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Introduction to the Wnt Family of Proteins
The Wnt genes constitute a large family of cysteine-rich, secreted glycoproteins that are involved in critical aspects of early embryonic development. The term wnt is an amalgam of wingless (Wg) and int. The Wg gene was identified as a locus in Drosophila required in each segment for the establishment of normal pattern. In the absence of Wg, posterior pattern elements (i.e. naked cuticle) are replaced by mirror image duplications of anterior denticle bands. The int loci (there were 2 termed int-1 and int-2) were originally identified in mice as sights of frequent integration (hence int loci) of the Moloney murine leukemia virus. Subsequently it was shown that the murine int-1 locus was homologous to Drosophila Wg and the family name was changed to Wnt to reflect both origins. To date there have been at least 18 mammalian members of the Wnt family (visit the Roeland Nusse lab for an extensive listing of Wnt genes). Several of the mouse Wnt genes are described in the Table below.
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Wnt Expression Patterns in Mice
GeneEmbryonic ExpressionAdult Expression
Wnt-1brain, spinal cordtestes
Wnt-2ventral-lateral mesoderm, heart, allantoislung, brain, heart
Wnt-3brain, spinal cord, limbsthalamus, Purkinje cells
Wnt-3abrain, primitive streaklung
Wnt-4not determinedbrain, lung
Wnt-5aventral brain, spinal cordheart, lung
Wnt-5bnot restrictedheart, brain, liver, lung
Wnt-6not determinedtestes
Wnt-7anot determinedbrain, lung
Wnt-8not determinedbrain

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Classification of Wnt Activities
Results from experiments designed to ectopically express Wnt proteins in mammary epithelial cells and in Xenopus embryos allowed for the loose division of the Wnts into two classes. One class are the axis inducing and transforming Wnt-1 class. All the Wnts of this class are capable of transforming mammalian cells in culture and of inducing a secondary neural axis when ectopically expressed in the future ventral side of early frog embryos. The Wnts in this first class are the mouse Wnt-1, -3a and -7a genes and the Xenopus Xwnt-1, -3a, -8 and -8b genes.
The second class of Wnts are the non-transforming Wnt-5a class. This class of Wnts cannot induce the transformed state in cells in culture and do not induce a secondary neural axis in frog embryos. The Wnts of this class include mouse the Wnt-4 and -5a genes and the Xenopus Xwnt-5a and -4 genes. Chimeric proteins generated by combining elements of Xwnt-8 (a Wnt-1 class protein) and Xwnt-5A (a Wnt-5a class protein) and then used for axis induction experiments in frog embryos, demonstrated that the different activities of the two Wnt classes is determined by the C-terminal portion of the proteins.
The Wnt proteins are capable of inducing cells to proliferate, to differentiate and to survive by signaling through both autocrine and paracrine pathways. The Wnt proteins exert these effects on cells and during early development by interacting with specific cell-surface receptors which in turn initiate a signaling cascade culminating with changes in gene expression. It is this Wnt-induced alteration in the expression of specific genes that leads to the multitude of effects observed for Wnt proteins.
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Wnt Genes and Central Nervous System Development
By using the technique of in situ hybridization, several studies have suggested that Wnt signaling plays an important role during the early patterning of the neural tube. In mouse embryos, the timing of Wnt gene expression in the central nervous system (CNS) indicates that these genes likely assist in the regional specification of the neural tube, in particular in the forebrain region and the spinal cord.
Wnt gene expression within the developing spinal cord exhibits particular patterns of restriction along the dorsal-ventral axis prior to the differentiation of neurons. Wnt-1, Wnt-3 and Wnt-3a are the earliest markers observed to be expressed along the dorsal midline of the developing spinal cord. Wnt-4 expression correlates with the region of the presumptive alar plate that is destined to produce sensory interneurons. Wnt-7a and Wnt-7b are expressed in the ventral spinal cord. Additionally, Wnt-4 is expressed in the ventral region of the floor plate. Since it has been shown that signals emanate from the notochord to induce the formation of the floor plate within the CNS (these are in the form of the inducing protein, sonic hedgehog, shh), the expression patterns of these Wnts suggest that they may play important roles in reinforcing the dorsal-ventral polarities of the CNS established by inducers such as shh. Overexpression of Wnt-1 within the spinal cord severely disrupts normal spinal cord morphology, most propably as a result of overprolifersation. However, this effect does not interfere with the primary dorsal-ventral patterning of the spinal cord indicating that Wnts likely regulate the patterns of cell proliferation within the spinal cord not the actual patterns induced by other genes.
