Figure 4.

Slit3^−/− mice have reduced skeletal vasculature and bone mass in vivo. (a–d) Representative images (a, c) and relative quantification of tube branch numbers (b, d) in a Matrigel tube formation assay with EPOCs. n = 5 per group. Scale bars, 200μm. (c, d) Where indicated, a SLIT3-neutralizing antibody or isotype control was added. Scale bars, 200μm. (e, f) Representative confocal images of CD31 or EMCN (red) and DAPI (blue) immunostained sections (e) and relative quantification (f) of CD31-postive or EMCN-positive vessel area in the BM cavity of the femurs from male Slit3^+/+ and Slit3^−/− mice at 2-weeks of age. n = 4 per group. Growth plate and cortical bone are marked with a dashed line. Scale bars, 300μm. (g, h) Representative confocal images (n = 3 total images per group) of CD31 (green) and EMCN (red) immunostained bone sections (g) flow cytometry dot plots (h, left), and relative frequency of CD31^hiEMCN^hi endothelial cells (h, right) of the femur in 2 week-old Shn3^+/+ Slit3^+/+ (n = 5), Shn3^+/+ Slit3^−/− (n = 3), Shn3^−/− Slit3^+/+ (n = 5) and Shn3^−/− Slit3^−/− mice (n = 5). Growth plate is marked with a dashed line. Scale bars, 100μm. (i) Representative histological images (left) and relative BV/TV (middle) of the L3 vertebrae (Slit3^+/+ = 9, Slit3^−/− = 7) and cortical thickness (right) of distal femoral metaphyseal regions (Slit3^+/+ = 7, Slit3^−/− = 6) at 6-weeks of age. Scale bars, 500μm. (j) Representative μCT images of the trabecular bone in the distal femur metaphysis (left) and relative BV/TV analysis of 6 week-old Shn3^+/+ Slit3^+/+(n = 7), Shn3^+/+ Slit3^−/−(n = 7), Shn3^−/− Slit3^+/+(n = 7) and Shn3^−/− Slit3^−/− mice (n = 6) (right). Scale bar, 1mm. (k) Representative μCT images of the trabecular bone in the distal femur metaphysis (left) and relative trabecular BV/TV analysis (middle) and cortical bone thickness in the femoral midshaft from male Slit3^+/+ mice (n = 8) and Slit3^−/− mice (n = 5) at 12-weeks of age. Scale bars, 1mm. (l, m) Representative images of calcein labelling (I) and relative histomorphometric quantification of MAR and BFR/BS in 6 week-old Shn3^+/+ Slit3^+/+(n = 5), Shn3^+/+ Slit3^−/−(n = 6), Shn3^−/− Slit3^+/+(n = 4) and Shn3^−/− Slit3^−/− male mice (n = 4). Scale bars, 100μm. Values represent mean ± s.e.m.; *P < 0.05, **P < 0.01 and ***P < 0.001 by an unpaired two-tailed Student’s t-test or one-way ANOVA followed by a Dunnett’s test in all panels.