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The Wnt genes encode a large family of secreted protein growth factors that have been identified in animals from hydra to humans. In humans, 19 WNT proteins have been identified that share 27% to 83% amino-acid sequence identity and a conserved pattern of 23 or 24 cysteine residues. Wnt genes are highly conserved between vertebrate species sharing overall sequence identity and gene structure, and are slightly less conserved between vertebrates and invertebrates. During development, Wnts have diverse roles in governing cell fate, proliferation, migration, polarity, and death. In adults, Wnts function in homeostasis, and inappropriate activation of the Wnt pathway is implicated in a variety of cancers.
In humans, 19 WNT genes have been identified and the chromosomal locations of each is known (see Table Table1)1) [1,2,3,4,5,6]. Several human WNT genes are located very close to each other in the genome [7,8]; these include WNT6 and WNT10a, which are located immediately adjacent to one another on chromosome 2 (about 6.4 kilobases (kb) apart), and WNT1 and WNT10b, which are located adjacent to each other on chromosome 12 (about 8.1 kb apart). WNT6 and WNT10a are transcribed in opposite directions, whereas WNT1 and WNT10b are expressed from the same strand of DNA. Several additional pairs of WNT genes are also clustered within the human genome, including WNT2 and WNT16 (about 4 megabases (Mb) apart), WNT3a and WNT14 (about 250 kb apart), and WNT3 and WNT15. In the mouse, there are at least 18 Wnt genes and the locations of all but two of them have been determined [1,2,3,5,6]. As in humans, the mouse Wnt1/Wnt10b, Wnt6/Wnt10a, and Wnt3/Wnt15 gene pairs are each located on the same chromosomes, and in the case of the Wnt1/Wnt10b and Wnt6/Wnt10a pairs the close proximity of these genes has been conserved from mouse to human. Interestingly, in the Drosophila genome, the paralogous genes wingless (wg), DWnt6 and DWnt10, are located immediately adjacent to one another on the second chromosome and are all transcribed in the same orientation. Thus, it is possible that there was an ancient cluster of Wnt genes consisting of Wnt1, Wnt6 and Wnt10 in a common ancestor of vertebrates and arthropods. In vertebrates, this cluster may have been duplicated with subsequent loss of Wnt1 from one cluster and Wnt6 from the other.
The majority of human WNT genes contain four coding exons, with exon 1 containing the initiation methionine (Figure (Figure1a)1a) . WNT genes that differ from this pattern include WNT14, with three exons, WNT2, WNT5b, and WNT11, with five exons, and WNT8b with six exons. Several WNTs - WNT2b/13, WNT8a/d, and WNT16 - have alternative amino or carboxyl termini, which result from the use of alternative 5' or 3' exons.
The deduced evolutionary relationships of 18 of the 19 known human WNT genes are shown in Figure Figure2.2. The majority of Wnt proteins share about 35% amino-acid sequence identity, although members of a subgroup (those with the same numeral, such as WNT3 and WNT3a) share increased sequence identity (from 58% to 83%) and some overlapping sites of expression. Members of subgroups are not closely linked within the genome, however, suggesting that they were generated by gene-translocation or genome-duplication events, not by local duplication events.
Wnt genes have been identified in vertebrates and invertebrates, but appear to be absent from plants, unicellular eukaryotes such as Saccharomyces cerevisiae and from prokaryotes. To date, in vertebrates, 16 Wnt genes have been identified in Xenopus, 11 in chick, and 12 in zebrafish ; in invertebrates, Drosophila has seven Wnt genes, Caenorhabditis elegans five and Hydra at least one . The apparent evolutionary relationships between selected invertebrate and vertebrate Wnt genes are shown in Figure Figure2b.2b. In vertebrates, the orthologs in different species are highly similar in sequence. For example, human WNT1 and mouse Wnt1 are 98% identical, and human WNT3a and Xenopus Wnt5a are 84% identical at the amino-acid level. Phylogenetic analyses of vertebrate and invertebrate Wnts demonstrate orthologous relationships between several human and Drosophila Wnts (Figure (Figure2b).2b). The sequence identity between orthologous proteins in humans and flies ranges from 21% between human WNT8a/d and Drosophila DWnt8 to 42% sequence identity between human WNT1 and Drosophila Wingless (Wg). The evolutionary relationship between the five C. elegans Wnt genes and human WNT genes is less apparent, making it difficult to determine which C. elegans Wnt genes may have orthologs in the human genome.
