Abstract
Blood vessels in the mammalian skeletal system control bone formation and support haematopoiesis by generating local niche environments. While a specialized capillary subtype, termed type H, has been recently shown to couple angiogenesis and osteogenesis in adolescent, adult and ageing mice, little is known about the formation of specific endothelial cell populations during early developmental endochondral bone formation. Here, we report that embryonic and early postnatal long bone contains a specialized endothelial cell subtype, termed type E, which strongly supports osteoblast lineage cells and later gives rise to other endothelial cell subpopulations. The differentiation and functional properties of bone endothelial cells require cell–matrix signalling interactions. Loss of endothelial integrin β1 leads to endothelial cell differentiation defects and impaired postnatal bone growth, which is, in part, phenocopied by endothelial cell-specific laminin α5 mutants. Our work outlines fundamental principles of vessel formation and endothelial cell differentiation in the developing skeletal system.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Kronenberg, H. M. Developmental regulation of the growth plate. Nature 423, 332–336 (2003).
Long, F. & Ornitz, D. M. Development of the endochondral skeleton. Cold Spring Harb. Perspect. Biol. 5, a008334 (2013).
Eshkar-Oren, I. et al. The forming limb skeleton serves as a signaling center for limb vasculature patterning via regulation of Vegf. Development 136, 1263–1272 (2009).
Conen, K. L., Nishimori, S., Provot, S. & Kronenberg, H. M. The transcriptional cofactor Lbh regulates angiogenesis and endochondral bone formation during fetal bone development. Dev. Biol. 333, 348–358 (2009).
Maes, C. et al. Impaired angiogenesis and endochondral bone formation in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Mech. Dev. 111, 61–73 (2002).
Maes, C. et al. Increased skeletal VEGF enhances β-catenin activity and results in excessively ossified bones. EMBO J. 29, 424–441 (2010).
Maes, C. et al. Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev. Cell 19, 329–344 (2010).
Ding, B. S. et al. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis. Nature 505, 97–102 (2014).
Ding, B. S. et al. Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration. Nature 468, 310–315 (2010).
Ding, B. S. et al. Endothelial-derived angiocrine signals induce and sustain regenerative lung alveolarization. Cell 147, 539–553 (2011).
Ramasamy, S. K., Kusumbe, A. P., Wang, L. & Adams, R. H. Endothelial Notch activity promotes angiogenesis and osteogenesis in bone. Nature 507, 376–380 (2014).
Hu, J. et al. Endothelial cell-derived angiopoietin-2 controls liver regeneration as a spatiotemporal rheostat. Science 343, 416–419 (2014).
Kusumbe, A. P., Ramasamy, S. K. & Adams, R. H. Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507, 323–328 (2014).
Xie, H. et al. PDGF-BB secreted by preosteoclasts induces angiogenesis during coupling with osteogenesis. Nat. Med. 20, 1270–1278 (2014).
Alford, A. I., Kozloff, K. M. & Hankenson, K. D. Extracellular matrix networks in bone remodeling. Int. J. Biochem. Cell Biol. 65, 20–31 (2015).
Marie, P. J., Hay, E. & Saidak, Z. Integrin and cadherin signaling in bone: role and potential therapeutic targets. Trends Endocrinol. Metab. 25, 567–575 (2014).
Olsen, B. R., Reginato, A. M. & Wang, W. Bone development. Annu. Rev. Cell Dev. Biol. 16, 191–220 (2000).
Humphries, J. D., Byron, A. & Humphries, M. J. Integrin ligands at a glance. J. Cell Sci. 119, 3901–3903 (2006).
Barczyk, M., Carracedo, S. & Gullberg, D. Integrins. Cell Tissue Res. 339, 269–280 (2010).
Lei, L. et al. Endothelial expression of β1 integrin is required for embryonic vascular patterning and postnatal vascular remodeling. Mol. Cell Biol. 28, 794–802 (2008).
Carlson, T. R., Hu, H., Braren, R., Kim, Y. H. & Wang, R. A. Cell-autonomous requirement for β1 integrin in endothelial cell adhesion, migration and survival during angiogenesis in mice. Development 135, 2193–2202 (2008).
Tanjore, H., Zeisberg, E. M., Gerami-Naini, B. & Kalluri, R. Beta1 integrin expression on endothelial cells is required for angiogenesis but not for vasculogenesis. Dev. Dyn. 237, 75–82 (2008).
