Table of contents

Wnt receptors

In Xenopus laevis embryos, the Wingless/Wnt-1 subclass of Wnt molecules, including mouse Wnt-1, Xenopus wnt-3A, Xwnt-8 and Drosophila Wingless, induces axis duplication, whereas the Wnt-5A subclass does not. Instead, dorsal injection of Xwnt-5A RNA generates head and tail defects that may result from perturbation of cell movements during gastrulation. This difference could be explained by distinct signal transduction pathways or by a lack of one or more Wnt-5A receptors during axis formation. Wnt-5A induces axis duplication and an ectopic Spemann organizer in the presence of human Fz5, a member of the Frizzled family of seven-transmembrane receptors (See Drosophila Frizzled 2. There is one notable difference between axes induced by Xwnt-8 and those induced by Xwnt-5A plus hFz5: whereas the ectopic axes induced by Xwnt-8 are often indistinguishable from the endogenous ones, the axes induced by Xwnt-5A and hFz5 are shorter in most cases, even when eyes and the cement gland are present. This might reflect the previously described ability of Xwnt-5A to inhibit cell movements during gastrulation. Wnt-5A/hFz5 signaling is antagonized by glycogen synthase kinase-3 and by the amino-terminal ectodomain of hFz5. These results identify hFz5 as a receptor for Wnt-5A (He, 1997).

To test the potential involvement of frizzled homologs in Wnt signaling, the effects of overexpressing rat frizzled-1 (Rfz-1) were examined on the subcellular distribution of Wnts and of Dishevelled, a cytoplasmic component of the Wnt signaling pathway. Ectopic expression of Rfz-1 recruits the Dishevelled protein as well as Xenopus Wnt-8 (Xwnt-8), to the plasma membrane (but not the functionally distinct Xwnt-5A). Rfz-1 is sufficient to induce theexpression of two Xwnt-8-responsive genes (siamois and Xnr-3) in Xenopus explants in a manner that is antagonized by glycogen synthase kinase-3, which also antagonizes Wnt signaling. When Rfz-1 and Xwnt-8 are expressed together, greater induction of these genes is observed, indicating that Rfz-1 can synergize with a Wnt. The results demonstrate that a vertebrate frizzled homolog is involved in Wnt signaling in a manner that discriminates between functionally distinct Wnts; this involves translocation of the Dishevelled protein to the plasma membrane, and works in a synergistic manner with Wnts to induce gene expression. These data support the likely function of frizzled homologs as Wnt receptors, or as components of a receptor complex (Yang-Snyder, 1996).

The Ryk receptor belongs to the atypical receptor tyrosine kinase family. It is a new member of the family of Wnt receptor proteins. However, the molecular mechanisms by which the Ryk receptor functions remain unknown. Mammalian Ryk, unlike the Drosophila Ryk homolog Derailed, functions as a coreceptor along with Frizzled for Wnt ligands. Ryk also binds to Dishevelled, through which it activates the canonical Wnt pathway, providing a link between Wnt and Dishevelled. Transgenic mice expressing Ryk siRNA exhibit defects in axon guidance, and Ryk is required for neurite outgrowth induced by Wnt-3a and in the activation of T cell factor (TCF) induced by Wnt-1. Thus, Ryk appears to play a crucial role in Wnt-mediated signaling (Lu, 2004).

Ryk siRNA mice have defects in axon guidance of craniofacial motor nerves, ophthalmic nerves, and other nerves, suggesting an essential role of Ryk in axon guidance. Although there is no obvious deficiency in dorsal root ganglion neurite outgrowth in Ryk siRNA transgenic mice, dorsal root ganglion explants isolated from Ryk siRNA mice exhibit defects in neurite outgrowth in response to Wnt-3a stimulation. The lack of deficiency in DRG neurite outgrowth in Ryk siRNA mice is probably because NGF and other growth factors are also involved in inducing neurite outgrowth in vivo. The fact that the Wnt-3a-induces neurite outgrowth of dorsal root ganglion explants is inhibited in Ryk siRNA mice provides strong evidence that there is a functional interaction between Wnt and Ryk in neurite outgrowth (Lu, 2004).

Role of low-density lipoprotein receptor (LDLR)-related protein (LRP) family genes in Wnt signaling

The Frizzled (Fz) family of serpentine receptors function as Wnt receptors, but how Fz proteins transduce signaling is not understood. Drosophila arrow encodes a transmembrane protein that is homologous to two members of the mammalian low-density lipoprotein receptor (LDLR)-related protein (LRP) family, LRP5 and LRP6. LRP6 functions as a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6 activates Wnt-Fz signaling, and induces Wnt responsive genes, dorsal axis duplication and neural crest formation. An LRP6 mutant lacking the carboxyl intracellular domain blocks signaling by Wnt or Wnt-Fz, but not by Dishevelled or beta-catenin, and inhibits neural crest development. The extracellular domain of LRP6 binds Wnt-1 and associates with Fz in a Wnt-dependent manner. These results indicate that LRP6 may be a component of the Wnt receptor complex (Tamai, 2000).

Human LRP5 and LRP6 share 71% amino-acid identity, and together with Arrow, form a distinct subgroup of the LRP family. Arrow, LRP5 and LRP6 each contain an extracellular domain with EGF (epidermal growth factor) repeats and LDLR repeats, followed by a transmembrane region and a cytoplasmic domain lacking recognizable catalytic motifs. An lrp6 mutation in mice results in pleiotropic defects recapitulating some, but not all, of the wnt mutant phenotype (Pinson, 2000). To study LRP5/LRP6 involvement in Wnt signaling, their function was examined in Wnt-induced axis and neural crest formation in Xenopus embryos (Tamai, 2000).

Wnt/beta-catenin signaling induces dorsal axis formation through activation of responsive genes, including nodal-related 3 (Xnr3) and siamois (sia). Ventral injection of LRP6 RNA into four-cell stage embryos results in dorsal axis duplication in a dose-dependent manner. In animal pole explants, LRP6 induces Xnr3/sia, but not brachyury (Xbra) expression, which is activated by mesoderm inducers like activin or basic fibroblast growth factor (bFGF). These results indicate that overexpression of LRP6 may specifically activate Wnt signaling. To examine whether LRP6 mediates Wnt effect, RNAs for LRP6 and Wnt-5a were co-injected. Neither Wnt-5a nor a low dose of LRP6 alone exhibits any effect, but Wnt-5a plus LRP6 synergistically induce axis duplication and ectopic Xnr3 expression in the embryo, and activate Xnr3/sia in explants. Synergy is also observed between Wnt-5a and hFz5, and between LRP6 and hFz5. Although LRP5 alone does not induce axes, co-injecting LRP5 and Wnt-5a does. LDLR alone or in combination with Wnt-5a does not induce axes or Xnr3/sia. Although Wnt-5a-hFz5 can induce complete axes including head and the notochord, Wnt-5a-LRP6 or LRP6 alone (higher doses) induce trunk axis with muscle and neural tissues but lacking head and the notochord. This may be explained by quantitative or qualitative differences between Wnt-5a/LRP6 and Wnt-5a/hFz5 co-injections (Tamai, 2000).

Because ectopic Wnt expression enhances, whereas lack of Wnt signaling inhibits, neural crest formation, the effect of LRP6 on neural crest development was analyzed. LDLR injection has no effect, but LRP6 expression significantly expands neural crest progenitors in the injected half of the embryo, as determined by the expression of a crest-specific marker, slug. Thus, overexpression of LRP6 also mimics Wnt signaling during neural crest formation (Tamai, 2000).

To distinguish whether LRP6 functions in Wnt-responding or Wnt-producing cells, Wnt-5a and LRP6 were injected separately into neighboring blastomeres at the four-cell stage. Induction of secondary axes in embryos and of Xnr3/sia in explants occurs even when Wnt-5a and LRP6 are expressed in different cells. Therefore, LRP6 is probably involved in responding to, rather than in enhancing, the production or secretion of the Wnt ligand (Tamai, 2000).

LRP6deltaC, which has most of its cytoplasmic domain deleted, was generated to inhibit the function of endogenous LRP6, which is expressed maternally and throughout embryogenesis. LRP6deltaC does not, either alone or in combination with Wnt-5a, induce axes or activate Xnr3/sia, but inhibits axis duplication and Xnr3/sia induction by the wild-type LRP6. This inhibition is counteracted by an increasing amount of co-injected LRP6. These data suggest that LRP6deltaC is a dominant interfering mutant for LRP6 or related molecules, and that LRP6 cytoplasmic domain is required for Wnt signaling. LRP6deltaC inhibits Xnr3/sia induction by several Wnt molecules, including Wnt-1, Wnt-2, Wnt-3a and Wnt-8. LRP6deltaC also inhibits Wnt-5a signaling through hFz5, showing that hFz5, and probably other endogenous Fz molecules mediating Wnt-1 or Wnt-8 signaling, depend on LRP6 or related proteins. LRP6deltaC does not affect Xbra induction by activin or bFGF, and thus interferes specifically with Wnt signaling (Tamai, 2000).

LRP6deltaC injected dorsally at the four-cell stage does not perturb endogenous axis formation. This may mean that the dorsal beta-catenin pathway is activated by mechanisms other than Wnt stimulation; alternatively, the dorsal Wnt-Fz signaling may occur early before LRP6deltaC can interfere. However, LRP6deltaC inhibits neural crest development as examined by slug expression, and suppresses ectopic crest formation induced by Wnt-3a DNA. Furthermore, co-injection of LRP6 rescues LRP6deltaC inhibition of crest formation. Thus, LRP6 or a related molecule is required for Wnt-dependent neural crest formation in vivo (Tamai, 2000).

In the current model of Wnt/beta-catenin signaling, Wnt stimulation of a Fz receptor activates the intracellular protein Dishevelled (Dsh or Dvl), thereby antagonizing the inhibitory action of the Axin/GSK-3 complex and stabilizing beta-catenin, which together with the transcription factor TCF/LEF activates responsive genes. To position LRP6 in this cascade, relationships between LRP6 and other Wnt signaling components were tested. LRP6deltaC inhibits Xnr3/sia induction by Wnts, but not by Dsh or beta-catenin, suggesting that LRP6deltaC interfers with Wnt signaling upstream of Dsh function. Supporting this epistasis, LRP6 induction of Xnr3/sia is antagonized by Axin, by a dominant-negative TCF, deltaNTCF, and by a dominant-negative Dsh, mDvl2-DIX. LRP6 activity is also inhibited by frizzled-related protein (FRP), a secreted Wnt antagonist, implying that LRP6 activation of Wnt signaling relies on endogenous Wnt molecules. Alternatively, FRP may directly inhibit LRP6. Thus, LRP6 acts between the extracellular Wnt, FRP and intracellular Dsh, possibly as a co-receptor for Wnt molecules (Tamai, 2000).

