Wingless

In Xenopus embryos, establishment of the dorsal-ventral axis can be traced to the post-fertilization cortical rotation and the subsequent activation of transplantable dorsal-determining information by the time the 8-16 cell stage has been reached. The initial indication that activation of a single signaling pathway is sufficient to trigger formation of a new dorsal axis came from the observation that ectopic expression of Wnt1 promotes duplication of this axis. Supporting the idea that the WNT1 pathway is normally involved in axis specification, transcripts encoding the known components of this pathway are present in Xenopus eggs, including beta-catenin (Drosophila homolog: Armadillo), dishevelled (See Drosophila Dishevelled), GSK3 (Drosophila homolog: Shaggy) and homologs of Drosophila Frizzled. Additionally, their ectopic expression (or inhibition, in the case of GSK3) elicits the expected duplication of the axis (Moon, 1997).

Although Xwnt8b mRNA is present maternally and is the only maternal Xwnt that induces a complete ectopic axis, it might not be required for axis formation, even if it normally participates in this process. This is supported by data showing that expression in fertilized Xenopus eggs of a dominant-negative WNT or a dominant-negative Dishevelled blocks formation of ectopic, but not endogenous, axes. However, ß-catenin is clearly necessary for the formation of the endogenous axis, as depletion of beta-catenin transcripts blocks formation of the endognous axis. ß-catenin promotes axis formation through interaction with the HMG-box transcription factor XTCF3 (Drosophila homolog: Pangolin), resulting in translocation of ß-catenin-XTCF3 complexes into the nucleus. This leads to induction of specific regulatory genes, such as the homeobox gene siamois and others, that are involved in axis formation (Moon, 1997).

Endogenous ß-catenin is enriched in the dorsal cytoplasm by the end of the first cell cycle, with further accumulation in nuclei of dorsal, but not ventral, blastomeres by the 16-cell stage. Remaining questions for investigation include determining whether fertilization of Xenopus activates this dorsal accumulation of ß-catenin in a WNT, or other ligand-dependent or -independent manner, and how the WNT pathway might interact with other maternal signaling pathways, such as Vg1 to initiate gene expression leading to formation of the Spemann gastrula organizer (Moon, 1997 and references).

In Xenopus embryos, overexpression of Wnts causes developmental effects that are strikingly similar to the effects of agents that modulate the phosphatidylinositol cycle, a signal transduction pathway stimulated by many Ca2+ mobilizing hormones and grow factors. These parallels led to a test of the hypothesis that Xwnt-5a might modulate PI cycle-mediated Ca2+ signaling in embryos. Ectopic expression of Xwnt-5A in zebrafish embryos enhances the frequency of intracellular Ca2+ transients in the enveloping layer of the blastodisc; Xwnt-8 does not. These transients are independent of extracellular Ca2+. Ligand-activated serotonin type 1C receptor (which stimulates PI cycle activity and Ca2+ signaling independent of Wnts) phenocopies embryonic responses to Xwnt-5A. This is consistent with the role played by the observed Ca2+ transients in the responses of embryos to Xwnt-5A. These results suggest that intercellular signaling by a subset of vertebrate Wnts involves modulation of an intracellular Ca2+ signaling pathway, possibly arising from phosphatidylinositol cycle activity. It is also possible that a Wnt-5a receptor, likely to be a member of the frizzled family, might activate phospholipase C in a ligand-dependent fashion, as do many Ca2+ mobilizing hormone receptors (Slusarski, 1997a).

In Drosophila, members of the frizzled family of tissue-polarity genes encode proteins that are likely to function as cell-surface receptors of the type known as Wnt receptors, and to initiate signal transduction across the cell membrane, although how they do this is unclear. The rat protein Frizzled-2 causes an increase in the release of intracellular calcium that is enhanced by Xwnt-5a, a member of the Wnt family. This release of intracellular calcium is suppressed by an inhibitor of the enzyme inositol monophosphatase and hence of the phosphatidylinositol signaling pathway; this suppression can be rescued by injection of the compound myo-inositol, which overcomes the decrease in this intermediate caused by the inhibitor. Agents that inhibit specific G-protein subunits, pertussis toxin, GDP-beta-S and alpha-transducin also inhibit the calcium release triggered by Xwnt-5a and rat Frizzled-2. These results indicate that some Wnt proteins work through specific Frizzled homologs to stimulate the phosphatidylinositol signaling pathway via heterotrimeric G-protein subunits (Slusarski, 1997b).

The apical ectodermal ridge (AER) is an essential structure for vertebrate limb development. Wnt3a is expressed during the induction of the chick AER, and misexpression of Wnt3a induces ectopic expression of AER-specific genes in the limb ectoderm. The genes beta-catenin and Lef1 can mimic the effect of Wnt3a, and blocking the intrinsic Lef1 activity disrupts AER formation. Hence, Wnt3a functions in AER formation through the beta-catenin/LEF1 pathway. In contrast, neither beta-catenin nor Lef1 affects the Wnt7a-regulated dorsoventral polarity of the limb. Thus, two related Wnt genes elicit distinct responses in the same tissues by using different intracellular pathways (Kengaku, 1998).

