Figure 2. The organization of the growth plate in mutant mice is disrupted by 3 weeks of age and shows marked hypocellularity along with the mislocalization of cartilage structural proteins. Representative images from the growth plates of 3-week-old wild-type and mutant mice. A and B: H&E staining showed disruption to the growth plate by 3 weeks of age in mice homozygous for the Comp D469del mutation. This disruption was characterized by fewer and more disorganized columns of chondrocytes in the proliferating zone, with distinct areas of hypocellularity (∗) and individual chondrocytes that were at right angles to their usual horizontal plane (red circles). IHC using COMP (C and D), and matrilin-3 (E and F) antibodies revealed less staining in the ECM between the proliferating columns in the growth plates of mice carrying the D469del mutation. Furthermore, there was intracellular staining for mutant COMP and matrilin-3 in chondrocytes from mice homozygous for D469del (insert) and chondrocytes in the mutant growth plates had lost the “clear” appearance of their pericellular matrix (black arrows). Extracellular staining for COMP and matrilin-3 in the resting zone of the mutant growth plates was restricted to the pericellular matrix surrounding chondrocytes (D and F; red boxes) compared to the wild-type growth plates (C and E; red boxes). mut/mut, mice homozygous for D469del. chondrocytes in the resting zone of the mutant growth plate that we had observed with mutant COMP (Fig. 2E and F; red boxes). Finally, a small amount of intracellular staining was also observed for type IX collagen in chondrocytes from the growth plates of mice homozygous for the mutation when compared to those of heterozygous mice and wild-type controls (Supp. Fig. S4A and B). Furthermore, in mice of all three genotypes staining for type IX collagen was more pronounced in the resting and early proliferative zones, but there were no differences in the localization of type IX collagen in the ECM of mutant mice as seen with both COMP and matrilin-3. The Growth Plates of Mutant Mice Also Have Mislocalization of Type II and Type X Collagen Type II and type X collagen are essential for correct skeletal de- velopment and are important cartilage markers; in particular type X collagen, which is expressed exclusively by hypertrophic chondro- cytes [Morrison et al., 1996]. Wild-type and mutant growth plates showed no overt differences in the extracellular staining for type II collagen (Supp. Fig. S4C and D) and as expected type X collagen staining was observed only in the ECM of the hypertrophic zone for mice of all three genotypes, however, staining in the growth plates of HUMAN MUTATION, Vol. 33, No. 1, 218–231, 2012 223