Skeletal muscle offers unique advantages for investigation of fundamental molecular genetic mechanisms of cell lineage determination and differentiation in the vertebrate embryo. A family of four myogenic regulatory genes, including myoD, have been shown to function as transcription factors regulating different processes in a pathway for skeletal muscle differentiation. Recent advances in the developmental analysis of gene expression and function in avian embryos, combined with technical advances in cDNA cloning methods, now make possible rapid progress toward defining genes that control the expression of myoD and the determination of skeletal muscle lineages in the somitic mesoderm of avian embryos. Two complementary experimental approaches will be pursued. First, promoter and enhancer regulatory sequences that control the expression of quail myoD (QmyoD) in myoblasts and somite myogenic lineages will be identified by mutagenesis, DNA binding, and expression studies. These findings will provide a basis for identification of transcription factors that regulate QmyoD by molecular and genetic screens of E. coli and yeast expression libraries of somite and myoblast cDNAs with target QmyoD regulatory sequences as probes. Second, genes that regulate somite myogenesis will be identified in molecular screens for genes differentially expressed in segmental plate mesoderm, somites and myoblasts, using PCR-based differential cDNA cloning methods. The developmental functions of cloned genes will be investigated by DNA sequence analysis to define their protein sequences; by in situ hybridization, RT-PCR and immunological techniques to determine their precise developmental timing and lineage specificity of expression in somites; by antisense inhibition of gene expression using phosphorothiolate-modified oligonucleotides to define essential functions in embryos; and by ectopic gene expression in somites and fibroblasts using plasmid and RCAS retroviral expression vectors to test their dominant myogenic regulatory activities. In these studies, we expect to identify novel genes involved in cellular signaling, transcriptional control of QmyoD, autoregulation and the establishment of determined myogenic lineages, and coordination of the initiation of differentiation with cell cycle control. These findings will be a basis for testing specific molecular models of cell lineage determination in the vertebrate embryo and for the development of gene and cellular therapies to treat human muscle wasting diseases, muscle injury, and muscle cancers.
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