Interactive Fly, Drosophila

ß-catenin and WNT signaling in Xenopus

The Wnt pathway regulates the early dorsal-ventral axis in Xenopus through a complex of beta-catenin and HMG box transcription factors of the Lef/Tcf family (see Drosophila Pangolin). The promoter of the dorsalizing homeo box gene siamois is a direct target for the beta-catenin/XTcf-3 complex, establishing a link between the Wnt pathway and the activation of genes involved in specifying the dorsal axis. By injecting siamois reporter constructs into the animal pole of Xenopus embryos, it has been shown that a 0.8-kb fragment of the siamois promoter is strongly activated by beta-catenin. The proximal 0.5 kb, which is also activated by beta-catenin, contains three Lef/Tcf-binding sites. Mutations in these sites eliminate the beta-catenin-mediated activation of siamois and show that siamois is regulated by the beta-catenin/XTcf-3 complex, in combination with additional transcriptional activators. When expressed at the equator of the embryo, the siamois promoter is activated to much higher levels on the dorsal side than the ventral side. Ectopic ventral expression of beta-catenin raises the ventral expression of the siamois promoter to the dorsal levels. Conversely, ectopic dorsal expression of dominant-negative XTcf-3 abolishes the dorsal activation of the siamois promoter. Elimination of the Lef/Tcf sites elevates the ventral expression of siamois, revealing a repressive role for XTcf-3 in the absence of beta-catenin. The endogenous siamois activator, although present throughout the dorsal side of the embryo, is most potent in the dorsal vegetal region. It is proposed that the dorsal activation of siamois by the beta-catenin/XTcf-3 complex combined with the ventral repression of siamois by XTcf-3 results in the restriction of endogenous siamois expression to the dorsal side of Xenopus embryos (Brannon, 1997).

The interaction between beta-catenin and LEF-1/TCF transcription factors plays a pivotal role in the Wnt-1 signaling pathway. The level of beta-catenin is regulated by partner proteins, including glycogen synthase kinase-3beta (GSK-3beta) and the adenomatous polyposis coli (APC) tumor suppressor protein. Genetic defects in APC are responsible for a heritable predisposition to colon cancer. APC protein and GSK-3beta bind beta-catenin, retain it in the cytoplasm, and facilitate the proteolytic degradation of beta-catenin. Abrogation of this negative regulation allows beta-catenin to translocate to the nucleus and to form a transcriptional activator complex with the DNA-binding protein lymphoid-enhancing factor 1 (LEF-1). This complex is thought to be involved in tumorigenesis. Covalent linkage of LEF-1 to beta-catenin and to transcriptional activation domains derived from the estrogen receptor or the herpes simplex virus protein VP16 generates transcriptional regulators that induce oncogenic transformation of chicken embryo fibroblasts. The chimeras between LEF-1 and beta-catenin or VP16 are constitutively active, whereas fusions of LEF-1 to the estrogen receptor are regulatable by estrogen. These experiments document the oncogenicity of transactivating LEF-1 and show that the transactivation domain normally provided by beta-catenin can be replaced by heterologous activation domains. These results suggest that the transactivating function of the LEF-1/beta-catenin complex is critical for tumorigenesis and that this complex transforms cells by activating specific LEF-1 target genes (Aoki, 1999).

Tcf/Lef transcription factors mediate signaling from Wingless/Wnt proteins by recruiting Armadillo/beta-catenin as a transcriptional co-activator. However, studies of Drosophila, Xenopus and Caenorhabditis elegans have indicated that Tcf factors may also be transcriptional repressors. Tcf factors are shown to physically interact with members of the Groucho family of transcriptional repressors. In transient transfection assays, the Xenopus Groucho homolog XGrg-4 inhibits activation of transcription of synthetic Tcf reporter genes. In contrast, the naturally truncated Groucho-family member XGrg-5 enhances transcriptional activation. Injection of XGrg-4 into Xenopus embryos represses transcription of Siamois and Xnr-3, endogenous targets of beta-catenin-Tcf. Dorsal injection of XGrg-4 has a ventralizing effect on Xenopus embryos. Secondary-axis formation induced by a dominant-positive Armadillo-Tcf fusion protein is inhibited by XGrg-4 and enhanced by XGrg-5. These data indicate that expression of Tcf target genes is regulated by a balance between Armadillo and Groucho (Roose, 1998).

