Journal: Nature cell biology

Article Title: Cell-matrix signals specify bone endothelial cells during developmental osteogenesis

doi: 10.1038/ncb3476

Figure Lengend Snippet: a , Immunostaining for Emcn (red) and the type E markers Caveolin 1 (green) and BCAM (white) in P6 femur. Panels on the right show higher magnification of corresponding insets. b , c , Transverse sections through P6 femur showing CD31 (green) and Caveolin 1-positive (white) vessels in compact bone (cb) (arrowheads) ( b ). By contrast, the bone marrow cavity (mc) contains Emcn-positive vessels with low CD31 signal (arrow). BCAM-positive vessels (white) in compact bone were associated with Osterix-expressing osteoprogenitors (green) ( c ). d , Quantitation of Osterix positive cells in proximity to type L vessels in the marrow cavity, metaphyseal type H vessels and to type E vessels in compact bone normalized to vessel length. Data represents mean± s.e.m. (n=6 mice), (p<0.001 between all compared groups, two-tailed unpaired t-test). e , RNA-seq-based expression levels of the indicated transcripts relative to expression in type L in freshly isolated P6 EC subpopulations. Data based on RPKM values obtained from RNA-seq. Data represents mean± s.e.m. (n=3 independent experiments). Statistics source data are shown in . f , Transcript expression in EC-C3H10T1/2 spheroid co-cultures (at day 7). Transcripts encoding Sox9, Runx2, Osteopontin ( Sp7 ), integrin binding sialoprotein ( Ibsp ), and Osteocalcin ( Bglap ) were upregulated in cultures containing primary type H or type E ECs. Data represents mean± s.e.m. (n=7 independent experiments), two-tailed unpaired t-test. g , Immunostaining of EC-C3H10T1/2 spheroids after 7 days in culture. Signals for Runx2 (white), Osteocalcin (green) and Alkaline phosphatase (red) were upregulated by CD31 hi (green) type H and type E ECs (arrowheads) but not in the presence of type L ECs. Nuclei, DAPI (blue). ECs were pre-labeled by DiI (red) at the onset of the experiments.

Article Snippet: The following primary antibodies were used: rat anti-Endomucin (sc-65495, Santa Cruz, diluted 1:100), goat anti-CD31 conjugated to Alexa Fluor (AF) 488 (FAB3628G, R&D Systems, 1:50), rabbit anti-Osterix (c-22536-R, Santa Cruz, 1:500), goat anti-VEGFR2 (AF644, R&D Systems, 1:50), goat anti-VEGFR3 (AF743, R&D Systems, 1:50), rabbit anti-Caveolin 1 (#3238, Cell Signaling, 1:50), goat anti-BCAM (AF8299, R&D Systems, 1:50), chicken anti-GFP (ab13970, Abcam, 1:500), rabbit anti-GFP conjugated to AF488 (A21311, Invitrogen, 1:250), goat anti-Integrin beta 1 (AF2405, R&D Systems, 1:50), rabbit anti-Laminin α4 (serum 377, 1:500), rat anti-Laminin α5 (clone 4G6, 1:10), rabbit anti-Fibronectin (F3648, Sigma, 1:100), rabbit anti-Collagen Type1 (AB765P, Millipore, 1:100), goat anti-Osteopontin (AF808, R&D Systems, 1:100), α-SMA-Cy3 (C6198, Sigma, 1:50), mouse anti-hypoxyprobe conjugated to fluorescein isothiocyanate (FITC) (#FITC-Mab, Hydroxyprobe, 1:50), rabbit anti-phosphoERK1/2 (D13.14.4E, Cell Signaling, 1:50), rabbit anti- Osteocalcin (LS-C17044, LifeSpan BioSciences, 1:300), rabbit anti-NG2 (AB5320, Millipore, 1:100), rat anti- Runx2 (MAB2006, R&D Systems, 1:50), rabbit anti-Calcitonin receptor (ab11042, Abcam , 1:50), rabbit anti-TRAP (sc-28204, Santa Cruz, 1:50).

