Fig 2: Representative images showing the bifurcation of the aorta and the innominate artery. No pericytic markers were found in atherosclerosis-prone sites (bifurcations of large arteries), except for NG2, CD146, and CD140b (which is a common marker for cells of mesenchymal origin): (a,b) staining with antibodies to CD140b (red) and to leptin receptor (green, arrow); (c) staining with antibodies to CD105 (red) and to NG2 (green); (d) GFP (green) and staining with antibodies to GFP (magenta) in nestin-GFP mice. In all images, nuclei are blue. Scale bars (a) 200 µm, (b,d) 50 µm, and (c) 20 µm.

Fig 3: PDGFRß+ cells require Wnt signalling for recruitment to the compact myocardium.(a) Maximum intensity projections showing the distribution of Pdgfrb(BAC)-CreERT2-labelled GFP+ cells (green; arrows) in Fzd4i?PC mutant and littermate control (Pdgfrb(BAC)-CreERT2+/T Fzd4lox/+) E14.5 heart sections. Note profound reduction of GFP+ cells in Fzd4i?PC compact myocardium. (b) Statistical analysis of Fzd4i?PC versus control ventricle size, number of GFP+ cells in E14.5 compact myocardium and relative coverage of ventricle by GFP+ cells (n=8 in each group). Error bars,±s.e.m. P values, two-tailed unpaired t-test; NS, no significance. (c) Maximum intensity projections showing Pdgfrb(BAC)-CreERT2-labelled, GFP+ cells (green; arrows) in Ctnnb1i?PC mutant and littermate control (Pdgfrb(BAC)- CreERT2+/T Ctnnb1lox/+) E14.5 heart sections. GFP+ cells showed reduced migration to the apex and were less abundant in the Ctnnb1i?PC compact myocardium. (d) Statistical analysis of Ctnnb1i?PC versus control ventricle size, number of GFP+ cells in E14.5 compact myocardium and relative coverage of ventricle by GFP+ cells (Ctnnb1i?PC: n=7; control: n=8). Error bars±s.e.m. P values, two-tailed unpaired t-test; NS, no significance.

Fig 4: Angiomodulin protein expression is specific to developing cerebral vasculature and not to highly angiogenic developing vasculature of peripheral organs. a AGM (green) could be detected as early as E12.5 in cortical vasculature (upper panel, arrow heads), co-labeled with CD31 positive endothelium (red). AGM expression was specific to CNS vasculature, as it was not detected in developing peripheral organs such as the heart or liver (middle and lower panels). Merge images includes DAPI nuclei staining. N = 3 mice; scale bar 100 µm for two upper panels and 200 µm for the lower panel. b AGM (green) co-localizes with CD31 positive endothelial cells (red, arrows) and PDGFR-beta pericytes (blue, arrow heads) shown in high magnification confocal imaging of E14.5 cortical vasculature. Scale bar 25 µm

Fig 5: AQP4 Endfeet Polarization Is Dependent on the Presence of Pericytes(A and E) Representative images of PDGFRb (pericytes; white) and AQP4 (magenta) immunostaining in cortex (A) and hippocampus (E). Scale bars, 50 µm.(B and F) Quantification of pericyte density in cortex (B) and hippocampus (F) (n = 12, all groups).(C and G) Percentage of pericyte cell bodies localized perivascularly in cortex (C) (n = 12, 12, and 8 for control, Pdgfbret/+, and Pdgfbret/ret, respectively) and hippocampus (G) (n = 12, 12, and 11 for control, Pdgfbret/+, and Pdgfbret/ret, respectively).(D and H) Mean AQP4 intensity in cortex (D) and hippocampus (H) (n = 12, all groups).(I and L) Representative images of AQP4 (magenta), PDGFRb (white), Lycopersicon esculentum lectin (green), and DAPI (blue) of WT, Pdgfbret/+, and Pdgfbret/ret in cortex (I) and hippocampus (L). Scale bar, 10 µm.(J and M) AQP4 polarization and (K and N) model of fluorescence intensity plot for mean baseline, variance, peak and intraluminal intensity (dotted line) in the cortex and hippocampus (n = 15, 12, and 12 for control, Pdgfbret/+, and Pdgfbret/ret, respectively).(O) Representative western blot of AQP4 and ß-tubulin parenchyma and vessel isolation fraction of WT, Pdgfbret/+, and Pdgfbret/ret mice. WT, wild-type; R/R, Pdgfbret/ret; R/+, Pdgfbret/+; CTR ag, AQP4 control antigen.(P and Q) Quantification of AQP4 western blot in parenchyma (par.) and vessel fraction (ves.) in cortex (P) and hippocampus(Q). N = 6, 7, and 6 for WT, Pdgfbret/+, and Pdgfbret/ret, respectively.In (C), (D), (G), and (H), one-way ANOVA with Tukey test for multiple comparisons for normally distributed data. In (B), (F), (J), and (M), Kruskal-Wallis with Dunn’s test for data not distributed normally. In (P) and (Q), two-way ANOVA with Dunnett test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Bar graphs represent mean ± SEM.