wingless

EVOLUTIONARY HOMOLOGS

Table of contents

Canonical and non-canonical Wnt signaling pathways

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).

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 (see Eisenmann's Wnt Signaling) 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).

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).

The non-canonical Wnt/cyclic GMP/Ca²⁺/NF-AT pathway operates via Frizzled-2, a member of the superfamily of G protein-coupled receptors. In scanning for signaling events downstream of the Frizzled-2/Gαt2/PDE6 triad activated in response to Wnt5a, a strong activation of the mitogen-activated protein kinase p38 was observed in mouse F9 teratocarcinoma embryonal cells. The activation of p38 is essential for NF-AT transcriptional activation mediated via Frizzled2. Wnt5a-stimulated p38 activation is rapid, sensitive to pertussis toxin, to siRNA against either Gαt2 or p38α, and to the p38 inhibitor SB203580. Real-time analysis of intracellular cyclic GMP using the Cygnet2 biosensor revealed p38 to act at the level of cyclic GMP, upstream of the mobilization of intracellular Ca²⁺. Fluorescence resonance energy transfer (FRET) imaging reveals the changes in cyclic GMP in response to Wnt5a predominate about the cell membrane, and likewise sensitive to either siRNA targeting p38 or to treatment with SB203580. Dishevelled is not required for Wnt5a activation of p38; siRNAs targeting Dishevelleds and expression of the Dishevelled antagonist Dapper-1 do not suppress the p38 response to Wnt5a stimulation. These novel results are the first to detail a Dishevelled-independent Wnt response, demonstrating a critical role of the mitogen-activated protein kinase p38 in regulating the Wnt non-canonical pathway (Ma, 2007).

This study reveals a novel role of the p38 MAPK in the Wnt/cyclic GMP/Ca²⁺/NF-AT transcriptional activation pathway mediated by Frizzled-2. Activation of the non-canonical Wnt/Ca²⁺ pathway promotes ventral cell fate in the Xenopus embryo. Wnt5a stimulates phosphatidylinositol signaling and Ca²⁺ transients that are essential to normal development in the zebrafish embryo. Mouse embryonic F9 cells were employed to probe the role of p38 MAPK in the signal linkage map from a proximal step (i.e. activation of Frizzled-2) downstream to the activation of the developmentally regulated, luciferase reporter gene sensitive to NF-AT. The results from these studies provided several key and novel insights about Wnt signaling in the non-canonical pathway (Ma, 2007).

First, although MAPK family members have been implicated in Wnt signaling, the current study is the first report to identify p38 MAPK as downstream in a Wnt-sensitive pathway. Earlier studies of the planar cell polarity pathway in Drosophila and Wnt pathways regulating convergent extension in vertebrate demonstrate the activation of N-terminal c-Jun protein kinase, JNK. Erk1/2 MAPK have not yet been implicated in Wnt signaling, but it is likely that cross-talk must exist between Wnt-sensitive pathways and the MAPK cascade of downstream signaling. For the Wnt5a/cyclic GMP/Ca²⁺/NF-AT-sensitive transcription pathway, p38 not only regulates the signaling, but is essential for the overall function of the pathway from Wnt5a to the activation of NF-AT (Ma, 2007).

Second, the activation of p38 by Wnt5a feeds into the Wnt5a/cyclic GMP/Ca²⁺/NF-AT pathway at the level of cyclic GMP, upstream of Ca²⁺ mobilization. The ability of Wnt5a to activate p38 MAPK itself is not sensitive to the elevation of intracellular cyclic GMP by addition of 8-bromo-cyclic GMP or by inhibition of PDE6 with zaprinast. Furthermore, inhibiting PKG activity does not alter the ability of Wnt5a to activate p38. What is clear is that inhibition of p38 MAPK interrupts the signaling of this pathway at the level of cyclic GMP. This important information was deduced both by read-outs of direct cyclic GMP measurement, as a reflection of PKG activity, and in live cells, making use of the Cygnet2.1 biosensor for cyclic GMP. Current understanding of how p38 MAPK modulates cGMP levels is not complete. Experimental results provide a line of evidence indicating that p38 MAPK is necessary for the PDE6 activation in response to Wnt5a. Although Wnt5a stimulation leads to the activation of Gαt and PDE6, mimicking the pathway in the visual system, the mechanism by which p38 MAPK regulates the PDE6 is not clear (Ma, 2007).