Techniques: Immunostaining, Expressing, Quantitation Assay, Two Tailed Test, RNA Sequencing Assay, Isolation, Binding Assay, Labeling

Journal: Nature cell biology

Article Title: Cell-matrix signals specify bone endothelial cells during developmental osteogenesis

doi: 10.1038/ncb3476

Figure Lengend Snippet: a , Representative 3D reconstruction from µCT measurements of tibial metaphysis of 3 week-old Itgb1 iΔEC and littermate control mice. Diagrams represent bone parameters measured in µCT analyses: bone volume/total volume (BV/TV) in percentage, trabeculae number in 1 per millimeter, trabecular thickness in millimeters, trabecular separation in millimeters, and connectivity density in 1 per cubic millimeter. Data represent mean± s.e.m. (n=6 mice), (p-values determined by two-tailed unpaired t-test). b , Quantitation of metaphyseal Osx+ cells in Itgb1 iΔEC mutant and Cre-negative littermate bone sections. Data represents mean± s.e.m. (n=4 individual femurs), (p=0.01, two-tailed unpaired t-test). Statistics source data are shown in . c , Tile scan and high magnification confocal images of Itgb1 iΔEC and littermate control femurs stained for Osterix (green). Dashed lines indicate borders of metaphysis to growth plate and marrow cavity, respectively. d , Itgb1 iΔEC and littermate control femurs stained for Osteocalcin (red) and counterstained with Hoechst (blue). e , High magnification of 2-photon second harmonic generation signals (white) of sections of P21 Itgb1 iΔEC and control femurs. Dashed line indicates adjacent growth plate (top). f , g , Maximum intensity projection of femoral sections from Itgb1 iΔEC mutants and littermate controls stained for NG2 ( f , green) or Runx2 ( g , red). Nuclei in ( g ), Hoechst (blue). h , RT-qPCR analysis of growth factor transcripts in freshly sorted Itgb1 iΔEC CD31 hi Emcn hi (orange) and control ECs (blue). Data represents mean± s.e.m. (n=9 mice per group), (p-values, two-tailed unpaired t-test). ns, not significant.

Article Snippet: The following primary antibodies were used: rat anti-Endomucin (sc-65495, Santa Cruz, diluted 1:100), goat anti-CD31 conjugated to Alexa Fluor (AF) 488 (FAB3628G, R&D Systems, 1:50), rabbit anti-Osterix (c-22536-R, Santa Cruz, 1:500), goat anti-VEGFR2 (AF644, R&D Systems, 1:50), goat anti-VEGFR3 (AF743, R&D Systems, 1:50), rabbit anti-Caveolin 1 (#3238, Cell Signaling, 1:50), goat anti-BCAM (AF8299, R&D Systems, 1:50), chicken anti-GFP (ab13970, Abcam, 1:500), rabbit anti-GFP conjugated to AF488 (A21311, Invitrogen, 1:250), goat anti-Integrin beta 1 (AF2405, R&D Systems, 1:50), rabbit anti-Laminin α4 (serum 377, 1:500), rat anti-Laminin α5 (clone 4G6, 1:10), rabbit anti-Fibronectin (F3648, Sigma, 1:100), rabbit anti-Collagen Type1 (AB765P, Millipore, 1:100), goat anti-Osteopontin (AF808, R&D Systems, 1:100), α-SMA-Cy3 (C6198, Sigma, 1:50), mouse anti-hypoxyprobe conjugated to fluorescein isothiocyanate (FITC) (#FITC-Mab, Hydroxyprobe, 1:50), rabbit anti-phosphoERK1/2 (D13.14.4E, Cell Signaling, 1:50), rabbit anti- Osteocalcin (LS-C17044, LifeSpan BioSciences, 1:300), rabbit anti-NG2 (AB5320, Millipore, 1:100), rat anti- Runx2 (MAB2006, R&D Systems, 1:50), rabbit anti-Calcitonin receptor (ab11042, Abcam , 1:50), rabbit anti-TRAP (sc-28204, Santa Cruz, 1:50).

Techniques: Two Tailed Test, Quantitation Assay, Mutagenesis, Staining, Quantitative RT-PCR