During myogenesis, reductions in trophic factor availability signal most myoblasts to fuse, up-regulate the expression of muscle-specific genes and form myotubes. Those cells failing to differentiate into myotubes initiate apoptosis and rapidly die. At present, the signal transduction molecules that determine if myoblasts should differentiate or die are largely unknown. In order to analyze the molecular mechanisms that mediate programmed cell death during development, we have cloned death-associated genes from the intersegmental muscles (ISMs) of the moth Manduca sexta. One of these genes encodes Death-Associated LIM-Only Protein (DALP). Forced expression of DALP in Drosophila results in skeletal muscle atrophy. Ectopic expression of DALP, or its mammalian paralog Hic-5, blocks differentiation and induces apoptosis in mouse C2Ct2 myoblasts. Both of these effects can be overcome by contact with normal myoblasts or by ectopic expression of the muscle-specific transcription factor MyoD. Hic-5 expression is specifically and dramatically induced in normal myoblasts that die following removal of trophic support. Taken together, these data suggest DALP and Hic-5 act upstream of MyoD and function as phylogenetically-conserved """"""""switches"""""""" to block muscle differentiation and induce death. Hic-5 shares high sequence identity with the focal adhesion protein paxillin. In the first aim of the proposal, they will test the hypothesis that Hic-5 acts in myoblasts to block focal adhesion kinase-dependent phosphorylation of paxillin. In the second aim, they use site-directed mutagenesis to identify essential motifs within Hic-5 and to generate possible dominant-negative regulators. In the third aim, they will test the hypothesis that blockade of the Hic-5 pathway enhances the survival of transplanted myoblasts in vivo. Lastly, they will target the expression of wild-type and dominant-negative (or antisense) Hic-5 to the muscle lineage of transgenic mice in order to determine its role in myogenesis. They hope to exploit Hic-5 in order to: 1) understand how myoblasts makes the decision to differentiate or die, and 2) develop strategies to enhance the utility of myoblast based transplantation strategies.
Showing the most recent 10 out of 30 publications
