Figure 5.

Altered chondrocyte development in VEGF^188/188 peripheral growth plates. (a) Von Kossa staining at P1.5, showing highly reduced calcification in the hypertrophic chondrocyte zone of VEGF^188/188 tibia compared with WT. (b and c) BrdU incorporation to detect proliferating cells at (b) P1.5 (hematoxylin counterstain) and (c) E16.5 (light green counterstain). The percentage of positive cells relative to total cell count was determined in peripheral resting/periarticular and proliferating/columnar zones, as indicated (boxed areas). Higher magnification of the resting chondrocyte area is shown (c). Values are means ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001 (n = 5–8). (d) Molecular analysis of chondrocyte development in WT and VEGF^188/188 peripheral and central sections of proximal tibia at P1.5 by in situ hybridization for Ihh, Ptc, PTHrP, PPR, and Cbfa-1. No signal is detected within the central region of VEGF^188/188 bones, consistent with the documented apoptosis. Note strongly enhanced and ectopic expression of Ptc in the resting/periarticular chondrocyte area and strong PTHrP expression near the bone ends of VEGF^188/188 mice. (e and f) Analysis of PTHrP expression level by qRT-PCR on (e) P1.5 WT and VEGF^188/188 mice (n = 12; **P < 0.01) and (f) E16.5 WT limbs cultured in normoxic versus hypoxic conditions (24 hours) (n = 5; *P < 0.05). Values represent the number of PTHrP mRNA copies per 1,000 copies of HPRT. Scale bars: (a–c) 100 μm; (d) 200 μm.