In situ hybridization and immunohistochemistry of versican, aggrecan and link protein, and histochemistry of hyaluronan in the developing mouse limb bud cartilage.

We investigated the expression pattern of versican, aggrecan, link protein and hyaluronan in the developing limb bud cartilage of the fetal mouse using in situ hybridization and/or immunohistochemistry. Versican mRNA and immunostaining were detected in the mesenchymal cell condensation of the future digital bone at E13. Versican mRNA expression rapidly disappeared from the tibial cartilage, as cartilage formation progressed during E13-15, but the immunostaining was gradually replaced by aggrecan immunostaining from the diaphysis. Immunostaining for both molecules thus had a 'nega-posi' pattern and consequently versican immunostaining was still detected at the epiphyseal end at E15. This result indicated that versican functions as a temporary framework in newly formed cartilage matrix. An aggrecan-positive region within the cartilage invariably had intense hyaluronan staining, whereas a versican-positive region also had affinity for hyaluronan within the cartilage, but not in the mesenchymal cell condensation. Therefore, the presence of versican aggregates was not confirmed in the developing limb bud cartilage. Furthermore, although link protein was more closely related with aggrecan than versican during limb bud cartilage formation, there was a discrepancy between the expression of aggrecan and link protein in tibial cartilage at E15. In particular, only a link protein-positive region was present in the marginal area of the metaphysis and the epiphysis at this stage. This finding may indicate a novel role for link protein.