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• W2000293457 abstract "•Gpr124 specifically enhances canonical signaling by Wnt7a and Wnt7b•Gpr124 signaling is partially redundant with Norrin/Fz4 signaling in CNS angiogenesis•Gpr124 signaling promotes postnatal blood-brain barrier integrity•Loss of Gpr124 can be rescued by artificially activating canonical Wnt signaling Canonical Wnt signaling in endothelial cells (ECs) is required for vascularization of the central nervous system (CNS) and for formation and maintenance of barrier properties unique to CNS vasculature. Gpr124 is an orphan member of the adhesion G protein-coupled receptor family that is expressed in ECs and is essential for CNS angiogenesis and barrier formation via an unknown mechanism. Using canonical Wnt signaling assays in cell culture and genetic loss- and gain-of-function experiments in mice, we show that Gpr124 functions as a coactivator of Wnt7a- and Wnt7b-stimulated canonical Wnt signaling via a Frizzled receptor and Lrp coreceptor and that Gpr124-stimulated signaling functions in concert with Norrin/Frizzled4 signaling to control CNS vascular development. These experiments identify Gpr124 as a ligand-specific coactivator of canonical Wnt signaling. Canonical Wnt signaling in endothelial cells (ECs) is required for vascularization of the central nervous system (CNS) and for formation and maintenance of barrier properties unique to CNS vasculature. Gpr124 is an orphan member of the adhesion G protein-coupled receptor family that is expressed in ECs and is essential for CNS angiogenesis and barrier formation via an unknown mechanism. Using canonical Wnt signaling assays in cell culture and genetic loss- and gain-of-function experiments in mice, we show that Gpr124 functions as a coactivator of Wnt7a- and Wnt7b-stimulated canonical Wnt signaling via a Frizzled receptor and Lrp coreceptor and that Gpr124-stimulated signaling functions in concert with Norrin/Frizzled4 signaling to control CNS vascular development. These experiments identify Gpr124 as a ligand-specific coactivator of canonical Wnt signaling. Central nervous system (CNS) angiogenesis begins with endothelial cell (EC) invasion from the perineural vascular plexus, followed by EC migration and vascular branching within the CNS. Concomitant with development of the CNS vasculature, brain ECs acquire the characteristics of a blood-brain barrier (BBB) (or in the eye, a blood-retina barrier [BRB]). Canonical Wnt signaling plays a central role in these processes. In the embryo, Wnt7a and Wnt7b produced by the neuroepithelium of the developing brain and spinal cord promote angiogenesis and BBB formation by activating canonical Wnt signaling in ECs (Stenman et al., 2008Stenman J.M. Rajagopal J. Carroll T.J. Ishibashi M. McMahon J. McMahon A.P. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature.Science. 2008; 322: 1247-1250Crossref PubMed Scopus (442) Google Scholar, Liebner et al., 2008Liebner S. Corada M. Bangsow T. Babbage J. Taddei A. Czupalla C.J. Reis M. Felici A. Wolburg H. Fruttiger M. et al.Wnt/beta-catenin signaling controls development of the blood-brain barrier.J. Cell Biol. 2008; 183: 409-417Crossref PubMed Scopus (553) Google Scholar, Daneman et al., 2009Daneman R. Agalliu D. Zhou L. Kuhnert F. Kuo C.J. Barres B.A. Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis.Proc. Natl. Acad. Sci. USA. 2009; 106: 641-646Crossref PubMed Scopus (497) Google Scholar). In the postnatal retina and cerebellum, an analogous response is mediated by glial-derived Norrin (a transforming growth factor β family member), which activates canonical Wnt signaling in ECs via the receptor Frizzled4 (Fz4) and coreceptors Lrp5 and Lrp6 (Xu et al., 2004Xu Q. Wang Y. Dabdoub A. Smallwood P.M. Williams J. Woods C. Kelley M.W. Jiang L. Tasman W. Zhang K. Nathans J. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.Cell. 2004; 116: 883-895Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, Ye et al., 2009Ye X. Wang Y. Cahill H. Yu M. Badea T.C. Smallwood P.M. Peachey N.S. Nathans J. Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization.Cell. 2009; 139: 285-298Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, Wang et al., 2012Wang Y. Rattner A. Zhou Y. Williams J. Smallwood P.M. Nathans J. Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity.Cell. 2012; 151: 1332-1344Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). In retinal ECs, the cell-surface Norrin/Fz4/Lrp5 signaling complex also includes the tetra-spanin protein Tspan12 (Junge et al., 2009Junge H.J. Yang S. Burton J.B. Paes K. Shu X. French D.M. Costa M. Rice D.S. Ye W. TSPAN12 regulates retinal vascular development by promoting Norrin- but not Wnt-induced FZD4/beta-catenin signaling.Cell. 2009; 139: 299-311Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). In humans and mice, mutations in the genes coding for each of these polypeptides cause retinal hypovascularization syndromes that impair or eliminate vision (Nikopoulos et al., 2010Nikopoulos K. Venselaar H. Collin R.W.J. Riveiro-Alvarez R. Boonstra F.N. Hooymans J.M.M. Mukhopadhyay A. Shears D. van Bers M. de Wijs I.J. et al.Overview of the mutation spectrum in familial exudative vitreoretinopathy and Norrie disease with identification of 21 novel variants in FZD4, LRP5, and NDP.Hum. Mutat. 