In studies of developmental signaling pathways stimulated by the Wnt proteins and their receptors, Xenopus Wnt-5A (Xwnt-5A) and a prospective Wnt receptor, rat Frizzled 2 (Rfz2), have been shown to stimulate inositol signaling and Ca2+ fluxes in zebrafish. Since protein kinase C (PKC) isoforms can respond to Ca2+ signals, it was asked whether expression of different Wnt and Frizzled homologs modulates PKC. Expression of Rfz2 and Xwnt-5A results in translocation of PKC to the plasma membrane, whereas expression of rat Frizzled 1 (Rfz1), which activates a Wnt pathway using beta-catenin but not Ca2+ fluxes, does not. Rfz2 and Xwnt-5A are also able to stimulate PKC activity in an in vitro kinase assay. Agents that inhibit Rfz2-induced signaling through G-protein subunits block Rfz2-induced translocation of PKC. To determine if other Frizzled homologs differentially stimulate PKC, mouse Frizzled (Mfz) homologs were tested for their ability to induce PKC translocation relative to their ability to induce the expression of two target genes of beta-catenin, siamois and Xnr3. Mfz7 and Mfz8 stimulate siamois and Xnr3 expression but not PKC activation, whereas Mfz3, Mfz4 and Mfz6 reciprocally stimulate PKC activation but not expression of siamois and Xnr3. These results demonstrate that some but not all Wnt and Frizzled signals modulate PKC localization and stimulate PKC activity via a G-protein-dependent mechanism. In agreement with other studies these data support the existence of multiple Wnt and Frizzled signaling pathways in vertebrates (Sheldahl, 1999).

RGS family members are GTPase activating proteins (GAPs) that antagonize signaling by heterotrimeric G proteins. Injection of Xenopus embryos with RNA encoding rat RGS4 (rRGS4), a GAP for Gi and Gq, results in shortened trunks and decreased skeletal muscle. This phenotype is nearly identical to the effect of injection of either frzb or dominant negative Xwnt-8. Injection of human RGS2, which selectively deactivates Gq, has similar effects. rRGS4 inhibits the ability of early Xwnt-8 but not Xdsh misexpression to cause axis duplication. This effect is distinct from axin family members that contain RGS-like domains but act downstream of Xdsh. Two Xenopus RGS4 homologs have been identified, one of which, Xrgs4a, is expressed as a Spemann organizer component. Injection of Xenopus embryos with Xrgs4a also results in shortened trunks and decreased skeletal muscle. These results suggest that RGS proteins modulate Xwnt-8 signaling by attenuating the function of a G protein (Wu, 2000).

The importance of a G protein in early pattern formation raises the question of the identity of G protein effectors. One possibility is that an embryonic G protein directly activates PLC-beta or indirectly activates PLC-gamma, enzymes that generate IP3 and diacylglycerol. Previous work has demonstrated that IP3 levels rise sharply at the 64-cell stage of Xenopus embryogenesis and that they remain elevated for several hours. Diacylglycerol production can lead to protein kinase C activation, which, in turn, can phosphorylate and inactivate GSK-3beta. Indeed, wingless-induced inactivation of GSK-3beta in murine fibroblasts is sensitive to pharmacological inhibitors of PKC. A second possibility is that a G protein activates phosphatidylinositol-3 kinase, an enzyme that generates phosphatidylinositol-3,4,5-phosphate (PIP3) and other phosphorylated lipid products. The levels of PIP3 in early embryos have not been previously determined. PIP3 is an activator of protein kinase B (Akt), which can phosphorylate and inactivate GSK-3beta. In these ways, it is possible to hypothesize how activation of a G protein might lead to inactivation of GSK-3beta and stabilization of beta-catenin (Wu, 2000).

Wnt ligands working through Frizzled receptors have a differential ability to stimulate release of intracellular calcium [Ca(2+)] and activation of protein kinase C (PKC). Since targets of this Ca(2+) release could play a role in Wnt signaling, the hypothesis that Ca(2+)/calmodulin-dependent protein kinase II (CamKII) is activated by some Wnt and Frizzled homologs was tested. Wnt and Frizzled homologs that activate Ca(2+) release and PKC also activate CamKII activity in Xenopus embryos, while Wnt and Frizzled homologs that activate beta-catenin function do not. This activation occurs within 10 min after receptor activation in a pertussis toxin-sensitive manner, concomitant with autophosphorylation of endogenous CamKII. Based on data that Wnt-5A and Wnt-11 are present maternally in Xenopus eggs, and activate CamKII, the hypothesis that CamKII participates in axis formation in the early embryo was tested. Measurements of endogenous CamKII activity from dorsal and ventral regions of embryos reveals elevated activity on the prospective ventral side, which is suppressed by a dominant negative Xwnt-11. If this spatial bias in CamKII activity were involved in promoting ventral cell fate one might predict that elevating CamKII activity on the dorsal side would inhibit dorsal cell fates, while reducing CamKII activity on the ventral side would promote dorsal cell fates. Results obtained by expression of CamKII mutants are consistent with this prediction, revealing that CamKII contributes to a ventral cell fate (Kuhl, 2000).

