The central goal of the proposed research is to illucidate the molecular events involved in the sequential cellular decisions that lead from the multipotential mesodermal precursor cells to the differentiated skeletal muscle. The experimental focus is on two biologically interrelated problems. I) Significance of the four member MRF family. In most in vitro biochemical assays and tissue culture transfections assays, the four MyoD family members are remarkably similar in function but their individual patterns of expression during development are significantly different from each other. Moreover, recent experiments in the investigator's lab (MRF4 knockout) and others have shown that individual disruption of each produces a different phenotype. What remains entirely unclear is whether distinct functions uncovered in vivo are intrinsic to individual family member proteins or are dependent instead on the timing and amount of expression. The investigator will address this problem directly by generating selected transplacements of one MRF family member for another: 1) MyoD transplaced into the myogenin locus and into the MRF4 locus with concomitant elimination of myogenin coding sequences. This tests whether the apparent uniqueness of myogenin for muscle differentiation can be taken over by the erstwhile dispensable determination member MyoD. 2) Myogenin transplace into the myf5 locus with concomitant deletion of myf5 coding sequences. From this they will learn whether expression of myogenin at the earliest times leads to premature differentiation or acts instead to complement the deficiency of myf5. II) Defining pre-MRF steps in embryonic myogenic lineages. It is now clear that expression of even the earliest MyoD family member is a relatively late event in the myogenic pathways in vivo. Therefore a major goal is to define the molecular and cellular basis for the steps that lead to the activation of the MRFs. The new approach proposed here begins with developing a system for nondestructively identifying, tracing, enriching and even isolating and culturing precursors cells of interest from the embryo. The investigator has chosen to begin with Pax3. Its expression marks precursors of axial and limb myogenic cells over an extended period prior to the onset of expression of myf5 , including cells that emigrate from the somite to found the limb musculature. Marking will be done by transplacing a vital marker and a selectable marker into the Pax-3 locus. This will permit the investigator to further define of the precursor cell population, assay and ultimately identify signals that induce or suppress their transition to myoblasts.