In Xenopus, the homeobox gene Siamois is activated prior to gastrulation in Spemann's organizer, and is capable of inducing a secondary body axis when ectopically expressed. To elucidate the function of endogeneous Siamois in dorsoventral axis formation, a dominant repressor construct (SE) was constructed in which the Siamois homeodomain was fused to an active repression domain of Drosophila Engrailed. Overexpression of 1-5 pg of this chimeric mRNA in the early embryo blocks axis development and inhibits activation of dorsal, but not in ventrolateral or marginal zone markers. Inhibition of several organizer genes, Xlim-1, chordin, and goosecoid, is observed in embryos overexpressing SE dorsally. In contrast, transcripts for the ventrolateral marker Xwnt8 may be slightly upregulated, consistent with ventralization of the normal dorsal side. Siamois can lead to transcriptional activation from the goosecoid PE promoter. PE is the proximal element of the goosecoid promoter, located between bases -155 to -105. Coexpression of mRNA encoding wild-type Siamois, but not a mutated Siamois, restores dorsal development to SE embryos. SE strongly blocks axis formation triggered by beta-catenin but not by the organizer product noggin. These results suggest that Siamois function is essential for beta-catenin-mediated formation of the Spemann organizer, and that Siamois acts prior to noggin in specifying dorsal development (Fan, 1997).

ß-Catenin, another vertebrate homolog of Armadillo, is a cytoplasmic cadherin-associated protein required for cadherin adhesive function. ß-Catenin is implicated in axial patterning of the early Xenopus embryo. Overexpression of ß-Catenin in the ventral side of the early Xenopus embryo induces the formation of a complete secondary body axis. The internal armadillo repeat region is both necessary and sufficient to induce axis duplication. This region interacts with C-cadherin and with the APC tumor suppressor protein, but not with alpha-Catenin, that requires the amino-terminal region of beta-Catenin to bind to the complex. ß-Catenin acts as an intracellular signaling molecule. All of the beta-Catenin constructs that contain the Armadillo repeat domain are present in both the soluble cytosolic and the membrane fraction. The Armadillo repeat region also accumulates in the nucleus (Funayama, 1995).

In Xenopus embryos, beta-catenin has been shown to be both necessary and sufficient for the establishment of dorsal cell fates. This signaling activity is thought to depend on the binding of beta-catenin to members of the Lef/Tcf family of transcription factors and the regulation of gene expression by this complex. To test whether beta-catenin must accumulate in nuclei to establish dorsal cell fate, various localization mutants were constructed that restrict beta-catenin to either the plasma membrane, the cytosol, or the nucleus. When overexpressed in Xenopus embryos, the proteins localize as predicted, but surprisingly, all forms induce an ectopic axis, indicative of inducing dorsal cell fates. Given this unexpected result, the membrane-tethered form of beta-catenin became the focus of study to resolve the apparent discrepancy between its membrane localization and the hypothesized role of nuclear beta-catenin in establishing dorsal cell fate. Overexpression of membrane-tethered beta-catenin elevates the level of free endogenous beta-catenin, which subsequently accumulates in nuclei. Consistent with the hypothesis that it is this pool of non-membrane-associated beta-catenin that signals in the presence of membrane-tethered beta-catenin, overexpression of cadherin, which binds free beta-catenin, blocks the axis-inducing activity of membrane- tethered beta-catenin. The mechanism by which ectopic membrane-tethered beta-catenin increases the level of endogenous beta-catenin likely involves competition for the adenomatous polyposis coli (APC) protein, which in other systems has been shown to play a role in degradation of beta-catenin. Consistent with this hypothesis, membrane-tethered beta-catenin coimmunoprecipitates with APC and relocalizes APC to the membrane in cells. Similar results are observed with ectopic plakoglobin, casting doubt on a normal role for plakoglobin in axis specification and indicating that ectopic proteins that interact with APC can artifactually elevate the level of endogenous beta-catenin, likely by interfering with its degradation. These results highlight the difficulty in interpreting the activity of an ectopic protein when it is assayed in a background containing the endogenous protein. Compared with nonphosphorylated beta-catenin, beta-catenin phosphorylated by glycogen synthase kinase-3 preferentially associates with microsomal fractions expressing the cytoplasmic region of N-cadherin (see Drosophila Cadherin-N). These results suggest that protein-protein interactions of beta-catenin can be influenced by its state of phosphorylation, in addition to prior evidence that this phosphorylation modulates the stability of beta-catenin (Miller, 1997).

