This project addresses several basic questions of skeletal muscle biology: (a.) how do mesodermal cells become determined to enter the skeletal muscle cell lineage? (b.) how are muscle satellite cells derived? (c.) how are skeletal muscle genes activated in response to growth factor deprivation? (d.) how are quantitative modulations in gene expression controlled with respect to fast and slow muscle fiber types? The specific aims are: [1.] Identification of M-creatine kinase (MCK) gene control elements. Major emphasis will be on a 206 nt enhancer which contains multiple control and factor-binding elements. Related studies will continue analysis of other regulatory regions, and will search for locus control regions. Complementary transgenic studies will evaluate what appear to be the more critical control elements in adult and embryonic mice. [2.] Analysis of muscle fiber type control of MCK expression. Preliminary results demonstrating differential expression of 3 MCK transgenes in fast and slow muscle fibers will be pursued in vitro using fast and slow muscle cell lines. Control elements defined in cell culture assays will be confirmed in the fully functional muscles of additional transgenic mice. [3.] Isolation and functional characterization of factors that bind MCK gene control elements. Factor binding analyses will continue with elements identified in Aims 1 & 2. Factors identified via cDNA library screening will be analyzed for their behavior during terminal differentiation, for their expression during development, and for their presence in fast and slow fiber types. Later studies will investigate the control of selected transcription factor genes. [4.] Analysis of skeletal muscle determination. Cellular and molecular environments required for activating myogenic determination genes in mesodermal cells will be studied in culture. [5.] Mechanism of satellite cell formation. Time-lapse videomicroscopy coupled with single cell assays for muscle gene activation, growth factor receptors and myogenic determination factors will be used to determine how myoblasts enter a quiescent satellite cell state. Results from this project should be applicable to skeletal muscle diseases, gene therapy, and the surgical reconstruction of injured muscle tissue.
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