Canonical WNT signaling components in vascular development and barrier formation
Yulian Zhou, … , Makoto M. Taketo, Jeremy Nathans
Yulian Zhou, … , Makoto M. Taketo, Jeremy Nathans
Published August 1, 2014
Citation Information: J Clin Invest. 2014;124(9):3825-3846. https://doi.org/10.1172/JCI76431.
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Canonical WNT signaling components in vascular development and barrier formation

Abstract

Canonical WNT signaling is required for proper vascularization of the CNS during embryonic development. Here, we used mice with targeted mutations in genes encoding canonical WNT pathway members to evaluate the exact contribution of these components in CNS vascular development and in specification of the blood-brain barrier (BBB) and blood-retina barrier (BRB). We determined that vasculature in various CNS regions is differentially sensitive to perturbations in canonical WNT signaling. The closely related WNT signaling coreceptors LDL receptor–related protein 5 (LRP5) and LRP6 had redundant functions in brain vascular development and barrier maintenance; however, loss of LRP5 alone dramatically altered development of the retinal vasculature. The BBB in the cerebellum and pons/interpeduncular nuclei was highly sensitive to decrements in canonical WNT signaling, and WNT signaling was required to maintain plasticity of barrier properties in mature CNS vasculature. Brain and retinal vascular defects resulting from ablation of Norrin/Frizzled4 signaling were ameliorated by stabilizing β-catenin, while inhibition of β-catenin–dependent transcription recapitulated the vascular development and barrier defects associated with loss of receptor, coreceptor, or ligand, indicating that Norrin/Frizzled4 signaling acts predominantly through β-catenin–dependent transcriptional regulation. Together, these data strongly support a model in which identical or nearly identical canonical WNT signaling mechanisms mediate neural tube and retinal vascularization and maintain the BBB and BRB.

Authors

Yulian Zhou, Yanshu Wang, Max Tischfield, John Williams, Philip M. Smallwood, Amir Rattner, Makoto M. Taketo, Jeremy Nathans

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Figure 11

Production of dnTCF4 in ECs mimics the phenotypes seen with loss of canonical WNT signaling in the retina and cerebellum.

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Production of dnTCF4 in ECs mimics the phenotypes seen with loss of cano...
(A) The R26-LSL-tdT-dnTcf4 knockin before and after Cre-mediated recombination (upper panel). EC accumulation of tdTomato in R26-LSL-tdT-dnTcf4 Tie2-Cre retina flat mounts (lower panel). Scale bar: 200 μm. (B) P7 R26-LSL-tdT-dnTcf4/+ Tie2-Cre retinas show a modest decrease in Claudin5 expression in veins and capillaries compared with WT controls. Scale bar: 500 μm (upper panels); 200 μm (lower panels). (C) Quantification of branch points from retinal veins and arteries at P7 (left panel) and vascular density in the 3 retinal layers at P16 (right panel). *P < 0.01. (D) R26-LSL-tdT-dnTcf4/R26-LSL-tdT-dnTcf4 Pdgfb-CreER retinas (i.e., 2 alleles expressing dnTcf4) from P9–P10 mice treated with 50 μg 4HT at P3 show sulfo-NHS-biotin leakage and reduced vessel density (top panels). Pdgfb-CreER mediates nearly complete recombination at R26 as assessed by tdTomato fluorescence (bottom panels). Scale bar: 400 μm. (E) Genetic interaction between Ndp and R26-LSL-tdT-dnTcf4 with EC expression of dnTcf4. At P18, PLVAP+ ECs increase in the vasculature of the NdpKO;R26-LSL-tdT-dnTcf4/+ Tie2-Cre cerebellum (lower panel; white arrows) compared with the NdpKO cerebellum (upper panel). Scale bar: 1 mm. (F) Synergistic effect of reducing or eliminating Tcf4 and expressing different levels of dnTcf4 in ECs. Flat-mount P9–P10 retinas from mice that had received 40 μg 4HT at P1. With each reduction in Tcf4 or increase in dnTcf4, there are greater PLVAP expression and lower Claudin5 expression in veins and capillaries. Scale bar: 500 μm.