Establishment of the dorso-ventral axis in Xenopus embryos is presaged by early asymmetries in ß-catenin modulated by the Wnt signaling pathway. ß-catenin displays greater cytoplasmic accumulation on the future dorsal side of the Xenopus embryo by the two-cell stage. This asymmetry persists and increases through early cleavage stages, with ß-catenin accumulating in dorsal but not ventral nuclei by the 16- to 32- cell stages. Steady-state levels and nuclear accumulation of ß-catenin increases in response to ectopic Xenopus Wnt-8 and to the inhibition of glycogen synthase kinase-3. As greater levels and nuclear accumulation of ß-catenin on the future dorsal side of the embryo correlate with the induction of specific dorsal genes, these data suggest that early asymmetries in ß-catenin presage and may specify dorso-ventral differences in gene expression and cell fate. These data further support the hypothesis that these dorso-vental differences in ß-catenin arise in response to the postfertilization activation of a signaling pathway that involves Xenopus glycogen synthase kinase-3. There is reason to believe, from the research literature, that any Xwnt is actually required for axis specification. Only Xwnt-8b is maternally expressed and thus a candidate for an endogenous regulator found in cleaving embryos. Since ectopic expression of a dominant negative Xwnt-8 blocks formation of ectopic but not endogenous axes and since overexpression of Xwnt-5A blocks ectopic axis formation by Xwnt-8 and Xwnt-8b, available data support the conclusion that even if Wnts are involved in endogenous axis formation, they are not strictly required. It is proposed that the dorsal-ventral asymmetry in ß-catenin in embryos before the mid-blastula transition is attributable to the broad synthesis of ß-catenin followed by its being targeted for degradation by Xgsk-3 on the ventral side to a greater extent than on the dorsal side. This is likely a very direct effect since Xgsk-3 directly phosphorylates ß-catenin in vitro and since deletion of the major in vitro phosphorylation site blocks most in vivo phosphorylation and leads to increased accumulation of ß-catenin (Yost, 1996). A prediction of this model is that there are dorsal-ventral differences in Xgsk-3 activities, or differences in other components of the catabolic pathway for ß-catenin. These differences arise in the process of postfertilization cortical rotation. Dorsal-determining information is present in the vegetal pole before cortical rotation. After cortical rotation, this dorsal-determining activity is displaced to the future dorsal side of the embryo. The nature of the oocytic dorsal-determinant has yet to be discovered (Larabell, 1997 and references).

The ßcatenin associated protein adenomatous polyposis coli has signaling activity in Xenopus embryos resulting in the induction of an ectopic dorsoanterior axis. When expressed in the future ventral side of four-cell embryos, APC induces a secondary axis and the induction of the homeobox gene Siamois. This is similar to the phenotype observed for ectopic ß-catenin expression. Axis induction by APC requires the availability of cytosolic ß-catenin. Signaling activity resides in the central domain of the protein, a part of the molecule that is missing in most of the truncating mutations in colon cancer. Signaling by APC in Xenopus is not accompanied by detectable changes in expression levels of ß-catenin, indicating that it has direct positive signaling activity in addition to its role in ß-catenin turnover. From these results it is proposed that APC acts as part of the Wnt/ß-catenin signaling pathway, either upstream of, or in conjunction with, ß-catenin (Vleminckx, 1997).

A component of the wingless pathway has been identified in Xenopus. A maternally expressed Xenopus homolog of the mammalian HMG box factors Tcf-1 and Lef-1 (homologs of Drosophila Pangolin) binds to the N-terminus of ß-catenin, containing the Armadillo repeat region. XTcf-3 is a transcription factor that mediates ß-Catenin-induced axis formation in Xenopus embryos. Microinjection of XTcf-3 mRNA into embryos results in nuclear translocation of ß-catenin. N-terminal deletion of XTcf-3 abrogates the interaction ß-catenin. It is proposed that the ßcatenin-XTcf-3 complex is responsible for activation of targets genes in response to upstream Wnt signals that allow cytoplasmic ß-catenin to interact with XTcf-3 (Molenaar, 1996).

The Xenopus LEF-1/ß-catenin complex, which undergoes nuclear translocation during Wnt signaling, binds to an E-cadherin promoter fragment. In mouse embryos during primitive streak formation, embryonic ectodermal cells, which represent a true epithelial cell layer, give rise to mesoderm. During primitive streak formation, some ectodermal cells lose E-cadherin expression and express LEF-1. From the Xenopus results, it is tempting to speculate that during this process, a complex of LEF-1 and ß-catenin is involved in down-regulating E-cadherin transcription. The observed interaction of LEF-1 with ß-catenin raises the possibility that LEF-1 might be involved in dorsal mesoderm formation. To test for this possibility, LEF-1 mRNA was overexpressed in Xenopus embryos. Overexpression of LEF-1 mRNA causes secondary axis formation, and this effect is enhanced with overexpression of ß-catenin (Huber, 1996).

In a yeast two-hybrid screen, the architectural transcription factor LEF-1 (for lymphoid enhancer-binding factor) interacts with beta-catenin. In mammalian cells, coexpressed LEF-1 and beta-catenin form a complex that is localized to the nucleus and can be detected by immunoprecipitation. LEF-1 and beta-catenin form a ternary complex with DNA that splays an altered DNA bend. Microinjection of LEF-1 into Xenopus embryos induces axis duplication, which is augmented by interaction with beta-catenin. Thus beta-catenin regulates gene expression by direct interaction with transcription factors such as LEF-1, providing a molecular mechanism for the transmission of signals to the nucleus from cell-adhesion components or WNT protein (Behrens, 1996).

