Fig. 4: Pericyte-derived nodal is required for brain vascular development.
From: Pericytes are organ-specific regulators of tissue morphogenesis
a Tile scan of coronal sections through P12 NodaliPCKO and littermate control brain cortex immunostained for ICAM2 (green). b Confocal images of ICAM2+ (green) and ERG+ (red) endothelial cells in NodaliPCKO and littermate control brain. c Graph showing the ICAM2-stained vascular density. Data represent mean ± s.e.m. (n = 8 mice per group); P-values, unpaired two-tailed Student t-test. Maximum intensity projections of P12 NodaliPCKO and littermate control brain sections. Images show PDGFRβ+ pericytes (gray) and ERG+ (red) ECs (d), and EdU+ (gray) proliferating cells together with ICAM2+ (green) ECs (e). Graphs showing the ratio of ERG+ EC nuclei to total cells (f), ratio of PDGFRβ+ cells to total cells (g), ratio of ERG+ cells to PDGFRβ+ cells (h), and the number of EdU+ ECs per area (283 × 283 × 22 µm) in P12 NodaliPCKO and littermate control brain cortex (i). Data represent mean ± s.e.m. (n = 6 in f–i); P-values, unpaired two-tailed Student t-test. j Western blot analysis of total and phosphorylated SMAD2 (p-SMAD2) in lysates of mouse brain endothelial cells (bEnd.3) treated with recombinant human Nodal (rhNodal) and SB431542 inhibitor, as indicated. Shown is a representative blot of three independent experiments. Molecular weight marker (kDa) is indicated. β-Actin is shown as a loading control. Quantitation of p-SMAD2/SMAD2 ratio is shown in the graph on the right. Data represent mean ± s.e.m. (n = 3 independent experiments); P-values, one-way ANOVA with Tukey’s test. k Confocal images of VE-cadherin+ (CDH5, green) and EdU+ (white) bEnd.3 cells treated with rhNodal in the presence/absence of SB431542 inhibitor. l Bright field images showing that rhNODAL increases bEnd.3 cell migration in vitro in a scratch wound assay, which is blocked by SB431542. Left column shows the start of the assay (0 h) and red lines indicate the edges of scratch wounds, whereas images on the right are taken after 26 h. Graphs showing the impact of rhNodal on bEnd.3 cell proliferation (m) and migration (n). Data represent mean ± s.e.m. (n = 6); P-values, one-way ANOVA with Tukey’s test. Source data are provided as a Source Data file.