2010; 31: 656-666Crossref PubMed Scopus (116) Google Scholar, Ye et al., 2010Ye X. Wang Y. Nathans J. The Norrin/Frizzled4 signaling pathway in retinal vascular development and disease.Trends Mol. Med. 2010; 16: 417-425Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). Gpr124, a member of the “adhesion G-protein-coupled receptor (GPCR)” family, was first linked to vascular biology based on the enrichment of its transcripts in human colorectal tumor vasculature (St. Croix et al., 2000St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Genes expressed in human tumor endothelium.Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1643) Google Scholar, Carson-Walter et al., 2001Carson-Walter E.B. Watkins D.N. Nanda A. Vogelstein B. Kinzler K.W. St. Croix B. Cell surface tumor endothelial markers are conserved in mice and humans.Cancer Res. 2001; 61: 6649-6655PubMed Google Scholar; Gpr124 is also referred to as tumor endothelial marker 5). Gpr124 is expressed in developing ECs, and targeted mutation of Gpr124 in mice, either throughout the body or specifically in ECs, leads to defects in embryonic CNS angiogenesis and BBB formation that closely resemble the defects caused by loss of canonical Wnt signaling (Kuhnert et al., 2010Kuhnert F. Mancuso M.R. Shamloo A. Wang H.T. Choksi V. Florek M. Su H. Fruttiger M. Young W.L. Heilshorn S.C. Kuo C.J. Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124.Science. 2010; 330: 985-989Crossref PubMed Scopus (214) Google Scholar, Anderson et al., 2011Anderson K.D. Pan L. Yang X.M. Hughes V.C. Walls J.R. Dominguez M.G. Simmons M.V. Burfeind P. Xue Y. Wei Y. et al.Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor.Proc. Natl. Acad. Sci. USA. 2011; 108: 2807-2812Crossref PubMed Scopus (110) Google Scholar, Cullen et al., 2011Cullen M. Elzarrad M.K. Seaman S. Zudaire E. Stevens J. Yang M.Y. Li X. Chaudhary A. Xu L. Hilton M.B. et al.GPR124, an orphan G protein-coupled receptor, is required for CNS-specific vascularization and establishment of the blood-brain barrier.Proc. Natl. Acad. Sci. USA. 2011; 108: 5759-5764Crossref PubMed Scopus (144) Google Scholar). Like many “adhesion GPCRs,” Gpr124 is currently classified as an orphan receptor since the ligand(s) that activate it and the signal transduction pathway(s) to which it couples are unknown. Starting with the clue that Gpr124 is essential for CNS angiogenesis, we demonstrate here that Gpr124 functions as a ligand-specific coactivator of canonical Wnt signaling in the CNS vasculature during both embryonic and postnatal life. As an initial screen, we asked whether transfection of a canonical Wnt signaling reporter cell line (Super Top Flash; STF) with Lrp5 and each of the 19 mammalian Wnts or Norrin could reveal an effect of coexpressed Gpr124 (Figure 1A). STF cells express multiple Wnt signal transduction components at a low level (Table S1 available online), which likely accounts for their responses to some Wnts in the absence of cotransfected Frizzleds, the high-affinity Wnt receptors. Gpr124 was observed to increase Wnt7a- and Wnt7b-dependent signaling ∼8-fold and ∼4-fold, respectively (Figure 1A; Figure S1A). Gpr124 had little or no effect on signaling by other Wnts or by Norrin. To determine which of the ten mammalian Frizzleds mediate the Gpr124 effect, we transfected STF cells with each Frizzled together with Lrp5 and Wnt7a or Wnt7b, with or without Gpr124 (Figure 1B). Among the five Frizzleds that exhibited a signal substantially above background, Fz1 and Fz4 showed the greatest enhancement by Gpr124 (up to ∼10-fold). We next used STF cells to compare Gpr124 activity to that of the closely related protein Gpr125 (Figure S1B). When Wnt7a or Wnt7b, Fz4, and Lrp5 were cotransfected with Gpr125, we observed an ∼2- to 3-fold depression in the STF signal relative to the vector control, whereas Gpr124 produced ∼15-fold and ∼2-fold increases in Wnt7a and Wnt7b signaling, respectively (Figure 1C). In a screen analogous to the one shown in Figure 1A (Lrp5 and each of the 19 mammalian Wnts or Norrin), Gpr125 showed little effect on signaling (Figure S1C). The data presented thus far are consistent with a model in which Gpr124 promotes the formation or enhances the activity of the Wnt/Fz/Lrp transmembrane signaling complex. Alternately, Gpr124 could increase the bioactivities of Wnt7a and Wnt7b by promoting their folding, intracellular trafficking, or secretion. To distinguish these possibilities, we cocultured STF cells transfected with Fz4 and Lrp5, with or without Gpr124, together with 293 cells that had been transfected with Wnt7a and/or Gpr124 (Figure 1D). This experiment showed that Gpr124 had no effect on Wnt7a production/secretion by cocultured 293 cells but instead exerted its effects only when expressed in STF cells, consistent with a role for Gpr124 as part of the Fz/Lrp signaling complex. Lrp5 and Lrp6, the closely related coreceptors for canonical Wnt signaling (Figure S1B), are largely interchangeable in a variety of biological contexts (Joiner et al., 2013Joiner D.M. Ke J. Zhong Z. Xu H.E. Williams B.O. LRP5 and LRP6 in development and disease.Trends Endocrinol. Metab. 