The proposed studies are intended to increase our understanding of the mechanisms underlying muscle differentiation. We previously demonstrated the existence of diffusible trans-acting activator(s) of muscle genes in studies involving heterokaryons, stable somatic cell hybrids formed between mouse muscle and human nonmuscle cells. Our goal is to characterize the macromolecule(s) responsible for muscle gene activation. Toward this end we will obtain nuclear and cytoplasmic subfractions from mouse muscle cultures at different stages of differentiation and microinject these cellular components into human nonmuscle cells. The activation of a specific human muscle gene coding for a cell surface antigen present throughout myogenesis will be monitored at the single cell level. Eventually requirements for the expression of five temporally distinct markers of human muscle differentiation will be examined. In this manner we hope to characterize the species of cytoplasmic macromolecule(s) responsible for muscle gene activation and whether the same macromolecules regulate the expression of muscle functions specific to different stages of differentiation.