Activation of the Wnt signaling pathway is important for induction of gene expression and cell morphogenesis throughout embryonic development. The subcellular localization of dishevelled, the immediate downstream component from the Wnt receptor, was examined in the embryonic mouse kidney. Dishevelled associates with actin fibers and focal adhesion plaques in metanephric mesenchymal cells. Stimulation of Wnt signaling leads to profound changes in metanephric mesenchymal cell morphology, including disruption of the actin cytoskeleton, increased cell spreading, and increased karyokinesis. Upon activation of Wnt signaling, dishevelled also accumulates in and around the nucleus. Casein kinase Iepsilon colocalizes with dishevelled along actin fibers and in the perinuclear region, whereas axin and GSK-3 are only present around the nucleus. These data indicate a branched Wnt signaling pathway comprising a canonical signal that targets the nucleus and gene expression, and another signal that targets the cytoskeleton and regulates cell morphogenesis (Torres, 2000).

The isolation and cloning of Wrch-1 (Wnt-1 responsive Cdc42 homolog) cDNA is reported. Wrch-1 is a novel gene whose mRNA level increases in response to Wnt-1 signaling in Wnt-1 transformed cells, Wnt-1 transgene induced mouse mammary tumors, and Wnt-1 retrovirus infected cells. Wrch-1 encodes a homolog of the Rho family of GTPases. It shares 57% amino acid sequence identity with Cdc42, but possesses a unique N-terminal domain that contains several putative PXXP SH3-binding motifs. Like Cdc42, Wrch-1 can activate PAK-1 and JNK-1, and induce filopodium formation and stress fiber dissolution. Active Wrch-1 stimulates quiescent cells to reenter the cell cycle. Moreover, overexpression of Wrch-1 phenocopies Wnt-1 in morphological transformation of mouse mammary epithelial cells. Taken together, Wrch-1 could mediate the effects of Wnt-1 signaling in the regulation of cell morphology, cytoskeletal organization, and cell proliferation (Tao, 2001).

The induction of downstream genes by the Wnt-1 signaling pathway occurs by multiple mechanisms. In the canonical model, Wnt-1 signaling elicits target gene transcription through ß-catenin/TCF-LEF transcription factors by stabilizing ß-catenin. Most of the identified Wnt-1-responsive genes are induced by this mode. Alternatively, the accumulated ß-catenin in the cytoplasm can stimulate the activation of other transcription factors, such as CREB, through the cAMP/PKA pathway, or associate with other transactivation partners, such as Teashirt, to induce gene expression. In addition, Wnt-1 can induce gene expression independently of ß-catenin. The promoter of BTEB2, one of the genes isolated in the same screen as Wrch-1, has been shown to be responsive to Wnt-1 signaling in a PKC-sensitive and ß-catenin-independent manner. Indeed, some Wnt family proteins, such as Xenopus Wnt-5A, use the PKC/calcium pathway rather than ß-catenin/TCF-LEF pathway to activate their target gene expression. Interestingly, Wrch-1 is up-regulated in Wnt-1-expressing cells but not in cells expressing the stabilized, transactivation-competent mutants of ß-catenin. These results suggest that Wnt-1 might regulate Wrch-1 expression independently of ß-catenin. The isolation and analysis of the Wrch-1 promoter would be required to confirm that Wrch-1 is a direct target of Wnt-1 signaling, and to elucidate the mechanism(s) by which Wrch-1 transcription is regulated (Tao, 2001).

Identification of Wrch-1 as a Wnt-1-regulated homolog of the Rho family of GTPases is likely to fill a gap between upstream Wnt-1 signaling and its effects on cell morphology, motility, and growth. The Rho-like GTPases act as molecular switches. In response to extracellular signals, they elicit coordinate changes in the organization of the actin cytoskeleton and in gene expression to regulate a variety of physiological processes, including morphogenesis, cell migration, axonal guidance, and cell cycle progression. Wrch-1 could regulate these processes in response to Wnt-1 signaling. Consistent with this hypothesis, Wrch-1 is predominantly expressed in the embryonic mouse nervous system, where Wnt-1 is also predominantly expressed. It has been shown that Wnt-1 plays a vital role in the development of the mouse nervous system. Homozygous deletion of the Wnt-1 alleles results in the absence of midbrain and cerebellum, and the Wnt-1^-/- mice die within 24 h after birth. In this scenario, Wrch-1 might act downstream of Wnt-1 signaling to control neural migration and axonal growth (Tao, 2001).

Wnt-1 is not normally expressed in the mouse mammary gland. However, aberrant activation of Wnt-1 expression in this tissue leads to tumor formation. Detection of Wrch-1 expression in Wnt-1 induces mouse mammary tumors, but not in wild-type mouse mammary glands, is consistent with the involvement of Wrch-1 in Wnt-1-induced transformation and tumor formation. The observation that Wrch-1 can mimic the effect of Wnt-1 in morphological transformation of C57MG cells further supports this notion. Indeed, overexpression or aberrant activation of Rho, Rac, or Cdc42 results in cell transformation, tumor formation, and metastasis (Tao, 2001).

