Fig. 4. BrdU labeling analysis. (A) BrdU-positive chondrocytes of E17.5 embryos were counted in the area indicated by red and black lines for the periarticular region and the columnar region, respectively. Insets are magnified views of the periarticular (red outline) and columnar (black outline) regions. The number of BrdU-positive cells was divided by the number of nuclei to calculate proliferation rate (shown on the right). At least nine sections from three animals for each genotype were counted. *, statistically significant by ANOVA with P<0.05. (B) BrdU pulse-chase assay. Representative pictures of proximal tibias of mice sacrificed 24 hours after single BrdU injection at E17.5. Hypertrophic cells were identified by morphological criteria. The blue lines indicating the beginning of the hypertrophic zone were drawn by connecting the points at which bone collars started to form. The distance between the lines and the furthest BrdU-positive hypertrophic cells (indicated by red arrowheads), as well as number of BrdU-positive hypertrophic chondrocytes were measured and counted (shown on the right). Insets are magnified views of BrdU-positive hypertrophic cells. The distance was 32% greater in Ppr^d/– than in controls. The absolute number of BrdU-positive cells was increased in Ppr^d/– by 2. At least ten sections from two independent animals for each genotype were subjected to the analysis. * and **, statistically significant by ANOVA with P<0.05 and P<0.01, respectively. (C) BrdU labeling ratio was similarly calculated in the periarticular chondrocytes of E17.5 mice with cartilage-specific PPR ablation (shown on the right). Insets are magnified views of the periarticular region. At least nine sections from three independent animals for each genotype were counted. *, statistically significant by ANOVA with P<0.05.