Fig 5. Mosaic local proliferation blockade in chondrocytes of the left limb results in systemic growth reduction.
(A) Right femur and tibia length (normalized to the average ePit-p21 littermate) from E17.5 embryos treated with Dox (n = 4 ePit-p21 and n = 11 ePit-Col-p21) or untreated (n = 5 and n = 6). Comparison by 2-way ANOVA with Genotype and Bone identity as variables. p-Values for Genotypes are shown below graphs; p-values for Sidak’s post hoc test shown on graph. (B–C) Box and whisker plots for normalized bone length (panel B) and weight (panel C) of ePit-Col-p21 and Col2a1-rtTA; Igs7TRE-tdT/+ embryos (eCol-tdT, expressing tdT in all cartilage elements), compared to their controls (ePit-p21 and ePit-tdT) by unpaired t tests. p-Values corrected for multiple comparisons (Holm-Sidak method) are shown. For panel B, n = 22 ePit-p21, n = 26 ePit-Col-p21, n = 25 ePit-tdT, and n = 5 eCol-tdT). For panel C, n = 4, 11, 24, and 5. (D) Model of the systemic growth response after local chondrocyte arrest triggers a stress signal. (E–F) qRT-PCR (panel E) and in situ hybridisation (panel F) for the indicated transcripts in the proliferative plus pre-hypertrophic zone from ePit-Col-p21 embryos. Panel E shows one of 2 independent experiments with 3 distinct biological replicates each (total n = 6). The –ΔCt (relative to Gapdh) for each stress-related transcript was compared by a paired t test (left versus right). In panel F, n = 2 E15.5, n = 4 E16.5, and n = 6 E17.5 embryos (arrowheads denote Il6 expression). For panel A–C and E, see also S3 Data. Dox, doxycycline; E, embryonic day; qRT-PCR, quantitative real-time polymerase chain reaction; tdT, tdTomato.