Most of the Rho family GTPases are constitutively expressed in many tissues. Their biological activities are induced by extracellular cues that activate their corresponding nucleotide exchange factors. However, expression of several Rho family members, such as RhoB and RhoG, is growth factor inducible, indicating an additional mechanism that regulates their activities, that is, controlling their transcription. Wrch-1 appears to be regulated by both mechanisms. Conceivably, Wnt-1 could elicit Wrch-1 activities by inducing its expression and activating its nucleotide exchange factor(s) simultaneously. Alternatively, the expressed Wrch-1 could be activated by other signaling molecules. Of note is the SH3 binding domain of Wrch-1. This domain could either mediate the association of Wrch-1 with an activator or effector protein, or target Wrch-1 to a specific subcellular compartment. Interestingly, another novel protein has been identified in a yeast two-hybrid screen that can specifically bind to Wrch-1. This protein contains three putative SH3 domains and might act as an adaptor for Wrch-1 to target it to an activator or effector. Elucidation of the pathways upstream and downstream of Wrch-1 should lead to a better understanding of the fundamental processes controlled by Wnt-1 signaling (Tao, 2001).

Rho GTPases are molecular switches that regulate many essential cellular processes, including actin dynamics, cell adhesion, cell-cycle progression, and transcription. The Xenopus homolog of Rho GTPase Cdc42 has been isolated and its potential role during gastrulation movements in early Xenopus embryos has been examined. XCdc42 is expressed in tissues undergoing extensive morphogenetic changes, such as the deep layers of involuting mesoderm and posterior neuroectoderm during gastrulation, and somitic mesoderm at neurula stages. Overexpression of either wild-type (WT) or dominant-negative (DN) XCdc42 interferes with convergent extension movements in intact embryos, activin-stimulated animal caps, and dorsal marginal zone explants. These effects occur without affecting mesodermal specification. Overexpression of WT or DN XCdc42 leads to the decrease and increase of cell adhesiveness of blastomeres, respectively, as demonstrated by the cell adhesion assay. In addition, when overexpressed, PKC-alpha, XWnt-5a, and Mfz-3 inhibit activin-induced convergent extension in animal cap explants. This inhibition can be rescued by coexpression of DN XCdc42, implying that XCdc42 acts downstream of the Wnt/Ca ²⁺ signaling pathway involving PKC activation. XCdc42 also lies downstream of XWnt-5a in the regulation of Ca ²⁺-dependent cell adhesion. Taken together, these results suggest that XCdc42 plays a role in the regulation of convergent extension movements during gastrulation through the protein kinase C-mediated Wnt/Ca²⁺ pathway (Choi, 2002).

It is thought that inositol-1,4,5-trisphosphate (Ins(1,4,5)P₃)-Ca²⁺ signalling has a function in dorsoventral axis formation in Xenopus embryos; however, the immediate target of free Ca²⁺ is unclear. The secreted Wnt protein family comprises two functional groups, the canonical Wnt and Wnt/Ca²⁺ pathways. The Wnt/Ca²⁺ pathway interferes with the canonical Wnt pathway, but the underlying molecular mechanism is poorly understood. The complementary DNA coding for the Xenopus homolog of nuclear factor of activated T cells (XNF-AT) has been cloned. A gain-of-function, calcineurin-independent active XNF-AT mutation (CA XNF-AT) inhibits anterior development of the primary axis, as well as Xwnt-8-induced ectopic dorsal axis development in embryos. A loss-of-function, dominant negative XNF-AT mutation (DN XNF-AT) induces ectopic dorsal axis formation and expression of the canonical Wnt signalling target molecules siamois and Xnr3. Xwnt-5A induces translocation of XNF-AT from the cytosol to the nucleus. These data indicate that XNF-AT functions as a downstream target of the Wnt/Ca²⁺ and Ins(1,4,5)P₃-Ca²⁺ pathways, and has an essential role in mediating ventral signals in the Xenopus embryo through suppression of the canonical Wnt pathway (Saneyoshi, 2002).

Since injected myo-inositol blocks the effect of dominant negative GSK3ß-induced secondary axis formation, these findings support the idea that there is cross-talk between phosphatidylinositide cycle signalling and the canonical Wnt pathway. The tyrosine kinase-linked receptor signalling pathway also activates Ins(1,4,5)P₃-Ca²⁺ signalling through phospholipase Cgamma activation. Gain of function of Ins(1,4,5)P₃-Ca²⁺ signalling on the dorsal side of the embryo leads to a dorso-anterior structure deficiency, whereas loss of function on the ventral side induces a partial ectopic dorsal axis. These findings suggest that the Ins(1,4,5)P₃-Ca²⁺ signalling pathway mediates ventral signals. One possible target of Ins(1,4,5)P₃-Ca²⁺ signalling is the Ca²⁺/calmodulin (CaM)-dependent protein phosphatase, calcineurin, and the transcription factor -- positioned further downstream -- NF-AT. XNF-AT might receive inputs from tyrosine kinase signalling pathways, therefore the activity of XNF-AT in the wild-type embryo may reflect the activity of Wnt pathway. A proposed model for dorsoventral axis formation and the interaction between the Wnt/Ca²⁺ and the canonical Wnt pathways suggests that XNF-AT is a direct target of the Ins(1,4,5)P₃-Ca²⁺ signal downstream of the Wnt/Ca²⁺ pathway, and that XNF-AT mediates ventralizing signal by suppression of canonical Wnt activity during axis formation of the Xenopus embryo (Saneyoshi, 2002).