The adenomatous polyposis coli gene (APC) is a tumor suppressor gene that is inactivated in most colorectal cancers. Mutations of APC cause aberrant accumulation of beta-catenin, which then binds T cell factor-4 (Tcf-4), causing increased transcriptional activation of unknown genes. The c-MYC oncogene has been identified as a target gene in this signaling pathway. Expression of c-MYC is repressed by wild-type APC and activated by beta-catenin; these effects are mediated through Tcf-4 binding sites in the c-MYC promoter. These results provide a molecular framework for understanding the previously enigmatic overexpression of c-MYC in colorectal cancers (He, 1998).

beta-Catenin is a multifunctional protein involved in cell adhesion and communication. In response to signaling by Wnt growth factors, beta-catenin associates with nuclear TCF factors to activate target genes. A transactivation domain identified at the C-terminus of beta-catenin can stimulate expression of artificial reporter genes. However, the mechanism of target gene activation by TCF/beta-catenin complexes and the physiological relevance of the beta-catenin transactivation domain still remain unclear. It was asked whether the beta-catenin transactivation domain can generate a Wnt-response in a complex biological system, namely axis formation during Xenopus laevis embryogenesis. A chimeric transcription factor consisting of beta-catenin fused to the DNA-binding domain of LEF-1 induces a complete secondary dorsoanterior axis when expressed in Xenopus. A LEF-1-beta-catenin fusion lacking the C-terminal transactivation domain is impaired in signaling while fusion of just the beta-catenin transactivator to the DNA-binding domain of LEF-1 is sufficient for axis-induction. The latter fusion molecule is blocked by dominant negative LEF-1 but not by excess cadherin, indicating that all events parallel or upstream of the transactivation step mediated by beta-catenin are dispensable for Wnt-signaling. Moreover, beta-catenin can be replaced by a heterologous transactivator. Apparently, the ultimate function of beta-catenin in Wnt signaling is to recruit the basal transcription machinery to promoter regions of specific target genes (Vleminckx, 1999).

Wnt signaling in very early embryos leads to a dorsalizing response, which establishes the endogenous dorsal axis. Only a few hours later in development, almost the opposite happens: Xwnt-8 functions to pattern the embryonic mesoderm by promoting ventral and lateral mesoderm. The specificity of the response could conceivably be carried out by differential use of different signal transduction pathways. However this dramatic shift in response to Wnt signaling in early Xenopus is not brought about by differential use of distinct signal transduction pathways. In fact ß-catenin, a downstream component of the canonical Wnt signal transduction pathway, functions not only in the early dorsalizing response but also in the later ventrolateral-promoting response. Interaction of ß-catenin with the XTcf-3 transcription factor is required for the early dorsalizing activity. In contrast, late Wnt signaling in the ventrolateral mesoderm does not require a similar dependency of ß-catenin function on XTcf-3. The most straightforward interpretation of these results is that the role of XTcf-3 is restricted to early dorsalizing Wnt signaling. Consequently, ß-catenin would be expected to function via an XTcf-3-independent nuclear mechanism to promote ventrolateral mesoderm. Different transcription factors might therefore interact with ß-catenin at late blastula and gastrula stages to accomplish ventrolateral-promoting Wnt signaling. These results highlight the potential versatility of the canonical Wnt pathway to interact with tissue-specific factors downstream of ß-catenin, in order to achieve tissue-specific effects (Hamilton, 2001).

Knowledge of when and where signaling pathways are activated is crucial for understanding embryonic development. This study systematically analyzes and compares the signaling pattern of four major pathways by localization of the activated key components ß-catenin (Wnt proteins), MAPK (tyrosine kinase receptors/FGF), Smad1 (BMP proteins) and Smad2 (Nodal/activin/Vg1). The distribution of these components has been determined at 18 consecutive stages in Xenopus development, from early blastula to tailbud stages. The image obtained is that of very dynamic and widespread activities, with very few inactive regions. Signaling fields can vary from large gradients to restricted areas with sharp borders. They do not respect tissue boundaries. This direct visualization of active signaling verifies several predictions inferred from previous functional data. It also reveals unexpected signal patterns, pointing to some poorly understood aspects of early development. In several instances, the patterns strikingly overlap, suggesting extensive interplay between the various pathways. To test this possibility, maternal ß-catenin signaling has been manipulated and the effect on the other pathways in the blastula embryo has been determined. The patterns of P-MAPK, P-Smad1 and P-Smad2 are indeed strongly dependent on ß-catenin at this stage: their dorsal accumulation is absent in UV-irradiated embryos. The highest levels are then found symmetrically in the vegetal-equatorial region, similar to ß-catenin. Upon LiCl treatment, P-MAPK and P-Smad2 are strongly activated also in the ventral side. A similar activation is observed at the site of ß-catenin overexpression. Despite extensive colocalization, P-MAPK and P-Smad2 activation appear, nevertheless, spatially more restricted than ß-catenin: in all conditions, high P-MAPK is limited to a broad equatorial ring, while P-Smad2 activation is most prominent in the vegetal hemisphere. These differences obviously reflect the differential distribution of other determinants, which limit activation of P-MAPK to the marginal zone and Smad2 to the vegetal pole. P-Smad1 has an opposite polarity, i.e. weakest in the dorsal animal region. In UV-irradiated embryos, P-Smad1 is also activated on the dorsal side. LiCl treatment or ventral ß-catenin overexpression causes a significant decrease in the ventral side. In conclusion, these data show that maternal ß-catenin signaling is an important factor in controlling intensity and pattern of the other pathways at blastula stages. While other parameters regulate the latitude of the activation fields, ß-catenin can entirely account for the dorsoventral polarity. Mechanistically, ß-catenin probably contributes to Smad2 activation by stimulating Xnrs expression. How ß-catenin controls MAPK and Smad1 remains to be investigated (Schohl, 2002).

