The hypoxia-inducible factor α pathway couples angiogenesis to osteogenesis during skeletal development
J. Clin. Invest. Ying Wang, et al. 117:1616 doi:10.1172/JCI31581 [Go to this article.]

Figure 7
Mice lacking Hif1a in osteoblasts have narrow, poorly vascularized long bones. (A) PCR analysis of Cre-mediated recombination in selected tissues from a ΔHif1a mouse. The recombined allele (Δflox) was present exclusively in bone tissue. (B) Representative histological sections of distal femurs from 6-week-old control and ΔHif1a mice after staining with antibodies against HIF-1α (left) or HIF-2α (right) as described in Methods. Sections were counterstained with hematoxylin. Red arrows indicate positive and black arrows negative staining in osteoblasts. Original magnification, ×400. (C) Representative images of femoral cross sections from control and ΔHif1a mice. Scale bars: 1.0 mm. (D) Representative μCT images of vasculature in Microfil-perfused femurs from 3-week-old ΔHif1a and control mice. Scale bar: 1.0 mm. (E and F) Confluent monolayers of Hif1a floxed primary osteoblasts were infected with either Ad-GFP or Ad-CreM1 (100 MOI). (E) Proteins in the cytoplasm and nucleus were extracted separately 48 hours after infection. Immunoblotting analysis was performed with antibodies against HIF-1α and HIF-2α. Immunoblots for TBP and α-tubulin were used as loading controls for nuclear and cytoplasmic proteins, respectively. (F) Total mRNA was extracted from confluent monolayers of osteoblasts 48 hours after infection. Hif1a, Hif2a, and Vegf mRNA expression was determined by quantitative real-time PCR. **P < 0.01.