Rho family members are key regulators of the actin cytoskeleton, and control transcriptional targets through the activation of the JNK/SAPK pathway. Evidence for the role of Rho GTPases downstream of frizzled first arose from the analysis of Drosophila mutants affecting the establishment of planar cell polarity (PCP) of epithelia in developing eyes, dorsal thorax, and wings. Genetic dissection of the PCP pathway has shown that the Rho GTPase RhoA and Rac are activated in a pathway involving Frizzled and Dishevelled. A growing amount of data support the idea that a vertebrate equivalent of the PCP pathway activated downstream of Wnt-11/Xfz7 controls at least some of the cellular behaviors involved during mediolateral intercalation of the cells in the dorsal marginal zone (DMZ). Wnt-11/Xfz7 signaling plays a major role in the regulation of convergent extension movements affecting the DMZ of gastrulating Xenopus embryos. In order to provide data concerning the molecular targets of Wnt-11/Xfz7 signals, the regulation of the Rho GTPase Cdc42 by Wnt-11 was analyzed. In animal cap ectoderm, Cdc42 activity increases as a response to Wnt-11 expression. This increase is inhibited by pertussis toxin, or sequestration of free Gßgamma subunits by exogenous Galphai2 or Galphat. Activation of Cdc42 is also produced by the expression of bovine Gß1 and Ggamma2. This process is abolished by a PKC inhibitor, while phorbol esther treatment of ectodermal explants activates Cdc42 in a PKC-dependent way, implicating PKC downstream of Gßgamma. In activin-treated animal caps and in the embryo, interference with Gßgamma signaling rescues morphogenetic movements inhibited by Wnt-11 hyperactivation, thus phenocopying the dominant negative version of Cdc42 (N¹⁷Cdc42). Conversely, expression of Gß1gamma2 blocks animal cap elongation. This effect is reversed by N¹⁷Cdc42. Together, these results strongly argue for a role of Gßgamma signaling in the regulation of Cdc42 activity downstream of Wnt-11/Xfz7 in mesodermal cells undergoing convergent extension. This idea is further supported by the observation that expression of Galphat in the DMZ causes severe gastrulation defects (Penzo-Mendez, 2003).

The c-myb proto-oncogene product (c-Myb) regulates both the proliferation and apoptosis of hematopoietic cells by inducing the transcription of a group of target genes. However, the biologically relevant molecular mechanisms that regulate c-Myb activity remain unclear. c-Myb protein is phosphorylated and degraded by Wnt-1 signal via the pathway involving TAK1 (TGF-beta-activated kinase), HIPK (homeodomain-interacting protein kinase), and NLK (Nemo-like kinase). Wnt-1 signal causes the nuclear entry of TAK1, which then activates HIPK and the mitogen-activated protein (MAP) kinase-like kinase NLK. NLK binds directly to c-Myb together with HIPK, which results in the phosphorylation of c-Myb at multiple sites, followed by its ubiquitination and proteasome-dependent degradation. Furthermore, overexpression of NLK in M1 cells abrogates the ability of c-Myb to maintain the undifferentiated state of these cells. The down-regulation of Myb by Wnt-1 signal may play an important role in a variety of developmental steps (Kanei-Oshii, 2004).

Wnt-signal transduction through beta-catenin is thought to require the inhibition of GSK3 by Frat/GBP. Frat1 was identified as a proto-oncogene that conveyed selective advantage to cells at later stages of murine T-cell lymphomagenesis. To investigate the role of Frat in mammalian development, mice were generated with targeted mutations in all three murine Frat homologs. Frat is shown to be normally expressed at sites of active Wnt signaling. Surprisingly, Frat-deficient mice do not display gross abnormalities. Moreover, canonical Wnt signaling in primary cells is unaffected by the loss of Frat. These studies show that Frat is not an essential component of the canonical Wnt pathway in higher organisms, despite the strict requirement of Frat/GBP for maternal Wnt signaling in Xenopus (van Amerongenn, 2005).

JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates

A pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the planar cell polarity (PCP) pathway. However, it is not known whether the JNK pathway functions in this non-canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. This study shows that Wnt5a is capable of activating JNK in cultured cells, and evidence is presented that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. These results thus demonstrate that the non-canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and establish that JNK has an activity that regulates morphogenetic cell movements (Yamanaka, 2002).

