Figure 3. Multiple signaling pathways related to chondrocyte proliferation and maturation are altered in distinct regions of the left P1-Pit::DTR growth plate.

(A–A’) In situ hybridization for Ihh and the HH target Gli1, in the left and right proximal tibia of Pit::DTR mice, 2dpi. (B, C) Immunohistochemical staining for phosphorylated ribosomal protein S6 [(B), readout of mTORC1 activity] or for the canonical WNT target LEF1 (C), in the left and right proximal tibia of Pit::DTR mice, 2dpi. Note left-specific downregulation of p-S6 in the same region where Ihh is reduced. Arrowheads= ectopic expression, asterisks= reduced expression. See also associated Figure 3—figure supplements 1 and 2.

DOI: http://dx.doi.org/10.7554/eLife.27210.007

Figure 3—figure supplement 1. Bone growth impairment in P1-Pit::DTR animals is not caused by increased FGF or EGF signaling in the HZ, nor by ablation of osteoprogenitors in the perichondrium or increased cell death in the osteochondral junction.

(A) Immunohistochemistry for p-EGFR did not reveal increased EGF signaling in the HZ at P3 (n = 3 PBS- and 3 DT-injected mice). Arrowheads= sparse activation of EGF signaling in the left articular cartilage. (B, B’) Immunohistochemistry for the FGF-signal transducer FRS2 and in situ hybridization for Fgfr3 did not reveal increased FGF signaling in the left HZ (n = 8, 1, and two for P3, P4 and P5). (C) Co-staining for the osteoblast lineage markers COLI or SP7 (OSX) (Maes et al., 2010) and TUNEL were done to determine whether cell death is increased in osteoprogenitors of Pit::DTR long bones. DT-mediated ablation of osteoprogenitors (also derived from the lateral plate mesoderm) could contribute to the Pit::DTR bone growth defect by impairing the generation of new bone, but no increase in osteoblast death was detected. The approximate regions of (C) are indicated in (A) as a reference. (D–F) To further rule out a possible contribution of early osteoblast precursor ablation to the phenotype, we crossed Sp7-tTA,tetO-EGFP/Cre mice (Rodda and McMahon, 2006) with R26^LSL-DTR animals to generate Sp7-tTA,tetO-EGFP/Cre; R26^LSL-DTR mice (Osx::DTR). These animals were injected with either DT or PBS at P1. DT-injected Osx::DTR mice showed an almost complete depletion of osteoprogenitors 2dpi (D) and reduced thickness of the cortical bone as compared to PBS-injected animals (E, insets show a 2.5x magnification of the bracketed area; E’ shows µCT images and cortical thickness quantification of the midshaft femoral region, color-coded by litter of origin, n = 6 PBS-treated and 6 DT-treated mice, p-value for unpaired Mann Whitney test is shown. See associated Source Data 1). However, no consistent reduction of bone length was observed at P5 (F n = 8 PBS- and 7 DT-injected pups), ruling out a major contribution of osteoprogenitor ablation to the Pit::DTR phenotype. Note that although DT-injected mice weighed significantly less than PBS-injected ones, their bones were not significantly shorter. (G) Quantification of the density of apoptotic cells at the osteochondral junction of Pit::DTR pups 2-3dpi. The aim of the experiment was to test whether an increase in cell death at the osteochondral junction (where hypertrophic chondrocytes die by apoptosis and the cartilage is replaced by bone) could contribute to the observed reduction in the height of the HZ due to chondrocytes were being eliminated faster than produced. Note, no significant differences were detected between left and right GPs (n = 5 pups, 2–3 sections per limb). PS= primary spongiosa. The graph shows left and right paired data (TUNEL⁺ cells per area) for each animal, which were compared by a ratio paired t-test.

DOI: http://dx.doi.org/10.7554/eLife.27210.009

Figure 3—figure supplement 2. Characterization of the role of IHH and mTOR in the signaling and growth defects of P1-Pit::DTR mice.

(A) X-gal staining to detect Pthlh expression, in the left and right proximal tibia of Pit::DTR; Pthlh^lacZ/+ mice (Chen et al., 2006), 2dpi (n = 3 at P3 and 2 at P4). (B–C’) Treatment of P1-Pit::DTR pups with the SHP2 inhibitor NSC-87877 (5 mg/kg s.c. daily from P1) was found to rescue Ihh expression in the left GP at P3 (B and B’, n = 2 and 6 with partial and full rescue, respectively) but it did not rescue bone growth by P5 (C, frontal view of the tibiae, and C’, n = 5 control and three experimental animals). p-values for unpaired Mann Whitney test (SHP2i vs. vehicle for each bone) are shown. (D, D’) p-S6 expression in proximal tibiae from vehicle-treated (D) or rapamycin-treated (D’) P1-Pit::DTR pups, showing dependence on mTORC1 activity. n = 2 at P3, two at P4. (E) Length of the right (control) bones of P4 Pit::DTR pups, treated with vehicle (n = 3) or rapamycin (n = 3) from P1 to P4. Analysis was done by 2-way ANOVA (alpha = 0.05, Treatment and Bone as variables, p=0.0079 for Treatment) followed by Sidak’s posthoc multiple comparisons test (p-values shown in Figure).

DOI: http://dx.doi.org/10.7554/eLife.27210.010