To function as a Wnt co-receptor, LRP6 should bind Wnt or Fz or both. Used to examine the issue were a secreted form of mFz8, mFz8CRD-IgG, comprising the cysteine-rich domain (CRD) of mFz8 N-terminal extracellular region fused with the immunoglobulin-gamma (IgG) Fc epitope, and a secreted LRP6N-Myc, consisting of the LRP6 extracellular domain tagged by the Myc epitope. mFz8CRD-IgG and LRP6N-Myc proteins were incubated with or without Wnt-1. mFz8CRD-IgG co-precipitates LRP6N-Myc only in the presence of Wnt-1; the secreted IgG fusion partner fails to do so regardless of Wnt-1. A reciprocal precipitation was also performed using secreted LRP6N-IgG and mFz8CRD-Myc, which were generated by swapping the two epitopes. LRP6N-IgG co-precipitates mFz8CRD-Myc, again only in the presence of Wnt-1, whereas the control IgG does not. LRP6N-IgG also co-precipitates Wnt-1-Myc, a tagged Wnt-1 protein. These results suggest that the extracellular domain of LRP6 can bind Wnt-1 and form a complex with Fz in a Wnt-dependent fashion (Tamai, 2000).

Thus, in two developmental processes dependent on the Wnt pathway in Xenopus -- secondary axis and neural crest formation -- LRP6 activates Wnt signaling, but a dominant-negative LRP6 inhibits Wnt signaling, providing compelling evidence that LRP6 is critical in Wnt signal transduction. LRP6 functions upstream of Dsh in Wnt-responding cells, synergizes with either Wnt or Fz, and importantly, is able to bind Wnt-1 and to associate with Fz in a Wnt-dependent manner. The simplest interpretation of these findings is that LRP6 is a component of the Wnt-Fz receptor complex. Genetic studies of arrow in Drosophila and lrp6 in mice (Pinson, 2000) strongly support this hypothesis. The binding data also raise the possibility that Wnt-induced formation of the Fz-LRP6 complex assembles LRP6, Fz and their associated proteins, thereby initiating cytoplasmic signaling. Consistent with this notion, Wnt signal transduction requires intracellular regions of both Fz and LRP6, which harbours candidate protein-protein interaction motifs. Notably, arrow does not exhibit fz planar polarity phenotype, implying that Arrow-LRP6 may specify Wnt-Fz signaling towards the beta-catenin pathway. How Fz, LRP6 and proteoglycan molecules such as Dally interact to mediate Wnt recognition/specificity and signal transduction remains to be studied. In addition, whether other LRPs and LRP-binding proteins participate in or modulate different Wnt-Fz signaling pathways needs evaluation (Tamai, 2000).

Although many components of the Wnt signaling pathway have been identified, little is known about how Wnts and their cognate Frizzled receptors signal to downstream effector molecules. Evidence is presented that a new member of the low-density lipoprotein (LDL)-receptor-related protein family, LRP6, is critical for Wnt signaling in mice. Embryos homozygous for an insertion mutation in the LRP6 gene exhibit developmental defects that are a striking composite of those caused by mutations in individual Wnt genes. Furthermore, a genetic enhancement of a Wnt mutant phenotype has been shown in mice lacking one functional copy of LRP6. These results support a broad role for LRP6 in the transduction of several Wnt signals in mammals (Pinson, 2000).

In a screen for recessive lethal phenotypes in mice caused by gene trap insertions in cell-surface proteins, an insertion mutation was recovered that joined the first 321 amino acids of the LRP6 protein in-frame with the betageo reporter gene. Embryos homozygous for the insertion in LRP6 die at birth, and exhibit a variety of severe developmental abnormalities including a truncation of the axial skeleton, limb defects, microophthalmia and malformation of the urogenital system. Northern blot analysis shows a complete absence of LRP6 transcripts in LRP6-/- embryos and embryonic fibroblasts. Southern blot analysis indicates a simple insertion event in which LRP6 exons downstream of the vector are retained. On the basis of betageo reporter activity, which accurately reports endogenous gene expression, the LRP6 gene appears to be expressed in all cells of the developing embryo (Pinson, 2000).

The specific developmental defects that are observed in homozygous embryos are remarkably similar to mice carrying mutations in Wnt genes, specifically Wnt-3a, Wnt-1 and Wnt-7a. Both the targeted null mutation and a classical hypomorphic allele of Wnt-3a, vestigial tail (vt), cause caudal truncations of the body axis. A similar axial truncation was observed in LRP6-/- neonates in which vertebrae caudal to the lumbar regions are absent. This was first evident as a reduction in the size of the tailbud at 8.5 days of development, with the loss of paraxial mesoderm and caudal somites at later stages. Sections through the tailbud of LRP6-/- embryos revealed an excess of neural tissue and a corresponding loss of paraxial mesoderm, similar to what has been observed in the targeted Wnt-3a mutant. As in vt mutants, Wnt-3a transcripts are reduced in the tailbud. By 10.5 days of development, Wnt-3a transcripts are completely absent from the tail, probably reflecting the depletion of mesodermal precursors at this stage. The extent of axial truncations in vt and Wnt-3a null mutants is sensitive to the dose of Wnt-3a. On the basis of the level at which the axial skeleton is truncated, it is concluded that LRP6-/- embryos display a severely hypomorphic Wnt-3a phenotype (Pinson, 2000).

To determine whether the vestigial tail phenotype is modified by the dose of LRP6, vt;LRP6 double heterozygous animals were crossed to vt/vt animals and embryos were collected at 11.5 and 13.5 days of development. The expected ratio of phenotypic classes was observed: half showed a normal tail length (corresponding to the vt/+;+/+ and vt/+;LRP6-/+ genotypes) and half exhibited a truncation of the tail (representing the vt/ vt;+/+ and vt/vt;LRP6-/+ genotypes). The tail defects in vt/vt embryos were clearly more pronounced among all eight embryos that inherited the LRP6 mutant allele. The enhancement of the vestigial tail phenotype upon elimination of one functional copy of the LRP6 gene provides genetic evidence that LRP6 and Wnt-3a function in the same pathway (Pinson, 2000).

Mutant embryos were examined for mid/hindbrain defects, a hallmark of Wnt-1-deficient mice. The phenotypes of a classical Wnt-1 allele, swaying, and a targeted mutation of Wnt-1 have variable expressivity, exhibiting a range of malformations of the midbrain and cerebellum. About one-half of the LRP6 mutants exhibit neural tube closure defects (exencephaly and/or spina bifida), and among those embryos that were not exencephalic morphological changes at the mid/hindbrain junction were observed. At 10.5 days, the mid/hindbrain boundary is less distinct in LRP6 mutant embryos, and by 14.5 days, a deletion of the caudal midbrain is evident. At birth, the inferior colliculus is absent and the cerebellum is disorganized. Furthermore, an elevation and expansion of Wnt-1 transcripts observed in swaying embryos is also seen in LRP6-/- embryos. Thus, LRP6-deficient mice phenocopy the less severe class of mid/hindbrain defects observed in Wnt-1 mutant mice (Pinson, 2000).

LRP6 mutant embryos display both anteroposterior and dorsoventral patterning defects in the limbs. These defects are reminiscent of those observed in Wnt-7a mutant mice that fail to maintain Shh expression in the zone of polarizing activity (ZPA), leading to the variable loss of posterior digits. Wnt-7a is also required to establish dorsal cell fates in the limb: in its absence, a biventral limb is formed. Because hindlimb defects in the LRP6 mutants may be exacerbated by a general disruption of caudal development, an analysis was focused on forelimb development. The forelimbs of LRP6 -/- embryos show a loss of one or more posterior digits that is presaged at earlier stages by a gradual loss of Shh expression. Moreover, some of the remaining digits contained ectopic tendons diagnostic of a ventralized limb. A distinguishing feature of the LRP6 limb phenotype not observed in Wnt-7a mutants was a failure to maintain the apical ectodermal ridge (AER), as judged by discontinuous expression of Fgf8. Discontinuity of the AER may explain the variable loss of anterior digits observed in LRP6 mutant embryos. The added complexity may indicate that, in addition to Wnt-7a, LRP6 mediates signaling by other Wnts in the limb (Pinson, 2000).

Clearly, LRP6 mutant mice do not exhibit all of the Wnt phenotypes reported to date (for example, the early embryonic lethality associated with Wnt-3 mutants and the limb outgrowth defects in Wnt-5a mutants; therefore, LRP6 may be involved in signaling by only a subset of vertebrate Wnts. Furthermore, the defects in LRP6 mutant embryos are generally less severe than those exhibited by individual Wnt mutants. Notably, a second highly related family member, LRP5, is widely expressed in embryos and may limit the range and severity of Wnt phenotypes observed in LRP6-/- mice (Pinson, 2000).

Members of the LDL receptor family are classically known for their involvement in the endocytic pathway, mediating the uptake through clathrin-coated pits of macromolecules such as apolipoproteins, lipases and proteases. Mice carrying mutations in two members of the LDL receptor family (the VLDL and ApoER2 receptors) have been shown to phenocopy mouse mutations in reelin or disabled , providing the first indication that LDL receptors can participate in specific signaling pathways. Given the central importance of the LDL receptor family in the transport of lipoproteins and other macromolecules into the cell, the relationship between nutrient uptake and the response of cells to developmental signals such as Wnts warrants further investigation (Pinson, 2000).

The Wnt family of secreted glycoproteins mediate cell- cell interactions during cell growth and differentiation in both embryos and adults. Canonical Wnt signalling by way of the ß-catenin pathway is transduced by two receptor families. Frizzled proteins and lipoprotein-receptor-related proteins 5 and 6 (LRP5/6) bind Wnts and transmit their signal by stabilizing intracellular ß-catenin. Wnt/ß-catenin signalling is inhibited by the secreted protein Dickkopf1 (Dkk1), a member of a multigene family, which induces head formation in amphibian embryos. Dkk1 has been shown to inhibit Wnt signalling by binding to and antagonizing LRP5/6. The transmembrane proteins Kremen1 and Kremen2 are high-affinity Dkk1 receptors that functionally cooperate with Dkk1 to block Wnt/ß-catenin signalling. Kremen2 forms a ternary complex with Dkk1 and LRP6, and induces rapid endocytosis and removal of the Wnt receptor LRP6 from the plasma membrane. The results indicate that Kremen1 and Kremen2 are components of a membrane complex modulating canonical Wnt signalling through LRP6 in vertebrates (Mao, 2002).