Human WNT proteins are all very similar in size, ranging in molecular weight from 39 kDa (WNT7a) to 46 kDa (WNT10a) . Drosophila Wnt proteins are also similar to this, with the exception of Wg, which is approximately 54 kDa and has an internal insert not found in vertebrate Wnts, and DWnt3/5, which is about 112 kDa . Very little is known about the structure of Wnt proteins, as they are notoriously insoluble, but all have 23 or 24 cysteine residues, the spacing of which is highly conserved (Figure (Figure1b),1b), suggesting that Wnt protein folding may depend on the formation of multiple intramolecular disulfide bonds. Analysis of the signaling activities of chimeric Wnt proteins has shown that the carboxy-terminal region of Wnt proteins may play a role in determining the specificity of responses to different Wnts . Furthermore, deletion mutants lacking the carboxy-terminal third of a Wnt protein can act as dominant-negatives in a cell-non-autonomous manner , suggesting that the amino-terminal region may mediate interactions with Wnt receptors but requires the carboxyl terminus to activate these receptors.
Wnt proteins have an amino-terminal signal sequence, can act in a cell non-autonomous manner, and are present in the secretory pathway, indicating that they are secreted proteins . In addition, genetic analyses of Wg signaling in Drosophila uncovered mutations in the porcupine gene that show a lack of Wnt activity due to the retention of Wg protein in the endoplasmic reticulum [12,13,14]. The porcupine gene is predicted to encode a protein with eight transmembrane domains and has a perinuclear localization in transfected cells ; overexpression of porcupine does not increase levels of secreted Wg but does change the pattern of Wg glycosylation . In worms, mom-1 encodes a porcupine homolog and, when mutated, phenocopies mutants of mom-2, which encodes a Wnt, suggesting that the function of porcupine is conserved [15,16]. Although size chromatography suggests that Wg is secreted as a multimer, it remains unclear whether Wnt proteins in general are secreted as monomers, oligomers, or as part of a multi-protein complex . Wnt proteins are glycosylated, but mutation of some or all of the predicted glycosylation sites in mouse Wnt1 does not abolish its activity in cultured cells ; these modifications may thus be unimportant for Wnt function.
Once secreted, Wnt proteins associate with glycosaminoglycans in the extracellular matrix and are bound tightly to the cell surface [19,20]. Although Wnts are found in tight association with the plasma membrane, it is possible to collect active Wnt from the medium of cultured cells [21,22]. Beyond this information, the localization of Wnt proteins in vertebrates is poorly understood. Examination of the localization of Wg in Drosophila, however, has provided critical insights into the subcellular distribution of Wnt proteins and the importance of this distribution for signaling activity. In the embryonic epidermis, Wg is found inside cells that secrete Wg and in association with the plasma membrane of secreting cells and non-secreting cells several cell diameters from the Wg source . Wg is also prevalent in vesicles and multi-vesicular bodies of non-Wg-producing cells anterior to the source of Wg, suggesting that Wg is endocytosed [23,24]. This idea is supported by examination of shibire embryos, which have a mutation in dynamin, a critical component of the endocytic machinery; these mutants have defects in Wg distribution, and Wg signaling activity is compromised . Similarly, expression of a dominant-negative form of shibire also reduces Wg activity . Endocytosis may also help to limit the distribution of Wg signal. In contrast to cells anterior to the Wg source, cells posterior to Wg-producing cells have much lower levels of Wg in endocytic vesicles, and this asymmetry in distribution mirrors the observation that Wg acts over a much shorter range towards the posterior than towards the anterior. This difference in Wg distribution appears to be due to rapid degradation of endocytosed Wg in posterior cells . The spatially restricted pattern of Wg degradation is regulated by signals through the epidermal growth factor (EGF) receptor that hasten the destruction of Wg in posterior cells .
Association of Wg with specific membrane microdomains also appears to play a role in controlling the distribution of Wg signals during Drosophila development. In imaginal discs, Wg is found in specialized membrane vesicles called argosomes, which are thought to be derived from lipid raft microdomains . Incorporation of Wg into argosomes requires heparan sulfate proteoglycans, suggesting that proteoglycans play a role in sorting Wg to specialized membrane microdomains in Wg-producing cells or, alternatively, may play a role localizing Wg in distinct endocytic compartments in receiving cells.
