Separation of Precursor Myogenic Early Limb Bud Mesenchyme by a and Chondrogenic Cells 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 antibodyinduced 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. The concept of cell lineages has provided a model for understanding cell diversification in developing somites and limb buds (1, 20, 28-36). According to this view, the generation of terminal phenotypes follows an invariant pattern such that the fate of each descendent cell is predictable from its ancestry or position in the lineage tree. Early development involves the rapid emergence of a succession of early founder cells for multipotential lineages (31). These in turn yield secondary, tertiary, etc. lineages that proliferate extensively yielding first bipotential and finally unipotential lineages. This concept contrasts with other views that hypothesize the existence of populations in somites and limb buds of uncommitted, undifferentiated, and multipotent cells that, under specific environmental conditions, can be induced to differentiate into muscle, cartilage, or connective tissue cells (2, 3, 7-10, 22, 25, 40, 43, 50-52, 54, 56, 59, 60, 62). There is much evidence supporting the lineage concept in the developing limb bud. In vitro cloning experiments first suggested this concept as a viable model. Single cells from early limb buds in the same culture dish yielded either chondrogenic-fibrogenic or myogenic-fibrogenic clones. Mixed clones of definitive chondrogenic and myogenic cells were never observed (19, 20, 28, 36). Subsequent in vivo experiments using quail-chick grafts demonstrated that cells from transplanted quaff somites migrated into the chick limb buds when the latter first form. Somite-derived cells (e.g., myotome) appear to be the source of all future striated muscle that eventually appears in the mature limb, whereas all future cartilage and connective tissue cells appear to be derived from the somatopleure (13, 58). The lineage hypothesis predicts that one should be physically able to separate and fractionate mesenchymal precursors THE JOURNAL OF CELL BIOLOGY • VOLUME 99 DECEMBER 1984 1856-1866 1 8 5 6 © The Rockefeller University Press . 0021-9525/84112/1856111 $1.00 on A uust 8, 2017 jcb.rress.org D ow nladed fom from the early limb into distinct subpopulations that, upon further differentiation, will express their unique predetermined phenotypes. In the present work, we fractionate the cell population from early chick limb buds into the major subpopulationswone containing predominantly precursor myogenic cells and the other precursor chondrogenic cells. This separation is based on the property of a monoclonal antibody, called CSAT, that has been shown to detach myogenic cells from extracellular matrices (17, 37, 38, 45). The basis for this separation appears to be a difference in the adhesion between these two subpopulations leading to a preferential rounding and detachment of the cells in the myogenic lineage by the CSAT antibody. These findings suggest that the ongoing differentiation program and phenotypic options of precursor limb bud cells are a function of their position in their respective lineages rather than the consequence of inductive interactions resulting from their position in the limb