Drosophila has neither dkk nor krm but rather an LRP6 homolog, arrow, which functions in Wnt signalling. To determine if they could inhibit Wnt signalling in the fly, Xenopus dkk1 and mouse krm2 were expressed as heterologous transgenes in Drosophila and the GAL4/UAS system was used with a scalloped (sd)-GAL4 driver to direct their expression to the wing disc. Development of the wing critically depends on Wnt signalling, and interference with wingless or components of the Wnt pathway characteristically results in loss of wing structures. Even though Dkk1 protein is produced in transgenic flies, it does not affect wing development by itself. However, coexpression of dkk1 and krm2 results in almost complete loss of wings, whereas expression of krm2 alone has no effect. These results indicate that Krm is required for inhibition of Wnt signalling by Dkk1, presumably by interacting with arrow. Indeed, Dkk1 binds to and functionally interacts with Drosophila arrow transfected in 293T cells. Furthermore, inhibition of krm1 and krm2 by antisense Morpholino oligonucleotides reveals that they are required for Wnt inhibition during embryonic head formation and interact with dkk1 in Xenopus. These results suggests a model whereby Dkk1 inhibits Wnt signalling by acting in concert with its receptor Kremen to form a ternary complex with LRP6, which is rapidly endocytosed. This eliminates the Wnt receptor from the plasma membrane, thus preventing Wnt–LRP6 interaction (Mao, 2002).

Multiple signaling pathways, including Wnt signaling, participate in animal development, stem cell biology, and human cancer. Although many components of the Wnt pathway have been identified, unresolved questions remain as to the mechanism by which Wnt binding to its receptors Frizzled and Low-density lipoprotein receptor-related protein 6 (LRP6) triggers downstream signaling events. With live imaging of vertebrate cells, this study shows that Wnt treatment quickly induces plasma membrane-associated LRP6 aggregates. LRP6 aggregates are phosphorylated and can be detergent-solubilized as ribosome-sized multiprotein complexes. Phospho-LRP6 aggregates contain Wnt-pathway components but no common vesicular traffic markers except caveolin. The scaffold protein Dishevelled (Dvl) is required for LRP6 phosphorylation and aggregation. It is proposed that Wnts induce coclustering of receptors and Dvl in LRP6-signalosomes, which in turn triggers LRP6 phosphorylation to promote Axin recruitment and beta-catenin stabilization (Bilic, 2007).

The scaffold protein Dvl was previously thought to act downstream of LRP6 because dsh overexpression activates ß-catenin signaling in Drosophila LRP6 (arrow) mutants and because the constitutively active Dfz2-Arrow fusion protein is inactive in dsh mutants. The explanation for this discrepancy may be that overexpressing Dsh/Dvl leads to artificial sequestration of Axin or that the protein has multiple functions in the Wnt pathway (Bilic, 2007).

Taken together, the results suggest that Dvl-mediated co-aggregation triggers LRP6 phosphorylation by CK1γ. In this model, upon Wnt signaling Dvl aggregates form at the plasma membrane, where they co-cluster LRP6 with other pathway components including Fz, Axin, and GSK3ß, in a 'LRP6-signalosome.' The role of Wnt would be to bridge LRP6 and Fz, which copolymerize on a Dvl platform. Clustering of LRP6 then provides a high local receptor concentration that triggers phosphorylation by CK1γ and Axin recruitment (Bilic, 2007).

Predictions of this model are as follows: (1) artificial oligomerization of LRP6 should activate the receptor and (2) oligomerized LRP6 should signal independent of Dvl. Indeed, forced oligomerization of LRP6 using a synthetic multimerizer is sufficient to induce Wnt signaling, and this oligomerization bypasses the need for Dvl. (3) Constitutively active LRP6 should signal independently of Dvl because its self-aggregation should bypass the need for Dvl polymers. This is also the case as shown in reporter assays with Dvl siRNA knockdown, which supports previous findings. (4) If LRP6 aggregation is a prerequisite for phosphorylation by CK1γ rather than its consequence, LRP6 aggregates should form even when the kinase is blocked. This is the case: Nonphosphorylated LRP6 aggregates were observed in response to Wnt treatment in cells transfected with dominant-negative CK1γ. The model of LRP6-signalosomes not only provides a mechanism for Wnt signal transduction but may also be relevant for the understanding of intracellular transport of maternal Wnt determinants in the fertilized Xenopus egg (Bilic, 2007).

Wnt and Dickkopf (Dkk) regulate the stabilization of beta-catenin antagonistically in the Wnt signaling pathway; however, the molecular mechanism is not clear. In this study, Wnt3a was foud to act in parallel to induce the caveolin-dependent internalization of low-density-lipoprotein receptor-related protein 6 (LRP6), as well as the phosphorylation of LRP6 and the recruitment of Axin to LRP6 on the cell surface membrane. The phosphorylation and internalization of LRP6 occurred independently of one another, and both were necessary for the accumulation of beta-catenin. In contrast, Dkk1, which inhibits Wnt3a-dependent stabilization of beta-catenin, induced the internalization of LRP6 with clathrin. Knockdown of clathrin suppressed the Dkk1-dependent inhibition of the Wnt3a response. Furthermore, Dkk1 reduced the distribution of LRP6 in the lipid raft fraction where caveolin is associated. These results indicate that Wnt3a and Dkk1 shunt LRP6 to distinct internalization pathways in order to activate and inhibit the beta-catenin signaling, respectively (Yamamoto, 2008).

Heparan sulfate proteoglycans and Wnt signaling

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. The non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. The function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family, has been analyzed. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analyses with different Dsh mutants and Wnt11 demonstrate that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/ß-catenin pathway, to regulate gastrulation movements. Evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, the results suggest that Xgly4 functions is a positive regulator in non-canonical Wnt/PCP signaling during gastrulation (Ohkawara, 2003).

In amniotes, it is widely accepted that WNTs secreted by the dorsal neural tube form a concentration gradient that regulates early somite patterning and myotome organization. This study demonstrates in the chicken embryo that WNT protein is not secreted to act at a distance, but rather loaded onto migrating neural crest cells that deliver it to somites. Inhibiting neural crest migration or ablating their population has a profound impact on the WNT response in somites. Furthermore, it was shown that a central player in the efficient delivery of WNT to somites is the heparan sulfate proteoglycan GPC4, expressed by neural crest. Together, these data describe a novel mode of signaling whereby WNT proteins hitch a ride on migratory neural crest cells to pattern the somites at a distance from its source (Serralbo, 2014).

Frzb(s), proteins that interfere with Wnt signalling by binding to Wnt receptors

A Xenopus homolog of Frzb, a newly described mammalian protein containing an amino-terminal Frizzled motif has been isolated. Frzb dorsalizes Xenopus embryos and is expressed in the Spemann organizer during early gastrulation. Unlike Frizzled proteins, endogenous Frzb is soluble. Frzb is secretable and can act across cell boundaries. In several functional assays, Frzb antagonizes Xwnt-8, a proposed ventralizing factor, with an expression pattern complementary to that of Frzb. Frzb blocks induction of MyoD, an action reported recently for a dominant-negative Xwnt-8. Frzb coimmunoprecipitates with Wnt proteins, providing direct biochemical evidence for Frzb-Wnt interactions. These observations implicate Frzb in axial patterning and support the concept that Frzb binds and inactivates Xwnt-8 during gastrulation, preventing inappropriate ventral signaling in developing dorsal tissues (Wang, 1997).

An expression cloning screen was used to isolate a novel gene homologous to the extracellular cysteine-rich domain of frizzled receptors. The gene (which has been called sizzled, for 'secreted frizzled') encodes a soluble secreted protein, containing a functional signal sequence but no transmembrane domains. Sizzled (Szl) is capable of inhibiting Xwnt8 as assayed by (1) a dose-dependent inhibition of siamois induction by Xwnt8 in animal caps, (2) rescue of embryos ventralized by Xwnt8 DNA and (3) inhibition of XmyoD expression in the marginal zone. Szl can dorsalize Xenopus embryos if expressed after the midblastula transition, strengthening the idea that zygotic expression of wnts, and in particular of Xwnt8, plays a role in antagonizing dorsal signals. It also suggests that inhibiting ventralizing wnts parallels the opposition of BMPs by noggin and chordin. szl expression is restricted to a narrow domain in the ventral marginal zone of gastrulating embryos. szl thus encodes a secreted antagonist of wnt signaling likely involved in inhibiting Xwnt8 and XmyoD ventrally and whose restricted expression represents a new element in the molecular pattern of the ventral marginal zone (Salic, 1997).

Wnts are highly conserved developmental regulators that mediate inductive signaling between neighboring cells and participate in the determination of embryonic axes. Frizzled proteins constitute a large family of putative transmembrane receptors for Wnt signals. FrzA is a novel protein that shares sequence similarity with the extracellular domain of Frizzled. The Xenopus homolog of FrzA is dynamically regulated during early development. At the neurula stages, XfrzA mRNA is abundant in the somitic mesoderm, but later becomes strongly expressed in developing heart, neural crest derivatives, endoderm, otic vesicle and other sites of organogenesis. To evaluate possible biological functions of FrzA, its effect on early Xenopus development was analyzed. Microinjection of bovine or Xenopus FrzA mRNA into dorsal blastomeres results in a shortened body axis, suggesting a block of convergent extension movements. Consistent with this possibility, FrzA blocks elongation of ectodermal explants in response to activin, a potent mesoderm-inducing factor. FrzA inhibits induction of secondary axes by Xwnt8 (see Drosophila Wnt8) and human Wnt2, but not by Xdsh, supporting the idea that FrzA interferes with Wnt signaling. Furthermore, FrzA suppresses Wnt-dependent activation of the early response genes in ectodermal explants and in the marginal zone. Finally, immunoprecipitation experiments demonstrate that FrzA binds to the soluble Drosophila Wingless protein in cell culture supernatants in vitro. These results indicate that FrzA is a naturally occurring secreted antagonist of Wnt signaling. Xenopus FrzB, a related protein is confined to the stomodeal-hypophyseal anlage at tailbud stages, while FrzA is strongly expressed in the developming myocardium, otic vesicle, endoderm, pronephros and neural crest. Thus, tissue distribution of FrzA and FrzB in Xenopus embryos suggest that these molecules may locally control Wnt activities, and that the specificity of their effects could be primarily determined by their expression patterns (Xu, 1998).

