Separation of precursor myogenic and chondrogenic cells in early limb bud mesenchyme by a monoclonal antibody

We have addressed the problem of the segregation of cell lineages during the development of cartilage and muscle in the chick limb bud. The following experiments demonstrate that early limb buds consist of at least two independent subpopulations of committed precursor cells--those in (a) the myogenic and (b) the chondrogenic lineage--which can be physically separated. Cells obtained from stage 20, 21, and 22 limb buds were cultured for 5 h in the presence of a monoclonal antibody that was originally isolated for its ability to detach preferentially myogenic cells from extracellular matrices. The detached limb bud cells were collected and replated in normal medium. Within 2 d nearly all of the replated cells had differentiated into myoblasts and myotubes; no chondroblasts differentiated in these cultures. In contrast, the original adherent population that remained after the antibody-induced detachment of the myogenic cells differentiated largely into cartilage and was devoid of muscle. Rearing the antibody-detached cells (i.e., replicating myogenic precursors and postmitotic myoblasts) in medium known to promote chondrogenesis did not induce these cells to chondrify. Conversely, rearing the attached precursor cells (i.e., chondrogenic precursors) in medium known to promote myogenesis did not induce these cells to undergo myogenesis. The definitive mononucleated myoblasts and multinucleated myotubes were identified by muscle-specific antibodies against light meromyosin or desmin, whereas the definitive chondroblasts were identified by a monoclonal antibody against the keratan sulfate chains of the cartilage-specific sulfated proteoglycan. These findings are interpreted as supporting the lineage hypothesis in which the differentiation program of a cell is determined by means of transit through compartments of a lineage.