Dorsal axis formation in Xenopus embryos is dependent upon asymmetrical localization of β-catenin, a transducer of the canonical Wnt signaling pathway. Recent biochemical experiments have implicated protein kinase CK2 as a regulator of members of the Wnt pathway including β-catenin. The role of CK2 in dorsal axis formation was examined. CK2 was present in the developing embryo at an appropriate time and place to participate in dorsal axis formation. Overexpression of mRNA encoding CK2 in ventral blastomeres was sufficient to induce a complete ectopic axis, mimicking Wnt signaling. A kinase-inactive mutant of CK2α was able to block ectopic axis formation induced by XWnt8 and β-catenin and was capable of suppressing endogenous axis formation when overexpressed dorsally. Taken together, these studies demonstrate that CK2 is a bona fide member of the Wnt pathway and has a critical role in the establishment of the dorsal embryonic axis (Dominguez, 2004).

In conclusion, experiments carried out in this study demonstrate that CK2 plays an essential role in the complex process of dorsal axis determination. The current model of dorsal axis formation proposes that in the fertilized embryo, β-catenin degradation is promoted by GSK3β phosphorylation. When the dorsal determinants move dorsally during cortical rotation, they promote signaling that stabilizes β-catenin. In cells in culture, β-catenin is regulated by a destruction complex that involves kinases, phosphatases, and scaffold proteins. In the Xenopus embryo, it is not know how β-catenin is dorsally up-regulated, since the nature of the dorsal determinants is still unknown. However, recent experiments have proposed that the dorsal determinants might include dsh and GBP, two Wnt transducers implicated in dorsal axis formation. These translocate to the dorsal side of the embryo where GSK3β is down-regulated, β-catenin accumulates, and dsh is phosphorylated. CK2, expressed most highly in the medial part of the embryo where β-catenin up-regulation occurs, may contribute to Wnt signaling by phosphorylating and stabilizing β-catenin itself, perhaps through the major CK2 phosphorylation site at T393 identified in mammalian cells. Alternatively, CK2 may act upon dsh, GBP, or other as yet uncharacterized dorsal determinants. Indeed, CK2 inhibition in mammalian cells leads to dsh instability (Dominguez, 2004).

The lack of CK2 expression in the vegetal part of the embryo is consistent with a model in which the constitutively active CK2 kinase does not act until the physical process of cortical rotation brings the dorsal determinants to the waiting enzyme in the medial portion of the embryo. This model is consistent with vegetal cortex transplantation studies that suggest that it is the combination of the dorsal determinants plus uncharacterized factors in the cytoplasm of the equatorial (medial) part of the embryo that are required for dorsal determination. Future experiments will validate this hypothesis through a more detailed analysis of the signaling pathway and of biochemical studies of functional protein complexes and their localization in the developing embryo (Dominguez, 2004).

Progenitor cells in the central nervous system must leave the cell cycle to become neurons and glia, but the signals that coordinate this transition remain largely unknown. Wnt signaling, acting through Sox2, promotes neural competence in the Xenopus retina by activating proneural gene expression. This study reports that Wnt and Sox2 inhibit neural differentiation through Notch activation. Independently of Sox2, Wnt stimulates retinal progenitor proliferation and this, when combined with the block on differentiation, maintains retinal progenitor fates. Feedback inhibition by Sox2 on Wnt signaling and by the proneural transcription factors on Sox2 mean that each element of the core pathway activates the next element and inhibits the previous one, providing a directional network that ensures retinal cells make the transition from progenitors to neurons and glia (Agathocleous, 2009).

