Wnt/β-Catenin Signaling, Disease, and Emerging Therapeutic Modalities

The WNT signal transduction cascade is a main regulator of development throughout the animal kingdom. Wnts are also key drivers of most types of tissue stem cells in adult mammals. Unsurprisingly, mutated Wnt pathway components are causative to multiple growth-related pathologies and to cancer. Here, we describe the core Wnt/β-catenin signaling pathway, how it controls stem cells, and contributes to disease. Finally, we discuss strategies for Wnt-based therapies. The WNT signal transduction cascade is a main regulator of development throughout the animal kingdom. Wnts are also key drivers of most types of tissue stem cells in adult mammals. Unsurprisingly, mutated Wnt pathway components are causative to multiple growth-related pathologies and to cancer. Here, we describe the core Wnt/β-catenin signaling pathway, how it controls stem cells, and contributes to disease. Finally, we discuss strategies for Wnt-based therapies. Since the initial discovery of the first member of the Wnt family 35 years ago (Nusse and Varmus, 1982Nusse R. Varmus H.E. Many tumors induced by the mouse mammary tumor virus contain a provirus integrated in the same region of the host genome.Cell. 1982; 31: 99-109Abstract Full Text PDF PubMed Scopus (949) Google Scholar), interest in Wnt signaling has steadily risen. In fields ranging from cancer and development to early animal evolution, Wnt signaling has emerged as a fundamental growth control pathway. Details about the mechanisms of Wnt signaling have been revealed, including structural information on the main molecular players. In this review, we will present an update (Clevers and Nusse, 2012Clevers H. Nusse R. Wnt/β-catenin signaling and disease.Cell. 2012; 149: 1192-1205Abstract Full Text Full Text PDF PubMed Scopus (1863) Google Scholar) on recent insights into Wnt signaling in various contexts, during normal physiology as well as in disease. Wnt signaling represents one of a handful of pathways, including Notch-Delta, Hedgehog, transforming growth factor β (TGF-β)/bone morphogenetic protein (BMP) and Hippo, which are all implicated in developmental processes. Each of these signaling pathways is conserved in evolution and widespread in its activity; it could be asked what is unique about the Wnt system compared to others? What are the effects of Wnt signals on cells and why is this pathway so ubiquitously active in growing tissues? Fundamentally, Wnts are growth stimulatory factors, leading to cell proliferation (Niehrs and Acebron, 2012Niehrs C. Acebron S.P. Mitotic and mitogenic Wnt signalling.EMBO J. 2012; 31: 2705-2713Crossref PubMed Scopus (99) Google Scholar). In doing so, Wnt signals impact the cell cycle at various points. Compared to other growth factors, a distinctive aspect of Wnt signaling is the ability to giving shape to growing tissues while inducing cells to proliferate, acting in the process as directional growth factors (Goldstein et al., 2006Goldstein B. Takeshita H. Mizumoto K. Sawa H. Wnt signals can function as positional cues in establishing cell polarity.Dev. Cell. 2006; 10: 391-396Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, Huang and Niehrs, 2014Huang Y.L. Niehrs C. Polarized Wnt signaling regulates ectodermal cell fate in Xenopus.Dev. Cell. 2014; 29: 250-257Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar, Schneider et al., 2015Schneider J. Arraf A.A. Grinstein M. Yelin R. Schultheiss T.M. Wnt signaling orients the proximal-distal axis of chick kidney nephrons.Development. 2015; 142: 2686-2695Crossref PubMed Scopus (6) Google Scholar, Kitajima et al., 2013Kitajima K. Oki S. Ohkawa Y. Sumi T. Meno C. 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Towards an integrated view of Wnt signaling in development.Development. 