One of the first recognizable steps during embryonic muscle development is the initiation of desmin expression in replicating presumptive myoblasts proceeding the expression of the myogenic regulator myoD and possibly myogenin. Terminal differentiation of skeletal muscle is associated with down regulation of vimentin and up regulation of desmin. We do not know the biological function of these two immediate filament proteins during these processes. The goal of the proposal is to use different approaches to study the function of desmin and vimentin during early and terminal muscle differentiation. Our data so far has linked overexpression of vimentin and inhibited differentiation. C2 myoblasts transfected with oncogenic ras genes fail to appropriately down regulate vimentin and up regulate desmin. We wish to address the association of this phenomenon with the inability of these cells to differentiate. The following lines of investigation are employed: The recently isolated and fully or partially characterized mouse desmin an vimentin cDNAs and gene will be used to manipulate the expression of vimentin and desmin during in vitro and in vivo myogenesis. Specifically, constructs designed to express a) high levels of vimentin or desmin at the wrong stages during myogenesis, b) high levels of antisense RNA that would inhibit mRMA accumulation and translation, or c) high levels of truncated polypeptides that will disrupt normal filament assembly are introduced into both myogenic cells and transgenic mice. This will allow us to study: a) any possible dependence of the expression of vimentin and desmin upon each other; b) any effect of these proteins on the expression or function of other muscle specific genes; and c) the functional consequences of such forced or inhibited expression on muscle differentiation. Alternatively, gene targeting constructs will be made to be used in homologous recombination experiments designed to disrupt the desmin gene and consequently study directly its functional role during early and late muscle development. For this purpose, further characterization of this mouse desmin gene will be performed and its precise expression during embryogenesis will be assessed by in situ hybridization. We also want to address the significance of the similarities that vimentin and desmin have recently shown to share with some growth (fos, jun) and myogenic (myoD, myogenin) transcription regulators, respectively, by investigating: a) possible association with these molecules, b) involvement in their transport, and c) modulation of their function.
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