This project is directed at two interrelated problems central to early cardiac development in vertebrate embryos: 1. The identification of cell lineages and developmental signals which regulate differentiation of cardiogenic precursor cells at pre-gastrula and gastrula stages of chick embryogenesis. 2. The assembly of striated myofibrils in the earliest myocytes that can be identified in the heart-forming region of chick embryos (stages 4-10). Lineage analysis will utilize a new replication- defective, retroviral vector (CXL), which contains beta-gal as a reporter gene. Derived from the spleen necrosis virus, it exhibits high levels of infectivity and beta-gal expression in virtually all cells of the chick embryo without altering normal development. After infection at stages 4 and 5 of development, embryos will be fixed at different ages, stained with X-gal, and serial sections used for computer-assisted, 3-dimensional image analysis. In other experiments, we will introduce genes such as myoDl. myogenin, ski, myc or ras into cardiac precursors to alter the differentiation of cardiac myocytes. The studies of myofibrillogenesis will focus on two sets of experiments: a) the role of sub-sarcolemmal adhesion plagues (SAPs) in sarcomere formation, and b) the roles of myosin-associated proteins (C-protein, 86 kD protein and titin) in both A-band alignment and linkage with I-Z-I complexes. The first project will involve analysis of SAPs in cultured myocytes by reflectance and DIC microscopy to record and measure the distance between SAPs which anchor myofibrils to the plasmalemma as the attached myofibrils elongate by sarcomere insertion. Also, myocytes will be microinjected with fluorescent antibodies to alpha-actinin, titin, vinculin or desmin, or with plasmids encoding truncated forms of these proteins to alter in vivo functions. Living cells will be visualized under video-enhanced fluorescence microscopy to follow myofibril assembly. The last set of experiments involve analysis of cDNAs encoding cardiac isoforms of C-protein and 86 kD protein, the skeletal muscle forms of which have been cloned and sequenced in our laboratory. These proteins contain type III fibronectin and C2 immunoglobulin features characteristic of the N-CAM family. We will isolate and sequence the cardiac cDNAs, express the proteins or selected domains in E. Coli, identify binding domains which interact with myosin or titin, and prepare mutant cDNAs for intracellular myocyte expression studies. These studies will contribute to our understanding of human cardiac malformations during early development.
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