Extended Data Fig. 8: Molecular signalling and metabolic regulation of rECs.
From: Specialized post-arterial capillaries facilitate adult bone remodelling
a. Violin plots showing selected genes related to reduction of bmEC markers and increased arterialization in Dach1OE and control. b. Heatmap of differentially expressed genes related to Notch signalling in Dach1OE and control bone ECs. c. Representative confocal images showing increased Delta-like 4 (DLL4) (green, white arrowheads) expression in vessels around Dach1OE trabecular bone (TB). Scale bars, 100μm. d. Scheme of tamoxifen-induced (TMX) EC-specific Dll4 inactivation with Cdh5-CreERT2 line. e, f, Tile scan confocal images of EMCN+ VEGFR3− ECs (white arrowheads) (e) and CAV1+ aECs (green arrowheads) (f) in 12-week-old Dll4iΔEC loss-of-function and control femur. Trabecular bone (TB) is indicated. Scale bars, 500μm. g, Quantitation of EMCN+ VEGFR3− vessel density (left), and number of CAV1+ arteries (right) in 12-week-old Dll4iΔEC and control femur (n =3 female mice per group). Mean ± SEM. P values, unpaired two-tailed t-test. h. Heatmap showing differentially expressed transcripts for secreted factors in bone EC subpopulations. i, j, Representative images (i) and quantitation (j) of calcified nodules in human mesenchymal stem cells (HMSC) cultured in osteogenic differentiation medium (ODM) supplemented with 100ng/ml Complement C1q and Tumour Necrosis Factor-Related Protein 9 (CTRP9), Neurotrophin 3 (NTF3), Platelet-Derived Growth Factor D (PDGFD), or Semaphorin 7A (SEMA7A) (n=3 independent experiments for each group). Scale bars, 100μm. Graph shows quantitation of absorbance at 405nm. Mean ± SEM. P values, ordinary one-way ANOVA and plotted with Dunnett’s multiple comparisons test. k, l, Representative images (k) and quantitation (m) of multinucleated TRAP+ osteoclasts generated from bone marrow-derived monocytes/macrophages treated with RANKL in osteoclast medium (OCM) supplemented with 100ng/ml CTRP9, NTF3, PDGFD, or SEMA7A n=8 (4 independent experiments) for each group. Scale bars, 500μm. Graph shows quantitation of TRAP+ multinucleated cells between OCM and treated conditions. Mean ± SEM. P values, ordinary one-way ANOVA and plotted with Dunnett’s multiple comparisons test. Panels a and b show separated conditions derived from the integrated mutant and control data, whereas panel h is based on the integrated scRNA-seq data.
