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

Abstract

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.

Chondroitin Sulfate N-Acetylgalactosaminyltransferase-1 Plays a Critical Role in Chondroitin Sulfate Synthesis in Cartilage

“…During differentiation, chondrocytes at the prehypertrophic stage show the highest level of aggrecan synthesis (30). Since the CS chains are attached to the core protein of aggrecan in cartilage, the expression of the key enzymes required for CS biosynthesis is probably up-regulated, correlating with the expression of aggrecan core protein during chondrocyte differentiation.…”

Isolation and chondroinduction of a dermis‐isolated, aggrecan‐sensitive subpopulation with high chondrogenic potential

“…Aggrecan has been found to play an essential role in the chondrogenesis process and the subsequent maintenance of the chondrocyte phenotype in vivo (33,34) and in vitro (35). Based on these observations, it was thus of interest in the present study to observe the effects of ACS on DIAS cell chondroinduction in terms of cell morphology, ECM gene expression and production, and cytoskeleton organization.…”

Positional changes in tendon insertions from bone to fascia: development of the pes anserinus and semimembranosus muscle insertion in human foetuses

“…For immunohistochemistry, the sections were pretreated with testicular hyaluronidase (25 mg/mL; Sigma type I-S; Sigma Chemicals, St. Louis, MO, USA) in phosphate-buffered saline for 30 min at 37°C to identify the two matrix substances [19]. The sections were subsequently incubated with mouse monoclonal anti-versican core protein antibody (12C5; Developmental Studies Hybridoma Bank), diluted 1:25; rabbit polyclonal anti-rat tenascin-c antibody (Chemicon, Temecula, CA, USA), diluted 1:100; and mouse monoclonal anti-human CD68 KP1 antibody (Dako M0814), diluted 1:100.…”

“…We speculated a difference in matrix substance between the perichondral and fascial insertions. Versican and tenascin-c are markers of chondrogenesis [19] as well as of the foetal cartilage-tendon interface under tensile stress [10]. Endochondral ossification occurs in versican-rich matrix, whereas perichondral ossification occurs in tenascin-positive fibrous tissues [3].…”