Zovein, A. C. et al. Beta1 integrin establishes endothelial cell polarity and arteriolar lumen formation via a Par3-dependent mechanism. Dev. Cell 18, 39–51 (2010).
Yamamoto, H. et al. Integrin β1 controls VE-cadherin localization and blood vessel stability. Nat. Commun. 6, 6429 (2015).
Zelzer, E. et al. Skeletal defects in VEGF(120/120) mice reveal multiple roles for VEGF in skeletogenesis. Development 129, 1893–1904 (2002).
Tammela, T. et al. VEGFR-3 controls tip to stalk conversion at vessel fusion sites by reinforcing Notch signalling. Nat. Cell Biol. 13, 1202–1213 (2011).
Benedito, R. et al. Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling. Nature 484, 110–114 (2012).
Lohela, M., Bry, M., Tammela, T. & Alitalo, K. VEGFs and receptors involved in angiogenesis versus lymphangiogenesis. Curr. Opin. Cell Biol. 21, 154–165 (2009).
Olsson, A. K., Dimberg, A., Kreuger, J. & Claesson-Welsh, L. VEGF receptor signalling—in control of vascular function. Nat. Rev. Mol. Cell Biol. 7, 359–371 (2006).
Zarkada, G., Heinolainen, K., Makinen, T., Kubota, Y. & Alitalo, K. VEGFR3 does not sustain retinal angiogenesis without VEGFR2. Proc. Natl Acad. Sci. USA 112, 761–766 (2015).
Nakayama, M. et al. Spatial regulation of VEGF receptor endocytosis in angiogenesis. Nat. Cell Biol. 15, 249–260 (2013).
Latonen, L., Taya, Y. & Laiho, M. UV-radiation induces dose-dependent regulation of p53 response and modulates p53-HDM2 interaction in human fibroblasts. Oncogene 20, 6784–6793 (2001).
Kikkawa, Y. & Miner, J. H. Review: Lutheran/B-CAM: a laminin receptor on red blood cells and in various tissues. Connect. Tissue Res. 46, 193–199 (2005).
Reznikoff, C. A., Brankow, D. W. & Heidelberger, C. Establishment and characterization of a cloned line of C3H mouse embryo cells sensitive to postconfluence inhibition of division. Cancer Res. 33, 3231–3238 (1973).
Liu, Q. et al. Genetic targeting of sprouting angiogenesis using Apln-CreER. Nat. Commun. 6, 6020 (2015).
Muzumdar, M. D., Tasic, B., Miyamichi, K., Li, L. & Luo, L. A global double-fluorescent Cre reporter mouse. Genesis 45, 593–605 (2007).
Luo, W., Friedman, M. S., Shedden, K., Hankenson, K. D. & Woolf, P. J. GAGE: generally applicable gene set enrichment for pathway analysis. BMC Bioinformatics 10, 161 (2009).
Yousif, L. F., Di Russo, J. & Sorokin, L. Laminin isoforms in endothelial and perivascular basement membranes. Cell Adh. Migr. 7, 101–110 (2013).
Wang, Y. et al. Ephrin-B2 controls VEGF-induced angiogenesis and lymphangiogenesis. Nature 465, 483–486 (2010).
Raghavan, S., Bauer, C., Mundschau, G., Li, Q. & Fuchs, E. Conditional ablation of β1 integrin in skin. Severe defects in epidermal proliferation, basement membrane formation, and hair follicle invagination. J. Cell Biol. 150, 1149–1160 (2000).
Miner, J. H., Cunningham, J. & Sanes, J. R. Roles for laminin in embryogenesis: exencephaly, syndactyly, and placentopathy in mice lacking the laminin α5 chain. J. Cell Biol. 143, 1713–1723 (1998).
Song, J. et al. Extracellular matrix of secondary lymphoid organs impacts on B-cell fate and survival. Proc. Natl Acad. Sci. USA 110, E2915–E2924 (2013).
Sorokin, L. M. et al. Developmental regulation of the laminin α5 chain suggests a role in epithelial and endothelial cell maturation. Dev. Biol. 189, 285–300 (1997).