Third, the activation of p38 appears to operate via two interacting signaling paradigms, a GPCR cascade and a traditional MAPK cascade. The Fz2/Gαt2/PDE6 triad operates down to the level of NF-AT-sensitive transcriptional activation, while the MEKK/MKK/MAPK cascade culminates in activation of p38, which is also required for the activation of NF-AT. This configuration has marked similarities to the Fz1-mediated regulation of planar cell polarity, operating in mammals and in flies through Fz1/Gαo/Dvl and downstream to a MEKK/MKK/JNK cascade. Thus GPCRs relay information from Wnt ligands to G proteins and their cognate effectors downstream to MEKKs that control MAPKs and the activity of transcription factors (Ma, 2007).

Finally, this study reveals for the first time the operation of a Wnt-sensitive signaling pathway that to the level of the effecter, p38, operates independent of the phosphoprotein Dvl. Knock-down of Dvl1, Dvl2, Dvl3, or the expression of the Dvl inhibitor Dapper-1 has no effect on the ability of Wnt5a to activate p38, although the signaling to the level of NF-AT does. Taken together, these novel observations reveal an essential role of p38 MAPK in Wnt-sensitive signaling via the non-canonical pathway (Ma, 2007).

The Wnt-β-catenin canonical signaling pathway is crucial for normal embryonic development, and aberrant expression of components of this pathway results in oncogenesis. Upon scanning for the mitogen-activated protein kinase (MAPK) pathways that might intersect with the canonical Wnt-β-catenin signaling pathway in response to Wnt3a, a strong activation of p38 MAPK was observed in mouse F9 teratocarcinoma cells. Wnt3a-induced p38 MAPK activation was sensitive to siRNAs against Gα_q or Gα_s, but not against either Gα_o or Gα₁₁. Activation of p38 MAPK is critical for canonical Wnt-β-catenin signaling. Chemical inhibitors of p38 MAPK (SB203580 or SB239063) and expression of a dominant negative-version of p38 MAPK attenuate Wnt3a-induced accumulation of β-catenin, Lef/Tcf-sensitive gene activation, and primitive endoderm formation. Furthermore, epistasis experiments pinpoint p38 MAPK as operating downstream of Dishevelleds. It was also demonstrated that chemical inhibition of p38 MAPK restores Wnt3a-attenuated GSK3β kinase activity. The involvement of G-proteins and Dishevelleds in Wnt3a-induced p38 MAPK activation was demonstrated, highlighting a critical role for p38 MAPK in canonical Wnt-β-catenin signaling (Bikkavilli, 2008).

Wnt signaling effectors direct the development and adult remodeling of the female reproductive tract (FRT); however, the role of non-canonical Wnt signaling has not been explored in this tissue. The non-canonical Wnt signaling protein van gogh-like 2 is mutated in loop-tail (Lp) mutant mice (Vangl2^Lp), which display defects in multiple tissues. Vangl2^Lp mutant uterine epithelium displays altered cell polarity, concommitant with changes in cytoskeletal actin and scribble (scribbled, Scrb1) localization. The postnatal mutant phenotype is an exacerbation of that seen at birth, exhibiting more smooth muscle and reduced stromal mesenchyme. These data suggest that early changes in cell polarity have lasting consequences for FRT development. Furthermore, Vangl2 is required to restrict Scrb1 protein to the basolateral epithelial membrane in the neonatal uterus, and an accumulation of fibrillar-like structures observed by electron microscopy in Vangl2^Lp mutant epithelium suggests that mislocalization of Scrb1 in mutants alters the composition of the apical face of the epithelium. Heterozygous and homozygous Vangl2^Lp mutant postnatal tissues exhibit similar phenotypes and polarity defects and display a 50% reduction in Wnt7a levels, suggesting that the Vangl2^Lp mutation acts dominantly in the FRT. These studies demonstrate that the establishment and maintenance of cell polarity through non-canonical Wnt signaling are required for FRT development (Vandenberg, 2009).

