a, Maximum intensity projections of Emcn (red) and CD31 (green) immunostained sections of 4-week-old tibia showing increase in CD31+ (type H) vessels in Fbxw7iΔEC mice. Mutants also displayed numerous small CD31+ Endomucin− arterioles (white arrows), which were associated with αSMA+ cells (blue arrows in bottom panels). b–d, Confocal images showing 4-week-old RbpjiΔEC or Fbxw7iΔEC mutant, or corresponding littermate control metaphysis after immunostaining for different VEGF receptors. VEGFR2 was highest on control arches, bulbs and arteries and staining was strongly reduced in the RbpjiΔEC metaphysis (b). VEGFR3 immunostaining decorated arches and bulb protrusion but was absent in the arteries. Staining was reduced in RbpjiΔEC mutants but enhanced in Fbxw7iΔEC vessels (c). Expression of VEGFR1 was predominantly found on perivascular mesenchymal and osteoprogenitor cells. Expression in these populations was not appreciably altered (d). e, f, qPCR analysis of sorted endothelial cells from Notch loss-of-function (RbpjiΔEC, e) and gain-of-function (Fbxw7iΔEC, f) mice. In endothelial cells, Notch positively regulated transcripts for the receptors VEGFR2 (Kdr), VEGFR3 (Flt4) and membrane-anchored VEGFR1 (mFlt1). In contrast, expression of soluble Flt1 (sFlt1), a known antagonist of VEGF signalling, was increased in RbpjiΔEC endothelial cells and significantly reduced in Fbxw7iΔEC cells (n = 4 mice from 4 independent litters). Error bars, ± s.e.m. P values, two-tailed unpaired t-test. g, Increased formation of CD31+ (red) vessels in the metaphysis of 4-week-old Notch gain-of-function mice after endothelial-cell-specific overexpression of active Notch (NICDiOE-EC). Nuclei, blue (DAPI). h, Confocal images showing extensive formation of CD31+ (green) Emcn− (red) arterioles (arrows) in the NICDiOE-EC metaphysis. Nuclei, DAPI (blue).