2013; 24: 31-39Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). In particular, prenatal CNS angiogenesis is unaffected by loss of either Lrp5 or Lrp6 but is severely compromised by the combined loss of both receptors (Zhou et al., 2014Zhou Y. Wang Y. Tischfield M. Williams J. Smallwood P.M. Rattner A. Taketo M.M. Nathans J. Canonical WNT signaling components in vascular development and barrier formation.J. Clin. Invest. 2014; Google Scholar). Surprisingly, Gpr124 does not enhance Wnt7a/Fz4/Lrp6 signaling in STF cells (Figure 1E, left panel). A control experiment shows that both Lrp5 and Lrp6 mediate Tspan12 enhancement of signaling in response to Norrin, albeit with different levels of basal activity (Figure 1E, right panel). In a titration experiment (Figure 1F), Lrp6 showed no Gpr124 stimulation at any dose tested, whereas each component in the putative Wnt7a/Fz4/Lrp5/Gpr124 signaling complex showed well-behaved dose-response curves. We note that in the Wnt7a, Fz4, and Lrp5 titrations, Gpr124 stimulation was observed at all concentrations tested. Earlier work demonstrated that Lrp5 and Lrp6 differ quantitatively in signal transduction efficiency (MacDonald et al., 2011MacDonald B.T. Semenov M.V. Huang H. He X. Dissecting molecular differences between Wnt coreceptors LRP5 and LRP6.PLoS ONE. 2011; 6: e23537Crossref PubMed Scopus (55) Google Scholar), but the difference between Lrp5 and Lrp6 in Gpr124 responsiveness in the STF assay is striking, as it represents an all-or-none functional difference between the two Lrp coreceptors. The severe defects in CNS angiogenesis in Gpr124−/− embryos imply that loss of Gpr124 in vivo impairs both Lrp5 and Lrp6 signaling. We tested the idea that Gpr124 might enhance Wnt7a or Wnt7b signaling via Lrp6 in the context of Frizzleds other than Fz4 by surveying all ten Frizzleds in STF cells. This survey revealed only a modest (∼2-fold) enhancement for Fz1 (Figure S1D). We hypothesize that other still-unknown components facilitate Gpr124 stimulation of Wnt/Fz signaling via Lrp6 in CNS ECs and that these components are missing from STF cells. Current evidence indicates that Tspan12 is an integral part of the Norrin/Fz4/Lrp5 signaling complex and that Tspan12 enhances Norrin-induced but not Wnt-induced canonical signaling through Fz4 (Junge et al., 2009Junge H.J. Yang S. Burton J.B. Paes K. Shu X. French D.M. Costa M. Rice D.S. Ye W. TSPAN12 regulates retinal vascular development by promoting Norrin- but not Wnt-induced FZD4/beta-catenin signaling.Cell. 2009; 139: 299-311Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). To compare the ligand specificities of Tspan12 and Gpr124 in the context of Fz4/Lrp5 signaling, we measured canonical Wnt signaling in response to Wnt7a or Norrin (Figures 1G and 1H). These experiments show that Gpr124 enhances Wnt7a signaling but not Norrin signaling and that Tspan12 enhances Norrin signaling but not Wnt7a signaling. Thus, Tspan12 and Gpr124 function as ligand-specific coactivators of canonical Wnt signaling in the context of the Fz4/Lrp5 complex. The components of these two signaling complexes are diagrammed in Figure 1I. To test whether Gpr124 participates in canonical Wnt signaling in vivo, we examined the expression of a Tcf/Lef-nLacZ canonical Wnt signaling reporter transgene (BAT-gal; Maretto et al., 2003Maretto S. Cordenonsi M. Dupont S. Braghetta P. Broccoli V. Hassan A.B. Volpin D. Bressan G.M. Piccolo S. Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors.Proc. Natl. Acad. Sci. USA. 2003; 100: 3299-3304Crossref PubMed Scopus (687) Google Scholar) in wild-type (WT) versus Gpr124−/− embryos. In the Gpr124−/− CNS between embryonic day (E)11.5 and E13.5, the number of beta-galactosidase+ EC nuclei was dramatically reduced in multiple regions, including the medial ganglionic eminence (MGE), hindbrain, and spinal cord compared to WT controls (Figures 2A and 2C ; Figures S2A and S2B), indicating a reduction in canonical Wnt signaling. Embryos with an EC-specific knockout of beta-catenin exhibit defective CNS angiogenesis throughout the entire neuraxis (Stenman et al., 2008Stenman J.M. Rajagopal J. Carroll T.J. Ishibashi M. McMahon J. McMahon A.P. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature.Science. 2008; 322: 1247-1250Crossref PubMed Scopus (442) Google Scholar, Liebner et al., 2008Liebner S. Corada M. Bangsow T. Babbage J. Taddei A. Czupalla C.J. Reis M. Felici A. Wolburg H. Fruttiger M. et al.Wnt/beta-catenin signaling controls development of the blood-brain barrier.J. Cell Biol. 2008; 183: 409-417Crossref PubMed Scopus (553) Google Scholar, Daneman et al., 2009Daneman R. Agalliu D. Zhou L. Kuhnert F. Kuo C.J. Barres B.A. Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis.Proc. Natl. Acad. Sci. USA. 2009; 106: 641-646Crossref PubMed Scopus (497) Google Scholar, Zhou et al., 2014Zhou Y. Wang Y. Tischfield M. Williams J. Smallwood P.M. Rattner A. Taketo M.M. Nathans J. Canonical WNT signaling components in vascular development and barrier formation.J. Clin. Invest. 2014; Google Scholar), whereas Gpr124−/− embryos exhibit CNS angiogenesis defects in the forebrain and ventral spinal cord but not in the midbrain, hindbrain, or dorsal spinal cord (Kuhnert et al., 2010Kuhnert F. Mancuso M.R. Shamloo A. Wang H.T. Choksi V. Florek M. Su H. Fruttiger M. Young W.L. Heilshorn S.C. Kuo C.J. Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124.Science. 2010; 330: 985-989Crossref PubMed Scopus (214) Google Scholar, Anderson et al., 2011Anderson K.D. Pan L. Yang X.M. Hughes V.C. Walls J.R. Dominguez M.G. Simmons M.V. Burfeind P. Xue Y. Wei Y. et al.Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor.Proc. Natl. Acad. Sci. USA. 2011; 108: 2807-2812Crossref PubMed Scopus (110) Google Scholar, Cullen et al., 2011Cullen M. Elzarrad M.K. Seaman S. Zudaire E. Stevens J. Yang M.Y. Li X. Chaudhary A. Xu L. Hilton M.B. et al.GPR124, an orphan G protein-coupled receptor, is required for CNS-specific vascularization and establishment of the blood-brain barrier.Proc. Natl. Acad. Sci. USA. 2011; 108: 5759-5764Crossref PubMed Scopus (144) Google Scholar). In embryos lacking Wnt7a and Wnt7b, the pattern of CNS angiogenesis defects resembles the pattern seen in Gpr124−/− embryos (Stenman et al., 2008Stenman J.M. Rajagopal J. Carroll T.J. Ishibashi M. McMahon J. McMahon A.P. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature.Science. 2008; 322: 1247-1250Crossref PubMed Scopus (442) Google Scholar, Daneman et al., 2009Daneman R. Agalliu D. Zhou L. Kuhnert F. Kuo C.J. Barres B.A. Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis.Proc. Natl. Acad. Sci. USA. 2009; 106: 641-646Crossref PubMed Scopus (497) Google Scholar). Intriguingly, at midgestation, Ndp (Norrie disease protein; the gene coding for Norrin) is expressed in the dorsal spinal cord and in the hindbrain but is not detectable in the forebrain (Ye et al., 2011Ye X. Smallwood P. Nathans J. Expression of the Norrie disease gene (Ndp) in developing and adult mouse eye, ear, and brain.Gene Expr. Patterns. 2011; 11: 151-155Crossref PubMed Scopus (43) Google Scholar), suggesting that Norrin/Fz4 signaling may play a complementary and/or partially redundant role with Wnt7a/Wnt7b/Gpr124 signaling. To test this hypothesis, we compared CNS angiogenesis in NdpKO, Gpr124−/−, and Gpr124−/−;NdpKO embryos at E11.5 (Figures 2B–2F, S2A–S2C, and S2E; Ndp is an X-linked gene, and we refer to both Ndp−/− females and Ndp−/Y males as NdpKO). The CNS vasculature in NdpKO embryos is indistinguishable from WT, and canonical Wnt signaling in ECs appears to be normal in NdpKO embryos as judged by Tcf/Lef-nLacZ expression (Figures 2A, 2C, and S2A). Unlike NdpKO or Gpr124−/− embryos, Gpr124−/−;NdpKO embryos show a severe defect in hindbrain vascularization with numerous glomeruloid-like vascular bodies (Figures 2E and S2A; multiple examples are shown in Figure S2E). Gpr124−/−;NdpKO embryos also show an enlarged avascular zone in the ventral spinal cord relative to Gpr124−/− embryos (Figures 2B, 2C, S2A, and S2C). These data are summarized in Figure 2F and Table S2. Consistent with these architectural defects, the fraction of EC nuclei in these regions that are beta-galactosidase+ is smaller in Gpr124−/−;NdpKO embryos than in Gpr124−/− embryos (Figures 2C and S2A). Despite widespread postnatal expression of Gpr124 in CNS and non-CNS vasculature, early postnatal and EC-specific elimination of Gpr124 in Gpr124CKO/−;Pdgfb-CreER mice produces no detectable vascular phenotype in the brain or retina (Kuhnert et al., 2010Kuhnert F. Mancuso M.R. Shamloo A. Wang H.T. Choksi V. Florek M. Su H. Fruttiger M. Young W.L. Heilshorn S.C. Kuo C.J. Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124.Science. 2010; 330: 985-989Crossref PubMed Scopus (214) Google Scholar; Figures 2G, S2G, and S2H; data not shown). Constitutive loss of Ndp leads to CNS vascular phenotypes that are confined to the retina (severe hypovascularization) and cerebellum (a mild BBB defect), despite the expansion of the Ndp expression domain around the time of birth to include astrocytes throughout the CNS in addition to Bergman glia in the cerebellum and Muller glia in the retina (Ye et al., 2010Ye X. Wang Y. Nathans J. The Norrin/Frizzled4 signaling pathway in retinal vascular development and disease.Trends Mol. Med. 2010; 16: 417-425Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). The broad spatiotemporal expression domains observed for Ndp and Gpr124 suggest that these genes may play wider roles in postnatal CNS vascular development/homeostasis than previously appreciated, but these roles may have been masked by redundancy. Consistent with this idea, eliminating both Gpr124 and Ndp postnatally in Gpr124CKO/−;NdpKO;Pdgfb-CreER mice produced widespread loss of