Myogenesis is the result of a complex interplay among multiple regulatory factors which determines where, when, and how far differentiation may proceed. Molecular regulatory differences will be detected in two cell lines which have different myogenic capacities and resemble myogenic cells from different locations and developmental stages in vivo. C2C12s form multinucleated myotubes, exit the cell cycle irreversibly, and express a broad array of muscle-specific genes at high levels. BC3H1s remain mononucleated, differentiate reversibly, fail to express some muscle specific genes and underexpress others, such as the skeletal muscle actin gene. C2C12s came from skeletal muscle and are mesodermal. BC3H1 cells came from a cranial tumor; the contribution of lineage to BC3H1 myogenic capacity is unclear. C2C12s express all four myogenesis determination genes; BC3H1s express only two. Initial work will establish baselines of differentiation: the % of nuclei which are expressing myogenin in differentiated cultures of BC3H1 and C2C12 will be determined by immunocytochemistry and the level of accumulation of myogenin mRNA relative to the level of accumulation of skeletal muscle actin mRNA will be determined and compared between lines. Skeletal muscle actin mRNA stability will be compared in the two lines using pulse-chase and pharmacological methods. MEF2s and the members of the thyroid hormone receptor family are important influences in myogenesis. MEF2 gene expression in the two lines will be compared by Northern and RT-PCR analysis and binding activity will be compared by gel shift analysis using probes from the skeletal muscle actin gene promoter. Proteins bound to DNA will be further characterized. The effect of specific MEF2 isoforms on BC3H1 myogenic capacity and the effect of forced BC3H1 terminal differentiation on MEF2 expression will be addressed with gene transfer studies. The hypothesis that in BC3H1 cells retinoic acid will inhibit rather than enhance myogenesis will be tested. The lineage of BC3H1 cells will be characterized with assays for Mhox and beta-tubulin Ill, mesodermal and neural markers, respectively. If appropriate, the effect of added Mhox expression on BC3H1 myogenic capacity will be tested. Results of this work will aid in understanding the balance of factors required for successful myogenesis and will thus contribute to the understanding of problems of abnormal development or loss of the differentiated state.

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