Scoazec, J. Y., Racine, L., Couvelard, A., Flejou, J. F. & Feldmann, G. Endothelial cell heterogeneity in the normal human liver acinus: in situ immunohistochemical demonstration. Liver 14, 113–123 (1994).
Rajotte, D. et al. Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display. J. Clin. Invest. 102, 430–437 (1998).
Scott, R. P. & Quaggin, S. E. Review series: the cell biology of renal filtration. J. Cell Biol. 209, 199–210 (2015).
Molema, G. & Aird, W. C. Vascular heterogeneity in the kidney. Semin. Nephrol. 32, 145–155 (2012).
Palmer, T. D., Willhoite, A. R. & Gage, F. H. Vascular niche for adult hippocampal neurogenesis. J. Comp. Neurol. 425, 479–494 (2000).
Culver, J. C., Vadakkan, T. J. & Dickinson, M. E. A specialized microvascular domain in the mouse neural stem cell niche. PLoS One 8, e53546 (2013).
Gumbiner, B. M. Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84, 345–357 (1996).
Danen, E. H. & Sonnenberg, A. Integrins in regulation of tissue development and function. J. Pathol. 201, 632–641 (2003).
Felcht, M. et al. Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling. J. Clin. Invest. 122, 1991–2005 (2012).
Hakanpaa, L. et al. Endothelial destabilization by angiopoietin-2 via integrin β1 activation. Nat. Commun. 6, 5962 (2015).
Emre, Y. & Imhof, B. A. Matricellular protein CCN1/CYR61: a new player in inflammation and leukocyte trafficking. Semin. Immunopathol. 36, 253–259 (2014).
Ivaska, J. & Heino, J. Cooperation between integrins and growth factor receptors in signaling and endocytosis. Annu. Rev. Cell Dev. Biol. 27, 291–320 (2011).
Forlino, A., Cabral, W. A., Barnes, A. M. & Marini, J. C. New perspectives on osteogenesis imperfecta. Nat. Rev. Endocrinol. 7, 540–557 (2011).
Tosi, L. L. & Warman, M. L. Mechanistic and therapeutic insights gained from studying rare skeletal diseases. Bone 76, 67–75 (2015).
Tranquilli Leali, P. et al. Bone fragility: current reviews and clinical features. Clin. Cases Miner. Bone Metab. 6, 109–113 (2009).
Tian, X. et al. Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries. Cell Res. 23, 1075–1090 (2013).
Kisanuki, Y. Y. et al. Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo. Dev. Biol. 230, 230–242 (2001).
Thyboll, J. et al. Deletion of the laminin α4 chain leads to impaired microvessel maturation. Mol. Cell Biol. 22, 1194–1202 (2002).
Liaw, L. et al. Altered wound healing in mice lacking a functional osteopontin gene (spp1). J. Clin. Invest. 101, 1468–1478 (1998).
Kim, D. et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013).
Anders, S., Pyl, P. T. & Huber, W. HTSeq–a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169 (2015).
Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).
Wold, S., Esbensen, K. & Geladi, P. Principal component analysis. Chemometr. Intel. Lab. Syst. 2, 37–52 (1987).
Acknowledgements
We thank H.-W. Jeong for his support in RNA-seq experiments, K. Kato for experimental guidance, and M. Stehling for expert advice in flow cytometry experiments. The Max Planck Society, the University of Muenster, the Cells in Motion (CiM) graduate school, the DFG cluster of excellence ‘Cells in Motion’ (L.S., J.M.V. and R.H.A), and the European Research Council (AdG 339409 AngioBone) have supported this study.
Author information
Authors and Affiliations
Contributions
U.H.L., M.E.P., J.M.V. and R.H.A. designed the study. R.E.-G., A.S. and J.M.V. performed bioinformatics analyses, M.G.B. the two-photon microscopy, K.K.S. the MG132 inhibition experiments, J.M.K. the spheroid assays, and A.P.K. the ELISA experiments. A.P.K. also developed the FACS protocol for the isolation of bone ECs. J.D.R. and L.S. provided Lama4 and 5 mutant tissues, B.Z. the Apln-CreER mice. All other experiments were performed by U.H.L.; U.H.L., J.M.V. and R.H.A. wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Long bone development and vascularization.