2009; 136: 3205-3214Crossref PubMed Scopus (625) Google Scholar), or maintaining tissue architecture in adult life. In this overview of the field, we will mostly discuss the Wnt/β-catenin (a.k.a. "canonical") pathway, its nuclear effects, and implications for diseases, recognizing that to cover all aspects of Wnt signaling is beyond our scope. There are multiple Wnt genes in any animal genome—19 in mammals for example (http://web.stanford.edu/group/nusselab/cgi-bin/wnt/)—raising the question of specificity: do individual Wnts have unique or overlapping functions? An argument for unique roles for each Wnt comes from loss-of-function genetic data: most Wnt genes, when eliminated from the genome, have distinct phenotypes. For example, mice mutant for Wnt1 have a midbrain defect (McMahon et al., 1992McMahon A.P. Joyner A.L. Bradley A. McMahon J.A. The midbrain-hindbrain phenotype of Wnt-1-/Wnt-1- mice results from stepwise deletion of engrailed-expressing cells by 9.5 days postcoitum.Cell. 1992; 69: 581-595Abstract Full Text PDF PubMed Scopus (0) Google Scholar) while Wnt4 mutants are compromised in the development of the kidney (Stark et al., 1994Stark K. Vainio S. Vassileva G. McMahon A.P. Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4.Nature. 1994; 372: 679-683Crossref PubMed Google Scholar). There are numerous other unique or partially overlapping phenotypes associated with loss of Wnt genes (http://web.stanford.edu/group/nusselab/cgi-bin/wnt/) and, not surprisingly, the morphological phenotypes correspond to where the Wnts are expressed. In addition to these genetic arguments, a case for inherent and important differences between individual Wnt signals comes from the high vertical evolutionary conservation of Wnt proteins. Orthologs within the Wnt family can be traced throughout all animal phyla: Wnt1 in mammals is the true ortholog of Wnt1 in Hydra and Wingless in Drosophila (Kusserow et al., 2005Kusserow A. Pang K. Sturm C. Hrouda M. Lentfer J. Schmidt H.A. Technau U. von Haeseler A. Hobmayer B. Martindale M.Q. et al.Unexpected complexity of the Wnt gene family in a sea anemone.Nature. 2005; 433: 156-160Crossref PubMed Scopus (0) Google Scholar). Strikingly, Hydra and other Cnidaria have a set of Wnt genes that correspond one-to-one to vertebrate counterparts (Kusserow et al., 2005Kusserow A. Pang K. Sturm C. Hrouda M. Lentfer J. Schmidt H.A. Technau U. von Haeseler A. Hobmayer B. Martindale M.Q. et al.Unexpected complexity of the Wnt gene family in a sea anemone.Nature. 2005; 433: 156-160Crossref PubMed Scopus (0) Google Scholar). Such a high degree of conservation and evolutionary constraint would argue that intrinsic properties of different Wnts are important for their functions. On the other hand, when it comes to biochemical signaling mechanisms or effects on target cells, different Wnts behave in a very similar way. With respect to binding of Wnts to the receptors, the Frizzleds (FZDs), there is extensive cross-reactivity (Yu et al., 2012Yu H. Ye X. Guo N. Nathans J. Frizzled 2 and frizzled 7 function redundantly in convergent extension and closure of the ventricular septum and palate: evidence for a network of interacting genes.Development. 2012; 139: 4383-4394Crossref PubMed Scopus (48) Google Scholar, Dijksterhuis et al., 2015Dijksterhuis J.P. Baljinnyam B. Stanger K. Sercan H.O. Ji Y. Andres O. Rubin J.S. Hannoush R.N. Schulte G. Systematic Mapping of WNT-Frizzled Interactions Reveals Functional Selectivity by Distinct WNT-Frizzled Pairs.J Biol Chem. 2015; 290: 6789-6798Crossref PubMed Scopus (21) Google Scholar). In addition, most Wnt proteins will lead to elevated levels of β-catenin in cells or increases in signaling reporter activity (Alok et al., 2017Alok A. Lei Z. Jagannathan N.S. Kaur S. Harmston N. Rozen S.G. Tucker-Kellogg L. Virshup D.M. Wnt proteins synergize to activate β-catenin signaling.J. Cell Sci. 2017; 130: 1532-1544Crossref PubMed Scopus (0) Google Scholar). These assays however, mostly done in cell culture, may not reveal the whole spectrum of signaling activity or receptor-binding finesses of different Wnts. As we will show below, there are various co-receptors for Wnts that may modulate signaling outcome. Taking all these observations together, we suggest that, by and large, the differences between loss-of-function Wnt phenotypes can