a, Confocal images showing immunostaining for mesenchymal lineage markers Osx (green) and Runx2 (red) in Fbxw7iΔEC and littermate control tibiae, as indicated. Nuclei, DAPI (blue). Note reduction of Osx+ cells and increased Runx2+ population after targeting of Fbxw7 in endothelial cells. b, Analysis of differentiation capacity of primary mesenchymal cells isolated from RbpjiΔEC or control bone. Alizarin Red S staining after 10 days in vitro (10 div) showed that mesenchymal cells isolated from endothelial-cell-specific RbpjiΔEC mutants generated mineral nodules (red arrows) prematurely in comparison to controls. c, d, Maximum intensity projection of stained RbpjiΔEC and control tibiae. Labelling of hypoxic cells with pimonidazole (Pimo, green) showed no appreciable differences between control and RbpjiΔEC samples (c). Likewise, anti-HIF1α immunostaining (green) was comparable in the control and RbpjiΔEC metaphysis (d). Endothelial cells (Emcn antibody staining, red); nuclei, DAPI (blue). e, Representative confocal images showing that endothelial-cell-specific gene targeting of Jagged1 (Jag1) or Delta-like 1 (Dll1) did not lead to appreciable alterations in Jag1iΔEC or Dll1iΔEC mutant metaphyseal vasculature at 4 weeks. Dashed lines indicate vessel columns (C) and distal arches (A). Endothelial cells, CD31 (red); nuclei, DAPI (blue). f, Smaller size of freshly isolated femurs from 4 week-old Dll4iΔEC mutants relative to littermate controls. g, Disrupted organization of vessel arches (A) and columns (C) in the Dll4iΔEC metaphyseal vasculature. Endothelial cells, CD31 (red); nuclei, DAPI (blue). h, Quantitative analysis of size of chondrocyte proliferating zone in littermate control and RbpjiΔEC mutants (n = 6 mice from 4 independent litters). Error bars, ± s.e.m. P values, two-tailed unpaired t-test. i, j, Quantification of Sox9 (i) and VEGF-A (j) fluorescence intensity in immunostained sections of RbpjiΔEC mutants showing decreased signals in mutant growth plates (n = 4 mice from 3 independent litters). Error bars, ± s.e.m. P values, two-tailed unpaired t-test. k, Maximum intensity projection of stained RbpjiΔEC and littermate control tibiae showing decreased VEGF-A immunosignals.