Polarized distribution of wg transcripts in embryonic epithelial cells is also required for optimal signaling activity. High-resolution in situ hybridization analyses demonstrate that wg transcripts are localized apically in the embryonic epidermis and that this distribution is mediated by two cis-acting elements found in the 3' UTR of the wg mRNA . Mutation of these elements results in uniform localization of wg transcripts and impaired Wg protein distribution and signaling. The asymmetric distribution of wg transcripts is dependent on dynein-mediated microtubule transport .
Reception and transduction of Wnt signals involves binding of Wnt proteins to members of two distinct families of cell-surface receptors, members of the Frizzled (Fzd) gene family and members of the LDL-receptor-related protein (LRP) family [31,32]. The canonical Fzd receptor has an amino-terminal cysteine-rich domain (CRD) that binds Wnt, seven transmembrane domains and a short cytoplasmic tail containing a consensus PDZ domain binding motif (S/T-X-V in the single-letter amino-acid code) at the carboxyl terminus. The CRD forms a novel protein fold with a conserved dimerization interface that may be important for Wnt binding . Fzd receptors have been identified in vertebrates and invertebrates; there are ten known members in humans and mice, four in flies, and three in worms. The general structure of Fzd receptors resembles that of seven-transmembrane G-protein-coupled receptors, suggesting that Fzd proteins may use heterotrimeric G proteins to transduce Wnt signals. Several recent studies provide evidence consistent with this idea, showing that a subgroup of Fzd receptors can signal through the pertussis-toxin-sensitive subclass of heterotrimeric G proteins to stimulate an increase in intracellular Ca2+ and activate protein kinase C (PKC) [34,35,36,37,38]. Heterotrimeric G proteins do not appear to be involved in transducing Wnt/Fzd signals that regulate the cytoskeleton-associated protein (β-catenin, however (see below).
Two members of the vertebrate LRP family, LRP-5 and LRP-6, can bind Wnts and may form a ternary complex with a Wnt and a Fzd . Mutations in LRP-6 in mice result in developmental defects similar to those seen in mice deficient for several individual Wnt genes , and overexpression of LRP in Xenopus can activate the Wnt pathway . In Drosophila, arrow, the ortholog of LRP5 and LRP6, is required for optimal Wg signaling . Although the mechanism of LRP signaling is unclear, recent evidence suggests that binding of the cytoplasmic domain of LRP to the Wnt antagonist Axin may play a role in Wnt pathway activation .
In addition to the Fzd and LRP receptors, cell-surface proteoglycans also appear to have a role in the reception of Wnt signals. For example, genetic analyses in Drosophila have shown that several genes required for optimal Wg signaling encode cell-surface proteoglycans of the glypican family [43,44] and proteins involved in proteoglycan synthesis [45,46,47]. Furthermore, QSulf1, an avian protein related to heparan-specific N-acetyl glucosamine sulfatases, has also been shown to regulate heparan-dependent Wnt signaling in cultured cells . It is unclear at this time how proteoglycans modulate Wnt signaling, but current suggestions include concentrating Wnt proteins at the cell surface or presenting Wnt ligands to cell-surface receptors.
Wnt signals are modulated extracellularly by diverse secreted proteins, including members of the Frizzled-related protein (FRP or FrzB) family , Wnt-inhibitory factor-1 (WIF-1) , Cerberus , and Dickkopf (Dkk) . FRPs, WIF-1, and Cerberus can bind Wnt proteins directly and are thought to antagonize Wnt function by preventing their interaction with Fzd receptors. FRPs can also interact with Fzds, suggesting that a second way in which FRPs might antagonize Wnt signaling is through the formation of a nonfunctional complex with Fzd receptors. Humans have at least five FRP genes, and the specificity of each FRP for different Wnts remains to be determined. Dkk does not bind Wnts but instead interacts with the extracellular domain of LRPs, thereby blocking activation of Wnt signaling [42,53,54]. Four Dkk genes have been identified in vertebrates, including Dkk2, which does not act as a Wnt antagonist but rather can stimulate Wnt signaling .
Wnt signals are transduced through at least three distinct intracellular signaling pathways including the canonical 'Wnt/β-catenin' pathway, the 'Wnt/Ca2+' pathway, and the 'Wnt/polarity' pathway (also called the 'planar polarity' pathway) [5,56,57,58,59,60,61,62]. Distinct sets of Wnt and Fzd ligand-receptor pairs can activate each of these pathways and lead to unique cellular responses. The Wnt/β-catenin pathway primarily regulates cell fate determination during development, whereas the major function of the Wnt/polarity pathway is regulation of cytoskeletal organization. The biological function of the Wnt/Ca2+ pathway is unclear.
