Figure 2. Reduced chondrocyte proliferation and maturation underlie the growth defect of the left bones of P1-Pit::DTR mice.

(A) The fraction of EdU+ nuclei in the PZ was calculated for left and right proximal tibia GPs 2dpi and represented as L/R ratio (mean ± SD). p-value for unpaired two-tailed Mann-Whitney test between control and experimental ratios is shown. (B, D) The PZ and HZ of left and right GPs were measured on hematoxylin and eosin-stained sections of P1-Pit::DTR proximal tibia GPs, 4dpi (D), and represented as L/R ratios (B) 3–4 sections per GP, n = 4 mice, mean ± SD are shown). 2-way ANOVA (variables: Genotype and GP region, alpha = 0.05, p-value=0.0118 for Genotype) followed by Sidak’s posthoc multiple comparisons test (p-values shown) was used. Insets in (D) show magnifications of the boxed regions. (C) RNA in situ hybridization for Col10a1 in the proximal tibia GP at 3dpi. (E, F) The secondary ossification centers (SOCs, arrowheads in E) appear later and their subsequent area (quantified in F) is reduced in the left P1-Pit::DTR skeletal elements. Analysis was done by 2-way ANOVA (variables: SOC location and Side, alpha = 0.05, p-value=0.0003 for Side) followed by Sidak’s posthoc multiple comparisons test (p-values shown in Figure). Asterisk= reduced expression. See also associated Figure 2—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.27210.005

Figure 2—figure supplement 1. Bone growth impairment in Pit::DTR animals takes place on top of a systemic growth delay likely caused by injury-induced hypophagia.

(A) Comparison of body weight (mean ± SD) between control and P1-Pit::DTR pups at P5 (n = 18 controls, five experimental). (B) Relative length of the right tibia from control (black), DT-injected (red) and PBS-injected (green) Pit::DTR animals (mean ± SD, n = 4, 2 and 7, respectively, at early stages; 9, 7 and 6 long-term), normalized against the average length of the right tibia in control animals. (C and C’) DTR expression and TUNEL staining (arrowheads) in a heart section of a P1-Pit::DTR pup, 1dpi (n = 3). A, V= atrium, ventricle. Inset (C’) shows magnification of the boxed area in (C). (D–E’) Hematoxylin and eosin staining of the right GPs (internal control) from control and experimental animals at P5 (n = 4 control and four experimental mice). Note the striking difference in size, including the HZ (D’ and E’). (F) In situ hybridization for the indicated ECM components in control and experimental animals (n = 2 animals for each marker and genotype), showing the systemic delay affects ECM synthesis equally in left and right proximal tibia GPs of experimental animals.

DOI: http://dx.doi.org/10.7554/eLife.27210.006