Fig. 6. Chondrocyte development and differentiation at E18.5. (A,B,G-I) Proximal tibia; (C-F) distal tibia. (A-C) Marker analysis by RNA in situ hybridization in wild-type (+/+) and C4st1^gt/gt (gt/gt) growth plates. Developmental markers for proliferating chondrocytes (collagen II, A), hypertrophic chondrocytes (collagen X, B) as well as columnar chondrocytes (Fgfr3, C) were expressed normally in mutant growth plates (ii) when compared with their wild-type counterparts (i), with the size of their expression domains reduced in relation to the overall shortening of the growth plate. (D) BrdU labeling of proliferating chondrocytes was detected by immunofluorescence (green). In wild-type growth plates (i), the zone of proliferation (double-headed arrow) excludes the hypertrophic layer. This proliferative zone is reduced in size in mutant cartilage (ii). (E) TUNEL staining (green) to identify chondrocytes undergoing apoptosis. In wild-type cartilage (i), low numbers of only the most differentiated hypertrophic cells (white bar) showed TUNEL staining, whereas in mutant cartilage, an increased number of cells in all zones (double-headed arrow) showed TUNEL staining (ii). (F) Immunofluorescence (red) using an -Bcl2 antibody. Wild-type growth plates (i) show widespread Bcl2 staining in the columnar zone (double-headed arrow), whereas staining in gt/gt growth plates (ii) is severely reduced and present only in lateral areas (arrows). (G) Immunofluorescence (red) using an -Bax antibody. Wild-type growth plates (i) show strong staining in prehypertrophic and hypertrophic chondrocytes (double-headed arrow), but not in columnar and proliferating cells (arrowhead). Homozygous mutant growth plates (ii), however, display staining in hypertrophic cells (arrow) and proliferating cells (arrowhead). (H,I) Higher magnification of regions labeled by white squares in G. Bax expression in wild-type growth plates is restricted to prehypertrophic and hypertrophic cells (`h' in H, i), and is not present in columnar cells (`c' in H, i) or proliferating cells (I, i). White line represents the border between columnar and prehypertrophic and hypertrophic zones. Bax expression in mutant growth plates is observed in hypertrophic cells (arrowhead in H, ii), but also in columnar cells (arrow in H, ii) and proliferating cells (green arrow in H, ii; arrowhead in I, ii). (J) Quantitation of BrdU-labeled cells in proliferating (prol.), columnar (column.) and hypertrophic (hypertr.) chondrocytes. Cartilage in mutant (gt/gt) shows an approximate twofold increase in BrdU-labeled cells in both proliferating and columnar chondrocytes compared with wild type. (K) Quantitation of TUNEL-labeled (apoptotic) cells in all three layers of the growth plate. Mutant growth plates showed a 3.5-fold increase in the number of cells undergoing apoptosis in the hypertrophic zone and apoptotic cells in both proliferating and columnar chondrocytes. Scale bar: 150 µm.