Tenascin-C is associated with early stages of chondrogenesis by chick mandibular ectomesenchymal cells in vivo and in vitro

Abstract

Tenascin-C is an extracellular matrix protein thought to be involved in skeletogenesis. We have examined the distribution of tenascin-C in the developing chick mandibular arch between stages 18-36, and during in vitro chondrogenesis of mandibular ectomesenchymal cells in micromass cultures using a probe and antibody that correspond to the portion of the tenascin-C transcript conserved in all of the three known chick splice variants. In situ hybridization and immunohistochemical analyses demonstrate that tenascin-C is predominantly expressed in the condensing mesenchyme of developing cartilage, and in the perichondrium of differentiated cartilage. Tenascin-C expression, although detected in differentiating chondroblasts, was not detected in differentiated cartilage. Tenascin-C was also expressed in the developing membranous bones. In addition, the expression of tenascin-C transcripts during in vitro chondrogenesis of mandibular ectomesenchymal cells in micromass cultures was compared to the patterns of expression of aggrecan core protein and alpha 1(I) collagen transcripts. Our in situ hybridization analyses of micromass cultures demonstrate the expression of tenascin-C and aggrecan core protein mRNAs by pre-chondrogenic aggregates in the 1-day cultures and by chondroblasts in differentiating cartilage nodules in 2-day cultures. In 4- and 9-day cultures, the pattern of expression of tenascin-C mRNA was different from the patterns of expression of aggrecan core protein mRNA, and appeared to be more closely related to the expression of alpha 1(I) collagen mRNA. Aggrecan core protein mRNA was expressed by chondrocytes in cartilage nodules in 4- and 9-day cultures. On the other hand, tenascin-C and alpha 1(I) collagen mRNAs, in addition to being expressed in the loose connective tissues in the inter-nodular spaces, were predominantly expressed by the elongated, flattened, and fibroblast-like cells around the cartilage nodules. These results indicate that during the in vitro chondrogenesis of mandibular ectomesenchymal cells, expression of tenascin-C mRNA identifies chondrocytes in their early stages of differentiation. The patterns of expression of tenascin-C mRNA in 4- and 9-day cultures further suggest that tenascin-C is expressed in the perichondrium-like structures that form around the cartilage nodules in micromass cultures. Therefore, our in vitro studies, in agreement with our in vivo studies, suggest an association of tenascin-C with the initial or early stages of chondrogenesis in the chicken mandibular arch.