Wnt/β-catenin signaling acting through Sox2 activates proneural gene expression in the frog retina. This study shows that Wnt and Sox2 inhibit proneural action through Notch, thereby blocking neuronal differentiation. In addition, Wnt signaling stimulates proliferation independently of Sox2, maintaining the progenitor fate, while Sox2 pushes retinal progenitors to Müller glial fates. Concurrent activation of Sox2 and the cell cycle can recapitulate the effects of Wnt in maintaining the retinal precursor cell (RPC) fate. Finally, inhibition of Wnt signaling by Sox2, and of Sox2 by the proneural transcription factors, facilitates a transition from proliferation to differentiation, thereby ensuring that progenitors progress forwards to a differentiated state (Agathocleous, 2009).

These results tie together disparate strands in the function of Wnt/β-catenin and Sox2 signaling as investigated in various vertebrate models. Sox2 both sets up neural potential and inhibits terminal neuronal differentiation. The present study shows that Sox2 plays a central role in suppressing retinal neurogenesis downstream of Wnt/β-catenin signaling, but it enhances Müller glial differentiation and does not maintain progenitors. Similarly, Sox2 overexpression increases Müller cells in mouse retinal explants and promotes the in vitro differentiation of neocortical progenitors into astroglial cells. Notch activation by Sox2 may be involved in this gliogenic effect, as activated Notch signaling promotes gliogenesis. Therefore, either the absence of proneural gene expression or the suppression of proneural activity allows retinal progenitors to adopt the glial fate (Agathocleous, 2009).

The Wnt pathway is activated in the peripheral retina near the ciliary marginal zone in other species besides Xenopus. Yet, Wnt activation in the chick causes cells to be blocked in a proneural-negative progenitor state and in the mouse they assume non-neuronal peripheral fates. In chick and mouse, Wnt signaling does not appear to regulate Sox gene expression; however, the suppression of neurogenesis via activation of Wnt/β-catenin is common to the frog, chick and mammalian retina (Agathocleous, 2009).

There is strong evidence for connections between Wnt/β-catenin, SoxB1 and proneural genes in the regulation of neural differentiation in other tissues. In the zebrafish hypothalamus, canonical Wnt signaling, acting via Sox3, is necessary for the expression of proneural and neurogenic genes. LRP mutant mice exhibit dramatic hypoplasia of the developing neocortex owing to a reduction in neurogenesis as well as in proliferation. Similarly, in the adult hippocampus, Wnt activation promotes both neurogenesis and stem cell proliferation in a dissociable manner, which fits with the explanation that Wnt/β-catenin signaling sets up neuronal potential but then suppresses differentiation and maintains progenitor cells (Agathocleous, 2009).

The results suggest two parallel aspects of the progenitor cell fate: the suppression of neuronal differentiation and the maintenance of proliferative ability, controlled by two branches of Wnt signaling, one of which is Sox2 dependent. This model fits with findings in the spinal cord that Wnt activates proliferation, whereas Sox2 does not. The parallel control of differentiation and proliferation might be a more general feature of Wnt signaling; for example, in the developing limb, Wnt/β-catenin signaling and Sox9 interact to couple proliferation and chondrocyte differentiation (Agathocleous, 2009).

If Sox2 is not mediating the proliferative effects of Wnt/β-catenin signaling, other effectors must be involved. Although exogenous Cyclin E1 was able to cooperate with Sox2 in progenitor maintenance, little or no change was detected in Cyclin E1 retinal expression after Wnt signaling perturbations, nor in the expression of other cell cycle activators including Cyclin D1, Cyclin A2, n-Myc and c-Myc, suggesting that these genes might not be transcriptional targets in the frog retina. Perhaps other genes might function as Wnt-dependent effectors of proliferation here, or perhaps proliferation is regulated through post-transcriptional mechanisms or by changing the mode of progenitor division (Agathocleous, 2009).

Müller cells are transcriptionally very similar to neuroepithelial progenitor cells. They can divide after injury or provision of growth factors, at which point they may return to a neuroepithelial-like state, perhaps through a Wnt-dependent mechanism. These and results therefore suggest that a crucial distinction between RPCs and Müller cells is a Wnt-mediated capacity to proliferate (Agathocleous, 2009).

For the progression from a progenitor to a neuronal fate, both Wnt/β-catenin signaling and Sox2 must be switched off, relieving the inhibition of proneural activity and stopping proliferation. The inhibition of Wnt by Sox2 is likely to take place during retinogenesis, as Sox2 injections do not result in early defects in the specification of retinal progenitor identity. This therefore suggests a negative-feedback mechanism of Sox2 on Wnt signaling. Interestingly, mutations in human SOX2 associated with anophthalmia have been mapped to the C-terminal domain, which normally interacts with β-catenin, resulting in an inability of Sox2 to inhibit canonical Wnt signaling in vitro (Agathocleous, 2009).

