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Fig. 5. Analysis of growth plate extracellular matrix (ECM) markers of the proximal
tibia at E18.5. (A) Fluorophore-assisted carbohydrate electrophoresis (FACE)
analysis of ECM glycosaminoglycans (GAGs). (B) Chondroitin sulfate (CS)
immunohistochemistry showing ECM staining (black arrow) in all three growth
plate layers (p, proliferating chondrocytes; c, columnar chondrocytes; h,
hypertrophic chondrocytes) of wild-type cartilage (+/+), whereas CS staining
in mutant (gt/gt) proliferating and columnar layers is restricted to the
pericellular space (arrowhead), with very little staining in the ECM (black
arrow). However, in the columnar and hypertrophic layers of mutants, ECM
staining of CS was observed (white arrows). (C) C6S was detected by
immunofluorescence staining and is distributed in the outer-most layers of the
proliferative zone in wild-type cartilage (+/+), whereas in mutant cartilage
(gt/gt), low pericellular C6S staining was observed in both proliferative and
columnar layers. (D) Distribution of aggrecan was analyzed by
immunofluorescence, which revealed localization to the ECM in all layers of
the wild-type cartilage (+/+), as well as in the proliferative layer of mutant
cartilage (gt/gt). However, in the mutant, aggrecan was increasingly
restricted to the pericellular space in columnar and hypertrophic (arrowhead)
layers. (E) Detection of collagen II by immunofluorescence shows strong
staining of the ECM in both wild-type (+/+) and mutant (gt/gt) proliferative
and columnar layers, and decreased staining in the ECM of the hypertrophic
layer. Scale bar: 10 µm.
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