The goals are to: 1. establish the cell lineages of cardiac myocytes in the chick embryo; 2. explore changes of cell polarity of the presumptive myocytes which accompany the transition from an epithelial to a muscle organization; 3. define the function of cytoskeleton and intercellular junctions during myofibrillogenesis; 4. establish the posttranslational steps in protein assembly which regulate sarcomere assembly. Lineage will be followed in clonal cell cultures of the blastodisc from pregastrula, 5-8 hr-old chick embryos. Beating colonies of cardiac muscle differentiate in such cultures from epithelioid cells of the blastodisc after a limited number of, probably no more than 4, divisions. Using antibodies to distigunish cell-types in these cultures, in combination with 3H-TdR, we will define the mitotic history of the myocytes, and relate the appearance or disappearance of muscle- or nonmuscle-specific proteins to stages of this lineage. We will attempt to """"""""immortalize"""""""" cells of the cardiac lineage with transforming viruses, e.g. temperature-sensitive Rous sarcoma virus. Lineages of cells distined for subcompartments (e.g. atrium vs. ventricle) of the heart will be followed in ovo, in organ cultures and clonal cells cultures using McAbs to the myosin heavy chain which delineate cells of the atrium, ventricle and conduction systems. Cell polarity of the presumptive myocytes will be investigated in organ cultures of the heart using influenza and vesicular stomatitis viruses which bud respectively from the apical or basolateral surfaces of epithelia. We will localize the Na-K ATPase (an epithelial basolateral marker) using immunocytochemistry. Alterations in these indices of polarity will be followed during myocardial differentiation. Myofibril assembly will be exaimined by: 1. immunocytochemistry at both light and electron microscopic levels with a panel of antibody probes specific for nonmuscle and muscle contractile proteins, components of the cytoskeleton, proteins of intercellular junctions, and elements of the centrosphere; 2. microinjection of selected antibodies or anti-sense recombinant probes into cultured myoctyes to disrupt myofibrillogenesis and the living cells followed with image enchancement and video recording techniques; 3. following the effects of the phorbol ester, TPA, on myocyte mitosis and fibrillogenesis; 4. posttranslational steps of contractile protein insertion into the sarcomere will be explored in a cell-free system recently developed in the P.I.'s laboratory.
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