(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 R26LSL-DTR animals to generate Sp7-tTA,tetO-EGFP/Cre; R26LSL-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.