These results suggest that the appropriate activation of JNK is necessary for correct convergent extension. It has previously been shown that overexpressed Dishevelled inhibits the convergent extension. Antisense JNK MO cancels significantly the overexpressed Dishevelled-induced inhibition of the convergent extension movements of explants, suggesting that Dishevelled lies upstream of JNK. Since recent reports indicate that in zebrafish and Xenopus embryos, the activity of Wnt11, a member of the Wnt5a class ligands, is required for cells to undergo correct convergent extension movements, the JNK pathway in vivo may lie downstream of Wnt11 rather than Wnt5a, or both Wnt5a and Wnt11 may function redundantly. In any case, it can be concluded that the non-canonical Wnt/JNK pathway is conserved evolutionarily in both vertebrates and invertebrates, since previous studies have demonstrated that a pathway regulating convergent extension in developing vertebrate embryos is equivalent to the PCP pathway (also known as the non-canonical Wnt pathway) in Drosophila, and that the PCP pathway in Drosophila signals via the JNK pathway to control cell polarity. The mechanism by which JNK regulates convergent extension movements in vertebrates remains to be established. Preliminary data suggest that activated JNK affects cell-cell adhesion. This is in good agreement with the previous report indicating that Wnt5a is able to decrease the cell-cell adhesion. Furthermore, a study in Drosophila has suggested a role for the transcription factor c-Jun, one of major targets of JNK, in the PCP pathway. It is thus possible that the JNK/c-Jun-mediated gene expression is also important for regulation of the morphogenetic cell movements. Identifying molecular components upstream and downstream of the MKK7/JNK cascade in the non-canonical Wnt pathway in vertebrates is a priority for future research (Yamanaka, 2002 and references therein).

Wnt11/β-catenin signaling in both oocytes and early embryos acts through LRP6-mediated regulation of axin

Current models of canonical Wnt signaling assume that a pathway is active if β-catenin becomes nuclearly localized and Wnt target genes are transcribed. In Xenopus, maternal LRP6 is essential in such a pathway, playing a pivotal role in causing expression of the organizer genes siamois and Xnr3, and in establishing the dorsal axis. Evidence iis provided that LRP6 acts by degrading axin protein during the early cleavage stage of development. In the full-grown oocyte, before maturation, axin levels are also regulated by Wnt11 and LRP6. In the oocyte, Wnt11 and/or LRP6 regulates axin to maintain β-catenin at a low level, while in the embryo, asymmetrical Wnt11/LRP6 signaling stabilizes β-catenin and enriches it on the dorsal side. This suggests that canonical Wnt signaling may not exist in simple off or on states, but may also include a third, steady-state, modality (Kofron, 2007)

Distinct PAR-1 proteins function in different branches of Wnt signaling during vertebrate development

The kinase PAR-1 plays conserved roles in cell polarity. PAR-1 has also been implicated in axis establishment in C. elegans and Drosophila and in Wnt signaling, but its role in vertebrate development is unclear. PAR-1 has two distinct and essential roles in axial development in Xenopus mediated by different PAR-1 isoforms. Depletion of PAR-1A or PAR-1BX causes dorsoanterior deficits, reduced Spemann organizer gene expression, and inhibition of canonical Wnt-β-catenin signaling. By contrast, PAR-1BY depletion inhibits cell movements and localization of Dishevelled protein to the cell cortex, processes associated with noncanonical Wnt signaling. PAR-1 phosphorylation sites in Dishevelled are required for this translocation, but not for canonical Wnt signaling. It is concluded that PAR-1BY is required in the PCP branch and mediates Dsh membrane localization while PAR-1A and PAR-1BX are essential for canonical signaling to β-catenin, possibly via targets other than Dishevelled (Ossipova, 2005).

Since the identification of PAR-1 as a Dishevelled-associated kinase, its precise role in Wnt signaling has been unclear. The question remains as to whether phosphorylation of Dsh by PAR-1 kinase is an essential process in Wnt-β-catenin signal transduction. The results show that the role of PAR-1 in Wnt signaling is complex and isoform dependent. Isoforms PAR-1A and PAR-1BX were identified that exert effects primarily on canonical Wnt signaling: they are physiologically essential in the Xenopus embryo for establishment of the organizer. This is an important finding because although PAR-1 has previously been implicated in canonical Wnt signaling, any understanding of its importance for endogenous Wnt signaling has remained ambiguous. A mutated Dishevelled that lacks PAR-1 sites is capable of driving a canonical β-catenin signal, which is still further potentiated by overexpressed PAR-1. This raises the possibility that PAR-1 acts on other components of the Wnt pathway that remain to be identified. Consistent with this notion, three other Dsh kinases in the canonical Wnt pathway, namely CKIα, CKIε, and CKII, have alternative substrates in addition to Dsh, namely β-catenin, APC, and TCF3, respectively (Ossipova, 2005).

A third PAR-1 isoform, PAR-1BY, differs from the other two in its role in vivo, being required for convergent extension but not for canonical Wnt signaling or organizer formation. The PAR-1B isoforms differ by only 10% at the amino acid level, but most of this difference is concentrated at the N terminus and in the spacer region around serine 646. The amino terminus of PAR-1BX is present in the Xenopus tropicalis homolog but is not conserved outside the Xenopus genus. The spacer differences are seen in sequenced vertebrates as alternative splicing of exons that are conserved, although whether this is the case in Xenopus laevis in particular will remain unknown until its genome is sequenced (Ossipova, 2005).