(a) Emcn (red) immunostained embryonic hindlimb sections at the indicated developmental stages. Nuclei, Hoechst (blue). (b) Overview (left) and high magnification images of P10 wild-type femur stained for Emcn (red). Nuclei, Hoechst (blue). Lines mark metaphysis (mp, orange) with columnar vessels, diaphysis (dp, green) containing highly branched sinusoidal vessels, and the transition zone (tz, blue) interconnecting metaphyseal and diaphyseal vessels. (c) Tile scan confocal images of Osterix-immunostained (green) femoral sections at the indicated developmental stages.
Supplementary Figure 2 Gene expression analysis of the bone vasculature.
(a) MA-plots of differentially regulated genes in P6 bone EC subpopulations. The x-axis represents the mean normalized counts and the y-axis shows the log2 fold change between EC subtypes. Differentially regulated genes are represented by red colored points (FDR-adjusted P-value < 0.01 and absolute log2 fold change <1). Data points outside of the range of the y-axis are represented as triangles. (b) Experimental validation of differentially regulated genes by RT-qPCR. Pairwise comparison of gene expression by RT-qPCR and RNA-seq in DESeq2 between different conditions (E versus L, H versus L and E versus H). The correlation coefficients are ∼0.98, ∼0.93 and ∼0.90. Data represents mean ± s.e.m. (n = 3 for RNA-seq and n = 3 for qPCRs; n represents individual experiments). Statistics source data are shown in Supplementary Table 6 (c,d) Sections of wild-type femur at the indicated developmental stages stained for VEGFR2 (c, white) or VEGFR3 (d, white). Dashed lines mark area of high/low staining. (e,f) Immunostaining for VEGFR2 (e, green) or VEGFR3 (f, green) on femoral sections of 3-week-old wild-type mice after 3 h of vehicle (DMSO) or proteasome inhibitor (MG132) treatment. Nuclei, DAPI (blue). Note strongly increased VEGFR2 and VEGFR3 signals in metaphyseal ECs but not in the diaphysis after MG132.
Supplementary Figure 3 Bone vessel subtypes and their osteogenic potential.
(a) Graph illustrating expression of selected genes in type E endothelium relative to type L or type H ECs, respectively. Data based on RPKM values obtained from RNA-seq of sorted cells at P6. Data represents mean ± s.e.m. (n = 3). Statistics source data are shown in Supplementary Table 6 (b) Longitudinal section of E16.5 femur stained for Emcn (red) and Caveolin 1 (green). White arrowheads indicate double positive vessels. (c) Transverse section of P6 mouse femur stained for Caveolin 1 (green). Nuclei, Hoechst (blue). White arrowheads mark Caveolin 1-positive vessels in endosteum and compact bone. (d) Light microscopic appearance of spheroids at day 1 and day 7 of culture. Note disaggregation of cells in the presence of type L ECs. (e) Visualization of DiI-labeled bone ECs (arrowheads) in C3H10T1/2 spheroids day 1 and day 7 of culture. Remodelling of C3H10T1/2 spheroids (formation of internal cavities) was induced by type H and E ECs but not by type L ECs or in absence of ECs. Nuclei, DAPI (blue).
Supplementary Figure 4 Hierarchy of EC subtypes and cell-matrix interactions.
(a–c) Overview pictures of femoral sections from Apln-CreER R26-mT/mG mice treated with 4-OHT at E15.5 (a), P0 (b) or P6 (c) and analysis at the indicated stages. Note colocalization of GFP + (green) cells and Emcn + (red) capillaries. Insets in the rightmost panel of (a) show higher magnifications of metaphysis (top) and diaphysis (bottom) at P6. Arrows in (c) indicate expansion of GFP + vessels into the marrow cavity. (d) Expression of selected matrix molecule transcripts identified by RNA sequencing of sorted P6 type L (green), type H (orange) and type E (blue) ECs. Data represents mean ± s.e.m. (n = 3 independent experiments). (e) Expression (RNA-seq) of transcripts encoding integrin α subunits in type L, type H and type E ECs. Data represents mean ± s.e.m. (n = 3 independent experiments). (f) RNA-seq results for Itgb1 transcript expression in bone ECs at P6. Data represents mean ± s.e.m. (n = 3 independent experiements). (g) RT-qPCR analysis of Itgb1 expression in P21 type L (green) and type H (orange) ECs. Data represents mean ± s.e.m. (n = 3 independent experiments). Statistics source data are shown in Supplementary Table 6 (h) Confocal images showing Emcn (red) and integrin β1 (white) protein expression in the P21 femoral metaphysis. Arrowheads mark endothelial integrin β1 signal. (i) Confocal images of integrin β1 (white), Osterix (green) and CD45 (red) staining in wild-type P21 metaphysis and diaphysis. (j) Maximum intensity projection confocal images of 3 week-old femoral metaphysis immunostained for Emcn (red) and Laminin α4, Laminin α5, Fibronectin, Collagen 1 or Osteopontin (white). Arrowheads mark endothelial expression of matrix proteins.
