Extended Data Fig. 5: Immunostaining reveals ME capillary boundary and validates differential gene expression across regions.

(a) Co-immunostaining with anti-CD31 (white) and anti-SMA (red) antibodies to visualize arteries in the cortex and ME. Scale bar 200 µm. (b) High magnification images of blood vessels (CD31, white), highlighting vessels interacting with SMA-positive smooth muscle cells (red) in cortex (top) and ME (bottom). Scale bar 20 µm. (c) Co-immunostaining with anti-CD31 (white) and anti-PLVAP (red) antibodies in the ME. Yellow arrows indicate arteries in this region. Scale bar 50 µm. (d) Co-immunostaining of SPOCK2 (white), GLUT1 (green) and EMCN (red) in the cortex and ME of P5 wild type mouse. Scale bar 10 µm. (e) Heatmap showing significantly upregulated Reactome pathways for the top 115 differentially expressed genes in cortex-derived cECs 1 and 2 and ME cECs. Differentially expressed genes were determined by two-sided Wilcoxon test in Seurat comparing cortex-derived cECs 1 and 2 with ME cECs (minimum percentage = 25%, log2-fold change > 0.6, adjusted p-value < 0.05). p-value was calculated with a one-sided Fisher’s exact test, and -log(FDR) values are shown. Upregulated pathways from other pathway databases can be found in Supplementary Table 1. (f) Co-immunostaining for CD31 (white), endothelial nuclei marker ERG (red), and GFP in cortex and ME of TCF/LEF-GFP Wnt-signaling reporter mice. GFP expression (green) indicates activation of the Wnt-signaling pathway. Arrows indicate ERG and GFP double positive nuclei. Scale bar 50 µm.