A central problem in developmental biology is to understand the regulation of cell fate determination. In vertebrate mesoderm, some multipotential precursor cells will become restricted to the myogenic lineage. The resulting unipotential cells are now myoblasts, a proliferating population of precursors that will become muscle upon differentiation, but that do not yet express the repertoire of specialized gene products characteristic of muscle. Upon differentiation, myoblasts cease proliferation and become myocytes. In skeletal muscle they express the full range of muscle specific gene products and fuse to form multinucleate myotubes. The goal of the proposed work is to gain a molecular level understanding of these determination and differentiation events that define the myogenic lineage. The experimental approach centers on a family of four closely related genes, MyoD, myogenin, myf-5 and herculin. Each of these is capable of initiating the myogenic determination step in some otherwise nonmyogenic cells. They also appear to play a central role in executing muscle differentiation by binding to specific cis-acting DNA sequences found in many muscle specific genes. The regulatory activities of these molecules differ in important ways in the undifferentiated myoblast environment and in the differentiated myocyte environment, as they interact with different sets of target genes. The questions to be answered by the experiments proposed are: How are the myogenic regulators themselves regulated? To begin to answer this question, our experiments will focus on regulation of herculin using transfection assays, in vivo footprinting and in vitro protein:DNA binding assays. When and where is herculin expressed in the developing animal? Knowing the temporal and spatial pattern of expression for these regulators is important for understanding their roles in determination and differentiation in the animal. This information will allow us to constrain and interpret data from cell culture studies and will provide the intellectual framework for testing the effects of ectopic expression of herculin in transgenic mice. How do tissue specific enhancers work? Studies of several muscle specific enhancers have raised an important set of questions about how they are activated during differentiation, and how that activation differs in muscle cell types expressing different subsets of the MyoD family, or in the case of cardiac muscle, none of the known members of this family. We will use the well-studied creatine kinase (MCK) E1 enhancer to address these questions. The experiments will include use of a new in vivo footprinting technology which permits us to compare the activity of the enhancer with occupancy of specific binding sites in the cell in various myoblasts and myocytes. These results are further compared with in vitro studies of protein:DNA interactions at the same target sites.