The Mediator complex is commonly seen as a molecular bridge that connects DNA-bound transcription factors to the RNA polymerase II (Pol II) machinery. It is a large complex of 30 subunits that is present in all eukaryotes. The Med12 subunit has been implicated not only in the regulation of Pol II activity, but also in the binding of transcription factors to the bulk of the Mediator complex. Med12 was targeted in mouse embryonic stem cells to investigate the in vivo function of this subunit. This study reports the developmental defects of Med12 hypomorphic mutants that have a drastic reduction in Med12 protein levels. These mutants fail to develop beyond embryonic day 10 and have severe defects in neural tube closure, axis elongation, somitogenesis and heart formation. In Med12 hypomorphic embryos, the Wnt/planar cell polarity pathway is disrupted, and canonical Wnt/beta-catenin signaling is impaired. In agreement with this, embryos that are incapable of Med12 expression failed to establish the anterior visceral endoderm or activate brachyury expression, and did not complete gastrulation (Rocha, 2010).

Wnt ligands signal through β-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through β-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. This study demonstrates that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). Ror2 Ser 864 was identified as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, evidence is provided that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways (Grumolato, 2010).

Neural crest (NC) cells are multipotent progenitors that form at the neural plate border, undergo epithelial-mesenchymal transition and migrate to diverse locations in vertebrate embryos to give rise to many cell types. Multiple signaling factors, including Wnt proteins, operate during early embryonic development to induce the NC cell fate. Whereas the requirement for the Wnt/β-catenin pathway in NC specification has been well established, a similar role for Wnt proteins that do not stabilize β-catenin has remained unclear. Gain- and loss-of-function experiments implicate Wnt11-like proteins in NC specification in Xenopus embryos. In support of this conclusion, modulation of β-catenin-independent signaling through Dishevelled and Ror2 causes predictable changes in premigratory NC. Morpholino-mediated depletion experiments suggest that Wnt11R, a Wnt protein that is expressed in neuroectoderm adjacent to the NC territory, is required for NC formation. Wnt11-like signals might specify NC by altering the localization and activity of the serine/threonine polarity kinase PAR-1 (also known as microtubule-associated regulatory kinase or MARK), which itself plays an essential role in NC formation. Consistent with this model, PAR-1 RNA rescues NC markers in embryos in which noncanonical Wnt signaling has been blocked. These experiments identify novel roles for Wnt11R and PAR-1 in NC specification and reveal an unexpected connection between morphogenesis and cell fate (Ossipava, 2011).

Noncanonical Wnt ligands, such as Wnt5a and Wnt11, do not stabilizeα-catenin or activate TCF-dependent transcription, but regulate morphogenetic processes that involve changes n cell shape and motility, which are sometimes referred to as planar cell polarity (PCP). The signaling from Wnt5 or Wnt11 is thought to involve Ror and Ryk receptors, small Rho GTPases, Rho-associated kinase, c-Jun N-terminal kinases and intracellular calcium. Although noncanonical Wnt pathways have been shown to function in NC cell migration, their importance for NC specification has remained unclear (Ossipava, 2011).

Craniofacial defects in Wnt5a knockout mice, and in wnt11 (silberblick) and wnt5 (pipetail) zebrafish mutant embryos suggest possible roles for noncanonical Wnt signaling in NC development. The results of this study support the view that noncanonical signaling from Wnt11R is essential for NC specification in Xenopus embryos and that it might act by changing the localization and activity of the polarity kinase PAR-1 (Ossipava, 2011).

PAR proteins are conserved regulators of cell polarity that interact with several embryonic signaling pathways, including the Wnt pathway. PAR-1 associates with Dishevelled (Dvl, or Dsh) and participates in Frizzled-dependent Dvl recruitment. This study shows that PAR-1 is itself required for NC specification and can rescue NC defects in embryos with inhibited Wnt5 and Wnt11 signaling. These findings identify PAR-1 as a molecular target for noncanonical Wnt signaling and reveal an unexpected causal connection between cell polarization and the NC cell fate (Ossipava, 2011).

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