message; and 2) by immunohistochemistry using the monoclonal polyclonal antibodies prepared against mouse integrin alpha subunits. Third, we will determine the functional significance of integrin expression in the developing heart of cultured mouse embryos by 1) disrupting early morphogenesis with biologically active anti-integrin antibodies; 2) blocking integrin synthesis in early myocardial or endocardial cells using retrovirus vectors carrying antisense constructs specific for mouse integrin alpha subunits; and 3) perturbing normal cellular interactions by inducing inappropriate integrin expression using defective retroviruses carrying complete integrin alpha subunit cDNA's. Upon completion of this work, we will have determined precisely the importance of integrins to various stages of heart development. As these experiments will be evaluated histologically, we will know the role of integrins in the formation of the myocardium, the endocardium, primitive heart tube and early A-V valves. We will have tested the hypothesis that there is a precise program of integrin expression in the developing heart and determined at what point the adult pattern of integrin expression is established. Finally, we will have determined the functional importance of both integrin expression and the program of integrin expression during mammalian heart development.
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