Analysis of the myogenic lineage in chick embryos. III. Quantitative evidence for discrete compartments of precursor cells

Probabilistic and programmed lineage models for the generation of terminally differentiated skeletal muscle cells were tested in a clonal culture assay. Myogenic cells from the breast muscles of 10-day chick embryos were plated at an initial density of 250-1000 cells per 60 mm dish. Well-isolated individual cells were marked with a ring on the underside of the dishes, and clones arising from only these cells were followed. The presence of post-mitotic myoblasts in clones was assayed by peroxidase-antiperoxidase (PAP) and fluorescence immunocytochemical staining for both M-type creatine kinase (MCK) and skeletal muscle myosin heavy chain (MHC). Clones were fixed at intervals up to 76 h and were scored for the number of cells per clone and the number of MCK + and MHC + cells per clone. Quantitative and kinetic data were obtained indicating that post-mitotic myoblasts occurred overwhelmingly in homogeneous clones (all cells MCK + and MHC +) which contained 2" cells (n = 0, 1, 2, 3, 4). This result does not support either probabilistic models of myogenesis or the existence of 'proliferative' mitoses at the end stages of differentiation. Rather, it indicates that myogenic precursor cells are a heterogeneous population, within which individual cells are predetermined to undergo a set number of symmetrical mitoses prior to yielding terminally differentiated progeny. These findings are strong evidence for a programmed, cell cycle-dependent lineage in the end stages of muscle differentiation.