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.

Fig. 1

The anlage of the future digital bone at E13. Mesenchymal cell condensation (arrow in a) with no matrix metachromasia is seen by toluidine blue staining (TB). Versican mRNA (arrow in b) as well as immunostaining (arrow in c) are expressed in this mesenchymal cell condensation. There is strong HA staining in the mesenchyme around the condensation (arrowheads in d) but not in the mesenchymal condensation (arrow in d). Width of view = 100 µm.

Fig. 2

Tibial cartilage at E13. (a) A metachromatically stained matrix with toluidine blue staining is seen. The diaphysis (Dia) and the epiphysis (Epi) are distinguishable. (b) Versican mRNA is strongly expressed in the mesenchyme around the epiphysis (arrow) and only slightly in flattened chondrocytes of the epiphysis (arrowhead). (c,d) Aggrecan (c) and link protein (d) mRNA is expressed throughout the tibial cartilage (arrows). (e) There is clear versican immunostaining in the tibial cartilage (arrow) and in the mesenchyme around it (arrowhead). (f,g) There is weak aggrecan (arrow in f) and link protein (arrow in g) immunostaining in the diaphysis of this cartilage. (h) There is HA staining within the tibial cartilage (arrow) and mesenchyme around it (arrowhead). Width of view = 100 µm.

Fig. 3

Tibial cartilage at E14. (a) Diaphysis (Dia), metaphysis (Meta) and epiphysis (Epi) are distinguishable. Toluidine blue staining. (b) Aggrecan mRNA expression is detected in the metaphysis and the epiphysis (arrow), but reduced in the diaphysis (arrowhead). (c) Link protein mRNA shows an expression pattern similar to aggrecan. (d) There is clear versican immunostaining in the mesenchyme around the cartilage (*) and in the epiphysis (arrowheads), with less toward the diaphysis (arrow). (e) There is strong aggrecan immunostaining in the diaphysis (arrow) with less toward the epiphysis (arrowhead). (f) Link protein immunostaining shows an expression pattern similar to aggrecan. (g) HA staining is detected throughout the cartilage (arrows) and mesenchyme around it (*). Width of view = 100 µm.

Fig. 4

Tibial cartilage at E15. (a) Endochondral bone formation has started from the diaphysis (thin arrow). Toluidine blue staining. (b) Aggrecan mRNA is expressed in the epiphysis and metaphysis (thin arrow) but not in the diaphysis (thick arrow). (c) Link protein mRNA is strongly expressed in the metaphysis (thin arrow), weakly in the epiphysis (thick arrow) but not in the diaphysis (*). The marginal area of metaphysis and epiphysis shows relatively strong expression (arrowheads). (d) There is versican immunostaining in the mesenchyme around the tibial cartilage (*) and still in the epiphyseal end of this cartilage (thin arrow), but not in the metaphysis and diaphysis (thick arrow). (e) There is strong aggrecan immunostaining in the diaphysis (thin arrow) but there is less toward the epiphysis and none at the epiphyseal end (thick arrow). (f) There is strong link protein immunostaining in the diaphysis (thin arrow), but very weak in the metaphysis and epiphysis (thick arrow), but the marginal area of this region has moderate immunostaining (arrowheads). (g) HA staining is detected in the mesenchyme around the cartilage (*) and throughout the cartilage (thin arrow). Note that the marginal area shows no versican and aggrecan immunostainings (arrowheads in d and e, respectively) and HA staining (arrowheads in g). Width of view = 100 µm.