The canonical Wnt/β-catenin pathway is intensely studied, and on the basis of current literature I propose the model illustrated in Figure Figure3a3a [59,63,64]. Signaling through this pathway depends on the levels of β-catenin in the cell. In the absence of Wnt, β-catenin is targeted for degradation by a multi-protein destruction complex. Wnt signaling antagonizes the destruction complex, leading to the accumulation of β-catenin and activation of target genes. Up-to-date lists of proteins involved in Wnt/β-catenin signaling and the potential roles of each of these proteins can be found on the worldwide web [5,60,62].
The Wnt/Ca2+ pathway involves an increase in intracellular Ca2+ and activation of PKC; it can be activated by a distinct group of Wnt ligands and Fzd receptors from those that activate other pathways, including Wnt5a, Wnt11 and Fzd2 (Figure (Figure3b)3b) [58,61,62]. The Wnt/Ca2+ pathway involves activation of a heterotrimeric G protein, an increase in intracellular Ca2+, and activation of calcium/calmodulin-regulated kinase II (CamKII) and PKC [34,35,37]. The downstream targets of CamKII and PKC are currently unknown, but it has been shown that activation of the Wnt/Ca2+ pathway can antagonize the Wnt/β-catenin pathway in Xenopus, although it is unclear at what level this interaction occurs .
Wnt/polarity signaling regulates the polarity of cells through regulation of their cytoskeletal organization (Figure (Figure3c)3c) [56,57,62]. In vertebrates, Wnt/polarity signaling is thought to control polarized cell movements during gastrulation and neurulation [66,67,68,69,70]. In Drosophila, Wnt/polarity signaling is required for the appropriate orientation of trichomes - or hairs - of the adult wing and for appropriate chirality of ommatidia in the eye, and may regulate asymmetric cell divisions of certain neuroblasts [56,71,72]. The only molecules known to function in both the vertebrate and the invertebrate Wnt/polarity pathways are members of the Fzd family and the cytoplasmic scaffold protein Dsh. The regulation of gastrulation movements in vertebrates also requires the activity of Wnt11, which may signal through Fzd7 to regulate protrusive activity during convergent extension [66,67]. In flies, genetic analyses have identified a number of potential components of the Wnt/polarity pathway in addition to DFzd1 and Dsh, including the small GTPase DrhoA, Drosophila rho-associated kinase (Drok), Jun N-terminal kinase (JNK), myosin II, myosin VIIA, and the products of the novel genes flamingo/starry night, fuzzy, inturned, and strabismus/van gogh [56,72]. A Wnt ligand for the Wnt/polarity pathway has not been identified in flies, however, and it remains to be seen how much of the intracellular signaling mechanism has been conserved between vertebrates and invertebrates.
Several studies have suggested that distinct classes of Wnts signal through either the Wnt/β-catenin pathway or the Wnt/Ca2+ pathway ; for example, overexpression studies in Xenopus have shown that XWnt1, XWnt3a, XWnt8, and XWnt8b can stimulate the Wnt/β-catenin pathway whereas XWnt4, XWnt5a, and XWnt11 can stimulate the Wnt/Ca2+ pathway . Furthermore, the separation of Wnts into these two distinct functional classes is mirrored by the classification of Fzd proteins into similar functional groups on the basis of their ability to activate one or other pathway in overexpression assays. Although this classification of Wnts, which partially mirrors their evolutionary relationships, may provide a useful tool for predicting the function of Wnts and Fzds, the relationship between specific Wnts and the intracellular pathway they use is not fixed. For example, overexpression of XWnt5a in combination with human FZD5 in Xenopus embryos results in activation of the Wnt/β-catenin pathway , suggesting that the activity of Wnts in vivo will be determined by the repertoire of Fzd receptors present at the cell surface.
Loss-of-function mutations in 9 of the 18 mouse Wnt genes have been generated, and the phenotypes of mutant embryos demonstrate the diverse functions of Wnt genes during embryogenesis (Table (Table2).2). For example, knocking out Wnt1 results in a dramatic loss of a portion of the midbrain and deletion of the rostral cerebellum [74,75]. Inactivation of Wnt4 results in the absence of kidneys , masculinization of mutant females (absence of the Müllerian duct and continued development of the Wolffian duct) , and defects in mammary gland morphogenesis during pregnancy . Targeted knockout of Wnt7a also has pleiotropic effects, including ventralization of the limbs , female infertility due to failure of Müllerian-duct regression , and a delay in the morphological maturation of glomerular rosettes in the cerebellum .