BBB integrity, as assessed by leakage of sulfo-NHS-biotin from the intravascular space to the parenchyma (Figures 2G and S2F). Vascular leakage was associated with suppression of the tight junction protein claudin5 and induction of plasmalemma vesicle-associated membrane protein (PLVAP), a structural component of EC fenestrations. This phenotype was observed in the cerebral cortex, hippocampus, colliculus, brain stem, and cerebellum, but it was largely absent from the thalamus and the roof of the anterior midbrain (Figures 2G, S2F, and S2G; data not shown). Presumably, CNS regions that were less affected express other activators of canonical Wnt signaling. As a further test of the hypothesis that Gpr124 participates in canonical Wnt signaling in vivo, we asked whether the combined loss of Fz4 and Gpr124 produced a more severe phenotype than loss of either gene alone (Figure 3). In midgestation embryos, Fz4 and Gpr124 are expressed throughout the CNS vasculature (Ye et al., 2009Ye X. Wang Y. Cahill H. Yu M. Badea T.C. Smallwood P.M. Peachey N.S. Nathans J. Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization.Cell. 2009; 139: 285-298Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, Kuhnert et al., 2010Kuhnert F. Mancuso M.R. Shamloo A. Wang H.T. Choksi V. Florek M. Su H. Fruttiger M. Young W.L. Heilshorn S.C. Kuo C.J. Essential regulation of CNS angiogenesis by the orphan G protein-coupled receptor GPR124.Science. 2010; 330: 985-989Crossref PubMed Scopus (214) Google Scholar, Anderson et al., 2011Anderson K.D. Pan L. Yang X.M. Hughes V.C. Walls J.R. Dominguez M.G. Simmons M.V. Burfeind P. Xue Y. Wei Y. et al.Angiogenic sprouting into neural tissue requires Gpr124, an orphan G protein-coupled receptor.Proc. Natl. Acad. Sci. USA. 2011; 108: 2807-2812Crossref PubMed Scopus (110) Google Scholar, Cullen et al., 2011Cullen M. Elzarrad M.K. Seaman S. Zudaire E. Stevens J. Yang M.Y. Li X. Chaudhary A. Xu L. Hilton M.B. et al.GPR124, an orphan G protein-coupled receptor, is required for CNS-specific vascularization and establishment of the blood-brain barrier.Proc. Natl. Acad. Sci. USA. 2011; 108: 5759-5764Crossref PubMed Scopus (144) Google Scholar). Fz4−/− embryos have no vascular defects, and Gpr124−/− embryos survive through late gestation, despite their CNS vascular defects. When we generated various combinations of Fz4 and Gpr124 alleles, the following phenotypes were observed (listed in order of increasing severity): (1) Fz4+/−;Gpr124+/− embryos, fetuses, and postnatal mice showed no vascular defects; (2) Fz4−/−;Gpr124+/− embryos showed no vascular defects, but postnatal Fz4−/−;Gpr124+/− mice showed an enhanced conversion of cerebellar ECs from a PLVAP− to a PLVAP+ state relative to Fz4−/− controls (Figure 3B); (3) Fz4+/−;Gpr124−/− embryos showed variable defects in hindbrain angiogenesis at E11.5 (Figure 3C); and (4) Fz4−/−;Gpr124−/− embryos exhibited a severe and lethal growth retardation by E10.5 (Figure 3D). As summarized in Figure 3A, among 97 embryos harvested at E11.5 from a Gpr124+/−;Fz4+/− × Gpr124+/−;Fz4+/− intercross, 6 were Gpr124−/−;Fz4−/−. In this cohort, 6 of 97 embryos were growth retarded and/or dead, and this phenotype coincided precisely with the Gpr124−/−;Fz4−/− genotype. If genotype and lethality were uncorrelated, the probability of this coincidence would be 1.36 × 10−6. This dosage experiment shows that with a progressive reduction in the number of WT copies of both Gpr124 and Fz4, there is a progressive increase in phenotypic severity. These experiments also imply that Gpr124 does not act exclusively through Fz4. If it did, then Fz4−/− embryos would have a vascular phenotype at least as severe as that of Gpr124−/− embryos. Presumably, Gpr124 also enhances canonical Wnt signaling in ECs via one or more other Frizzleds. We note that multiple Frizzleds in addition to Fz4, are expressed in ECs (Goodwin et al., 2006Goodwin A.M. Sullivan K.M. D’Amore P.A. Cultured endothelial cells display endogenous activation of the canonical Wnt signaling pathway and express multiple ligands, receptors, and secreted modulators of Wnt signaling.Dev. Dyn. 2006; 235: 3110-3120Crossref PubMed Scopus (96) Google Scholar) and are, therefore, candidates for interacting with Gpr124 in vivo, despite their lower synergy with Gpr124 in the STF assay. All of the data presented up to this point support the hypothesis that Gpr124 acts in the CNS vasculature by enhancing canonical Wnt signaling. A critical test of this hypothesis would be to determine whether the vascular phenotype of Gpr124−/− embryos can be rescued by artificially increasing canonical Wnt signaling. We have performed this test in two ways: (1) by stabilizing beta-catenin in ECs and (2) by overexpressing Norrin in the forebrain. Beta-catenin communicates the canonical Wnt signal from cytoplasm to nucleus, and its artificial stabilization—by Cre-mediated excision of glycogen synthase kinase-3 phosphorylation sites within exon 3 of the beta-catenin gene (Ctnnb1) in Ctnnb1flex3/+;Pdgfb-CreER mice (Harada et al., 1999Harada N. Tamai Y. Ishikawa T. Sauer B. Takaku K. Oshima M. Taketo M.M. Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene.EMBO J. 1999; 18: 5931-5942Crossref PubMed Scopus (968) Google Scholar)—should override canonical Wnt signaling defects at the plasma membrane. The Norrin overexpression experiment, which utilizes a Cre-activated Norrin knockin at the Ubiquitin-B locus (Z/Norrin; Ye et al., 2009Ye X. Wang Y. Cahill H. Yu M. Badea T.C. Smallwood P.M. Peachey N.S. Nathans J. Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization.Cell. 2009; 139: 285-298Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, Wang et al., 2012Wang Y. Rattner A. Zhou Y. Williams J. Smallwood P.M. Nathans J. Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity.Cell. 2012; 151: 1332-1344Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar) and an early-embryonic-forebrain-specific Cre driver (Foxg1-Cre; Hébert and McConnell, 2000Hébert J.M. McConnell S.K. Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures.Dev. Biol. 2000; 222: 296-306Crossref PubMed Scopus (432) Google Scholar), tests whether expanding the domain of Norrin/Fz4 signaling in the early embryo can compensate for the loss of Wnt/Gpr124 signaling. Figure 4B shows that E13.5 Gpr124−/− embryos exhibit forebrain and lower spinal cord hemorrhage (column 1; white arrows), severe defects in vascularization of the MGE (columns 2–4) and cerebral cortex (columns 2, 3, and 5), and loss of CNS vascular expression of Glucose transporter-1 (Glut-1; an EC marker for the BBB state; columns 2–5). The beta-catenin rescue experiment (Figures 4D, 4G, and S2D) shows that E13.5 Gpr124−/−;Ctnnb1flex3/+;Pdgfb-CreER embryos—treated at E10.5 with a maternal intraperitoneal injection of 1.5 mg tamoxifen—exhibit almost complete suppression of the forebrain hemorrhage but not the spinal cord hemorrhage (column 1; white arrow), partial rescue of the MGE (columns 2–4) and cerebral cortical (columns 2, 3, and 5) vascularization defects, and partial rescue of the Glut-1 expression defect (columns 2–5). In E13.5 Gpr124−/−;Ctnnb1flex3/+;Pdgfb-CreER ventral spinal cords, vascularization is largely rescued and Glut-1 expression is partially restored (Figure S3A). The incomplete rescue of the spinal cord hemorrhage most likely reflects the temporal overlap between spinal cord angiogenesis, which begins at ∼E10, and the initiation of Pdgfb-CreER-dependent recombination following tamoxifen administration at E10.5. Rescue of Gpr124−/−;Ctnnb1flex3/+;Pdgfb-CreER embryos did not occur without tamoxifen treatment (data not shown). The Norrin rescue experiment (Figure 4F and S3D) shows that Gpr124−/−;Z/Norrin;Foxg1-Cre embryos exhibit a nearly complete rescue of all Gpr124−/− defects except for the spinal cord hemorrhage, a result that is expected based on the forebrain-specific expression of Foxg1-Cre. When examined at different time points, the Gpr124−/−;Z/Norrin;Foxg1-Cre cerebral cortex shows retarded vascularization at E11.5 but nearly normal vascular architecture at E12.5 and E13, and the Gpr124−/−;Z/Norrin;Foxg1-Cre MGE showed normal pericyte coverage of the rescued vessels at E12.5, as judged by the distribution of NG2 (Figures S3B–S3D). We note that increasing canonical Wnt signaling on a WT background in control Ctnnb1flex3/+;Pdgfb-CreER and Z/Norrin;Foxg1-Cre embryos had little or no effect on embryonic vascular development at or prior to E13.5 (Figures 4C, 4E, 4G, S3A, S3B and S3D). The experiments reported here establish Gpr124 as a ligand-specific coactivator of canonical Wnt signaling in developing and mature ECs, yet they also suggest that additional components of this system remain to be discovered (Figure 4H). We speculate that Gpr124 evolved to ensure a high signal-to-noise ratio for Wnt7a- and Wnt7b-induced signaling in brain vascular development and homeostasis, analogous to the role of Tspan12 in the context of Norrin/Fz4 signaling. Coactivators of canonical Wnt signaling might be especially important in the context of CNS vascular biology because signal strength in this system appears to be close to the minimal threshold required for normal development and function: in both mice and humans, modest decrements in canonical Wnt signaling in ECs have been shown to subtly retard vascular development and/or BBB/BRB maintenance (Nikopoulos et al., 2010Nikopoulos K. Venselaar H. Collin R.W.J. Riveiro-Alvarez R. Boonstra F.N. Hooymans J.M.M. Mukhopadhyay A. Shears D. van Bers M. de Wijs I.J. et al.Overview of the mutation spectrum in familial exudative vitreoretinopathy and Norrie disease with identification of 21 novel variants in FZD4, LRP5, and NDP.Hum. Mutat. 2010; 31: 656-666Crossref PubMed Scopus (116) Google Scholar, Zhou et al., 2014Zhou Y. Wang Y. Tischfield M. Williams J. Smallwood P.M. Rattner A. Taketo M.M. Nathans J. Canonical WNT signaling components in vascular development and barrier formation.J. Clin. Invest. 