Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskeleton

Abstract

Cell shape is known to influence the chondrogenic differentiation of cultured limb bud mesenchyme cells (Solursh, M., T. F. Linsenmayer, and K. L. Jensen, 1982, Dev. Biol., 94: 259-264). To test whether specific cytoskeletal components mediate this influence of cell shape, we examined different cytoskeleton disrupting agents for their ability to affect chondrogenesis. Limb bud cells cultured at subconfluent densities on plastic substrata normally become flattened, contain numerous cytoplasmic microtubules and actin bundles, and do not undergo spontaneous chondrogenesis. If such cultures are treated with 2 micrograms/ml cytochalasin D during the initial 3-24 h in culture, the cells round up, lose their actin cables, and undergo chondrogenesis, as indicated by the production of immunologically detectable type II collagen and a pericellular Alcian blue staining matrix. Cytochalasin D also induces cartilage formation by high-density cultures of proximal limb bud cells, which normally become blocked in a protodifferentiated state. In addition, cytochalasin D was found to reverse the normal inhibition by fibronectin of chondrogenesis by proximal limb bud cells cultured in hydrated collagen gels. Agents that disrupt microtubules have no apparent effect on the shape or chondrogenic differentiation of limb bud mesenchymal cells. These results suggest an involvement of the actin cytoskeleton in controlling cell shape and chondrogenic differentiation of limb bud mesenchyme. Interactions of the actin cytoskeleton and extracellular matrix components may provide a regulatory mechanism for mesenchyme cell differentiation into cartilage or fibrous connective tissue in the developing limb.