be attributed to discrete and unique expression patterns of the Wnt genes. Because of the fact that Wnt proteins signal very close to where they are produced, it seems that the overall phenotypes caused by loss of Wnt gene function are primarily due to local expression domains of each Wnt. In addition, intrinsic differences between Wnts, their binding to receptors and co-receptors are no doubt consequential for the various developmental processes as well. Wnt proteins act as intercellular signals but there are several unresolved questions on the nature of the extracellular form of Wnts and the mechanisms of export. During synthesis, Wnt proteins, 40 kDa in size and rich in cysteines, are modified by attachment of a lipid, an acyl group termed palmitoleic acid (Willert et al., 2003Willert K. Brown J.D. Danenberg E. Duncan A.W. Weissman I.L. Reya T. Yates 3rd, J.R. Nusse R. Wnt proteins are lipid-modified and can act as stem cell growth factors.Nature. 2003; 423: 448-452Crossref PubMed Scopus (1421) Google Scholar, Rios-Esteves et al., 2014Rios-Esteves J. 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Resh M.D. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins.Cell Rep. 2013; 4: 1072-1081Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The lipid functions primarily as a binding motif the Wnt receptor, FZD (see below) (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 (262) Google Scholar), but it also renders the Wnt protein hydrophobic and may tether it to cell membranes. The lipid may therefore contribute to restricting Wnt spreading and its range of action. During maturation of the Wnt protein, the transmembrane protein Wntless/Evi (Wls) (Bartscherer et al., 2006Bartscherer K. Pelte N. Ingelfinger D. Boutros M. Secretion of Wnt ligands requires Evi, a conserved transmembrane protein.Cell. 2006; 125: 523-533Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, Bänziger et al., 2006Bänziger C. 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A uniform human Wnt expression library reveals a shared secretory pathway and unique signaling activities.Differentiation. 2012; 84: 203-213Crossref PubMed Scopus (51) Google Scholar) and is required for ferrying Wnts to the plasma membrane to become secreted (Figure 1). How extracellular Wnt signals are transferred to target cells remains mysterious, but available evidence suggests that the proteins are not present in a free form. More likely, Wnt proteins are incorporated into secretory vesicles or exosomes (Gross et al., 2012Gross J.C. Chaudhary V. Bartscherer K. Boutros M. Active Wnt proteins are secreted on exosomes.Nat. Cell Biol. 2012; 14: 1036-1045Crossref PubMed Scopus (246) Google Scholar, Korkut et al., 2009Korkut C. Ataman B. Ramachandran P. Ashley J. Barria R. Gherbesi N. Budnik V. 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Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless.Cell. 2009; 139: 393-404Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar) (Figure 1),in such a form that the Wnt protein is present on the outside of the vesicle, available for binding to receptors. In another model, Wnt transfer involves direct contact between cells mediated by receptors FZD and the transmembrane E3 ligases Rnf43/Znrf3 (Farin et al., 2016Farin H.F. Jordens I. Mosa M.H. Basak O. Korving J. Tauriello D.V.F. de Punder K. Angers S. Peters P.J. Maurice M.M. Clevers H. Visualization of a short-range Wnt gradient in the intestinal stem-cell niche.Nature. 2016; 530: 340-343Crossref PubMed Scopus (294) Google Scholar) (Figure 1). Although it is sometimes assumed that secreted Wnt signals are long-range morphogens, there is little evidence that this is the prevailing mode. In most tissues, Wnt signaling occurs between neighboring cells that contact each other. Even in the best studied example of long-range signaling by a Wnt—that is, by the Wnt ligand Wingless in Drosophila—recent evidence has made a case that the requirements for the gene can be largely provided by a membrane-tethered form of the protein which, in principle, cannot diffuse (Alexandre et al., 2014Alexandre C. Baena-Lopez A. Vincent J.