For neuronal differentiation to proceed, Sox2 must also be switched off to relieve the inhibition of proneural activity. In the Xenopus retina, it was found that the proneural bHLH transcription factor Xath5 induced a dramatic reduction of the Sox2 protein. In the cortex, a serine protease cleaves Sox2 specifically in neuronal but not glial precursors, thus relieving the block on neurogenesis. It will be interesting to see whether in the retina, proneural genes feed back on Sox2 through this mechanism or through transcriptional repression (Agathocleous, 2009).

Wnt, Sox2 and the proneural genes appear to form a modular circuit in which each step activates the subsequent step and is in turn inactivated by it, driving cells towards differentiation, while limiting the ability of an external proliferation signal, such as Wnt, to continue signaling indefinitely. The relative levels of Wnt, Sox2 and proneural genes determine where a cell lies along the pathway from proliferation to differentiation and whether it assumes a progenitor, glial or neuronal fate. Understanding fully the function of each interaction in the cascade must await a more quantitative analysis of the relationship between the participating factors. This mechanism of transition from one cell state to another by the integration of directional interactions and feedback loops resembles that reported in diverse systems; for example, during sporulation of Bacillus subtilis, where a circuit with five basic nodes displays successive hierarchical gene activations, coupled with negative-feedback loops that switch off the previous state. Further investigations will reveal whether general aspects of the mechanism that is described here are at work in other neural and non-neural tissues, and how this directional pathway integrates with other factors that help to coordinate neuronal proliferation and differentiation (Agathocleous, 2009).

beta-Catenin primes organizer gene expression by recruiting a histone H3 arginine 8 methyltransferase, Prmt2

An emerging concept in development is that transcriptional poising presets patterns of gene expression in a manner that reflects a cell's developmental potential. However, it is not known how certain loci are specified in the embryo to establish poised chromatin architecture as the developmental program unfolds. This study found that, in the context of transcriptional quiescence prior to the midblastula transition in Xenopus, dorsal specification by the Wnt/beta-catenin pathway is temporally uncoupled from the onset of dorsal target gene expression, and that beta-catenin establishes poised chromatin architecture at target promoters. beta-catenin recruits the arginine methyltransferase Prmt2 to target promoters, thereby establishing asymmetrically dimethylated H3 arginine 8 (R8). Recruitment of Prmt2 to beta-catenin target genes is necessary and sufficient to establish the dorsal developmental program, indicating that Prmt2-mediated histone H3(R8) methylation plays a critical role downstream of beta-catenin in establishing poised chromatin architecture and marking key organizer genes for later expression (Blythe, 2010).

The targets of the maternal Wnt/beta-catenin pathway demonstrate different latencies between dorsal specification and the onset of gene expression. The preMBT genes xnr5 and xnr6 require additional input from the maternal transcription factor VegT. Thus, VegT could function as a 'release factor' for these genes, recruiting elongation-promoting factors to the xnr5 and xnr6 loci downstream of β-catenin. In contrast, factors that regulate the global activation of the zygotic genome at the MBT could be responsible for the release of the later responding siamois and xnr3 genes. Indeed, transcriptional poising is an attractive mechanism to account for the synchronous activation of large-scale zygotic gene expression at the MBT. While such global activating factors have yet to be identified in Xenopus, in Drosophila, both Smaug and Zelda have been shown to be essential factors for zygotic genome activation. Smaug activity is essential for the establishment of elongating (CTD pSer2) RNA Pol II at the MBT, and could thereby promote 'release' of such preMBT poised loci, albeit indirectly. The mechanism of action for Zelda is unknown, but it binds DNA sequences present in the majority of immediate-early zygotic transcripts. Further investigation is needed to determine the extent of transcriptional poising at immediate-early zygotic loci and the mechanism of action for such global zygotic gene activators at the MBT (Blythe, 2010).

While preMBT Xenopus embryos are transcriptionally competent, several overlapping mechanisms dominantly suppress zygotic gene expression. Interfering with these repressive activities can reveal a suppressed protranscriptional activity. Depleting embryos of the DNA methyltransferase Dnmt1 causes precocious expression of many genes, suggesting that these genes are poised for activation prior to the MBT but are repressed by Dnmt1-mediated DNA methylation. Also, embryos generated from transplantation of transcriptionally active nuclei will display preMBT expression of genes that were active in the original donor cells. This transcriptional memory is linked to chromatin modifications that correlate with active transcription, particularly the incorporation of the histone variant H3.3. These observations demonstrate the competency of preMBT embryos to establish and maintain active-but-repressed chromatin. It is further speculated that transcriptional poising is a major mechanism underlying the activation of the zygotic genome at the MBT (Blythe, 2010).