In apparent contrast to the dorsalizing activity of szl, the gene is mainly expressed in the ventral blastopore lip, where it occupies a sector that becomes narrower as the blastopore closes and involutes. szl responds to lithium and UV treatments in a manner consistent with its ventral expression. A similar situation is encountered in the case of the Anti-Dorsalizing Morphogenetic Protein, a molecule with ventralizing activity expressed in the organizer. These two examples of genes with expression patterns contrasting with their ectopic activity point to the existence of both positive and negative regulators of dorsal and ventral development, respectively (Salic, 1997)

Frzb-1 is a secreted mammalian protein containing a domain similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, Xenopus frzb-1 is expressed and regulated as a typical Spemann organizer component. frzb-1 is a downstream target of organizer homeobox genes and is activated, directly or indirectly, by goosecoid, Xlim-1 and siamois. Injection of frzb-1 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Thus frzb-1 blocks muscle and trunk formation. Frzb-1 antagonizes the effects of Xwnt-8 ectopic expression in a non-cell-autonomous manner. Cultured cells transfected with a membrane-tethered form of Wnt-1 bind epitope-tagged Frzb-1 in the 10(-10) M range. The results strengthen the view that the Spemann organizer is a source of secreted inhibitory factors (Leyns, 1997).

Fritz, a mouse (mfiz) and human (hfiz) gene, codes for a secreted protein that is structurally related to the extracellular cysteine-rich portion of the frizzled genes from Drosophila and vertebrates. The overall identity between the extracellular domains of various frizzled-like proteins and hfiz is only in the range of 10-38%. The Fritz protein antagonizes Wnt function when both proteins are ectopically expressed in Xenopus embryos. In early gastrulation, mouse fiz mRNA is expressed in all three germ layers. Later in embryogenesis fiz mRNA is found in the central and peripheral nervous systems, nephrogenic mesenchyme and several other tissues, all of which are sites where Wnt proteins have been implicated in tissue patterning. A model is proposed in which Fritz protein can interfere with the activity of Wnt proteins via their cognate frizzled receptors and thereby modulate the biological responses to Wnt activity in a multitude of tissue sites (Mayr, 1997).

The Wnt family of secreted proteins has been shown to have multiple roles in embryonic development. Wnt signals are thought to be propagated by binding to the cysteine-rich extracellular domain (CRD) of Frizzled, a seven-transmembrane-domain cell surface receptor. Secreted Frizzled-related proteins (generally denoted Frzb or Sfrp) possess a domain with a high degree of sequence identity and structural similarity with the CRD of Frizzled. Current data indicate that the cysteine-rich domain of secreted Frzb proteins can bind Wnt proteins, suggesting the possibility that Frzbs compete with membrane-bound Frizzled for Wnt binding and consequently act as competitive inhibitors of Wnt signaling. In order to gain a better understanding of the potential roles of Frzb-1 in chick development, polymerase chain reaction was used to isolate a partial cDNA of the chick ortholog of frzb-1, cfrzb-1; its expression pattern to that of Wnt-1, Wnt-3a, Wnt-5a, Wnt-7a, and Wnt-8c was compared. Whole-mount in situ hybridizations reveal three major phases of expression for cfrzb-1 in the developing chick. The earliest expression of cfrzb-1 is in cells fated to become neural ectoderm in streak-stage embryos. Expression of cfrzb-1 in the neural ectoderm continues up through stage 8. After stage 8, cfrzb-1 expression is gradually attenuated in the closing neural tube of the trunk and is concomitantly up-regulated in neural crest cells. Finally, cfrzb-1 appears in the condensing mesenchyme of the bones in both the limb and the trunk in stage 251 embryos. Comparative analysis of the cfrzb-1 and the Wnt gene expression patterns suggests possible interactions between cFrzb-1 and all of the Wnt family members examined (Baranski, 2000).

Dorsoventral polarity of the somitic mesoderm is established by competitive signals originating from adjacent tissues. The ventrally located notochord provides the ventralizing signals to specify the sclerotome, while the dorsally located surface ectoderm and dorsal neural tube provide the dorsalizing signals to specify the dermomyotome. Noggin and SHH-N (the amino-terminal cleavage product of Sonic Hedgehog) have been implicated as the ventralizing signals produced by the notochord. However, the members of the WNT family of proteins have been implicated as the dorsalizing signals derived from the ectoderm and dorsal neural tube. When presomitic explants are confronted with cells secreting SHH-N and WNT1 simultaneously, competition to specify the sclerotome and dermomyotome domains within the naive mesoderm can be observed. Using these explant cultures, evidence is provided that SHH-N competes with WNT1, not only by upregulating its own receptor Ptc1, but also by upregulating Sfrp2 (Secreted frizzled-related protein 2), which encodes a potential WNT antagonist. Among the four known Sfrps, Sfrp2 is the only member expressed in the sclerotome and upregulated by SHH-N recombinant protein. SFRP2-expressing cells can reduce the dermomyotome-inducing activity of WNT1 and WNT4, but not that of WNT3a. Together, these results support the model that SHH-N at least in part employs SFRP2 to reduce WNT1/4 activity in the somitic mesoderm (Lee, 2000).

The Wnt genes are known to play fundamental roles during patterning and development of a number of embryonic structures. Receptors for Wnts are members of the Frizzled family of proteins containing a cysteine-rich domain (CRD) that binds the Wnt protein. Recently several secreted frizzled-related proteins (Sfrps) that also contain a CRD have been identified and some of these can both bind and antagonize Wnt proteins. In this paper the expression patterns of the chick homologs of Frzb (a known Wnt antagonist) and Sfrp-2 are reported. Both genes are expressed in areas where Wnts are known to play a role in development, including the neural tube, myotome, cartilage, and sites of epithelial-mesenchymal interactions. Initially, Sfrp-2 and Frzb are expressed in overlapping areas in the neural plate and neural tube, whereas later, they have distinct patterns. In particular Sfrp-2 is associated with myogenesis while Frzb is associated with chondrogenesis, suggesting that they play different roles during development. Finally, the early Xenopus embryo was used as an in vivo assay to show that Sfrp-2, like Frzb, is a Wnt antagonist. These results suggest that Sfrp-2 and Frzb may function in the developing embryo by modulating Wnt signaling (Ladher, 2000).

Wnt-4 signaling plays a critical role in kidney development and is associated with the epithelial conversion of the metanephric mesenchyme. Furthermore, secreted Frizzled-related proteins (sFRPs) that can bind Wnts are normally expressed in the developing metanephros, and function in other systems as modulators of Wnt signaling. sFRP-1 is distributed throughout the medullary and cortical stroma in the metanephros, but is absent from condensed mesenchyme and primitive tubular epithelia of the developing nephron where wnt-4 is highly expressed. In contrast, sfrp-2 is expressed in primitive tubules. To determine their role in kidney development, recombinant sFRP-1, sFRP-2 or combinations of both were applied to cultures of 13-dpc rat metanephroi. Both tubule formation and bud branching are markedly inhibited by sFRP-1, but concurrent sFRP-2 treatment restores some tubular differentiation and bud branching. sFRP-2 itself shows no effect on cultures of metanephroi. In cultures of isolated, induced rat metanephric mesenchymes, sFRP-1 blocks events associated with epithelial conversion (tubulogenesis and expression of lim-1, sfrp-2 and E-cadherin); however, it has no demonstrable effect on early events (compaction of mesenchyme and expression of wt1). sFRP-1 binds Wnt-4 with considerable avidity and inhibits the DNA-binding activity of TCF, an effector of Wnt signaling, while sFRP-2 has no effect on TCF activation. These observations suggest that sFRP-1 and sFRP-2 compete locally to regulate Wnt signaling during renal organogenesis. The antagonistic effect of sFRP-1 may be important either in preventing inappropriate development within differentiated areas of the medulla or in maintaining a population of cortical blastemal cells to facilitate further renal expansion. However, FRP-2 might promote tubule formation by permitting Wnt-4 signaling in the presence of sFRP-1 (Yoshino, 2001).

The Xenopus trunk organizer recruits neighboring tissues into secondary trunk axial and paraxial structures and itself differentiates into notochord. The inductive properties of the trunk organizer are thought to be mediated by the secretion of bone morphogenetic protein (BMP) antagonists. Ectopic repression of BMP signals on the ventral side is sufficient to mimic the inductive properties of the trunk organizer. Resultant secondary trunks contain somite and neural tube, but no notochord. Excess BMP signaling on the dorsal side results in an expansion of epidermis and ventrolateral mesoderm. Conversely, inhibition of BMP signaling on the ventral side of embryos at the onset of gastrulation leads to the formation of a secondary trunk that includes a neural tube and segmented somites. During late gastrulation, however, this program is lost, due to an invasion of secreted Wnts from neighboring tissues. Maintenance of this program requires co-repression of BMP and Wnt signaling within the presumptive notochord region. To shed light on the molecular cascade that leads to the repression of the Wnt pathway, individual organizer genes were sought whose overexpression could complement the inhibition of BMP signaling to promote notochord formation in the secondary trunks. Two genes, gsc and Xnot, were thus identified and shown to act in different ways. Xnot acts as a transcriptional repressor within the mesodermal region. Gsc acts in deeper vegetal cells, where it regulates Frzb expression to maintain Xnot expression in the neighboring notochord territory. These results suggest that, during gastrulation, the necessary repression of Wnt/ß-catenin signaling in notochord precursors is achieved by the action of secreted inhibitors, such as Frzb, emitted by gsc-expressing dorsal vegetal cells (Yasuo, 2001).

The Wnt antagonist Frzb-1 is expressed during limb skeletogenesis, but its roles in this complex multistep process are not fully understood. To address this issue, Frzb-1 gene expression patterns were determined during chick long bone development and gain- and loss-of-function studies were carried out by misexpression of Frzb-1, Wnt-8 (a known Frzb-1 target), or different forms of the intracellular Wnt mediator LEF-1 in developing limbs and cultured chondrocytes. Frzb-1 expression is quite strong in mesenchymal prechondrogenic condensations and then in characterized epiphyseal articular chondrocytes and prehypertrophic chondrocytes in growth plates. Virally driven Frzb-1 misexpression causes shortening of skeletal elements, joint fusion, and delayed chondrocyte maturation, with consequent inhibition of matrix mineralization, metalloprotease expression, and marrow/bone formation. In good agreement, misexpression of Frzb-1 or a dominant-negative form of LEF-1 in cultured chondrocytes maintains the cells at an immature stage. Instead, misexpression of Wnt-8 or a constitutively active LEF-1 strongly promotes chondrocyte maturation, hypertrophy, and calcification. Immunostaining has revealed that the distribution of endogenous Wnt mediator ß-catenin changes dramatically in vivo and in vitro, from largely cytoplasmic in immature proliferating and prehypertrophic chondrocytes to nuclear in hypertrophic mineralizing chondrocytes. Misexpression of Frzb-1 prevents ß-catenin nuclear relocalization in chondrocytes in vivo or in vitro. The data demonstrate that Frzb-1 exerts a strong influence on limb skeletogenesis and is a powerful and direct modulator of chondrocyte maturation, phenotype, and function. Phases of skeletogenesis, such as terminal chondrocyte maturation and joint formation, appear to be particularly dependent on Wnt signaling and thus very sensitive to Frzb-1 antagonistic action (Enomoto-Iwamoto, 2002).

