Figure 1. Mutant COMP mice are normal at birth but develop short-limb dwarﬁsm by 9 weeks of age. A: Individual mice were radiographed at 3, 6, and 9 weeks of age; radiographs of 9-week-old mice are shown. B: Bone measurements of male littermates at 9 weeks of age with inner canthal distance (ICD) used as a marker of intramembranous ossiﬁcation, and the femur, tibia, and pelvis lengths used as markers of endochondral ossiﬁcation. The differences in femur, tibia, and pelvis lengths between wild-type males and males homozygous for the mutation were >6%, >5%, and >7%, respectively (n ≥ 7 mice per genotype; One-way ANOVA, ∗P < 0.05). C: The angle (◦) between the tuberosity of the ischium and the pelvic region was measured at 3, 6, and 9 weeks of age in mice of all three genotypes. D: The hip angle was >57% greater for males homozygous for the mutation compared to their wild-type littermates at 9 weeks (n ≥ 7 mice per genotype; One-way ANOVA, ∗∗P < 0.005). wt, wild-type; wt/m, mice heterozygous for the mutation; m/m, mice homozygous for the mutation. regions of hypocellularity were observed within the proliferative zone indicating an intermediate phenotype (data not shown). Mutant COMP is Retained within Growth Plate Chondrocytes Along with Matrilin-3 and to a Lesser Extent Type IX Collagen IHC was performed on the tibia growth plates of 3-week-old mice to determine the localization of key structural components, in particular COMP, matrilin-3 and type IX collagen, which are known to interact with each other. Extracellular staining for COMP was observed in the growth plates of wild-type mice (Fig. 2C) and mice heterozygous for Comp D469del (not shown). However, ex- tracellular staining of COMP was markedly reduced in the growth plates of mice homozygous for the mutation, in particular within the proliferative zone, where chondrocytes were also seen to have lost the “white” appearance of their pericellular matrix (Fig. 2D; ar- 222 HUMAN MUTATION, Vol. 33, No. 1, 218–231, 2012 rows). In contrast, chondrocytes within the resting zone of mutant growth plates appeared to have more intense staining for COMP in the pericellular matrix compared to wild-type (Fig. 2C and D; red squares). Most notable, however, was the intracellular staining for mutant COMP, which was most pronounced in chondrocytes within the hypertrophic zone (Fig. 2D; insert), although a few pro- liferative chondrocytes also had retained mutant COMP (Fig. 2D; blue circles). Extracellular staining for matrilin-3, which was comparable to COMP staining, was observed in wild-type mice and in mice het- erozygous for the mutation (Fig. 2E, not shown). Although ECM staining for matrilin-3 was present in the growth plates of mice homozygous for the Comp D469del mutation, there was also some intracellular staining seen in the hypertrophic chondrocytes, al- though this staining was less intense than that seen for mutant COMP (Fig. 2E; insert). Interestingly, there was a similar concen- tration of matrilin-3 staining in the pericellular matrix surrounding