Gene expression in wild-type and MyoD-null satellite cells: regulation of activation, proliferation, and myogenesis

Regeneration is the process of renewal or repair of damaged cells and tissue. In skeletal muscle, regeneration is accomplished by satellite cells, which are rare, mononucleate, mitotically quiescent myogenic precursor cells normally present in undamaged muscle tissue. When stimulated by injury, overuse, or disease, satellite cells will become activated to proliferate and form a pool of replacement myoblasts which will differentiate to replace necrotic muscle fibers. These cells may also have the quality of self-renewal associated with stem cells. Due mainly to technical difficulties caused by their rarity, difficulty of isolation, and lack of identifying markers, satellite cells have not been as well studied as other myogenic cells. Here I present work in which I establish a reliable means of isolating and culturing mouse satellite cells resident on single explanted myofibers; a molecular marker for satellite cells which also yields information about their mechanism of activation, and a method of multiplex single-cell RT-PCR which allows simultaneous detection of six genes from a single satellite cell. Using these techniques, I have determined the temporal coexpression pattern of the four myogenic regulatory factors (MRFs) in single activated satellite cells over the first four days of a regeneration response in vitro. I have also assayed satellite cell cDNA pools for expression of genes important in regulating myogenesis, cell cycling, and cell fate decisions in other myogenic lineages. Finally, I have performed these analyses on MyoD-null satellite cells, which are differentiation-deficient in vivo, and present possible mechanisms for this based on gene expression; this analysis also suggested a potential marker for activated satellite cells which will return to the reserve satellite cell population and may act as myogenic stem cells