-P. Patterning and growth control by membrane-tethered Wingless.Nature. 2014; 505: 180-185Crossref PubMed Scopus (92) Google Scholar). While the conclusion of this result might be that Wingless does not act as a long-range morphogen, it could still be that Wingless bound to membranous vesicles or filopodia (Stanganello et al., 2015Stanganello E. Hagemann A.I.H. Mattes B. Sinner C. Meyen D. Weber S. Schug A. Raz E. Scholpp S. Filopodia-based Wnt transport during vertebrate tissue patterning.Nat. Commun. 2015; 6: 5846Crossref PubMed Scopus (38) Google Scholar) would operate over longer distances. In support of the vesicle model, it has been shown that vesicles containing Wingless and its transporter protein Wntless/Evi are present at neuromuscular junctions in Drosophila and interact with FZD receptors (Korkut et al., 2009Korkut C. Ataman B. Ramachandran P. Ashley J. Barria R. Gherbesi N. Budnik V. Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless.Cell. 2009; 139: 393-404Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). Further alternatives to explain long-range activities by Wnts include sequential signaling between Wnt target cells and their neighbors, mediated by various Wnt family members. Indeed, Cnidaria embryos display staggered expression of various Wnt family members across the primary axis (Kusserow et al., 2005Kusserow A. Pang K. Sturm C. Hrouda M. Lentfer J. Schmidt H.A. Technau U. von Haeseler A. Hobmayer B. Martindale M.Q. et al.Unexpected complexity of the Wnt gene family in a sea anemone.Nature. 2005; 433: 156-160Crossref PubMed Scopus (0) Google Scholar). In yet another context, stem cell niches of the intestinal crypts, Wnt protein bound to FZD receptor-expressing cells can become diluted as cells move and divide (Farin et al., 2016Farin H.F. Jordens I. Mosa M.H. Basak O. Korving J. Tauriello D.V.F. de Punder K. Angers S. Peters P.J. Maurice M.M. Clevers H. Visualization of a short-range Wnt gradient in the intestinal stem-cell niche.Nature. 2016; 530: 340-343Crossref PubMed Scopus (294) Google Scholar), a mode of Wnt transport that can also be directly visualized in intestinal organoid cultures (Figure 1). These results add to—but do not—resolve the continuing debate on the Wnt signaling landscape and the existence of morphogens. On the surface of cells, Wnt proteins bind to a receptor complex of two molecules, FZD (FZD) and LRP5/6 (Figure 2). FZD proteins have 7-transmembrane (7TM) and an extracellular N-terminal cysteine-rich domain (CRD) (Bhanot et al., 1996Bhanot P. Brink M. Samos C.H. Hsieh J.C. Wang Y. Macke J.P. Andrew D. Nathans J. Nusse R. A new member of the frizzled family from Drosophila functions as a Wingless receptor.Nature. 1996; 382: 225-230Crossref PubMed Scopus (1045) Google Scholar). The CRD is the primary interacting module for Wnt binding with affinities in the nM range (Hsieh et al., 1999Hsieh J.C. Rattner A. Smallwood P.M. Nathans J. Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein.Proc. Natl. Acad. Sci. USA. 1999; 96: 3546-3551Crossref PubMed Scopus (246) Google Scholar). The structure of the CRD as bound to Wnt demonstrates that there are multiple interacting surfaces, including a hydrophobic pocket in the CRD that binds to the lipid on Wnt (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 (262) Google Scholar). In addition, the C terminus of Wnt makes contact with the CRD (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 (262) Google Scholar). During signaling, FZDs cooperate with the single-pass transmembrane molecule LRP5/6, in such a way that binding of the Wnt protein leads to dimerization of the two receptors (Figure 2) (Janda et al., 2017Janda C.Y. Dang L.T. You C. Chang J. de Lau W. Zhong Z.A. Yan K.S. Marecic O. Siepe D. Li X. et al.Surrogate Wnt agonists that phenocopy canonical Wnt and β-catenin signalling.Nature. 