MOLECULAR BASIS OF MYOGENIC RESPONSE IN DEVELOPMENT

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The proposed experiments are a continuation of our studies designed to understand the mechanisms responsible for establishment of myogenic cell lineage and the development and differentiation of muscle progenitor cells. While the major impetus in understanding of the skeletal myogenic regulation was brought by the discovery of the activator myogenic regulatory factors (MRFs), MyoD, myf-5, myogenin, MRF-4, it is now becoming clear that other mechanisms, wherein the myogenic regulation can be attributed to the synergy between various classes of activators and inhibitors of transcription, are involved in crucial steps of myogenic programming. Using the chicken embryos, which offer a highly useful experimental system for functional analysis of developmentally important genes, we have isolated and cloned a regulatory protein (Nished) in chicken skeletal muscle whose expression in early embryos appears to be a prerequisite for accurate programming of skeletal myogenesis. This notion received further experimental support by the definitive results in our laboratory that the myogenic gene markers, desmin, myosin and myogenin, are activated in fibroblasts 10T + stably transfected with Nished along with the acquisition of distinct morphological characteristics of the muscle cell. The facility with this experimental paradigm determines the immediate future direction of further studies, proposed herein, which is to carry out a rigorously controlled assessment whether the activation of the skeletal muscle genes is linked with the Nished mediated derepression of any known myogenic regulatory network. The proposal will test the hypothesis that Nished has a direct consequence on the expression of the endogenous network of responsive genes and that alterations in the physiological level of Nished in embryonic tissues will have an impact on development of the muscle tissue phenotype within the whole animal context. Prediction of the precise function of Nished will be done by a variety of approaches including controlled ectopic expression and gene targeting experiments. Our specific aims are to: (i) undertake a full spectrum study to test the structure-function correlates to be explored in gain-in-function type experiments, (ii) establish the functional significance of Nished expression in vivo by identification of the endogenous network of Nished-responsive gene network and (iii) determine the physiological role of Nished in transgenic mice and via targeted ablation of selected loci in mice. The outcome of this study is expected to lend significant insights into the mechanism(s) responsible for establishment and maintenance of skeletal muscle phenotype.