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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Research Article Vascular biology

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 7

Production of stabilized β-catenin in ECs rescues NdpKO and Fz4–/– cerebellar BBB defects.

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Production of stabilized β-catenin in ECs rescues NdpKO and Fz4–/– cereb...
(A) BBB integrity assessed by i.p. injection of Evans blue at P50. Brains were collected 1 day after Evans blue injection. Fz4–/– Ctnnb1flex3/+ Pdgfb-CreER mice received 4 i.p. injections of a total of 330 μg 4HT/injection between P10 and P45. (B) Cerebellar BBB integrity assessed by sulfo-NHS-biotin leakage in Fz4–/– and NdpKO mice (left panels) and Fz4–/– Ctnnb1flex3/+ Pdgfb-CreER and NdpKO Ctnnb1flex3/+ Pdgfb-CreER mice (right panels). β-Catenin stabilization rescues the cerebellar BBB defect. In B–E, mice carrying the Ctnnb1flex3 Pdgfb-CreER allele received 3 to 4 i.p. injections of 20 to 100 μg 4HT/injection at intervals of 2 to 3 days between P2 and P10, a regimen that leads to nearly complete EC recombination of the Ctnnb1flex3 allele in the presence of Pdgfb-CreER. Scale bar: 1 mm (B). (C and D) Neuronal death, measured by cleaved caspase 3 immunostaining, is abundant in the Fz4–/– cerebellum and is suppressed in the Fz4–/– Ctnnb1flex3/+ Pdgfb-CreER cerebellum at P18. Box and whisker plot parameters are described in Methods. Scale bar: 200 μm. (E) BBB integrity assessed by extravasation of mouse IgG and by sulfo-NHS-biotin leakage in WT, Fz4–/–, and Fz4–/– Ctnnb1flex3/+ Pdgfb-CreER cerebella at P19. Although β-catenin stabilization largely rescues the cerebellar BBB defect, rare sites of leakage remain (arrows, right panels). Scale bar: 200 μm.