2017; https://doi.org/10.1038/nature22306Crossref Scopus (4) Google Scholar). This mechanism would lead to a conformational change of the receptors. As a consequence, the cytoplasmic tail of LRP, after phosphorylation by several protein kinases, recruits the scaffold protein Axin. One of these phosphorylations on LRP is mediated by GSK3 on a serine in a PPPSP motif. The same motif is found in a number of Wnt signaling components including β-catenin, Axin, and APC (Stamos et al., 2014Stamos J.L. Chu M.L. Enos M.D. Shah N. Weis W.I. Structural basis of GSK-3 inhibition by N-terminal phosphorylation and by the Wnt receptor LRP6.eLife. 2014; 3: e01998Crossref PubMed Google Scholar). While LRP has a relatively well-understood function in signaling, there is still little known about the role of FZD in Wnt reception. The cytoplasmic part of FZD can bind to Dishevelled (DVL) (Tauriello et al., 2012Tauriello D.V.F. Jordens I. Kirchner K. Slootstra J.W. Kruitwagen T. Bouwman B.A.M. Noutsou M. Rüdiger S.G.D. Schwamborn K. Schambony A. Maurice M.M. Wnt/β-catenin signaling requires interaction of the Dishevelled DEP domain and C terminus with a discontinuous motif in Frizzled.Proc. Natl. Acad. Sci. USA. 2012; 109: E812-E820Crossref PubMed Scopus (0) Google Scholar) (Figure 4) that would then provide a platform for the interaction between the LRP tail and Axin, through the DIX domain on DVL and Axin (Schwarz-Romond et al., 2007Schwarz-Romond T. Fiedler M. Shibata N. Butler P.J. Kikuchi A. Higuchi Y. Bienz M. The DIX domain of Dishevelled confers Wnt signaling by dynamic polymerization.Nat. Struct. Mol. Biol. 2007; 14: 484-492Crossref PubMed Scopus (195) Google Scholar, Fiedler et al., 2011Fiedler M. Mendoza-Topaz C. Rutherford T.J. Mieszczanek J. Bienz M. Dishevelled interacts with the DIX domain polymerization interface of Axin to interfere with its function in down-regulating β-catenin.Proc. Natl. Acad. Sci. USA. 2011; 108: 1937-1942Crossref PubMed Scopus (72) Google Scholar). Multimers of receptor-bound DVL and Axin molecules might support the formation of the LRP-FZD dimer. In line with this model, higher-order complexes containing Wnts, receptors, and DVL as well as small particles of multimerized DVL molecules have been detected in cells (Schwarz-Romond et al., 2005Schwarz-Romond T. Merrifield C. Nichols B.J. Bienz M. The Wnt signalling effector Dishevelled forms dynamic protein assemblies rather than stable associations with cytoplasmic vesicles.J. Cell Sci. 2005; 118: 5269-5277Crossref PubMed Scopus (0) Google Scholar, Gammons et al., 2016Gammons M.V. Renko M. Johnson C.M. Rutherford T.J. Bienz M. Wnt signalosome assembly by DEP domain swapping of Dishevelled.Mol. Cell. 2016; 64: 92-104Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar, Jiang et al., 2015Jiang X. Charlat O. Zamponi R. Yang Y. Cong F. Dishevelled promotes Wnt receptor degradation through recruitment of ZNRF3/RNF43 E3 ubiquitin ligases.Mol. Cell. 2015; 58: 522-533Abstract Full Text Full Text PDF PubMed Google Scholar). Wnts are not the only ligands of the FZD receptors. The cysteine-knot protein Norrin, encoded by the NDP gene, can also bind and activate Wnt receptors (Figure 3). In humans, NDP mutations cause Norrie disease, an X-linked disorder characterized by hypovascularization of the retina and a severe loss of visual function. Norrin binds with high affinity and specificity to FZD-4 (Ke et al., 2013Ke J. Harikumar K.G. Erice C. Chen C. Gu X. Wang L. Parker N. Cheng Z. Xu W. Williams B.O. et al.Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex.Genes Dev. 2013; 27: 2305-2319Crossref PubMed Scopus (0) Google Scholar, Chang et al., 2015Chang T.H. Hsieh F.L. Zebisch M. Harlos K. Elegheert J. Jones E.Y. Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan.eLife. 