Several Wnts have been shown to be expressed in the developing forebrain. Wnt-5a and Wnt-7a are expressed in overlapping domains in the ventral and lateral diencephalon. Wnt-1, Wnt-3, Wnt-3a and Wnt-4 are expressed in the dorsal diencephalon. Wnt-7b is expressed throughout much of the diencephalon and the optic stalks. Wnt-3b and Wnt-7b expression extends into the telencephalon along the dorsal regions. These patterns of expression indicate a role for the Wnts as important regulators of the developing forebrain. The pattern of segmentation in the vertebrate hindbrain is divided into regions identified as rhombomeres. Evidence on the expression of numerous genes suggests the the forebrain may be similarly identifiable by specific patterns of segmentation. Wnt gene expression patterns generally coincide with these predicted segments of the forebrain. As an example, an early patch of Wnt-3a expression in the diencephalon appears to correspond to the presumptive pretectum (synencephalon). A domain of Wnt-3 expression correlates with the future dorsal thalamus. Expression of Wnts in the developing embryonic CNS clearly demarcates dorsal-ventral zones whose subdivisions are comparable to the roof, alar and basal plates of the more posterior neural tube.
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Wnt Genes and Axis Specification
As indicated above the Wnts are divided into two classes determined in part on an ability, or lack thereof, to induce a secondary neural axis when ectopically expressed in the future dorsal side of frog embryos. The earliest experiments with Wnts and axis duplication were performed with Wnt-1. Subsequently, numerous Xenopus Wnt genes (identified as Xwnts) have been isolated and characterized. Three possible functions for Wnts in the establishemnt of axial pattern have been suggested.
One mechanism suggests that Wnts could assist in the initial establishment of dorsal versus ventral sides of the embryo very early following fertilization and be components of the dorsalizing or ventralizing centers of mesoderm induction. Induction of mesoderm has been established as being critically important in future neuralization of the embryo. A second mechanism has the Wnts acting as parts of the dorsal or ventral signals that modify the inherent character of pre-existing mesoderm. Lastly, Wnts could function by assisting in the production of specified mesoderm, as the primary axis information is established during gastrulation. Strong evidence more closely links Wnts to the latter two models.
Both Xwnt-8 and Xwnt-11 have the capacity to modify the character of mesoderm that has been induced by other signals. Xwnt-8 has the capability to dorsalize regions of the embryo when ectopically expressed. However, it is normally expressed on the ventral side of the embryo during the blastula and gastrula stages. When Xwnt-8 is expressed dorsally it has the capability to ventralize the embryo indicating that it is a good candidate for a ventralizing signal. Conversely, Xwnt-11 is capable of dorsalizing previoulsy induced mesoderm and the RNA is localized to the marginal zone (future mesodermal tissue is derived from this region of the embryo) with highest levels being found dorsally. When embryos are exposed to uv-irradiation on the vegetal hemisphere they are completely ventralized and form no dorsal structures. Xwnt-11 is capable of partially rescuing the dorsal axis in uv-ventralized embryos. Both Xwnt-8 and Xwnt-11 are incapable of inducing mesoderm on their own, therefore, the above described results indicate that both Wnts must play a role in the modification of pre-existing mesoderm.
Wnt proteins are important mediators of axis establishment when the primary patterns of the embryo are determined during gastrulation. Direct evidence for this has been shown in mice in which Wnt genes have been knocked out by targeted disruption. Expression of Wnt-3a is first detected throughout the primitive streak of the mouse embryo in regions fated to generate dorsal mesoderm. Targeted disruption of the mouse Wnt-3a gene results in a severe truncation of the body axis posterior to the forelimbs. The most affected tissue being dorsal mesoderm; the notochord is disrupted and the tailbud fails to form. These data demonstrate that Wnt-3a is required for the production of somitic mesoderm and for the generation of all new embryonic mesoderm by the late primitive streak stage.
The Wnt-5a and -5b genes are also expressed in the primitive streak during gastrulation in the mouse. Overexpression of Xwnt-5a in frog embryos leads to complex head and tail malformations. Xwnt-5a does not ectopically induce mesoderm or change the type of pre-existing mesoderm but can inhibit the normal morphogenetic movements that occur during gastrulation. The chicken Cwnt-8c gene is expressed in the posterior marginal zone during gastrulation and later in the primitive streak and Hensen's node. When Cwnt-8c is ectopically expressed in frog embryos it can dorsalize mesoderm and induce axis duplication.
Although no Wnts are candidates for endogenous mesoderm induction or initial patterning, it is clear that this family of proteins is capable of modifying pre-existing mesoderm (Xwnt-8, Xwnt-11 and Cwnt-8c) and affecting gastrulation (Wnt-3a, Wnt-5a, Wnt-5b and Cwnt-8c).
