Fig. 3: Type S vessels are highly plastic and crucial for osteogenesis.
From: Spatial heterogeneity of bone marrow endothelial cells unveils a distinct subtype in the epiphysis
a, Protocol for 4OHT injection in neonates. b–d, Femur sections (b) and whole-mount Alizarin Red staining (control (c) and Vegfr2iΔEC (d)) at P11. Vegfr2iΔEC mice show severely impaired angiogenesis (open arrowheads) and osteogenesis (arrows) in the epiphysis, although metaphyseal ossification is largely intact (asterisks). e–j, Immunohistochemistry of femur sections stained with Osx and DAPI (e,f) or Runx2 and Emcn (g–j). Panels i and j how the high magnification views of the area indicated in g and h. Panels e, g and i are from control, and panels f, h and j are from Vegfr2iΔEC mice. Osx+ and Runx2+ cells are found around vessels, their numbers are reduced (arrows) and they are detected only around surviving vessels (arrowhead) in Vegfr2iΔEC mice. k, Quantification of the SOC area (n = 3, biologically independent experiments) and Osx+ cells (n = 4, biologically independent experiments) in the epiphysis. l, Protocol for 4OHT injection in neonates. m–o, Femur sections (m) and whole-mount Alizarin Red staining (control (n) and Dll4iΔEC (o)) at P11. Dll4iΔEC mice have an increased vessel density but a reduced vascularized area (open arrowheads) and reduced osteogenesis (arrows) in the epiphysis, although metaphyseal ossification is largely intact (asterisks). p–u, Immunohistochemistry of femur sections stained with Osx and DAPI (p,q) or Runx2 and Emcn (r–u). Panels t and u show the high magnification views of the area indicated in r and s. Panels p, r and t are from control, and panels q, s and u are from Dll4iΔEC mice. Osx+ and Runx2+ cells are found around vessels and their numbers are reduced (arrows) in Dll4iΔEC mice. v, Quantification of the SOC area (n = 3, biologically independent experiments) and Osx+ cells (control: n = 4, Dll4iΔEC: n = 6, biologically independent experiments) in the epiphysis. Data are presented as the mean ± s.d. ***P < 0.001; two-tailed Student’s t-test. Scale bars, 1 mm (c–h and n–s); 200 µm (b,i,j,m,t,u). The comparisons between the averages of two groups were evaluated using the two-tailed Student’s t-test.
