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 /Ag/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.An apparent correlation between cell shape and differentiation in a number of different experimental systems has supported the hypothesis that cell shape and the cytoskeleton may affect the expression of certain genes (8,14,17) . In the cases examined so far, the actin cytoskeleton undergoes reorganization during cell shape changes that are coupled to altered gene expression . For example, cultured corneal epithelial cells respond to certain matrix molecules by reorganizing their basal surface and doubling their production of collagen (34,35). Reorganization of the basal cytoplasm involves the formation of an orderly meshwork of actin bundles. Spiegelman and his associates (31,32,33) have found that when certain sublines of 3T3 cells undergo adipogenesis, the acquisition of a spherical cell shape and loss of actin filaments are prerequisites to initiation of biosynthesis of lipogenic enzymes . Other systems in which altered gene expression appears linked to reorganization of the actin cytoskeleton include the production of procollagenase by cultured fibroblasts (1) and cAMP-dependent hormone-induced

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