Recent studies have postulated that distinct regulatory cascades control myogenic differentiation in the head and the trunk. However, although the tissues and signaling molecules that induce skeletal myogenesis in the trunk have been identified, the source of the signals that trigger skeletal muscle formation in the head remains obscure. Although myogenesis in the trunk paraxial mesoderm is induced by Wnt signals from the dorsal neural tube, myogenesis in the cranial paraxial mesoderm is blocked by these same signals. In addition, BMP family members that are expressed in both the dorsal neural tube and surface ectoderm are also potent inhibitors of myogenesis in the cranial paraxial mesoderm. Evidence is provided suggesting that skeletal myogenesis in the head is induced by the BMP inhibitors, Noggin and Gremlin, and the Wnt inhibitor, Frzb. These molecules are secreted by both cranial neural crest cells and by other tissues surrounding the cranial muscle anlagen. These findings demonstrate that head muscle formation is locally repressed by Wnt and BMP signals and induced by antagonists of these signaling pathways secreted by adjacent tissues (Tzahor, 2003).

Inductive interactions between gut endoderm and the underlying mesenchyme pattern the developing digestive tract into regions with specific morphology and functions. The molecular mechanisms behind these interactions remain largely unknown. Expression of the conserved homeobox gene Barx1 is restricted to the stomach mesenchyme during gut organogenesis. Using recombinant tissue cultures, it has been shown that Barx1 loss in the mesenchyme prevents stomach epithelial differentiation of overlying endoderm and induces intestine-specific genes instead. Additionally, Barx1 null mouse embryos show visceral homeosis, with intestinal gene expression within a highly disorganized gastric epithelium. Barx1 directs mesenchymal cell expression of two secreted Wnt antagonists, sFRP1 and sFRP2, and these factors are sufficient replacements for Barx1 function. Canonical Wnt signaling is prominent in the prospective gastric endoderm prior to epithelial differentiation, and its inhibition by Barx1-dependent signaling permits development of stomach-specific epithelium. These results define a transcriptional and signaling pathway of inductive cell interactions in vertebrate organogenesis (Kim, 2005).

Cell identity and tissue morphogenesis are tightly orchestrated during organogenesis, but the mechanisms regulating this are poorly understood. This study shows that interactions between Wnt11 and the secreted Wnt antagonist secreted frizzled-related protein 5 (Sfrp5) coordinate cell fate and morphogenesis during Xenopus foregut development. sfrp5 is expressed in the surface cells of the foregut epithelium, whereas wnt11 is expressed in the underlying deep endoderm. Depletion of Sfrp5 results in reduced foregut gene expression and hypoplastic liver and ventral pancreatic buds. In addition, the ventral foregut cells lose adhesion and fail to form a polarized epithelium. The cell fate and epithelial defects are due to inappropriate Wnt/β-catenin and Wnt/PCP signaling, respectively, both mediated by Wnt11. Evidence is provided that Sfrp5 locally inhibits Wnt11 to maintain early foregut identity and to allow an epithelium to form over a mass of tissue undergoing Wnt-mediated cell movements. This novel mechanism coordinating canonical and noncanonical Wnt signaling may have broad implications for organogenesis and cancer (Li, 2009).

Frizzled-related proteins (sFRPs) are a family of secreted proteins that bind to Wnts extracellularly and are so-called Wnt inhibitors. Although Drosophila does not have sFRP genes, at least one has been found in the Nematostella genome, suggesting that the regulatory interactions between sFRP and Wnt existed in the ancestral eumetazoan. Although Wnt proteins are thought to diffuse extracellularly and act as morphogens, little is known about the diffusibility of either Wnts or sFRPs. This study shows that Frzb and Crescent (Cres), which are members of the sFRP family, have the ability to regulate the diffusibility and signalling areas of the Wnt ligands Wnt8 and Wnt11. It was found, using the Xenopus embryo, that Wnts do not diffuse effectively, whereas Frzb and Cres spread very widely. Interestingly, Frzb and Cres substantially promoted the diffusion of Wnt8 and Wnt11 through extracellular interactions. Importantly, Wnt8 conveyed by sFRPs can activate canonical Wnt signalling despite the function of sFRPs as Wnt inhibitors, suggesting a novel regulatory system for Wnts by sFRPs (Mii, 2009).

Secreted frizzled-related proteins (Sfrps) are considered Wnt signalling antagonists but recent studies have shown that specific family members enhance Wnt diffusion and thus positively modulate Wnt signalling. Whether this is a general and physiological property of all Sfrps remains unexplored. It is equally unclear whether disruption of Sfrp expression interferes with developmental events mediated by Wnt signalling activation. This study addressed these questions by investigating the functional consequences of Sfrp disruption in the canonical Wnt signalling-dependent specification of the mouse optic cup periphery. Compound genetic inactivation of Sfrp1 and Sfrp2 prevents Wnt/β-catenin signalling activation in this structure, which fails to be specified and acquires neural retina characteristics. Consistent with a positive role of Sfrps in signalling activation, Wnt spreading is impaired in the retina of Sfrp1-/-;Sfrp2-/- mice. Conversely, forced expression of Sfrp1 in the wing imaginal disc of Drosophila, the only species in which the endogenous Wnt distribution can be detected, flattens the Wg gradient, suppresses the expression of high-Wg target genes but expands those typically activated by low Wg concentrations. All this occurs in spite of the observation that there is no apparent SFRPs homologues have been identified in the Drosophila genome. Collectively, these data demonstrate that, in vivo, the levels of Wnt signalling activation strongly depend on the tissue distribution of Sfrps, which should be viewed as multifunctional regulators of Wnt signalling (Esteve, 2011).

Other secreted antagonists of Wnt signaling

dickkopf-1 (dkk-1), encodes Dkk-1, a secreted inducer of Spemann's organizer in Xenopus and a member of a new protein family. The protein has 259 amino acids that consist of a signal sequence and two cysteine-rich domains. Mouse sequence database searches reveal other members of the protein family. The gene is expressed in the Spemann organizer. In comparision, the head inducer cerberus is expressed in a wider dorsolateral arc. Within the organizer, expression is detected in the deep cells at stage 10 but excluding the endoderm. At late gastrula stage, dkk-1 is expressed in three domains, with the most anterior expression being in the leading edge cells of the involuting cylinder, which will give rise to the liver. Strongest expression is found in a wing-shaped middle domain of endomesoderm corresponding to the prechordal plate. The Wnt antagonist frzb is strongly expressed at the same anterior level. This tissue is the most potent head inducer. In a posterior third domain dkk-1 is expressed in two longitudinal stripes, flanking the anterior chordamesoderm, partly overlapping chordin expression. Prospective chordamesoderm induces predominantly trunk structures. These posterior expressing cells may correspond to adaxial muscle pioneer precursors. At late-neurula stage, dkk-1 shows strongest expression in the prechordal plate adjacent to prospective forebrain and eyes. In addition, expression occurs in a stripe corresponding to the forming somites. Injections of mRNA and antibody indicate that dkk-1 is sufficient and necessary to cause head induction. dkk-1 s a potent antagonist of Wnt signaling, suggesting that dkk genes encode a family of secreted Wnt inhibitors (Glinka, 1998).

Wnts are secreted glycoproteins implicated in diverse processes during embryonic patterning in metazoans. They signal through seven-transmembrane receptors of the Frizzled (Fz) family to stabilize ß-catenin. Wnts are antagonized by several extracellular inhibitors including the product of the dickkopf1 (dkk1) gene, which was identified in Xenopus embryos and is a member of a multigene family. The dkk1 gene acts upstream of the Wnt pathway component dishevelled but its mechanism of action is unknown. Although the function of Dkk1 as a Wnt inhibitor in vertebrates is well established, the effect of other Dkks on the Wnt/ß-catenin pathway is unclear. A related family member, Dkk2, activates rather than inhibits the Wnt/ß-catenin signaling pathway in Xenopus embryos. Dkk2 strongly synergises with Wnt receptors of the Fz family to induce Wnt signaling responses. The study identifies Dkk2 as a secreted molecule that is able to activate Wnt/ß-catenin signaling. The results suggest that a coordinated interplay between inhibiting dkk1 and activating dkk2 can modulate Fz signaling (Wu, 2000).

An important conclusion from this work is that Dkk2 represents a novel secreted factor capable of activating the Wnt/ß-catenin pathway. The only other secreted proteins known to synergise with Fz receptors are the Wnt glycoproteins themselves. Dkk2 can act in an opposite fashion to that of Dkk1, suggesting that the proteins function as mutual antagonists. In the mouse, the dkk1 and dkk2 genes are coordinately expressed during organogenesis in a multitude of organs, and their expression frequently is found in adjacent or partially overlapping domains. This suggests that, in these tissues, the interplay of dkk1 and dkk2 coordinately modulates the Wnt/beta-catenin pathway (Wu, 2000).

The Wnt proteins constitute a large family of extracellular signaling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. This paper describes Wnt-inhibitory factor-1 (WIF-1), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1 is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm, notochord, branchial arches and neural crest derivatives. Xenopus embryos are used to show that WIF-1 overexpression affects somitogenesis (the generation of trunk mesoderm segments), in agreement with its normal expression in paraxial mesoderm. In vitro, WIF-1 binds to Drosophila Wingless and Xenopus Wnt8, produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled-related proteins, these results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity (Hsieh, 1999).

WNT-7a induces axonal spreading and branching in developing cerebellar granule neurons. This effect is mediated through the inhibition of GSK-3beta, a serine/threonine kinase and a component of the WNT pathway. Lithium, an inhibitor of GSK-3beta, mimics WNT-7a in granule cells. The effect of GSK-3beta inhibition on cytoskeletal re-organization has been examined further. Lithium induces axonal spreading and increases growth cone area and perimeter. This effect is associated with the absence or reduction of stable microtubules in spread areas. Lithium induces the loss of a phosphorylated form of MAP-1B, a microtubule associated protein involved in axonal outgrowth. Down-regulation of the phosphorylated MAP-1B, MAP-1B-P, from axonal processes occurs before axonal remodelling is evident. In vitro phosphorylation assays show that MAP-1B-P is generated by direct phosphorylation of MAP-1B by GSK-3beta. WNT-7a, like lithium, also leads to loss of MAP-1B-P from spread axons and growth cones. These data suggest that WNT-7a and lithium induce changes in microtubule dynamics by inhibiting GSK-3beta, which in turn leads to changes in the phosphorylation of MAP-1B. These findings suggest a novel role for GSK-3beta and WNTs in axonal remodelling and identify MAP-1B as a new target for GSK-3beta and WNT (Lucas, 1998b).