2015; 4https://doi.org/10.7554/eLife.06554Crossref Scopus (13) Google Scholar), while coexpression of Norrin, FZD-4, and LRP5 potently activates Wnt/β-catenin signaling (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 (480) Google Scholar). Biochemical evidence and analyses of mice carrying mutations in the tetraspanin family member, Tspan12, provide evidence that Tspan12 is a Norrin-specific co-receptor (Figure 3) (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 (152) Google Scholar) that may act by forming a ternary complex with FZD4 (Ke et al., 2013Ke J. Harikumar K.G. Erice C. Chen C. Gu X. Wang L. Parker N. Cheng Z. Xu W. Williams B.O. et al.Structure and function of Norrin in assembly and activation of a Frizzled 4-Lrp5/6 complex.Genes Dev. 2013; 27: 2305-2319Crossref PubMed Scopus (0) Google Scholar). Interestingly, FZD can also act as a receptor for the Clostridium difficile toxin B (TcdB) (Tao et al., 2016Tao L. Zhang J. Meraner P. Tovaglieri A. Wu X. Gerhard R. Zhang X. Stallcup W.B. Miao J. He X. et al.Frizzled proteins are colonic epithelial receptors for C. difficile toxin B.Nature. 2016; 538: 350-355Crossref PubMed Scopus (5) Google Scholar), a toxin known to be a critical virulence factor in causing diseases after infection by C. difficile infection. TcdB can bind to the CRD of FZD, with different affinities for several FZD family members. As TcdB can actually compete with Wnt for binding to FZDs and blocks Wnt signaling, the pathology underlying C. difficile infection could be caused by loss of Wnt signaling in the intestine, a supposition that offers hope for therapeutic intervention in C. difficile infections (Tao et al., 2016Tao L. Zhang J. Meraner P. Tovaglieri A. Wu X. Gerhard R. Zhang X. Stallcup W.B. Miao J. He X. et al.Frizzled proteins are colonic epithelial receptors for C. difficile toxin B.Nature. 2016; 538: 350-355Crossref PubMed Scopus (5) Google Scholar). In addition to the core receptors FZD and LRP5/6, there are several other transmembrane molecules implicated in Wnt signaling. These include the ROR and RYK tyrosine kinase receptors, able to bind to Wnt ligands using a CRD or WIF domain respectively (Figure 3). Once activated, these receptors feed into other signaling pathways in cells. Each of them has also been shown to interact with DVL, leading to the phosphorylation of this common Wnt pathway component. The consequences of these DVL modifications are otherwise unknown (Ho et al., 2012Ho H.-Y.H. Susman M.W. Bikoff J.B. Ryu Y.K. Jonas A.M. Hu L. Kuruvilla R. Greenberg M.E. Wnt5a-Ror-Dishevelled signaling constitutes a core developmental pathway that controls tissue morphogenesis.Proc. Natl. Acad. Sci. USA. 2012; 109: 4044-4051Crossref PubMed Scopus (0) Google Scholar, Huang et al., 2013Huang X. McGann J.C. Liu B.Y. Hannoush R.N. Lill J.R. Pham V. Newton K. Kakunda M. Liu J. Yu C. et al.Phosphorylation of Dishevelled by protein kinase RIPK4 regulates Wnt signaling.Science. 2013; 339: 1441-1445Crossref PubMed Scopus (32) Google Scholar). Yet another receptor, GPR 124, is required for correct Wnt signaling in establishing the blood brain barrier (Zhou and Nathans, 2014Zhou Y. Nathans J. Gpr124 controls CNS angiogenesis and blood-brain barrier integrity by promoting ligand-specific canonical wnt signaling.Dev. Cell. 2014; 31: 248-256Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, Posokhova et al., 2015Posokhova E. Shukla A. Seaman S. Volate S. Hilton M.B. Wu B. Morris H. Swing D.A. Zhou M. Zudaire E. et al.GPR124 functions as a WNT7-specific coactivator of canonical β-catenin signaling.Cell Rep. 2015; 10: 123-130Abstract Full Text Full Text PDF PubMed Google Scholar). Here, Wnt7 is the locally acting ligand, working through FZD and LRP, but whether Wnt7A binds directly to the multiple pass transmembrane protein GPR124 is not clear (Figure 3) (Zhou and Nathans, 2014Zhou Y. Nathans J. Gpr124 controls CNS angiogenesis and blood-brain barrier integrity by promoting ligand-specific canonical wnt signaling.Dev. Cell. 2014; 31: 248-256Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Whether all of these Wnt receptors, including ROR, RYK and GPR124 cooperate on cells, forming higher order structures, or operate independently is a major question that would require the development of new assays. Going back to the structure of the Wnt-FZD complex, it is striking that there is extensive surface left between the two separate binding domains on Wnt for FZD, suggesting that other molecules, including other receptors could par