Fig 2: (A) TRAP staining of raw264.7 and osteoclasts. (B) Cellular immunofluorescence of Netrin-1, Unc5B and DCC on osteoclasts. Arrow: the expression of Netrin-1 and Unc5B on the membrane of the osteoclasts.

Fig 3: Wnt /ß-catenin signaling regulation by Unc5B-blocking antibodies.a Immunofluorescence staining of Human IgG, Claudin-5 and PLVAP and confocal imaging on brain sections from mice i.v. injected with anti-Unc5B-3 (10 mg/kg) for 1 h, 8 h or 24 h, reproduced on n = 3 untreated brains, n = 4 anti-Unc5B-3 treated brains for 1 h, n = 3 anti-Unc5B-3 treated brains for 8 and n = 3 anti-Unc5B-3 treated brains for 24 h. b CTRL IgG or Unc5B immunoprecipitation of brain protein extracts from mice i.v. injected with anti-Unc5B-3 and protein quantification (c), n = 3 control IgG treated brains, n = 4 anti-Unc5B-3 treated brains for 1 h and n = 3 anti-Unc5B-3 treated brains for 8 and n = 3 anti-Unc5B-3 treated brains for 24 h. Each dot represents one mouse. One control mouse was set as 1. All data are shown as mean ± SEM. NS non-significant. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups. Source data are provided as a Source Data file.

Fig 4: Netrin-1 controls BBB integrity.a, b Quantification of cadaverine content in P67 brains, 30 min after i.v. cadaverine injection. Ntn1 gene deletion was induced by tamoxifen injection between P60 and P64, n = 4 Ntn1fl/fl, n = 4 Ntn1iko, n = 5 Robo4+l+ and n = 4 Robo4+l- brains. Each dot represents one mouse. Western blot (c) and quantification (d) of brain protein extracts, n = 7 Ntn1fl/fl and n = 10 Ntn1iko brains. Each dot represents one mouse. One control mouse was set as 1. Western blot (e) and quantification (f) of mouse brain ECs treated with scrambled CTRL or Unc5B siRNA for 48 h and treated with recombinant mouse Netrin-1 (500 ng/ml) or not (-) for the indicated times. Each dot represents one independent experiment, n = 4 independent experiment. g CTRL IgG or Unc5B immunoprecipitation of brain protein extracts and Western blot for LRP6. h quantification of LRP6 pulldown with Unc5B, n = 3 Ntn1fl/fl and n = 6 Ntn1iko brains. Each dot represents one mouse. One control mouse was set as 1. Western blot (i) and quantification (j) of mouse brain ECs treated with recombinant mouse Netrin-1 (500 ng/ml) or not (-) for the indicated times. Each dot represents one independent experiment, n = 4 independent experiment. Western blot (k) and quantification (l) of mouse brain ECs treated with CTRL DMSO or FAK inhibitor (5 uM) for 30 min followed by recombinant mouse Netrin-1 treatment (500 ng/ml) or not (-) for 1 h. Each dot represents one independent experiment, n = 3 independent experiment (pFAK-Y397(/ß-actin): exact p-value between DMSO + Netrin1 (1 h) and FAKi + Netrin1 (1 h) = 0.000031). All data are shown as mean ± SEM. NS non-significant. Two-sided Mann–Whitney U test was performed for statistical analysis between two groups. ANOVA followed by Bonferroni’s multiple comparisons test was performed for statistical analysis between multiple groups. Source data are provided as a Source Data file.

Fig 5: Endothelial Unc5B controls BBB integrity.a Unc5B gene deletion strategy using tamoxifen injection in adult mice. b Immunofluorescence staining with the indicated antibodies and confocal imaging of adult brain sections at P67, 30 min after i.v cadaverine injection and reproduced on n = 4 Unc5Bfl/fl and n = 5 Unc5BiECko brains. c Quantification of dye content in brains and peripheral organs at P67, 30 min after i.v. cadaverine injection, n = 11 Unc5Bfl/fl and n = 10 Unc5BiECko brains (exact p-value = 0.0000397); n = 5 Unc5Bfl/fl and n = 10 Unc5BiECko lungs, kidneys and hearts; n = 6 Unc5Bfl/fl and n = 5 Unc5BiECko GI tracts. Each dot represents one mouse. d Immunofluorescence staining with the indicated antibodies and confocal imaging of adult brain sections, reproduced on n = 4 Unc5Bfl/fl and n = 4 Unc5BiECko brains. e Quantification of endomucin+ vascular density. Each dot represents the mean of several images, n = 6 Unc5Bfl/fl and n = 7 Unc5BiECko brains. One control mouse value was set as 1. f Tile-scan confocal imaging of adult Unc5BiECko brain sections at P67, 30 min after i.v cadaverine injection and reproduced on n = 3 Unc5Bfl/fl and n = 3 Unc5BiECko brains. g Immunofluorescence staining of Unc5B and tile-scan confocal imaging of brain sections, reproduced on n = 4 mice. Boxes show higher magnifications of cortical areas. h Immunofluorescence staining with the indicated antibodies and confocal imaging of adult brain sections, reproduced on n = 4 Unc5Bfl/fl and n = 4 Unc5BiECko brains. Arrowhead: Unc5B+/CD13+ pericyte. i, j Unc5Bfl/fl was crossed with PDGFRßCreERT2 and BBB permeability was assessed at P67, 30 min after i.v. cadaverine injection, n = 6 Unc5Bfl/fl and n = 10 Unc5BiPCko brains. Each dot represents one mouse. All data are shown as mean ± SEM. NS non-significant, RSP Retrosplenial cortex, PTL Posterior parietal association areas, SSp Primary somatosensory cortex, PIR Piriform cortex, HI Hippocampus, HY Hypothalamus, TH Thalamus, ST Striatum, CB Cerebellum, M Medulla. Two-sided Mann–Whitney U test was performed for statistical analysis. Source data are provided as a Source Data file.