Dickkopf1 (Dkk1) is a secreted protein that acts as a Wnt inhibitor and, together with BMP inhibitors, is able to induce the formation of ectopic heads in Xenopus. Dkk1 null mutant embryos lack head structures anterior of the midbrain. Analysis of chimeric embryos implicates the requirement of Dkk1 in anterior axial mesendoderm but not in anterior visceral endoderm for head induction. In addition, mutant embryos show duplications and fusions of limb digits. Characterization of the limb phenotype strongly suggests a role for Dkk1 both in cell proliferation and in programmed cell death. These data provide direct genetic evidence for the requirement of secreted Wnt antagonists during embryonic patterning and implicate Dkk1 as an essential inducer during anterior specification as well as a regulator during distal limb patterning (Mukhopadhyay, 2001).

Both the activation and inhibition of Wnt signaling are essential for proper anteroposterior axis formation in the vertebrate embryo for specification of caudal and rostral structures, respectively. A variety of secreted Wnt inhibitors has been identified that directly bind and inactivate Wnt proteins. Among them, sFRP, WIF, and Cerberus are related to the extracellular domains of Fz receptors. Cerberus is a multifunctional inhibitor of BMP, Nodal, and Wnt signals. Wif-1 contains domains with homology to EGF repeats. These Wnt inhibitors have been implicated in the repression of the canonical Wnt/ß-catenin signaling pathway. Dickkopf1 (Dkk1) is another secreted Wnt inhibitor and member of a distinct multigene family. The Dkk genes encode proteins that contain two conserved cysteine-rich domains. During vertebrate embryogenesis, Dkks are differentially expressed in various neural and mesenchymal tissues, suggesting that the proteins are involved in many inductive processes. In Xenopus, ectopic expression of Dkk1 results in the inhibition of all Wnts tested that transduce via ß-catenin including Wnt8, Wnt1, Wnt3a, and Wnt2b. In contrast, Dkk2 cooperates with Fz receptors to activate Wnt/ß-catenin signaling when overexpressed in early Xenopus embryos, suggesting that mutual antagonism between Dkk1 and Dkk2 may regulate Wnt/ß-catenin signaling (Mukhopadhyay, 2001 and references therein).

Wnt inhibitors have been implicated in the regulation of various embryonic patterning processes. Dkk1 and Crescent are capable of inducing the heart anlage in chick. sFRP2 is involved in the patterning of avian hindbrain rhombomeres. In Xenopus, Cerberus, Frzb, and Dkk1 are thought to antagonize Wnts that inhibit the Spemann organizer during embryonic head induction. That this pathway is capable of antagonizing the head organizer is suggested by the overexpression of Wnts during gastrulation, which leads to microcephaly in Xenopus and possibly in mouse. Conversely, the overexpression of Dkk1 results in enlarged heads in Xenopus and zebrafish embryos. Furthermore, in Xenopus, coexpression of Dkk1 with inhibitors of the BMP signaling pathway leads to ectopic head induction. The suggestion that Dkk1 acts as a head inducer in the Xenopus organizer is consistent with its expression in the anterior endomesoderm of the Spemann organizer, which characteristically harbors head-inducing activity. Hence, it has been proposed that the amphibian head organizer acts by simultaneous inhibition of Wnt and BMP signaling and that Dkk1 plays a key role in this process (Mukhopadhyay, 2001 and references therein).

In contrast to Xenopus, the role of Dkk1 in mammals is unknown. In the mouse, Dkk1 is first expressed in the anterior domain of the gastrulating embryo. In this domain, head induction is thought to be mediated by the anterior visceral endoderm (AVE), an extraembryonic tissue essential for initiating head formation in mammalian embryos, and the anterior mesendoderm (AME), a node-derived embryonic tissue involved in anterior specification. Murine Dkk1 inhibits the axis-inducing ability of XWnt8 in Xenopus embryos, indicating that the mouse gene functions as a Wnt inhibitor comparable to its Xenopus homolog. There has been no genetic evidence that Wnt inhibitors play a role in anteroposterior patterning in the mouse. For example, no axis defects have been noted in mice that lack the function of the Cerberus homolog Cer1. Also, there has been no genetic evidence implicating Wnt signaling in antagonizing head induction during gastrulation, although a requirement has been established for Wnt/ß-catenin signaling during early axis and node induction. Mice with a Dkk1 null mutation were generated in an effort to establish the function of this gene in the early mouse embryo. Dkk1 knockout mice have two major phenotypes: they lack anterior head structures and they display forelimb malformations. These results reveal a requirement for the inhibition of Wnt signaling during mouse axis formation and limb morphogenesis (Mukhopadhyay, 2001 and references therein).

No morphological defect can be detected in the Dkk1-/- embryos prior to the headfold stage. At the molecular level, the absence of Hesx1 gene expression in the prospective anterior neuroectodermal (ANE) cells of late streak mutant embryos is the earliest defect detected in this study. The complete absence of Hesx1 at E7.5 suggests that Dkk1 function is required for the proper expression of this gene in the AVE as well. Hesx1 expression in the ANE is required for forebrain development. Expression of Six3, another gene activated in the ANE at late streak stages, is also undetectable in Dkk1-/- mutants. Hesx1 and Six3 are the earliest known ANE markers in the mouse. Expression of these genes in the ANE of a wild-type embryo starts at E7.5 and continues through early somite stages. In the Hesx1-/- mutant, Six3 expression in the ANE appears normal at late streak stages but is reduced at the early somite stage. Therefore, the absence of Six3 expression at the early somite stage of Dkk1-/- mutants may be a consequence of the loss of Hesx1 expression (Mukhopadhyay, 2001).

Proper anterior positioning of the early Dkk1-expressing AVE cells appears to be controlled by Otx2, a marker which seems unaffected in these Dkk1-/- mutants. The severe anterior phenotype of Otx2-/- embryos suggests that Otx2 is a key factor in the head developmental process. Since Dkk1 acts downstream of Otx2, it most likely mediates forebrain induction pathways activated by Otx2 (Mukhopadhyay, 2001).

Forebrain patterning in the mouse is initiated by the inductive activity of the AVE and subsequently requires the function of the node-derived AME. Chimeric embryos, largely composed of Dkk1+/+ epiblast cells developing within the confines of a Dkk1-/- visceral endoderm, display a seemingly normal anterior morphology at E9.5. This shows that Dkk1 gene expression in the AVE is not required for head induction. Notably, a recent study has demonstrated that the function of Hesx1, the gene that is essential for forebrain development and acts downstream of Dkk1, is dispensable in the AVE. Thus, cells of the elongating axial mesendoderm are the likely source of the Dkk1 signal that mediates the rescue of head organization in the chimera. This conclusion is based on studies in other vertebrates showing that Dkk1 function in the AME is required for head development. In Xenopus, injection of Dkk1 mRNA, together with a BMP inhibitor, is able to induce the complete duplication of head structures. It is believed that the head duplication resulted from an ectopic expression of prechordal plate (AME) markers, such as Xhex and Xgsc. This work identifies Dkk1, a secreted factor associated with the role of AME, in early rostral specification of the mouse embryo (Mukhopadhyay, 2001).

It is proposed that Hesx1 function in the ANE is mediated through Dkk1, which is secreted by adjacent AME. Dkk1 fits the role of a ligand that interacts with a receptor to protect the prospective anterior neuroectoderm via Wnt inhibition from caudalizing effects of the node, thereby exerting an early and indispensable function in head induction. Interestingly, Dkk1 has recently been shown to bind the Wnt coreceptor LRP6 (LDL receptor-related protein 6), thus most likely repressing type I Wnt signaling. It is not yet known which type I Wnt protein is mediating negative control of head formation in the mouse. However, in Xenopus, Wnt8 has been implicated in this process. This study clearly shows that the ablation of Dkk1 function affects not only brain development but also that of surrounding head structures. Various degrees of head truncation have also been observed in mice carrying null mutations in other factors expressed during early stages of head induction, including Hesx1, Otx, and Lim1. This suggests that there may be a coordinated morphogenetic response of neural and nonneural precursor cells to head-inducing signals (Mukhopadhyay, 2001).

R-Spondin2 is a secreted activator of Wnt/ß-Catenin signaling and is required for Xenopus myogenesis

An expression screen was carried out for modulators of the Wnt/ß-catenin pathway and Xenopus R-spondin2 (Rspo2) was identified as a secreted activator of this cascade. Xenopus Rspo2 is predicted to encode a secreted protein with 243 amino acids (mature protein) and an isoelectric point of 9.8. All R-spondins contain an N-terminal signal peptide, two furin-like domains, one thrombospondin type1 domain, and a C-terminal low complexity region enriched with positively charged amino acids. Rspo2 is coexpressed with and is positively regulated by Wnt signals and synergizes with Wnts to activate ß-catenin. Analyses of functional interaction with components of the Wnt/ß-catenin pathway suggest that Rspo2 functions extracellularly at the level of receptor ligand interaction. In addition to activating the Wnt/ß-catenin pathway, Rspo2 overexpression blocks Activin, Nodal, and BMP4 signaling in Xenopus, raising the possibility that it may negatively regulate the TGF-ß pathway. Antisense Morpholino experiments in Xenopus embryos and RNAi experiments in HeLa cells reveal that Rspo2 is required for Wnt/ß-catenin signaling. In Xenopus embryos depleted of Rspo2, the muscle markers myoD and myf5 fail to be activated and later muscle development is impaired. Thus, Rspo2 functions in a positive feedback loop to stimulate the Wnt/ß-catenin cascade (Kazanskaya, 2004).

