During embryonic development, cells become committed to distinct cell lineages and differentiate to form the diverse types of cells that make up the animal. A striking example of cell commitment and diversification occurs during skeletal muscle formation. Myogenesis in the post-implantation mouse embryo proceeds through three stages. Cells first become committed to myogenesis and form muscle fibers in the somites. Commitment to myogenesis appears to require interaction with neural tissue and expression of the MyoD1 gene product. Myogenic cells subsequently migrate from the somites and form the primary (embryonic) and secondary (fetal) generations of muscle fibers in the developing trunk and limbs. By birth, the newly formed fibers are innervated and have diversified into distinct fiber types that express only fast, both fast and slow, or only slow myosin heavy chain isoforms. Proper development of these different muscle fiber types is crucial for muscle function, as patients with diseases characterized by poor differentiation of fiber types have highly impaired muscle function. The long-term goal of this laboratory is to understand the genetic mechanisms underlying the formation of these different types of muscle fibers. Multiple types of myoblasts, as well as multiple types of muscle fibers, are found at the different stages of myogenesis in the mouse. We propose that the anatomically and cytologically diverse types of muscle fibers are initially formed from the diverse types of myoblasts. The proposed experiments address questions about the genetic and cellular mechanisms underlying the commitment of myoblasts to diverse myogenic lineages and about the cell-specific expression of fast and slow myosin heavy chain isoforms. The experiments have three specific aims. (i) The role of the MyoD1 gene product in different types of myogenic cells will be analyzed by determining if myogenic cells committed to different lineages express MyoD1 in similar or different patterns and if the myogenic program of a cell can be changed by altering the levels of MyoD1 expression. (ii) Neural tissue regulation of myogenic commitment will be analyzed by determining which types of myoblast are formed in neural tissue/somite co-cultures and which cell types in neural tissue (neurons, glia, fibroblasts) can replace whole neural tissue explants in co-cultures to support myogenic differentiation of early somitic cells. (iii) Cell-specific expression of the two slow isoforms of myosin heavy chain will be analyzed by determining which somitic, embryonic, and fetal muscle cells express each isoform and which transcriptional and translational regulatory mechanisms underlay cell-specific expression. Methods will include clonal cell culture, neural tissue/somite co-culture, purification of cell types from neural tissue, immunocytochemistry, Western blotting, quantitative Northern blotting, and RNase protection assays.
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