Analysis of the myogenic lineage in chick embryos

Abstract. 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 2ⁿ 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.