Finally, the data reveal a critical mechanism of action of PAR-1 in regulating Dsh, namely its translocation from cytoplasmic vesicles to the cell cortex. This translocation is typically concomitant with noncanonical PCP signaling, and indeed it has been shown that PAR-1 is not just sufficient for JNK regulation, but is also necessary endogenously. PAR-1 is shown to be (1) sufficient and necessary to drive this translocation; (2) that PAR-1 kinase activity is activated by Frizzleds, and (3) that the kinase activity and its targets in Dishevelled are essential, at least for exit from vesicular structures. These findings reveal a pathway leading from Frizzled receptors to elevation of PAR-1 kinase to phosphorylation of Dsh on specific serine and threonine residues to translocation of Dsh from vesicles to the cell cortex. PAR-1 is thus a vertebrate missing link between Frizzled and Dsh. The C-terminal DEP domain of Dsh is sufficient for translocation. It therefore seems likely that PAR-1-dependent phosphorylation, acting elsewhere, regulates some protein-protein interaction that retains Dsh in the cytoplasm. In Drosophila, Dishevelled isoforms lacking the PAR-1 target sites are able to rescue Wingless but not PCP mutants. A mechanism for this phenomenon has been identified, namely that PAR-1 is necessary for Dsh localization to the membrane and therefore fails to localize such Dsh mutants properly within the cell. Thus, the 'noncanonical isoform' of PAR-1 has a role consistent with its classification as a polarity protein, albeit planar cell polarity rather than the previously reported apicobasal polarity. Whether the 'canonical PAR-1' isoforms have polarity roles at all remains to be seen (Ossipova, 2005).

A Wnt-CKI-Rap1 pathway regulates gastrulation by modulating SIPA1L1, a Rap GTPase activating protein

Noncanonical Wnt signals control morphogenetic movements during vertebrate gastrulation. Casein kinase I epsilon (CKIε) is a Wnt-regulated kinase that regulates Wnt/β-catenin signaling and has a β-catenin-independent role(s) in morphogenesis that is poorly understood. This study reports the identification of a CKIε binding partner, SIPA1L1/E6TP1, a GAP (GTPase activating protein) of the Rap small GTPase family. CKIε phosphorylates SIPA1L1 to reduce its stability and thereby increase Rap1 activation. Wnt-8, which activates CKIε, enhances the CKIε-dependent phosphorylation and degradation of SIPA1L1. In early Xenopus or zebrafish development, inactivation of the Rap1 pathway results in abnormal gastrulation and a shortened anterior-posterior axis. Although CKIε also transduces Wnt/β-catenin signaling, inhibition of Rap1 does not alter β-catenin-regulated gene expression. These data demonstrate a role for CKIε in noncanonical Wnt signaling and indicate that Wnt regulates morphogenesis in part through CKIε-mediated control of Rap1 signaling (Tsai, 2007).

Although previous studies of CKIε in Xenopus development suggested it functions in noncanonical Wnt signaling, details of the downstream signal transduction pathways have been unclear. Noncanonical Wnt pathways affect cell movements rather than gene transcription, making them more difficult to assay. Also, single-time-point assays in cell-based systems tend to miss dynamic effects of morphological progression. This study utilized cell-based systems to determine how CKIε regulates the SIPA1L1-Rap1 pathway, and then tested the biological effects in Xenopus and zebrafish development. The data suggest CKIε is involved in multiple pathways downstream of Wnt. Prior to gastrulation, CKIε enhances β-catenin-dependent gene expression, thus controlling cell fate specification. However, during gastrulation, CKIε regulates convergent extension through the SIPA1L1-Rap1 pathway. Consistent with this, DN-Rap1 or CKIε-resistant SIPA1L1 (SIPA1L1[CΔ]) do not affect β-catenin-dependent gene expression in early gastrula stages, but do inhibit convergent extension during gastrula and neurula stages of Xenopus development (Tsai, 2007).

Wnt-8 has been classified as a 'canonical' Wnt because it can induce axis duplication in Xenopus embryos and β-catenin-dependent gene expression. However, classifying Wnts by their ability to induce axis duplication or to activate a TCF-dependent promoter may be imprecise, as pathway activation also depends on receptor expression in target cells, and crosstalk between canonical and noncanonical signaling has been reported. These data indicate a role for Wnt-8 in a noncanonical Wnt pathway. Consistent with previous studies that find regulation of convergent extension does not require β-catenin, it was found that β-catenin could not moderate the CE defect caused by downregulation of Rap1. It is suggested that the CKIε/β-catenin and CKIε/Rap1 pathways together contribute to proper axis development. During axis development, Wnt-8 (and others) activates regional β-catenin distribution, initiating axis formation, and later activates the Rap1 pathway, facilitating morphogenic movements (Tsai, 2007).

