Figure 1. GPR126 is a target of Wnt/β-catenin signaling and is expressed in brain vasculature during BBB development.

(A) Volcano plot showing transcriptional changes in cBECs exposed to Wnt3a-conditioned medium versus control for 5 days. Genes with significant alterations (P < 0.05) are depicted (Fisher’s least significant difference test). Red dots, upregulated gene targets of Wnt/β-catenin; blue dots, downregulated genes. Gpr126 is highlighted in green. (B) Real-time qPCR of Axin2 and Gpr126 expression in cBECs and cLECs from adult WT mice treated with recombinant Wnt3a or control. (C) Real-time qPCR of Axin2 and Gpr126 in fBECs from mice at P18 (n = 6 WT, n = 9 dnTCF4^iECKI mice). (D) Real-time qPCR of Axin2 and Gpr126 in iBECs with or without primary neonatal astrocytes and treated with vehicle or Wnt-C59 (n = 3). (E) Real-time qPCR of Gpr126 expression in fBECs from WT mice during embryonic (E11–E16) and postnatal (P2–P30) stages and in the adult (P90) (n = 8 embryos, n = 5 postnatal, n = 3 adults). (F and G) Immunoblotting for GPR126 in fBECs from different postnatal stages and adulthood (P8–P30 and P90), quantified by GPR126/VE-cadherin ratios (n = 3 WT mice). (H and I) FISH confocal imaging for Gpr126 (red) and Cldn5 (green) mRNA in mouse cortex at P18, quantified by Gpr126 single-molecule RNA (smRNA) per vessel area (number of spots/μm²). Each symbol represents a field (3–4 fields per region, n = 4 WT mice). C, cortex; S, striatum; V, ventricle; H, hippocampus. (J) Electron microscopy of GPR126 immunogold-labeled (10 nm) cryosection of brain capillaries from WT mouse cortex at P18. Top: Luminal plasma membrane (PM). Bottom: Abluminal EC and pericyte plasma membrane (PM). Right: Late endosome (LE). L, lumen. Scale bars: 200 nm. Data are shown as means ± SD. (B and D–G) Each symbol represents an experiment; Brown-Forsythe and Welch’s ANOVA, Dunnett’s T3 multiple-comparison tests. (B and C) Unpaired t tests with Welch’s correction. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.