Increased cell diameter precedes chondrocyte terminal differentiation, whereas cell-matrix attachment complex proteins appear constant.

BACKGROUND

Chondrocytes in specific areas of chick sterna have different developmental fates. Cephalic chondrocytes become hypertrophic and secrete type X collagen into the extracellular matrix, whereas middle and caudal chondrocytes remain cartilagenous throughout development, continuing to secrete collagen types II, IX, and XI. In this report, we ask if the cell size and cytoarchitecture of chondrocytes differ in cephalic, middle, and caudal portions of whole sterna prior to and during hypertrophy. In addition, what is the distribution of integrin subunits and actin associate proteins in differentiating chondrocytes?

METHODS

Phalloidin was used to stain filamentous actin, and immunohistochemistry was used to localize the distribution of collagen molecules, integrin receptor subunits, and actin-associated proteins.

RESULTS

Chondrocytes stained for filamentous actin demonstrated that on day 14 cephalic chondrocytes had a significantly larger diameter than middle and caudal chondrocytes. Day 17 chondrocytes in nonhypertrophic cephalic and middle regions of sterna were significantly smaller than hypertrophic chondrocytes and significantly larger than caudal chondrocytes. In contrast to day 14 chondrocytes, day 17 chondrocytes in the hypertrophic region demonstrated similar diameters at all cartilagenous depths. The beta 1 integrin subunit appeared punctate and associated with cell membranes, allowing nonpolarized interactions with extracellular matrix molecules. The distribution of alpha integrin subunits was similar to the beta 1 integrin subunit, although alpha integrin subunits also appeared cytoplasmic. Actin-associated proteins, vinculin, and alpha-actinin, were associated with F-actin, but vinculin was more specifically localized to the ends of the actin filaments. Focal adhesion kinase was diffusely distributed throughout the cytoplasm but also demonstrated areas of colocalization with vinculin. Zyxin and paxillin demonstrated a punctate distribution, although paxillin was slightly more diffuse. Using immunohistochemical detection, no difference in integrin subunit or actin associated protein distribution could be determined between chondrocytes and hypertrophic chondrocytes.

CONCLUSIONS

The increased chondrocyte diameter observed in cephalic regions of sterna on day 14 suggests that intracellular changes may precede the specific hypertrophic marker, type X collagen, by several days. In addition, the presence of integrin subunits, which are known to interact with collagen and cytoskeletal proteins, suggests that communication may exist between chondrocytes and their extracellular matrix via these receptor molecules.