Supplementary Figure 5 Bone phenotype of EC-specific Itgb1 mutant mice.
(a) Scheme showing the time points of tamoxifen administration and analysis for the Itgb1iΔEC mutant mice. (b) Tile scan overview and high magnification images of femur from P21 Cdh5-CreERT2 R26-mT/mG mice treated with tamoxifen from P10 to P12. GFP signal is strictly confined to endothelium. ECs, Emcn (red). (c) Quantitative RT-qPCR analysis of Itgb1 expression in sorted bone ECs from P21 Itgb1iΔEC mutants and Cre-negative littermate controls (P < 0.001, two-tailed unpaired t-test). Data represents mean ± s.e.m. (n = 6 mice per group). (d) Picture of 3 week-old Itgb1iΔEC mutant and control littermate. Chart shows reduction of Itgb1iΔEC mutant body weight relative to control (P < 0.001, two-tailed unpaired t-test). Data represents mean ± s.e.m. (n = 10 mice per group). (e) Freshly dissected P21 Itgb1iΔEC and littermate control femurs. Ruler indicates length in centimeters. Chart shows length of femurs in millimeters (P = 0.028, two-tailed unpaired t-test). Data represents mean ± s.e.m. (n = 6 mice per group). (f) Tile scan confocal images of 3 week-old Itgb1iΔEC and control femurs stained for Emcn (red). (g) Confocal images of αSMA (red) staining in Itgb1iΔEC mice and littermate controls. Dashed line indicates border to adjacent growth plate (top). (h) Flow cytometry analysis of type L ECs per total ECs. Data represents mean ± s.e.m. (n = 16), (P < 0.001, two-tailed unpaired t-test). (i) Quantitative analysis of proliferating (EdU+) type L ECs per total type L ECs (P = 0.558, two-tailed unpaired t-test). Data represents mean ± s.e.m. (n = 7 mice per group). (j,k) Maximum intensity projections of Itgb1iΔEC and Cre- littermate controls femoral sections stained for Pimonidazole (green) (j) or phospho-ERK1/2 (pERJK1/2, green) (k). Nuclei, Hoechst (blue). Note reduction of the hypoxia-free zone (arrows) and of pERK1/2 signal in the Itgb1iΔEC metaphysis.
Supplementary Figure 6 Analysis of EC-specific Itgb1 mutant bone.
(a,b) Maximum intensity projections of Collagen 1 (a, white) and Osteopontin (b, white) immunostainings in 3 week-old Itgb1iΔEC and control femur. (c) 2-photon-generated second harmonic generation signal (white) in femoral sections of 3 week-old Itgb1iΔEC mice and littermate controls. (d,e) Immunostaining for Calcitonin receptor (d, green) or TRAP (e, red) in 3 week-old Itgb1iΔEC and control femur. Nuclei, Hoechst (blue). (f) Analysis of serum parathyroid hormone (PTH) and calcitonin levels in Itgb1iΔEC mutants and Cre- littermate controls. Data represent mean ± s.e.m (n = 5 mice per group) (P = 0.80 for PTH and P = 0.53 for calcitonin, two-tailed unpaired t-test).
Supplementary Figure 7 Apln-CreER-controlled Itgb1 mutants.