2014; Google Scholar). The ligand and receptor specificities of Gpr124 might provide a partial resolution to two apparent paradoxes: (1) that Wnt knockout phenotypes are generally specific to particular tissues and developmental process, whereas the distribution of individual Wnt ligands is often more widespread (http://www.stanford.edu/group/nusselab/cgi-bin/wnt/), and (2) that the 3D structure of a Wnt bound to a Frizzled ligand-binding domain shows that most ligand-receptor contacts involved residues (and a lipid) that are conserved across Wnt and Frizzled family members (Janda et al., 2012Janda C.Y. Waghray D. Levin A.M. Thomas C. Garcia K.C. Structural basis of Wnt recognition by Frizzled.Science. 2012; 337: 59-64Crossref PubMed Scopus (568) Google Scholar). We suggest that binding and signaling specificity between the 19 mammalian Wnts and the ten mammalian Frizzleds is sharpened by coactivators such as Gpr124, allowing cells such as ECs to exhibit greater discrimination in an environment in which multiple Wnts are competing for receptor binding. It would be of interest to test whether other members of the adhesion GPCR family function as coactivators in the context of other Wnt/Frizzled combinations or of other ligand-receptor systems. The finding that Wnt/Gpr124 and Norrin/Fz4 function in a largely redundant manner to maintain the BBB in postnatal life lends support to an emerging picture of the BBB as a metastable state of EC differentiation that is maintained by canonical Wnt signaling (Liebner et al., 2008Liebner S. Corada M. Bangsow T. Babbage J. Taddei A. Czupalla C.J. Reis M. Felici A. Wolburg H. Fruttiger M. et al.Wnt/beta-catenin signaling controls development of the blood-brain barrier.J. Cell Biol. 2008; 183: 409-417Crossref PubMed Scopus (553) Google Scholar, Wang et al., 2012Wang Y. Rattner A. Zhou Y. Williams J. Smallwood P.M. Nathans J. Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity.Cell. 2012; 151: 1332-1344Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). It would be of great interest to determine whether alterations in canonical Wnt signaling play a role in pathologic BBB breakdown, as observed in the contexts of neuroinflammation, stroke, and trauma (Obermeier et al., 2013Obermeier B. Daneman R. Ransohoff R.M. Development, maintenance and disruption of the blood-brain barrier.Nat. Med. 2013; 19: 1584-1596Crossref PubMed Scopus (1333) Google Scholar). Defining the molecules and pathways that control BBB integrity also suggests potential strategies for transient and/or localized modulation of BBB integrity to enhance CNS drug delivery (Paes et al., 2011Paes K.T. Wang E. Henze K. Vogel P. Read R. Suwanichkul A. Kirkpatrick L.L. Potter D. Newhouse M.M. Rice D.S. Frizzled 4 is required for retinal angiogenesis and maintenance of the blood-retina barrier.Invest. Ophthalmol. Vis. Sci. 2011; 52: 6452-6461Crossref PubMed Scopus (51) Google Scholar). The experiments reported here, together with the observation of high Gpr124 transcript levels in tumor vasculature (St. Croix et al., 2000St. Croix B. Rago C. Velculescu V. Traverso G. Romans K.E. Montgomery E. Lal A. Riggins G.J. Lengauer C. Vogelstein B. Kinzler K.W. Genes expressed in human tumor endothelium.Science. 2000; 289: 1197-1202Crossref PubMed Scopus (1643) Google Scholar, Carson-Walter et al., 2001Carson-Walter E.B. Watkins D.N. Nanda A. Vogelstein B. Kinzler K.W. St. Croix B. Cell surface tumor endothelial markers are conserved in mice and humans.Cancer Res. 2001; 61: 6649-6655PubMed Google Scholar), implicate canonical Wnt signaling in tumor angiogenesis. This inference is consistent with earlier observations that Wnt antagonists can inhibit both vascularization of tumors and differentiation of endothelial cell progenitors (Hu et al., 2009Hu J. Dong A. Fernandez-Ruiz V. Shan J. Kawa M. Martínez-Ansó E. Prieto J. Qian C. Blockade of Wnt signaling inhibits angiogenesis and tumor growth in hepatocellular carcinoma.Cancer Res. 2009; 69: 6951-6959Crossref PubMed Scopus (120) Google Scholar). It seems likely that canonical Wnt signaling in ECs activates similar proangiogenic programs during CNS and tumor angiogenesis. Taken together, these data support the idea that inhibition of canonical Wnt signaling could synergize with inhibition of vascular endothelial growth factor signaling as an antiangiogenic therapy. The following transgenic mouse alleles were used: Gpr124CKO (JAX 016881; Cullen et al., 2011Cullen M. Elzarrad M.K. Seaman S. Zudaire E. Stevens J. Yang M.Y. Li X. Chaudhary A. Xu L. Hilton M.B. et al.GPR124, an orphan G protein-coupled receptor, is required for CNS-specific vascularization and establishment of the blood-brain barrier.Proc. Natl. Acad. Sci. USA. 2011; 108: 5759-5764Crossref PubMed Scopus (144) Google Scholar), Tcf/Lef-nLacZ (JAX 005317; Maretto et al., 2003Maretto S. Cordenonsi M. Dupont S. Braghetta P. Broccoli V. Hassan A.B. Volpin D. Bressan G.M. Piccolo S. Mapping Wnt/beta-catenin signaling during mouse development and in colorectal tumors.Proc. Natl. Acad. Sci. USA. 2003; 100: 3299-3304Crossref PubMed Scopus (687) Google Scholar), NdpKO (JAX 012287; Ye et al., 2009Ye X. Wang Y. Cahill H. Yu M. Badea T.C. Smallwood P.M. Peachey N.S. Nathans J. Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization.Cell. 2009; 139: 285-298Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar), Fz4− (JAX 012823; Wang et al., 2001Wang Y. Huso D. Cahill H. Ryugo D. Nathans J. Progressive cerebellar, auditory, and esophageal dysfunction caused by targeted disruption of the frizzled-4 gene.J. Neurosci. 2001; 21: 4761-4771PubMed Google Scholar), Z-Norrin (JAX 011077; Ye et al., 2009Ye X. Wang Y. Cahill H. Yu M. Badea T.C. Smallwood P.M. Peachey N.S. Nathans J. Norrin, frizzled-4, and Lrp5 signaling in endothelial cells controls a genetic program for retinal vascularization.Cell. 2009; 139: 285-298Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar), Foxg1-Cre (JAX 004337; Hébert and McConnell, 2000Hébert J.M. McConnell S.K. Targeting of cre to the Foxg1 (BF-1) locus mediates loxP recombination in the telencephalon and other developing head structures.Dev. Biol. 2000; 222: 296-306Crossref PubMed Scopus (432) Google Scholar), Ctnnb1flex3 (Harada et al., 1999Harada N. Tamai Y. Ishikawa T. Sauer B. Takaku K. Oshima M. Taketo M.M. Intestinal polyposis in mice with a dominant stable mutation of the beta-catenin gene.EMBO J. 1999; 18: 5931-5942Crossref PubMed Scopus (968) Google Scholar), and Pdgfb-CreER (Claxton et al., 2008Claxton S. Kostourou V. Jadeja S. Chambon P. Hodivala-Dilke K. Fruttiger M. Efficient, inducible Cre-recombinase activation in vascular endothelium.Genesis. 2008; 46: 74-80Crossref PubMed Scopus (218) Google Scholar). Mice were handled and housed according to the approved Institutional Animal Care and Use Committee protocol MO13M469 of the Johns Hopkins Medical Institutions. Antibodies used in this study were as follows: chicken anti-beta-galactosidase (Abcam 9361); rabbit anti-GLUT-1 (Thermo Fisher Scientific RB-9052-P0); rabbit anti-NG2 (Millipore AB5320); rat anti-mouse CD102/ICAM2 (BD Biosciences 553326); rat anti-PLVAP/ MECA-32 (BD Biosciences 553849); mouse anti-Claudin-5, Alexa Fluor 488 conjugate (Invitrogen 352588), and Texas Red streptavidin (Vector Laboratories SA-5006). Alexa Fluor-labeled secondary antibodies and GS lectin (Isolectin GS-IB4) were from Invitrogen. Primary antibodies were used at 1:200 to 1:500 dilution. Luciferase assays were performed as described by Xu et al., 2004Xu Q. Wang Y. Dabdoub A. Smallwood P.M. Williams J. Woods C. Kelley M.W. Jiang L. Tasman W. Zhang K. Nathans J. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.Cell. 2004; 116: 883-895Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar. Tissue processing was performed as described by Wang et al., 2012Wang Y. Rattner A. Zhou Y. Williams J. Smallwood P.M. Nathans J. Norrin/Frizzled4 signaling in retinal vascular development and blood brain barrier plasticity.Cell. 2012; 151: 1332-1344Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar and Zhou et al., 2014Zhou Y. Wang Y. Tischfield M. Williams J. Smallwood P.M. Rattner A. Taketo M.M. Nathans J. Canonical WNT signaling components in vascular development and barrier formation.J. Clin. Invest. 2014; Google Scholar. RNA was extracted from STF cells using Trizol (Life Technologies) and purified with the RNeasy kit (QIAGEN). The complementary DNA library was synthesized using the TrueSeq kit (Illumina) and sequenced from one end on an Illumina HiSeq, with read lengths of 50 bases. A total of 103,453,057 reads were obtained, of which 103,441,162 could be aligned to the human genome (hg19) using TopHat (v2.0.8). Transcript abundance (fragments per kilobase of transcript per million mapped reads) was quantified using Cufflinks (v2.1.1). Box and whisker plots (RStudio) show the median (heavy horizontal bar) and the 25th–75th percentiles (box). The whiskers are located at the positions of the furthest flung data points above or below the mean that are within a distance of 1.5 times the 25th–75th percentile distance (i.e., height of the box) either below the 25th percentile position or above the 75th percentile position. Any data points beyond the whiskers are plotted individually. A detailed description of the Experimental Procedures is presented in the Supplemental Information. Y.Z. and J.N. designed experiments, analyzed data, and wrote the paper; Y.Z. conducted the experiments. The authors thank Dr. Maketo Taketo (Kyoto University) for the Ctnnb1flex3 mice, Dr. Calvin Kuo (Stanford University) for the Gpr124 expression plasmid, Dr. Amir Rattner for providing the STF RNAseq data, Phil Smallwood with assistance in plasmid construction and characterization, and Drs. Amir Rattner and Hao Wu for comments on the manuscript. This study was supported by the National Eye Institute (EY018637 to J.N.), the Ellison Medical Foundation, and the Howard Hughes Medical Institute. The Gene Expression Omnibus accession numbers for the RNA sequencing data reported in this paper are GSE60529 and GSM1481718. Download .pdf (9.46 MB) Help with pdf files Document S1. Supplemental Experimental Procedures, Figures S1–S3, and Tables S1 and S2" @default.
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