Fig. 4. Cartilage growth plate defects in the proximal tibia of C4st1^gt mutant embryos at E18.5. (A) RNA in situ hybridization using a C4st1-exon2+3-specific probe shows C4st1 expression in proliferating, but not hypertrophic chondrocytes in wild-type growth plates (i) and the reduction in C4st1 staining in mutant growth plates (ii). (B,C) Safranin O staining. (B) Safranin O staining shows a reduction in size of proliferating (p), columnar (c) and hypertrophic (h) zones, as well as less intense staining in mutant growth plates (ii) when compared with wild type (i). Brackets indicate areas shown in C. (C) Higher magnification of the transition zone between hypertrophic cartilage and primary bone, showing cartilage islands (arrow) in wild-type (i), but not mutant, bone (ii). (D-G) Hematoxylin and Eosin staining. (D) Mutant growth plates appear disorganized and contain ECM disruptions (arrowhead) and misoriented chondrocyte columns (arrow). (E,F) Higher magnification either wild-type (E) or mutant (F) growth plates (p, c and h are defined in B). Wild-type chondrocyte columns are oriented parallel to the longitudinal bone axis, whereas mutant columns are oriented almost perpendicular to it. In addition there is an increased thickness of the bone collar in the mutants (arrows). (G) Dark-field images of Hematoxylin and Eosin-stained wild-type (i) and mutant (ii) proliferating chondrocytes, showing fibrillation of ECM in mutant growth plates. Scale bar: 400 µm for A; 1 mm for B; 100 µm for C,D; 30 µm for E,F; 10 µm for G.