Fig. 4. Characterisation of cartilage in cellular nodules developing in neural crest cultures. (A,B) Brightfield in situ hybridisation for type II collagen mRNA. (A) Type II collagen mRNA is intensely expressed in a nodule. (B) Individual cells in the areas surrounding the nodule begin to express type II collagen at intermediate levels (arrows), with intensity of expression increasing as differentiation proceeds (arrowheads). (C-F) Confocal images of cultures stained immunohistochemically for type II collagen during progression of chondrogenic differentiation. Type II collagen, green; nuclei, red. (C) Exposure to 10 ng/ml FGF2 leads to clusters of cells beginning to express intracellular type II collagen. (D) These cells then aggregate to form nodules of cartilage, and (E) eventually type II collagen is secreted into the extracellular compartment (arrowheads). (F) Single cells expressing type II collagen appear in areas surrounding nodules. (G,H) Histological staining for cartilage. (G) Cartilage nodules stain positively with Alcian Blue and (H) sections through such nodules display metachromasia when stained with Toluidine Blue, indicating a cartilaginous matrix. (I,J) Examination of nodules by transmission electron microscopy, shows cellular ultrastructure typical of cartilage. (I) A typical chondroblast within the nodule has swollen ER (red asterisks), a scalloped plasma membrane profile and is surrounded by large areas of extracellular matrix. (J) A representative mature chondrocyte is enclosed within its own lacuna (arrowheads) and embedded in the extracellular matrix. Scale bars: 50 µm in A,B,G,H; 10 µm in C-F; 5 µm in I,J.