Overexpression and antisense 'knockdown' analyses in Xenopus have shown that the Wnt/β-catenin pathway is required for the specification of dorsal cell fates . A debate is ongoing, however, over whether a maternal Wnt ligand is required to activate this pathway in dorsal cells. In support of a role for a Wnt ligand, a recent study has shown that XFzd7 is important for establishing dorsal cell fates , thereby implicating a Wnt ligand in this process. Furthermore, targeted knockout of Wnt3 in mice results in defects in axis formation and gastrulation, suggesting a conserved role for Wnts in regulating the establishment of the dorsal-ventral axis in vertebrates . On the other hand, overexpression of a dominant-negative form of Xwnt8 in oocytes does not suppress formation of dorsal cell fates, arguing against the requirement for a maternal Wnt in axis specification . Further studies are necessary to resolve the role of Wnts in vertebrate early axial development.
In flies, Wnt signaling has a variety of functions during development. The wg gene is required for cell-fate choices in the ventral epidermis during embryogenesis, as well as for many other functions, and DWnt2 is required for testis and adult muscle development . In C. elegans, genetic analyses have defined a number of roles for Wnts, including establishment of polarity and endodermal cell fates in the early embryo and regulation of cell migration, among many others . A comprehensive list of Wnt genes and their mutant phenotypes in vertebrates and invertebrates can be found at the Wnt gene homepage .
In addition to the many roles for Wnt signaling during development and in adult tissues, it is also involved in tumorigenesis in humans [59,64]. Although mutation or misexpression of a Wnt gene has not been linked directly to cancer in humans, mutation of several intracellular components of the Wnt/β-catenin pathway is thought to be critical in many forms of cancer. Most notably, patients with familial adenomatous polyposis (FAP) develop multiple intestinal adenomas early in life and have germline mutations in the APC gene. In addition, mutation of APC is associated with more than 80% of sporadic colorectal adenomas and carcinomas. More than 95% of germline and somatic mutations of the APC gene are nonsense mutations that result in the synthesis of a truncated protein lacking the region of APC that is important for its function in the destruction complex. Significantly, these truncations in APC remove binding sites for β-catenin and Axin, as well as putative phosphorylation sites for GSK3; as a result, the mutant APC protein cannot efficiently promote degradation of β-catenin. Mutations in the third exon of the human β-catenin gene (CTNNb1) that make it refractory to phosphorylation-dependent degradation and lead to inappropriate accumulation of β-catenin have also been identified in a large number of primary human cancers (see  for a table of β-catenin mutations in human cancers). Interestingly, mutations in CTNNb1 and APC are rarely found in the same tumor; for example, in colon cancer, in which the vast majority of tumors have mutations in APC, the overall frequency of CTNNb1 mutations is relatively low, but colorectal tumors lacking APC mutations are much more likely to have mutations in CTNNb1. Recently, Axin has also been shown to act as a tumor suppressor; mutations in the Axin1 gene have been found in human hepatocellular cancers . Importantly, mutations in Axin1 and CTNNb1 found in hepatocellular carcinomas also show mutual exclusivity similar to that seen for APC and CTNNb1 in colon cancers. Together, these data strongly argue that mutations resulting in the stabilization of β-catenin can promote cancer in many tissue types.
The large number of Wnt genes and the many roles that Wnt signaling plays in development and human disease pose many unresolved issues for researchers. One of the major unanswered questions is the specificity of interactions between different Wnt ligands and Fzd receptors and also which downstream pathways these many different ligand-receptor pairs stimulate. It also remains unclear how Wnt signals are transduced by the Fzd-LRP receptor complex and what role proteoglycans play in this process. Inside the cell, many questions regarding the transduction of Wnt signals remain, including how receptor activation stimulates Dsh and how Dsh discriminates between different Wnt signals to activate either the Wnt/β-catenin or the Wnt/polarity pathway. Furthermore, many roles of Wnts during development remain to be determined. This challenge will require detailed analyses of knockout mice, in addition to biochemical, cell-biological and genetic analyses in other model systems, to characterize the functions of Wnts and the signaling pathways they use during embryogenesis. Finally, the identification and characterization of mutations in Wnt-pathway genes involved in human disease is ongoing and these studies, together with a greater knowledge of the molecular mechanism of Wnt signal transduction, promise future clinical therapies for devastating human afflictions such as colon cancer. Thus, although there is so much still to learn, the importance and widespread occurrence of Wnt signaling guarantees the rapid increase in our understanding of the normal and abnormal functions of the Wnts.