CCN family proteins: Extracellular-matrix constituents that modulate WNT signaling

Cyr61 is a secreted, heparin-binding, extracellular matrix-associated protein whose activities include the promotion of adhesion and chemotaxis, and the stimulation of fibroblast and endothelial cell growth. Cyr61 is a member of the CCN (Cyr61, CTGF, Nov) family. Members of the CCN family are versatile proteins, exhibiting properties that might well be expected of key regulators of gastrulation: they associate with the extracellular matrix; they can mediate cell adhesion, cell migration and chemotaxis; and they can augment the activity of peptide growth factors. Significantly, Cyr61 can also induce signalling events, such as activation of ERK and Rac, and the induction of gene expression in fibroblasts many, if not all, of these activities of Cyr61 are mediated through interactions with integrins. The role of Cyr61 in the early development of Xenopus laevis was explored. Gain- and loss-of-function experiments show that Xcyr61 is required for normal gastrulation movements. This role is mediated in part through the adhesive properties of Xcyr61 and its related ability to modulate assembly of the extracellular matrix. In addition, Xcyr61 can, in a context-dependent manner, stimulate or inhibit signalling through the Wnt pathway. These properties of Xcyr61 provide a mechanism for integrating cell signalling, cell adhesion and cell migration during gastrulation. Adhesion of blastomeres to Cyr61 requires the C-terminal (CT) region, which contains cystine knot domains, as does the inhibition of Wnt signalling, where this domain is sufficient to inhibit secondary axis induction by Xwnt8. By contrast, stimulation of Wnt signalling appears to be mediated by the IGFBP domain. Activation of Wnt signalling by Xcyr61 can occur through the IGFBP domain. It is not known if the Wnt-stimulating activity of domain 1 of Xcyr61 is related to its putative IGF binding activity, although it is noted that activation of the IGF receptor in Xenopus embryos inhibits the Wnt pathway. It is possible that domain 1 of Xcyr61 binds endogenous members of the insulin-like growth factor family and relieves this inhibition (Latinkic, 2003).

Connective-tissue growth factor (CTGF) is a member of the CCN family of secreted proteins. CCN family members contain four characteristic domains and exhibit multiple activities: they associate with the extracellular matrix; they can mediate cell adhesion, cell migration and chemotaxis, and they can modulate the activities of peptide growth factors. Many of the effects of CTGF are thought to be mediated by binding to integrins, whereas others may be mediated because of CTGF's recently identified ability to interact with BMP4 and TGFß. Using Xenopus embryos, it has been demonstrated that CTGF also regulates signalling through the Wnt pathway, in accord with its ability to bind to the Wnt co-receptor LDL receptor-related protein 6 (LRP6). This interaction is likely to occur through the C-terminal (CT) domain of CTGF, which is distinct from the BMP- and TGFß-interacting domain. These results define new activities of CTGF and add to the variety of routes through which cells regulate growth factor activity in development, disease and tissue homeostasis (Mercurio, 2004).

Wnt signaling is modified by Carboxypeptidase Z

Carboxypeptidase Z (CPZ) is a secreted Zn-dependent enzyme whose biological function is largely unknown. CPZ has a bipartite structure consisting of an N-terminal cysteine-rich domain (CRD) and a C-terminal catalytic domain. In the early chicken embryo CPZ is initially expressed throughout the somites and subsequently becomes restricted to the sclerotome. To initiate a functional analysis of CPZ, a CPZ producing retroviral vector was applied to the presomitic mesoderm at the level of the future wing. This resulted in a loss of the scapular blade and of rostral ribs. Such dysmorphogenesis is preceded by ectopic Pax3 expression in the hypaxial part of the dermomyotome, a region from which the blade of the scapula normally derives. A mutant CPZ, lacking a critical active site glutamate, fails to induce Pax3 expression and does not cause skeletal defects. The induction of Pax3, a Wnt-responsive gene in somites, and the presence of a CRD prompted an examination of whether CPZ affects Wnt signaling. In an in vitro assay, CPZ, but not its inactive mutant form, was found to enhance the Wnt-dependent induction of the homeobox gene Cdx1. In addition, immunoprecipitation experiments suggest that the CRD of CPZ acts as a binding domain for Wnt. Taken together these data provide the first evidence for CPZ playing a role in Wnt signaling. Although the data suggest that Wnt4 may represent a CPz substrate, definitive biochemical proof is still lacking. In addition, the relevance of proteolytic processing for binding of Wnts to their cognate receptors remains to be explored (Moeller, 2003).

Transcriptional regulation of Wnts

There is growing evidence that Gli proteins participate in the mediation of Hedgehog and FGF signaling in neural and mesodermal development. However, little is known about which genes act downstream of Gli proteins. The regulation of members of the Wnt family by Gli proteins in different contexts is shown in this study. These findings indicate that Gli2 regulates Wnt8 expression in the ventral marginal zone of the early frog embryo: activating Gli2 constructs induce ectopic Wnt8 expression in animal cap explants, whereas repressor forms inhibit its endogenous expression in the marginal zone. Using truncated Frizzled and dominant-negative Wnt constructs, the requirement of at least two Wnt proteins, Wnt8 and Wnt11, for Gli2/3-induced posterior mesodermal development is shown. Blocking Wnt signals, however, inhibits Gli2/3-induced morphogenesis, but not mesodermal specification. Gli2/3 may therefore normally coordinate the action of these two Wnt proteins, which regulate distinct downstream pathways. In addition, the finding that Gli1 consistently induces a distinct set of Wnt genes in animal cap explants and in skin tumors suggests that Wnt regulation by Gli proteins is general. Such a mechanism may link signals that induce Gli activity, such as FGFs and Hedgehogs, with Wnt function (Mullor, 2001).

Leptomeningeal glioneuronal heterotopias are a focal type of cortical dysplasia in which neural cells migrate aberrantly into superficial layers of the cerebral cortex and meninges. These heterotopias are frequently observed as microscopic abnormalities in the brains of individuals with central nervous system (CNS) malformations and epilepsy. The function of Emx2 is essential for development of the cortical preplate, which gives rise to the marginal zone and subplate. However, transcriptional targets of EMX2 during CNS development are unknown. Leptomeningeal glioneuronal heterotopias form in Emx2–/– mice that are equivalent to human lesions. Additionally, ectopic expression of Wnt1 is observed in the embryonic roofplate organizer region and dorsal telencephalon. To determine the phenotypic consequences of such Wnt1 misexpression, a putative EMX2 DNA-binding site was deleted from the Wnt1 enhancer and this was used to misexpress Wnt1 in the developing murine CNS. Heterotopias were detected in transgenic mice as early as 13.5 days postcoitum, consistent with a defect of preplate development during early phases of radial neuronal migration. Furthermore, diffuse abnormalities of reelin- and calretinin-positive cell populations were observed in the marginal zone and subplate similar to those observed in Emx2-null animals. Taken together, these findings indicate that EMX2 is a direct repressor of Wnt1 expression in the developing mammalian telencephalon. They further suggest that EMX2-Wnt1 interactions are essential for normal development of preplate derivatives in the mammalian cerebral cortex (Ligon, 2003).

The preplate forms as the initial wave of neural precursors leaves the ventricular zone and migrates radially to the margin of the developing telencephalon. The PP, marginal zone and subplate then serve as a framework for the subsequent influx of neurons from the ventricular zone, cortical hem and ventral forebrain, potentially along tangential and radial processes of cells that reside in these layers. Because ectopic Wnt1 expression was never detected in the preplate or marginal zone of Emx2-null or Wnt1-Tg mice, it is likely that ectopic Wnt1 acts principally on the early ventricular zone progenitors of the dorsomedial cortex. It is now known that cells from this region, especially the cortical hem, appear much more migratory than originally assumed. Several reports suggest that neurons from the hem migrate extensively into the adjacent cortical primordium. As a result, ectopic Wnt1 is expressed early on in the same progenitor cells that will later migrate into and throughout the neocortex thereby creating the potential for very long-range functional effects. Evidently, Wnt1 signaling inhibits development or initial migration of preplate cells and their processes (Ligon, 2003).

In keratinocytes, the cyclin/CDK inhibitor p21WAF1/Cip1 is a direct transcriptional target of Notch1 activation; loss of either the p21 or Notch1 genes expands stem cell populations and facilitates tumor development. The Notch1 tumor-suppressor function has been associated with down-regulation of Wnt signaling. This study shows that suppression of Wnt signaling by Notch1 activation is mediated, at least in part, by down-modulation of Wnts gene expression. p21 is a negative regulator of Wnts transcription downstream of Notch1 activation, independent of effects on the cell cycle. More specifically, expression of the Wnt4 gene is under negative control of endogenous p21 both in vitro and in vivo. p21 associates with the E2F-1 transcription factor at the Wnt4 promoter and causes curtailed recruitment of c-Myc and p300, and histone hypoacetylation at this promoter. Thus, p21 acts as a selective negative regulator of transcription and links the Notch and Wnt signaling pathways in keratinocyte growth control (Devgan, 2005).

Thus in keratinocytes, p21 functions as a transcriptional regulator that associates physically to the promoter of the Wnt4 gene. While increased p21 expression suppresses both Wnt3 and Wnt4 expression and endogenous p21 is required for the effective down-modulation of both genes by Notch1, in the skin of p21-/- mice and in primary p21-/- keratinocytes under basal conditions, only Wnt4 is up-regulated. This is likely a reflection of the fact that, biochemically, association of the endogenous as well as overexpressed p21 protein to the Wnt4 promoter is readily observed, while association to the Wnt3 promoter, if it occurs, is much weaker and harder to demonstrate. By ChIP assays, it was found that E2F-1 binds the same region of the Wnt4 promoter as p21, and that the two proteins can be recovered in association at this promoter. These findings are consistent with an elegant model, whereby E2F-1-p21 association provides a bridging mechanism for bringing p21 to target promoters. Importantly, however, in the cells used, p21 binding is specific for the Wnt4 promoter and does not occur at the promoter of another 'classical' E2F-1 target gene such as PCNA, the expression of which is unaffected by increased p21 expression. Concomitantly, p21 binding at the Wnt4 promoter is linked to curtailed recruitment of c-Myc and p300. By exogenous expression and promoter activity studies, p21 was previously reported to associate with the c-Myc protein suppressing its activity. The data are consistent with such a mechanism taking place at the Wnt4 promoter. Even in this case, however, there is an important element of selectivity, in that expression of other classical c-Myc target genes (such as that for ornithine decarboxylase), remains unaffected by p21 expression in keratinocytes (our unpublished observations). Thus, the findings are overall consistent with the emerging crucial role of chromatin configuration and promoter context in control of gene expression, with a physical and functional interplay between p21 and the specific transcription regulatory apparatus of individual genes such as that for Wnt4 (Devgan, 2005).

In summary, the common biological function of Notch1 and p21 as negative regulators of keratinocyte self renewal and tumorigenesis can be explained, in part, by one being a mediator of the other in transcriptional suppression of Wnt family members with consequent down-modulation of ß-catenin signaling. More specifically, p21 is directly involved in transcription regulation of the Wnt4 target gene, the control of which at the integrated chromatin level, remains an exciting topic for future studies (Devgan, 2005).

