Extended Data Figure 9: Endothelial-cell-dependent regulation of growth plate and bone. | Nature

Extended Data Figure 9: Endothelial-cell-dependent regulation of growth plate and bone.

From: Endothelial Notch activity promotes angiogenesis and osteogenesis in bone

Extended Data Figure 9

ac, Quantitative analysis of Fbxw7iΔEC mutant growth plates (a), proliferating zones (b) and maturation/hypertrophy zones (c) relative to control littermates (n = 4 mice from 3 independent litters). Error bars, ± s.e.m. P values, two-tailed unpaired t-test. d, e, Quantitative PCR analysis of Vegfa transcripts level in the bones (without marrow cells) of Notch mutants (RbpjiΔEC and Fbxw7iΔEC) and respective littermate controls (n = 4 mice from 3 independent litters). Error bars, ± s.e.m. P values, two-tailed unpaired t-test. f, g, Maximum intensity projection of stained Fbxw7iΔEC and littermate control tibiae. Note slight reduction of mutant growth plate and maturation/hypertrophy zone (MHZ). EdU (red) labelling (f) marks mitotic chondrocytes in the proliferating zone (PZ). Sox9 (green) immunosignals (g) label maturing and hypertrophic chondrocytes. Dashed lines mark borders of PZ and MHZ. Nuclei, DAPI (blue). h, Maximum intensity projection of tile scanned tibial section of Col1a1-CreERT2 ROSA26-mT/mG double transgenic mice. GFP expression (green) indicates Cre-mediated recombination. i, Maximum intensity projections showing overlapping GFP (green) and Col1α immunostaining (red). j, Maximum intensity projections showing osteoblasts expressing GFP (green). GFP signals were not seen in Emcn immunostained endothelial cells (red). k, Normal arrangement of CD31-stained endothelial columns (C) and arches (A) after Col1a1-CreERT2-mediated inactivation of Rbpj in cells of the osteoblast lineage (RbpjiΔOB). Nuclei, DAPI (blue). l, m, Maximum intensity projections of tile-scanned tibia sections after anti-Emcn (red) immunostaining. Nuclei, DAPI (blue). Note profound disorganization of the Dll4iΔEC metaphyseal vasculature (l) and restoration by simultaneous endothelial-cell-specific overexpression of active Notch in Dll4iΔEC/NICDiOE-EC double mutant mice (m). n, Tile scan confocal images of osteopontin (Opn, green) stained control and Dll4iΔEC/NICDiOE-EC tibiae showing that endothelial NICD expression can rescue trabecular bone defects in the Dll4iΔEC background (see Fig. 2c for comparison). o, Organization of the Emcn-stained metaphyseal vasculature and arrangement of osteoblasts (Osx) in tibia section from 4-week-old control, Dll4iΔEC, NICDiOE-EC, and Dll4iΔEC/NICDiOE-EC mice, as indicated. Nuclei, DAPI (blue). p, Sox9 immunostaining (green) in the epiphyseal region of 4-week-old tibia showing normalized maturing and hypertrophic chondrocytes zone (MHZ) in the growth plate of Dll4iΔEC/NICDiOE-EC mice. Dashed lines mark borders of MHZ. Nuclei, DAPI (blue).

Back to article page