Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis

Abstract

Despite the importance of CNS blood vessels, the molecular mechanisms that regulate CNS angiogenesis and blood-brain barrier (BBB) formation are largely unknown. Here we analyze the role of Wnt/beta-catenin signaling in regulating the formation of CNS blood vessels. First, through the analysis of TOP-Gal Wnt reporter mice, we identify that canonical Wnt/beta-catenin signaling is specifically activated in CNS, but not non-CNS, blood vessels during development. This activation correlates with the expression of different Wnt ligands by neural progenitor cells in distinct locations throughout the CNS, including Wnt7a and Wnt7b in ventral regions and Wnt1, Wnt3, Wnt3a, and Wnt4 in dorsal regions. Blockade of Wnt/beta-catenin signaling in vivo specifically disrupts CNS, but not non-CNS, angiogenesis. These defects include reduction in vessel number, loss of capillary beds, and the formation of hemorrhagic vascular malformations that remain adherent to the meninges. Furthermore, we demonstrate that Wnt/beta-catenin signaling regulates the expression of the BBB-specific glucose transporter glut-1. Taken together these experiments reveal an essential role for Wnt/beta-catenin signaling in driving CNS-specific angiogenesis and provide molecular evidence that angiogenesis and BBB formation are in part linked.

Fig. 1.

Wnt signaling is activated specifically in brain endothelial cells. (A) GeneChip analysis of Wnt signaling components in purified endothelial cells. FACS analysis was used to purify endothelial cells from the brain, liver and lung of Tie2GFP mice, and gene expression was analyzed using Affymetrix microarray analysis. The expression of several molecules that have been demonstrated to be downstream of Wnt/β-catenin signaling are enriched in brain endothelial cells compared with the liver and lung samples. For each probe set values are normalized to brain endothelial cells sample. (B-E) Tissue sections from the cerebral cortex (B and C higher magnification), heart (D) and lung (E) of an E12.5 TOP-Gal transgenic mouse were stained with an anti-LacZ antibody to indicate Wnt activity (i), the vessel marker BSL and the nuclear stain DAPI (ii). In merged images (iii), yellow arrows point to co-localization of LacZ and BSL signals. Wnt activity is observed in blood vessels in the brain, but not heart or lung at E12.5. [Scale bar, 100 μm (B, D, E) and 50 μm (C).]

Fig. 2.

Expression of Wnts in the developing mouse CNS. In situ hybridizations demonstrating Wnt ligand expression in the developing forebrain (A-H) and spinal cord (Ai-Hi) of E11.5 mice. Canonical Wnt ligands Wnt7a and Wnt7b are expressed by neural progenitors in the ventricular zone in the ventral-lateral spinal cord and cortical forebrain, whereas canonical Wnt ligands Wnt1, Wnt3, and Wnt3a are expressed by neural progenitors in the ventricular zone of the dorsal spinal cord and the hindbrain. Non-canonical Wnt ligands Wnt4, Wnt5a, and Wnt5b are also expressed by neural progenitors located in spatially distinct regions of the spinal cord and cortex. Double fluorescent in situ hybridizations in the developing forebrain (E10.5 I-K, E11.5 L-N) and spinal cord (E10.5 Ii-Ki, E11.5 Li-Ni) with Wnt7b (I, L, Ii, Li) and Claudin 5 (J, M, Ji, Mi) and merged (K, N, Ki, Ni) demonstrate that claudin 5 positive vessels vascularize Wnt7b positive regions of the developing CNS.

Fig. 3.

Conditional depletion of β-catenin in endothelial cells leads to CNS-specific vascular defects. (A) Cross sections of E11.5 (ii) endothelial-specific β-catenin mutants (β-cat ^flox/flox; Tek-cre) and (i) litter mate controls were stained with the nuclear marker DAPI (blue) and an antibody against the vascular marker CD31 (red). Normal vasculature was observed in peripheral tissues of both genotypes, whereas, angiogenesis defects were observed in the CNS of the endothelial-specific β-catenin mutants. White boxes outline developing neural tube. (Scale bar, 500 μm.) (B and C) Cross-sections of developing neural tube of an E11.5 (C) endothelial-specific β-catenin mutants and (B) litter-mate taken along the rostral to caudal axis (i-iii), were stained with the nuclear marker DAPI (blue) and an antibody against the vascular marker CD31 (red). The CNS of the endothelial-specific β-catenin mutants demonstrated a decrease in vascular density, a loss of capillary beds and the presence of malformed vessels (white arrows). (Scale bar, 100 μm.) (D) Sagittal sections through the developing neural tube of an E11.5 (ii) endothelial-specific β-catenin mutants and (i) litter-mate were stained with the nuclear marker DAPI (blue) and the vascular marker BSL (green). Large aggregates of endothelial cells were observed in the endothelial-specific β-catenin mutants. (Scale bar, 100 μm.)

Fig. 4.

Abnormal vasculature in the CNS of Wnt 7 mutants. (A-D) Coronal tissue sections of the E10.5 spinal cord in Wnt7a, Wnt7b double heterozygotes (A: Wnt 7a^+/−; Wnt 7b^+/−), Wnt 7a mutants (B: Wnt 7a^−/− ;Wnt 7b^+/−), Wnt 7b mutants (C: Wnt 7a^+/− ;Wnt 7b^−/−), and Wnt7a, Wnt 7b double mutants (D: Wnt 7a^−/− ;Wnt 7b^−/−) were stained with the nuclear marker DAPI (blue) and the vascular marker CD31 (red). Normal capillary beds were observed in the wild-type and Wnt7a mutants, whereas vascular malformations and thickened vascular plexus were observed in the Wnt7b mutants, and large vascular plexus dilations were observed in double mutants. Normal capillaries and normal vascular plexus are indicated with white and yellow arrows respectively, whereas vascular malformations and abnormal vascular plexus are indicated with white and yellow arrow heads respectively. (Scale bar, 100 μm.)

Fig. 5.

Wnt7a regulates CNS endothelial cell migration and expression of the BBB-specific transporter glut-1 (slc2a1). (A) Measurement of mouse brain endothelial cell (bEND3.0) migration across a fibronectin coated filter to a basal media, basal media containing VEGF, basal media containing Wnt, or basal media containing both VEGF and Wnt. Wnt7a is a potent migration factor for CNS endothelial cells. Error bars represent standard error of the mean. *<0.001 as analyzed by a two tailed standard T-test not assuming normal variance. (B) Table of GeneChip values, given in arbitrary units, for primary cultures of mouse brain endothelial cells grown in basal medium, or basal medium with Wnt7a. Wnt7a up-regulates expression of transcripts encoding molecular transporters (slc2a1, slc7a1, and slc7a5) but not tight junction molecules (Occludin, ZO-1) or pan-endothelial cell adhesion molecules (PECAM, VE-Cad). (C-F) Cross sections of the developing CNS of E11.5 endothelial-specific β-catenin mutants (E and F) and litter-mate controls (C and D) were stained with vascular marker BSL I (i, green) and an antibody directed against the BBB-specific glucose transporter glut-1 (ii, red). Glut-1 specifically stains vascular endothelial cells in the control animals, whereas glut-1 does not stain the vascular endothelial cells in the endothelial-specific β-catenin mutants but instead stains CNS parenchymal cells (iii, merged images). (Scale bar,100 μm.)