The zic1 gene plays an important role in early patterning of the Xenopus neurectoderm. While Zic1 does not act as a neural inducer, it synergizes with the neural inducing factor Noggin to activate expression of posterior neural genes, including the midbrain/hindbrain boundary marker engrailed-2. Since the Drosophila homologue of zic1, odd-paired (opa), regulates expression of the wingless and engrailed genes and since Wnt proteins posteriorize neural tissue in Xenopus, whether Xenopus Zic1 acted through the Wnt pathway was examined. Using Wnt signaling inhibitors, it was demonstrated that an active Wnt pathway is required for activation of en-2 expression by zic1. Consistent with this result, Zic1 induces expression of several wnt genes, including wnt1, wnt4 and wnt8b. wnt1 gene expression activates expression of engrailed in various organisms, including Xenopus, as demonstrated in this study. Together, these data suggest that zic1 is an upstream regulator of several wnt genes and that the regulatory relationships between opa, wingless and engrailed seen in Drosophila are also present in vertebrates (Merzdorf, 2006).

Integrin receptors for the extracellular matrix and receptor tyrosine kinase growth factor receptors represent two of the major families of receptors that transduce into cells information about the surrounding environment. Wnt proteins are a major family of signaling molecules that regulate morphogenetic events. There is presently little understanding of how the expression of Wnt genes themselves is regulated. This study demonstrates that alpha3beta1 integrin, a major laminin receptor involved in the development of the kidney, and c-Met, the receptor for hepatocyte growth factor, signal coordinately to regulate the expression of Wnt7b in the mouse. Wnt signals in turn appear to regulate epithelial cell survival in the papilla of the developing kidney, allowing for the elongation of epithelial tubules to form a mature papilla. Together, these results demonstrate how signals from integrins and growth factor receptors can be integrated to regulate the expression of an important family of signaling molecules so as to regulate morphogenetic events (Liu, 2009).

Lipid modification of Wnts

Wnt signalling is involved in numerous events in animal development, including the proliferation of stem cells and the specification of the neural crest. Wnt proteins are potentially important reagents in expanding specific cell types, but in contrast to other developmental signalling molecules such as hedgehog proteins and the bone morphogenetic proteins, Wnt proteins have never been isolated in an active form. Although Wnt proteins are secreted from cells, secretion is usually inefficient and previous attempts to characterize Wnt proteins have been hampered by their high degree of insolubility. Active Wnt molecules, including the product of the mouse Wnt3a gene, have been isolated in this study. By mass spectrometry, it has been found that the proteins are palmitoylated on a conserved cysteine. Enzymatic removal of the palmitate or site-directed and natural mutations of the modified cysteine result in loss of activity, and indicate that the lipid is important for signalling. The purified Wnt3a protein induces self-renewal of hematopoietic stem cells, signifying its potential use in tissue engineering (Willert, 2003).

It is interesting to note that the protein products of the Drosophila porcupine and C. elegans mom-1 genes have homology with acyl transferases and may catalyse Wnt acylation. Moreover, the Porcupine protein can bind to a domain in Wingless encompassing the acylation site. porcupine and mom-1 have phenotypes similar to Wnt alleles and are required in Wnt-producing cells, indicating that the lipid is an integral part of signalling activity. However, overexpression of Wingless in the Drosophila embryo can overcome the absence of Porcupine, just as high expression of Wnt3a(C77A) can lead to a modest increase in ß-catenin. This suggests that the lipid functions to increase the local concentration of Wnt on membranes, and that its absence can be overcome by high levels of expression. Although palmitoylation of secreted proteins seems unusual, there is an intriguing parallel between Wnt and Hedgehog signalling, since the Hedgehog protein is also palmitoylated (Willert, 2003).

The secretion and extracellular transport of Wnt protein are thought to be well-regulated processes. Wnt is known to be acylated with palmitic acid at a conserved cysteine residue (Cys77 in murine Wnt-3a), and this residue appears to be required for the control of extracellular transport. This study shows that murine Wnt-3a is also acylated at a conserved serine residue (Ser209). Of note, this residue is modified with a monounsaturated fatty acid, palmitoleic acid. Wnt-3a defective in acylation at Ser209 is not secreted from cells in culture or in Xenopus embryos, but it is retained in the endoplasmic reticulum (ER). Furthermore, Porcupine, a protein with structural similarities to membrane-bound O-acyltransferases, is required for Ser209-dependent acylation, as well as for Wnt-3a transport from the ER for secretion. These results strongly suggest that Wnt protein requires a particular lipid modification for proper intracellular transport during the secretory process (Takada, 2007).

Wntless transports Wnt from the Golgi network to the cell surface for release

Wnt proteins are lipid-modified glycoproteins that play a central role in development, adult tissue homeostasis and disease. Secretion of Wnt proteins is mediated by the Wnt-binding protein Wntless (Wls), which transports Wnt from the Golgi network to the cell surface for release. It has recently been shown that recycling of Wls through a retromer-dependent endosome-to-Golgi trafficking pathway is required for efficient Wnt secretion, but the mechanism of this retrograde transport pathway is poorly understood. This study reports that Wls recycling is mediated through a retromer pathway that is independent of the retromer sorting nexins SNX1-SNX2 and SNX5-SNX6. The unrelated sorting nexin, SNX3, has an evolutionarily conserved function in Wls recycling and Wnt secretion, and SNX3 interacts directly with the cargo-selective subcomplex of the retromer to sort Wls into a morphologically distinct retrieval pathway. These results demonstrate that SNX3 is part of an alternative retromer pathway that functionally separates the retrograde transport of Wls from other retromer cargo (Harterink, 2011).

The identification of the Wnt sorting receptor Wls has shown that the secretion of Wnt proteins is mediated by a specialized trafficking pathway that provides an important layer of regulation to Wnt signalling. A key step in this pathway is the retromer-dependent transport of Wls from endosomes to the TGN. This study reports that Wls retrieval is mediated by a retromer pathway that functions independently of the SNX-BAR retromer coat components (Harterink, 2011).

The retromer consists of a cargo-selective subcomplex that interacts with sorting nexins of the SNX-BAR family to segregate cargo into a tubular endosomal sorting pathway. The results show that the cargo-selective subcomplex also interacts with SNX3 as part of an alternative retromer pathway that mediates the recycling of Wls. Three lines of evidence suggest that these are functionally distinct retromer pathways. First, genetic analysis in C. elegans showed that snx-3 and the SNX-BAR sorting nexins function in parallel pathways. Thus, retrieval of the phagocytic receptor CED-1 is dependent on the SNX-BAR sorting nexins but independent of SNX3, whereas Wls recycling requires SNX3 but not the SNX-BAR sorting nexins. Second, co-immunoprecipitation experiments showed that the interaction of the cargo-selective subcomplex of the retromer with the SNX-BAR sorting nexins and SNX3 is mutually exclusive. Finally, live-cell imaging revealed that the SNX3 retromer pathway sorts Wls into a vesicular retrieval pathway that is morphologically distinct from the SNX-BAR-dependent tubular endosomal sorting pathway. On the basis of these results it is concluded that the SNX-BAR and SNX3 pathways are independent and mechanistically distinct retromer pathways (Harterink, 2011).

Studies in yeast have shown that the SNX3 orthologue Grd19p also functions in retromer-dependent endosome-to-Golgi retrieval, but, in contrast to SNX3, Grd19p functions together with the SNX-BAR sorting nexins Vps5p and Vps17p in the retrieval of cargo proteins such as the iron transporter Fet3p-Ftr1p. Grd19p physically interacts with a sorting sequence in the cytoplasmic tail of Ftr1p and with the SNX-BAR retromer complex, which led to the hypothesis that Grd19p acts as a cargo-specific adaptor that links Ftr1p to the SNX-BAR retromer complex. No interaction was observed between SNX3 and Wls in co-immunoprecipitation experiments and also co-precipitation of SNX3 with the SNX-BAR sorting nexins was not found. Furthermore, it was found that mutation of the SNX-BAR sorting nexins did not affect the SNX3-dependent retrieval of Wls, indicating that the function of SNX3 is fundamentally different from that of Grd19p in yeast (Harterink, 2011).

How do the two distinct SNX3- and SNX-BAR-retromer complexes regulate sorting of different endosomal cargo? One simple model to answer this question relies on the spatial segregation of SNX3 and the SNX-BAR sorting nexins along the endosomal maturation pathway. Although there is significant co-localization between these sorting nexins, SNX3 is predominantly localized to early endosomes by its high-affinity interaction with PtdIns(3)P, whereas the SNX-BAR retromer sorting nexins reside at the interface between early and late endosomes. Endocytosed Wls therefore initially enters SNX3-labelled early endosomes, where it engages the VPS26-VPS29-VPS35 trimeric complex (see Drosophila Vps35), recruited to this compartment by the interaction with SNX3. Through a vesicular pathway, possibly dependent on indirect binding to clathrin as well as further membrane-remodelling proteins, the SNX3 retromer complex sorts Wls for retrieval to the TGN. In the absence of SNX3, Wls can be missorted into intraluminal vesicles and hence lysosomal degradation, or can be recycled through SNX-BAR retromer to the TGN. The relative flux through these two distinct pathways therefore determines the steady-state level of Wls. As the level of Wls is severely reduced on loss of SNX3, the flux into the lysosomal degradative pathway seems to be dominant. Thus, although a proportion of Wls may undergo SNX-BAR retromer-mediated recycling in the absence of SNX3, this is insufficient to maintain the required level of Wls for Wnt gradient formation during iterative rounds of Wnt secretion and Wls retrieval from the cell surface (Harterink, 2011).

Interestingly, the steady-state trafficking of the classical SNX-BAR retromer cargo CI-MPR is primarily defined by intracellular cycling between the TGN and late endosomes with retrieval to the TGN by way of the SNX-BAR retromer. The spatial-segregation model therefore suggests that the lack of effect of SNX3 suppression on steady-state CI-MPR distribution arises from CI-MPR entering the endosomal network at a point downstream of SNX3. That said, the complexity of CI-MPR trafficking -- a proportion of this receptor traffics to the plasma membrane before undergoing endocytosis and retrograde transport to the TGN -- that such a simple spatial-segregation model may be an oversimplification. It is therefore speculated that, alongside spatial segregation, cargo binding to the VPS26-VPS29-VPS35 complex may be an important element in selecting the sorting nexin coat that specifies the subsequent retrograde trafficking route. Thus, binding of VPS26-VPS29-VPS35 to Wls may favour association with SNX3, whereas engagement with CI-MPR favours binding to the SNX-BAR coat complex (Harterink, 2011).

Table of contents

wingless continued: Biological Overview | Transcriptional regulation |Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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