(a) Scheme showing the time points of tamoxifen administration and analysis for Apln-CreER R26-mT/mG and Itgb1iΔApln mice. (b) Overview of P10 femur of Apln-CreER R26-mT/mG mice without tamoxifen-induced recombination. Note absence of GFP+ cells (green). Nuclei, Hoechst (blue). (c,d) Overview and high magnification confocal images of GFP signal (green) and Emcn staining (red) in femur, metaphysis and endosteum of Apln-CreER R26-mT/mG mice P13 (c) and P21 (d). Nuclei, Hoechst (blue). (e) Body weight of Itgb1iΔApln mutants relative to Cre- littermate controls. Data represents mean ± s.e.m. (n = 9) (P < 0.001, unpaired two-tailed t-test). (f) Quantitation of Itgb1iΔApln and Cre- littermate control femoral length. Data represents mean ± s.e.m. (n = 9) (P < 0.001, unpaired two-tailed t-test). (g) Representative 3D reconstruction from μCT measurements of 3 week-old Itgb1iΔApln and littermate control tibial metaphysis. (h) 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, and trabecular separation in millimeters. Data represent mean ± s.e.m. (n = 6 controls and 4 mutants), (P-values determined by two-tailed unpaired t-test). Statistics source data are shown in Supplementary Table 6 (i) Confocal images of TRAP immunosignal (green) in 3 week-old control or Itgb1iΔApln femur. Nuclei, Hoechst (blue).
Supplementary Figure 8 Characterization of matrix molecule mutants.
(a) High magnification confocal images of Emcn (red) staining in femoral metaphysis of Lama4KO and wild-type control mice. Dashed line indicates border to adjacent growth plate. (b,d,e) Confocal images of VEGFR3 immunosignal (white) in 3 week-old control and Lama4KO (b), Spp1KO (d) or Lama5ΔEC (e) femur. Dashed lines indicate upper/lower borders of metaphysis containing columnar vessels with low VEGFR3 signal. (c) Osterix (green) stained sections of 3 week-old wild-type control and Lama4KO femur. Dashed lines indicate upper/lower borders of trabecular region. (f) 2-photon-generated second harmonic generation signal (white) in femoral sections of 3 week-old Lama5ΔEC mice and littermate controls. (g) Quantitation of body weight of Lama5ΔEC mutant mice relative to Cre- littermate controls. Data represents mean ± s.e.m. (n = 13) (P = 0.46, unpaired two-tailed t-test). (h) Bar chart illustrating femoral length of Lama5ΔEC mutant mice and Cre- littermate controls. Data represents mean ± s.e.m. (n = 13) (P = 0.04, unpaired two-tailed t-test). (i) Representative 3D reconstruction from μCT measurements of 3 week-old Lama5ΔEC and littermate control tibial metaphysis. (j) Bone parameters measured in μCT analyses: bone volume/total volume (BV/TV) in percentage, trabeculae number in 1 per millimeter, trabecular thickness in millimeters, and trabecular separation in millimeters. Data represent mean ± s.e.m. (n = 4 controls and 3 mutants), (P-values determined by two-tailed unpaired t-test). Statistics source data are shown in Supplementary Table 6
Supplementary information
Supplementary Information
Supplementary Information (PDF 12146 kb)
Supplementary Table 1
Supplementary Information (XLSX 14 kb)
Supplementary Table 2
Supplementary Information (XLSX 14 kb)
Supplementary Table 3
Supplementary Information (XLSX 403 kb)
Supplementary Table 4
Supplementary Information (XLSX 556 kb)
Supplementary Table 5
Supplementary Information (XLSX 198 kb)
Supplementary Table 6
Supplementary Information (XLSX 120 kb)
Rights and permissions
About this article
Cite this article
Langen, U., Pitulescu, M., Kim, J. et al. Cell–matrix signals specify bone endothelial cells during developmental osteogenesis. Nat Cell Biol 19, 189–201 (2017). https://doi.org/10.1038/ncb3476
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ncb3476
This article is cited by
-
Hypoxia preconditioning of adipose stem cell-derived exosomes loaded in gelatin methacryloyl (GelMA) promote type H angiogenesis and osteoporotic fracture repair
Journal of Nanobiotechnology (2024)
-
Endothelial SMAD1/5 signaling couples angiogenesis to osteogenesis in juvenile bone
Communications Biology (2024)
-
The spatiotemporal heterogeneity of the biophysical microenvironment during hematopoietic stem cell development: from embryo to adult
Stem Cell Research & Therapy (2023)
-
Cellular niches for hematopoietic stem cells in bone marrow under normal and malignant conditions
Inflammation and Regeneration (2023)
-
Application of the neuropeptide NPVF to enhance angiogenesis and osteogenesis in bone regeneration
Communications Biology (2023)
