Figure 4. Cell proliferation is decreased and apoptosis is both increased and spatially dysregulated in 3-week-old tibias of mutant mice. A: Chondrocyte proliferation, assayed by 2 hr BrdU labeling, was signiﬁcantly reduced in growth plates of mice homozygous for the D469del mutation (by ∼17%; n = 36 sections from three mice per genotype, independent samples t-test, ∗∗P < 0.005). B: TUNEL positive (apoptotic) cells (white circles) were detected in the proliferative and hypertrophic zones of growth plates from mice homozygous for the D469del COMP mutation, but not in their wild-type littermates. The white lines mark the boundaries between the vascular invasion front, hypertrophic, and proliferative zones in the growth plate, and DAPI staining was used as a nuclear counterstain. C: Quantiﬁcation of the TUNEL assay results showed a signiﬁcant increase in apoptosis in the hypertrophic (>ﬁvefold) zone of growth plates from mice homozygous for the mutation (n = 36 sections from three mice per genotype, independent samples t-test, ∗∗∗P < 0.001). wt, wild-type; mut/mut, homozygous for D469del; VIF, vascular invasion front. levels of ﬁbril surface-associated material (such as COMP) as seen growth, we studied the relative rates of chondrocyte proliferation in the other mouse models of PSACH-MED [Leighton et al., 2007; and apoptosis in the growth plate. Pirog-Garcia et al., 2007]. Chondrocyte Proliferation Is Decreased in Mutant Mice While Apoptosis Is Increased and Spatially Dysregulated The spatial and temporal regulation of chondrocyte proliferation To determine the effect of mutant Comp expression on chon- drocyte proliferation, we used 2 hr BrdU labeling experiments on 3-week-old mice. Within the proliferative zone of the growth plates of wild-type mice 18% (±1.84%) of cells were labeled with BrdU, while only 14.8% (±1.95%) of cells were labeled in the growth plates of mice homozygous for Comp D469del, which corresponds and differentiation within the growth plate is crucial for correct to a decrease of 17% (P < 0.05) in the relative rates of chondrocyte bone growth. Therefore, to help identify a mechanistic link between proliferation in mutant growth plates (Fig. 4A). The relative levels the expression of Comp D469del and reduced longitudinal bone of cell proliferation within growth plates of mice heterozygous for HUMAN MUTATION, Vol. 33, No. 1, 218–231, 2012 225