MYOGENESIS SUBSEQUENT TO DENERVATION

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In vivo myogenesis occurs simultaneously with the development and maturation of the nervous system. Recently described techniques permit experimental manipulation of mouse fetuses without interfering with fetal viability and maturation. These techniques will be applied to laser ablate the spinal cords of young fetuses and to cut the sciatic nerves of older fetuses, permitting the evaluation of the effects of denervation (at various developmental periods) on the development and maturation of mammalian myofibers. Evaluation will be made with electron microscopy and morphometry. We also will evaluate the effect of fetal denervation on the expression of muscle specific genes encoding the various myosin and actin isoforms in mouse hindlimb muscles with in situ hybridization and with immunohistochemistry, using an immunohistochemical technique which will permit differentiation of isoforms present in primary versus secondary myotubes. The time course of expression of genes encoding the fetal and adult forms of creatine kinase and the genes coding for the various subunits of the acetylcholine receptor (both the fetal and adult receptors) in innervated hindlimb muscles will be determined with in situ hybridization. We then will evaluate the effects of denervation on the expression of these genes and the genes encoding the myogenic factors MyoD1 and myogenin. The proposed studies will allow us to assess the effect of environment on the innate genetic program of developing muscle: 1) It will permit assessment of cell interactions and of how the neuron may play a role in commitment, differentiation, or change the behavior of muscle cells. 2) I will permit determination of the extent to which nerve regulation is required for the maintenance of muscle phenotypes in the course of development. Information generated from the proposed studies will increase our "...understanding of the cellular and molecular processes involved in muscle development ..." and "... will be invaluable in the design and implementation of techniques to repair muscles that are damaged or nonfunctional due to genetic or environmental causes".