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Wnt Signaling Pathways
Wnts are soluble secreted factors that exert the effects described above through interaction with cell surface receptors. Wnts bind to receptors of the Frizzled family, named after the Drosophila tissue polarity gene frizzled. There are currently 8 mamalian frizzled proteins. Structurally the Frizzled receptors have an extracellular Wnt-binding domain, seven transmembrane-spanning regions and an intracellular C-terminal tail. The presence of the seven transmembrane-spanning domains places the Frizzled proteins in the class of receptors known as the serpentine receptors (see the Signal Transduction page). This class includes the receptors for serotonin (5-HT), angiotensin and glucagon for example. The serpentine receptors couple ligand binding to activation of tripartite G-proteins.
Additional components of the Frizzled receptor-Wnt interaction pathway are the soluble Wnt inhibitors which consist of secreted proteins containing a cysteine-rich domain (CRD) similar to that in the ligand binding domain of the Frizzled receptors. This class of Wnt inhibitory proteins are termed frizzled receptor-like proteins (FRPs). By binding Wnts, FRP proteins can sequester Wnts away from the cell-surface Frizzled receptors and thereby, reduce the effective concentration of available Wnt protein.
Direct evidence for the interaction of Wnts and Frizzled receptors came from several studies. When Drosophila frizzled-2 (Dfz2) was expressed in fly cells in culture, the cells attained the ability to bind Wg protein. When rat Frizzled 1 (RFz1) or human Frizzled 5 (Hfz5) were expressed in frog embryos, Xwnt-5a and Xwnt-8 activated axis duplication through interaction with these exogenous receptors. The presence of a large family of Frizzled receptors suggests that there may exist the ability to differentially bind various Wnt proteins. Indeed, evidence shows that Dfz1, Dfz2, MFz4 (mouse Frizzled 4), MFz7, MFz8 and HFz5 but not MFz3 were able to bind Wg in fly cell in culture. Since Wg can bind to at least 6 different Frizzled receptors it is likely that multiple Wnts can be bound by any given Frizzled receptor and that many Wnts will be able to bind to multiple Frizzled receptors. Since endogenous Wnt expression patterns overlap (particularly in the mouse hindbrain) there may be functional redundancy in Wnt expression.
It is also probable that overlapping patterns of Wnt expression may allow for different Wnts to antagonize each other during development. Evidence for this latter possibility has been most clearly demonstrated in studies of axis duplication in Xenopus. Prior expression of Xwnt-5a blocks the axis inducing activity of Xwnt-8. The precise mechanism for this interference is not clear. It could be that Xwnt-5a blocked Xwnt-8 action by receptor competition or by blocking the action of intracellular signaling components.
Since the Frizzled receptors have no enzymic motifs on their intracellular domains, the Wnt signals are likely transmitted through receptor coupling to G-proteins as for the other members of the serpentine receptor family. One candidate family of G-proteins, shown by genetic studies in Drosophila to be downstream of Wg, is that characterized by Dishevelled (dsh). Members of the Dishevelled family encode cytoplasmic proteins with no known enzymic functions. Three homologues have been identified in mice (Dvl-1, Dvl-2 and Dvl-3) and shown to have overlapping patterns of expression. This overlapping pattern of expression suggests redundancy of function for Dishevelled proteins. This supposition is supported by the fact that targeted deletion of Dvl-1 results in mice that are structurally normal.
There are three regions of sequence homology between the different Dishevelled family members. The N-terminally conserved domain is termed the Dsh homology domain. This domain is similar to the C-terminus of Axin (encoded by the Drosophila fused gene) a protein shown to interfere with the formation of the endogenous dorsal axis and to prevent Xwnt-8 from inducing an ectopic axis. The central portion of Dishevelled proteins contains a PDZ domain which is present in many other proteins and has been shown to bind C-terminal peptides of the sequence motif X-(S/T)-X-V (where X is any amino acid). The Drosophila Dsh PDZ domains have been shown to be essential for downstream signaling in frog embryos. The third homology domain in Dishevelled proteins is the DEP domain. This domain has similarity to GTPase activating proteins (GAPs) and gunaine nucleotide exchange factors (GEFs). The DEP domain may, therefore, be important for recruiting downstream targets in G-protein-coupled pathways.
