Figure 7. Induction of soft-tissue cell death at P14 also impairs bone growth.

(A) TUNEL staining (arrowheads) in sagittal section of P17 proximal tibia from Pit::DTR mice injected DT at P14 (P14-Pit::DTR). SOC= secondary ossification center (outlined). (B) Hematoxylin and eosin-stained sagittal sections of P18 distal femur from P14-Pit::DTR mice. Vertical bars indicate the length of the HZ. (C) Immunostaining for p-S6 in the GP of P14-Pit::DTR mice at P17. (D, E) In situ hybridization for Egr1 (D) and Igf1 (D, insets) and LEF1 immunostaining (E) in knees from P14-Pit::DTR mice. (F) Alizarin red staining of the skeletal elements from a representative P14-Pit::DTR mouse at P57. (G) Quantification of the left/right length ratio for femora and tibiae of P20 and P57 Control and P14-Pit::DTR mice. Analysis was done by 2-way ANOVA (alpha = 0.05, Bone Identity and Genotype as variables, p-value for Genotype was 0.1970 at P20 and < 0.0001 at P57) followed by Sidak’s posthoc multiple comparisons test (p-values shown in Figure). Arrowheads denote ectopic expression. AC= articular cartilage. RZ, PZ, HZ= resting, proliferative, hypertrophic zones. IPFP= infrapatellar fat pad. See also associated Figure 7—figure supplement 1.

DOI: http://dx.doi.org/10.7554/eLife.27210.019

Figure 7—figure supplement 1. Developmental description of infrapatellar fat pad formation and correlation with limb allometry.

(A–A’) Developmental progression of the knee interzone/infrapatellar fat pad region (A) at the indicated stages, and the elbow region at E17.5 (A’). Hematoxylin and eosin (H&E), Igf1 expression and FABP4 (adipocyte marker) and CD44 plus CD90 (mesenchymal cell markers) immunohistochemistry are shown in (A). At least two specimens per stage and technique were examined. Color-coded arrowheads point to cells expressing the marker of the corresponding color. Yellow and white dotted lines delineate, respectively, the fat pads and the cartilage anlagen. Asterisks denote lack of detectable expression. F, T, H, R, U= femur, tibia, humerus, radius, ulna. (B) Radius/Tibia length ratio in WT mice at the indicated stages (at least four animals per stage). Data were analyzed by one-way ANOVA with Stage as variable (alpha = 0.05, p<0.0001). p-values for Sidak’s multiple comparisons test (comparing each stage with the preceding one) are shown. Note that the ratio stabilizes between P8 and P14, coinciding with the cessation of Igf1 expression in the infrapatellar fat pad.

DOI: http://dx.doi.org/10.7554/eLife.27210.020