Extended Data Fig. 7: Effect of DACH1 overexpression in endothelial cells.
From: Specialized post-arterial capillaries facilitate adult bone remodelling
a, b. Confocal images of femur sections immunostained for EMCN (red) together with OSTERIX (OSX, green) (a) or ATP6V1B1/B2 (green) (b). Scale bars, 100μm. c. Quantitation of OSX+ osteoblast-lineage cells and ATP6V1B1/B2+ osteoclasts in Dach1OE and control femur (n = 3 in each group). Mean ± SEM. P values, unpaired two-tailed test with Welch’s correction. d-f. Injection scheme and time points of imaging after labelling of Dach1OE and control with Calcein Green (CG) and Alizarin Red (AZ) (d) and tile scan imaging of sectioned femur from P30 Dach1OE and control after double labelling (e). Scale bars, 500μm. Bottom panels in (e) show stained trabecular bone. Scale bars, 20μm. f. Quantitation showing extent of bone surface actively mineralizing as mineralizing surface/bone surface (MS/BS) in percentage, mineral apposition rate (MAR) as per micrometre/day, and bone formation rate (BFR) (n=3 mice per group). Mean ± SEM. P values, unpaired two-tailed t-test with Welch’s correction for MS/BS, MAR-trabecular bone and BFR. g. Maximum-intensity projections showing prominent HIF1α+ immunostaining (yellow arrowheads) in 6-week-old Sp7-mCherry+ trabecular osteoblasts, whereas HIF1α+ is strongly decreased after emergence of EMCN+ VEGFR3− type R vessels at 12 weeks. Scale bars, 50 μm. h. Maximum-intensity projections of 12-week-old femurs showing immunostaining for hypoxia-related markers in relation to EMCN+ VEGFR3− type R vessels. Shown are immunostaining for Glucose-6-Phosphate Isomerase (GPI), 5′-nucleotidase/CD73 and Haem Oxygenase-1 (HMOX1)-expressing macrophages (yellow arrowheads), all of which are elevated near juvenile trabecular bone relative to the equivalent region in adult. White arrowheads mark EMCN+ VEGFR3− type R vessels. i, j. UMAP plots of bone ECs (n=18383) with colour-coded subclusters (f), namely sinusoidal bmECs, metaphyseal mpECs, arterial aECs, remodelling rECs, and proliferating pECs. UMAP distribution of Dach1OE and control ECs, as indicated by colour (g). k. Bar plots showing proportion of cells in Dach1OE and control samples. l. UMAP plots comparing Dach1 expression in Dach1OE (bottom) and control (top) EC subclusters. m. Heatmap of differentially expressed genes between EC subclusters. n. Heatmap of differentially expressed genes related to hypoxia and, tissue oxygenation in Dach1OE and control bone ECs. o. Confocal images showing reduced HIF1α immunostaining (white arrowheads) in Dach1OE metaphysis and around trabecular bone relative to littermate control. Scale bars, 50μm. Results in panels i, j, m, and n show the integrated mutant and control scRNA-seq data, whereas panels k and l show separated samples derived from the integrated data.