Rap GTPases include Rap1 and Rap2, which share about 60% amino acid identity. While both are involved in cell migration and adhesion, their functions in Xenopus development appear to be distinct. Although Rap2 affects β-catenin-mediated dorsalization events, Rap1 is involved in a noncanonical Wnt pathway. Unlike Rap2, Rap1 knockdown causes a defect in gastrulation rather than a hyperventralized phenotype. Inhibition of Rap1 does not change the expression of Gsc, Chd, and Bmp4, suggesting that Rap1 signaling does not affect dorsal-ventral specification. Two prior observations are also consistent with the conclusion that a SIPA1L1-Rap1 pathway regulates morphogenetic movements in embryogenesis. First, Rap1 regulates cell migration in cell-based studies, consistent with a role in morphological movements during development. Second, SIPA1L1 induces actin reorganization in rat neurons via its GAP activity. Although SIPA1L1 can facilitate GTP hydrolysis both in Rap1 and Rap2, SIPA1L1 expression disrupts gastrulation rather than inducing a hyperventralized phenotype. SIPA1L1 may therefore preferentially affect Rap1 during development (Tsai, 2007).

Two small GTPases, Rho and Rac, are known Wnt-regulated small GTPases required for proper gastrulation during Xenopus development. Biochemical analysis of Rho and Rac regulation indicates that Wnt signaling activates these pathways through parallel mechanisms. Both distinct and overlapping functions of Rho and Rac are essential for directing CE. This study identifies Rap1 as an additional small GTPase downstream of Wnt. Rap1, Rho, and Rac may be cooperatively activated by a Wnt signal to mediate the complex series of movements involved in gastrulation. Rap1 has previously been found to be activated by changes in cytoskeletal tension. It is speculated that Rap1 activity facilitates changes in cell-cell adhesion required for proper morphogenetic movements (Tsai, 2007).

Wnt signaling, via CKIε and SIPA1L1, regulates Rap1, and Rap1 plays an essential role in embryogenesis. SIPA1L1/E6TP1 is also a target of the oncogenic human papillomavirus protein E6, suggesting its inactivation contributes to cancer development. E6-mediated SIPA1L1/E6TP1 degradation correlates with the immortalization of epithelial cells. In addition, mutations of SIPA1, a SIPA1L1-related protein, correlates with efficiency of breast cancer metastasis. The fact that Wnt signaling and CKIε activity also decrease SIPA1L1, combined with the fact that some Wnts are oncogenes, is consistent with the hypothesis that SIPA1L1 downregulation contributes to malignant transformation. The role of Rap1 in cell migration suggests Wnt signaling may therefore play a role not only in proliferation but in tumor invasion and metastasis as well (Tsai, 2007).

Noncanonical Wnt signaling through G protein-linked PKCΔ activation promotes bone formation

Wnt signaling regulates a variety of developmental processes in animals. Although the β-catenin-dependent (canonical) pathway is known to control cell fate, a similar role for noncanonical Wnt signaling has not been established in mammals. Moreover, the intracellular cascades for noncanonical Wnt signaling remain to be elucidated. This study delineate a pathway in which Wnt3a signals through the Gα_q/11 subunits of G proteins to activate phosphatidylinositol signaling and PKCδ in the murine ST2 cells. Gα_q/11-PKCδ signaling is required for Wnt3a-induced osteoblastogenesis in these cells, and PKCδ homozygous mutant mice exhibit a deficit in embryonic bone formation. Furthermore, Wnt7b, expressed by osteogenic cells in vivo, induces osteoblast differentiation in vitro via the PKCδ-mediated pathway; ablation of Wnt7b in skeletal progenitors results in less bone in the mouse embryo. Together, these results reveal a Wnt-dependent osteogenic mechanism, and they provide a potential target pathway for designing therapeutics to promote bone formation (Tu, 2007).

Distinct Wnt signaling pathways have opposing roles in appendage regeneration

In contrast to mammals, lower vertebrates have a remarkable capacity to regenerate complex structures damaged by injury or disease. This process, termed epimorphic regeneration, involves progenitor cells created through the reprogramming of differentiated cells or through the activation of resident stem cells. Wnt/ß-catenin signaling regulates progenitor cell fate and proliferation during embryonic development and stem cell function in adults, but its functional involvement in epimorphic regeneration has not been addressed. Using transgenic fish lines, it has been shown that Wnt/ß-catenin signaling is activated in the regenerating zebrafish tail fin and is required for formation and subsequent proliferation of the progenitor cells of the blastema. Wnt/ß-catenin signaling appears to act upstream of FGF signaling, which has recently been found to be essential for fin regeneration. Intriguingly, increased Wnt/ß-catenin signaling is sufficient to augment regeneration, as tail fins regenerate faster in fish heterozygous for a loss-of-function mutation in axin1, a negative regulator of the pathway. Likewise, activation of Wnt/ß-catenin signaling by overexpression of wnt8 increases proliferation of progenitor cells in the regenerating fin. By contrast, overexpression of wnt5b (pipetail) reduces expression of Wnt/ß-catenin target genes, impairs proliferation of progenitors and inhibits fin regeneration. Importantly, fin regeneration is accelerated in wnt5b mutant fish. These data suggest that Wnt/ß-catenin signaling promotes regeneration, whereas a distinct pathway activated by wnt5b acts in a negative-feedback loop to limit regeneration (Stoick-Cooper, 2007).

Table of contents

The Interactive Fly resides on the
Society for Developmental Biology's Web server.