MyoD(-/-) satellite cells in single-fiber culture are differentiation defective and MRF4 deficient

MyoD-deficient mice are without obvious deleterious muscle phenotype during embryogenesis and fetal development, and adults in the laboratory hare gro ssly normal skeletal muscle and Life span. However, a previous study showed that in the context of muscle degeneration on a mdx (dystrophin null) gene tic background, animals lacking MyoD have a greatly intensified disease phe notype leading to lethality not otherwise seen in mds mice. Here we have ex amined MyoD(-/-) adult muscle fibers and their associated satellite cells i n single myofiber cultures and describe major phenotypic differences found at the tissue, cellular, and molecular levels. The steady-state number of s atellite cells on freshly isolated MyoD(-/-) fibers was elevated and abnorm al branched fiber morphologies were observed, the latter suggesting chronic muscle regeneration in vivo. Single-cell RNA coexpression analyses were pe rformed for c-met, m-cadherin, and the four myogenic regulatory factors (MR Fs.) Most mutant satellite cells entered the cell cycle and upregulated exp ression of myf5, both characteristic early steps in satellite cell maturati on. However, they later failed to normally upregulate MRF4, displayed a maj or deficit in m-cadherin expression, and showed a significant diminution in myogenin-positive status compared with wildtype. MyoD(-/-) satellite cells formed unusual aggregate structures, failed to fuse efficiently, and showe d greater than 90% reduction in differentiation efficiency relative to wild type. A further survey of RNAs encoding regulators of growth and differenti ation, cell cycle progression, and cell signaling revealed similar or ident ical expression profiles for most genes as well as several noteworthy diffe rences. Among these, GDF8 and Msx1 were identified as potentially important regulators of the quiescent state whose expression profile differs between mutant and wildtype. Considered together, these data suggest that activate d MyoD(-/-) satellite cells assume a phenotype that resembles in some ways a developmentally "stalled" cell compared to wildtype. However, the MyoD(-/ -) cells are not merely developmentally immature, as they also display nove l molecular and cellular characteristics that differ from any observed in w ild-type muscle precursor counterparts of any stage. (C) 2000 Academic Pres s.