The next step in the Wnt signaling cascade is the inhibition of the serine/threonine kinase, glycogen synthase kinase-3b (GSK-3b) by Dishevelled. GSK-3b is a cytoplasmic kinase originally identified for its role in glycogen metabolism. GSK-3b has been shown to be the homolog of the Drosophila gene zeste-white-3 (zw3). Genetic studies showed that zw3 functioned downstream of dsh and probably inactivated Wg signaling. The mammalian GSK-3b protein can partially compensate for loss of zw3 during Drosophila embryogenesis. The fact that activation of Dishevelled leads to an inactivation of GSK-3b activity was demonstrated by the failure of GSK-3b to phosphorylate the microtubule-associated protein tau in Dvl-1 overexpression studies.
When a dominant negative form of Xenopus GSK-3b (dnGSK-3b), which lacks the kinase domain, is expressed in Xenopus embryos an ectopic axis forms which closely resembles the axis induced by ectopic expression of Xwnt-8 and Xdsh. In contrast, wild-type XGSK-3b is able to suppress the ectopic axis inducing activities of Xwnt-8 and Xdsh. This suggests that the normal function of GSK-3b is to suppress Wnt signaling from Dsh.
The consequences of Wnt-induced inhibition of GSK-3b activity is that the next protein identified in the cascade, b-catenin, is stabilized. b-Catenin is a member of a multigene family of proteins characterized by the presence of a domain found in the Drosophila Armadillo (Arm) gene. Genetic studies showed that Arm functions downstream of zw3/GSK-3b. Evidence indicates that Wnt inhibition of GSK-3b activity prevents the turnover of b-catenin leading to its accumulation. The loss of zw3 function or the use of dnGSK-3b stabilizes b-catenin and Arm levels in both Drosophila and Xenopus indicating that GSK-3b normally functions to lower b-catenin levels. Additionally, the loss of zw3 or the use of dnGSK-3b leads to a reduction in the phosphorylation of Arm and b-catenin. These observations indicate that phosphorylation of b-catenin leads to its degradation, by an as yet unidentified mechanism.
There are several structural domains in the b-catenins. The N-terminus is the site of GSK-3b-mediated phosphorylation. Another region binds to a-catenin, a cytoplasmic protein with similarity to vinculin which links actin filaments to the adherins junctions. The central region of the protein contains a positively charged groove that is suspected to be involved in interactions with acidic regions in the adenomatous polyposis coli (APC) locus encoded protein, the transcription factor TCF/LEF-1 (T-cell factor/Lymphocyte enhancer factor-1) and the cadherin family of calcium-dependent cell-adhesion molecules. The APC gene is a tumor-suppressor linked to inherited and sporadic forms of colon cancer (see the Tumor Suppressor page for more information on the connection between APC and Wnts).
Mutational analyses have shown that regions of b-catenin that are required for Wnt signaling are separable from those required for cell adhesion suggesting that cadherins are not required for Wnt signaling. However, changes in cadherin expression can affect Wnt signaling and conversely, Wnt signaling can affect cell adhesion. This could possibly lead to altered levels of free b-catenin and thus indirectly alter the level of b-catenin-TCF complexes.
It is not clear whether APC is directly involved in the Wnt signal cascade. However, b-catenin has been shown to directly interact with APC and mutations in APC that led to colon cancer result in a protein that does not bind to b-catenin. Complexes of APC and b-catenin bind to GSK-3b. These results suggest that APC functions as a molecular scaffold coordinating the activities of b-catenin and GSK-3b such that APC may assist in the ability of GSK-3b to phosphorylate b-catenin leading to its degradation. Lending confusion to the role of APC in Wnt signaling is the observation that overexpression of APC in frog embryos positively induced axis duplication instead of suppressing it as expected from the supposition that APC enhances GSK-3b activity.
The final link in the Wnt signal cascade is the interaction between b-catenin and the transcription factors of the TCF/LEF-1 family. Proteins of the TCF family bind to DNA and contain a high-mobility group (HMG) domain. Murine family members include LEF-1 and TCF-1 which function in lymphopoiesis and epithelial-mesenchymal interaction, respectively. There are three isoforms of the Xenopus XTCF3 gene. The pangolin gene in Drosophila and the pop-1 gene of C. elegans are family members. The N-terminus of TCF mediates the interaction with b-catenin. TCF:b-catenin complexes then enter the nucleus and interact with TCF DNA elements. Several candidate TCF:b-catenin target genes have thus far been identified. A few are the Xenopus homeobox genes siamois and Twin, the secreted protein nodal related 3 and fibronectin. The promoters of these genes all contain TCF/Lef binding sites. For a more complete updated listing, visit the Wnt Web site maintained by Roel Nusse which has a table of Wnt/b-catenin targets.
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This article has been modified by Dr. M. Javed Abbas.
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22:59 19/12/2002