The disturbed <i>Hif</i> associated signaling in <i>Tnni2^K175del</i> mice resulted in reduced angiogenesis and impeded formation of ossification centers of long bones.

<p>(A–B) Immunostaining analyses of the hif3a (A) and hif1a (B) expression in the hypertrophic cell zones (<i>arrows</i>) of radii and ulnae from <i>Tnni2^{K175del/K175del}</i> mice and wild-type littermates at P12. <i>Right panel</i> of A: Quantification of hif3a expression (n = 3). (C) Immunostaining analyses revealed a decreased vegf in the hypertrophic cell zones of growth plates of radii and ulnae (<i>arrows</i>) from P12 heterozygous mutant mice compared to that of wild-type littermates. <i>Right panel</i> of C: Quantification of vegf expression (n = 3). (D) HE staining showed that the number of blood vessels surrounding trabecular bones were markedly decreased in radii from P12 mutants. Red blood cells were stained as red color. (E) Alkaline phosphatase staining exhibited a retarded formation of the primary ossification centers in radii of E15.5 homozygous mutants. Osteoblasts (blue <i>arrows</i>) have entered the marrow cavity and replaced the apoptotic chondrocytes in wild-type embryos but not in homozygous embryos. (F) Immunostaining revealed a decreased vegf in the hypertrophic cell zones of growth plates of radii and ulnae (<i>arrows</i>) from E15.5 homozygous mutant mice compared to those of wild-type littermates. (G) The relative mRNA expression of <i>Vegf</i> (normalized to <i>Gapdh</i>) in E15.5 homozygous mutant radii and ulnae over those of wild-type littermates (WT/Homo) using qPCR analyses. (H) CD31 stained the vessel endothelial cells in bone marrow of E15.5 homozygous mutant radii and wild-type controls. Insets showed magnified views of the box regions. (I) Quantification of blood vessel density in (H). (J) HE staining showed the growth plate of radii at P6. All experiments were repeated using at least three pairs of samples. Quantification analyses in A, C, G and I: Student's <i>t</i>-test, mean±s.d.; Scale bar, 100 µm (A–F, H, and J).