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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

This study utilizes immunofluorescence to describe the distribution of several extracellular matrix molecules in the chick embryo during the process of limb outgrowth and the formation of precartilage condensations. A large chondroitin sulfate proteoglycan (PG-M) is detected at the wing level at Hamburger and Hamilton stage 14 in and under the dorsal ectoderm, and is associated with the basement membranes around the neural tube, notochord and pronephros, but not with other basement membranes. The galactose-specific lectin, peanut agglutinin (PNA), has a similar distribution except that it also binds to the dorsal side of the neural tube. PG-M is not detected in the limb mesenchyme until after stage 17, when it is present in the distal region, as is PNA-binding material. With further development of the wing bud, PG-M is present in the subectodermal mesenchyme, the mesenchyme at the distal tip and in the prechondrogenic core. After stage 22 PNA-binding material becomes localized in the prechondrogenic core, the basement membranes under the apical ectodermal ridge, and the ventral sulcus. The distribution of these components (PG-M and PNA binding material) overlaps, but differs from that of type I collagen and fibronectin and basement membrane components, such as laminin, basement membrane heparan sulfate proteoglycan, and type IV collagen. Tenascin, on the other hand, is not detected in the limb bud until stage 25, after the appearance of cartilage matrix components such as type II collagen and cartilage proteoglycan (PG-H). These results are considered in relation to the formation of precartilage aggregates, and indicate that PNA binds to components in precartilage aggregates other than PG-M or tenascin.

Phenotypic responses of rat calvarial osteoblast-like cells (RCOB) were evaluated on commercially pure titanium (cpTi) surfaces when cultured at high density (5100 cells/mm2). These surfaces were prepared to three different clinically relevant surface preparations (1-micron, 600-grit, and 50-microns-grit sand-blast), followed by sterilization with either ultraviolet light, ethylene oxide, argon plasma-cleaning, or routine clinical autoclaving. Osteocalcin and alkaline phosphatase, but not collagen expression, were significantly affected by surface roughness when these surfaces were altered by argon plasma-cleaning. In general, plasma-cleaned cpTi surfaces demonstrated an inverse relationship between surface roughness and phenotypic markers for a bone-like response. On a per-cell basis, levels of the bone-specific protein, osteocalcin, and the enzymatic activity of alkaline phosphatase were highest on the smooth 1-micron polished surface and lowest on the roughest surfaces for the plasma-cleaned cpTi. Detectable bone cell expression can be altered by clinically relevant surfaces prepared by standard dental implant preparation techniques.

The ultrastructural changes associated with primary mesenchyme cell formation in the sea UrchinLytechinuspictus were studied by transmission (TEM) and scanning electron microscopy (SEM). Early during ingression, one can recognize about 30 presumptive primary mesenchyme cells, which form a ring around eight central cells, located at the apex of the vegetal pole. The first steps involve the loss of the basal lamina in the vegetal plate region and the loss of cilia from the 30 presumptive primary mesenchyme cells. These cells then elongate into bottle-shaped cells which have a bulbous, appendage-free basal end that protrudes into the blastocoel, and a thin neck region that forms numerous apical cell processes. Longitudinally oriented microtubules are observed in the peripheral cytoplasm in the neck region of the bottle-shaped cells and in the adjacent cytoplasm of closely adherent neighboring non-mesenchymal blastomeres. After the disappearance of apical junctions, the neck regions of the bottle-shaped cells shorten, resulting in the transposition of the cells into the blastocoel as rounded primary mesenchyme. Mesenchyme cell displacement results in the temporary formation of fenestrae in the blastular wall, which are filled in by apical cell processes that extend from neighboring blastomeres. These morphological changes have several features in common with other examples of cells which exit from epithelia and are consistent with a morphological sequence involving the relaxation of the basal cytoplasm of the presumptive primary mesenchyme cells, followed by their active elongation and lateral compression by adjacent cells. PRIMARY MESENCHYME CELL INGRESSION IN SEA URCHIN