In vitro development of precursor cells in the myogenic lineage

Expression of the muscle-specific integral membrane protein H36 and the intermediate filament protein desmin, detected by immunofluorescence, was used to identify cells at distinct stages in the skeletal myogenic lineage. These proteins were coordinately expressed in cultures of rat hindlimb myoblasts from 17- and 19-day fetuses and newborn pups, and in satellite cells from juveniles. Both H36+ and desmin+ cells were present in cultures from 13.5- and 15-day embryonic hindlimbs, but desmin expression was more prevalent: H36-/desmin+ myoblasts predominate during this early stage of development. H36 was not detected in Day 12 embryo hindlimb bud cells in vivo nor in cultures soon after plating. Initially, only 1% of the Day 12 limb bud cells expressed desmin. When these cells were serially passaged every 3-4 days, cells with all three possible myogenic phenotypes developed: that is, H36+/desmin-, H36+/desmin+, and H36-/desmin+ cells. There was a progressive increase in the frequency of H36+ cells, with 75% of cells positive by passage 6 (Day 27 in vitro). The maximum frequency of cells that expressed desmin occurred in passage 5 (Day 23 in vitro). These results demonstrate that precursors to the cells that express H36 and desmin are present in the 12-day embryo hindlimb bud and that the transition from H36-/desmin- precursors to cells with a myogenic phenotype can occur in vitro. MyoD1 and myogenin were not detected in these cells, suggesting that the initial expression of H36 and desmin in the myogenic lineage may precede and/or is independent of these regulatory proteins. The conversion of precursor cells in the 12-day limb bud to a more advanced stage of development serves to define additional cells in the myogenic lineage. The ability to monitor in vitro these stages of development affords the opportunity to study how they are regulated.