Wnt pathway in Xenopus: The role of GSK3beta and PKC

The serine/threonine kinase Xgsk-3 and the intracellular protein beta-catenin are necessary for the establishment of the dorsal-ventral axis in Xenopus. Although genetic evidence from Drosophila indicates that Xgsk-3 is upstream of beta-catenin, direct interactions between these proteins have not been demonstrated. Phosphorylation of beta-catenin in vivo requires an in vitro amino-terminal Xgsk-3 phosphorylation site, which is conserved in the Drosophila protein Armadillo. beta-catenin mutants lacking this site are more active in inducing an ectopic axis in Xenopus embryos and are more stable than wild-type beta-catenin in the presence of Xgsk-3 activity, supporting the hypothesis that Xgsk-3 is a negative regulator of beta-catenin, acting through the amino-terminal site. Inhibition of endogenous Xgsk-3 function with a dominant-negative mutant leads to an increase in the steady-state levels of ectopic beta-catenin, indicating that Xgsk-3 functions to destabilize beta-catenin and thus decrease the amount of beta-catenin available for signaling. The levels of endogenous beta-catenin in the nucleus increases in the presence of the dominant-negative Xgsk-3 mutant, suggesting that a role of Xgsk-3 is to regulate the steady-state levels of beta-catenin within specific subcellular compartments (Yost, 1996).

The molecular nature of the primary dorsalizing inducing event in Xenopus is controversial and several secreted factors have been proposed as potential candidates: Wnts, Vg1, Activin and Noggin. However, recent studies have provided new insight into the activity of the dorsalizing region, called the Nieuwkoop Center. Two properties of the Nieuwkoop Center have been used to evaluate the dorsalizing activity of the four secreted factors Wnt8, Vg1, Activin and Noggin: (1) the activity of this dorsalizing center involves an entire signal transduction pathway that requires maternal ß-catenin, and (2) a transcription factor with potent dorsalizing activity, Siamois, is expressed within the Nieuwkoop Center (Fagotto, 1997).

The requirement for ß-catenin was tested by coexpressing a cadherin, which sequesters ß-catenin at the cell membrane and specifically blocks its intracellular signaling activity. Of the four growth factors, only Wnt is sensitive to inhibition of ß-catenin activity and only Wnt can induce Siamois expression. Therefore, Wnt is able to induce a bonafide Nieuwkoop Center, while Vg1, Activin and Noggin probably induce dorsal structures by a different mechanism. GSK acts upstream of ß-catenin, similar to the order of these components in the Wingless pathway in Drosophila. ß-catenin induces expression of Siamois and the free signaling pool of ß-catenin is required for normal expression of endogenous Siamois. It is concluded that the sequence of steps in the signaling pathway is initiated by Wnt, which acts to inhibit GSK. GSK in turn acts to inhibit ß-catenin which acts to activate Siamois (Fagotto, 1997).

Wnt signaling involves inhibition of glycogen synthase kinase-3beta (GSK-3beta) and elevation of cytoplasmic beta-catenin. This pathway is essential during embryonic development and oncogenesis. Previous studies on both Xenopus and mammalian cells indicate that lithium mimics Wnt signaling by inactivating GSK-3beta. Serum enhances accumulation of cytoplasmic beta-catenin induced by lithium in both 293 and C57MG cell lines and growth factors are responsible for this enhancing activity. Growth factors mediate this effect through activation of protein kinase C (PKC), not through Ras or phosphatidylinositol 3-kinase. In addition, Wnt-induced accumulation of cytoplasmic beta-catenin is partially inhibited by PKC inhibitors and by chronic treatment of cells with phorbol ester. Both calphostin C, a PKC inhibitor, and a dominant negative PKC exhibit partial inhibition on Wnt-mediated transcriptional activation. It is proposed that Wnt signaling to beta-catenin consists of two interactive components: one involves inhibition of GSK-3beta and is mimicked by lithium, and the other involves PKC and serves to augment the effects of GSK-3beta inhibition (Chen, 2000).

Interaction of Notch and ß-catenin in Xenopus

The blastula chordin- and noggin-expressing centre (BCNE) is the predecessor of the Spemann-Mangold's organiser and also contains the precursors of the brain. This signalling centre comprises animal-dorsal and marginal-dorsal cells and appears as a consequence of the nuclear accumulation of β-catenin on the dorsal side. This study proposes a role for Notch that was not previously explored during early development in vertebrates. Notch initially destabilises β-catenin in a process that does not depend on its phosphorylation by GSK3. This is important to restrict the BCNE to its normal extent and to control the size of the brain (Acosta, 2011).

Studies in the epithelium of the Drosophila wing disc have identified another endocytic pathway for Notch which requires neither the classical ligands nor cleavage by γ-secretase. Although it depends on structural motifs present in the intracellular domain of Notch, this pathway does not involve CSL-mediated transcription. Immunoprecipitation assays demonstrated that Notch and Arm/β-catenin are associated in the same protein complex. Notch associates near the adherens junctions with hypophosphorylated Arm/β-catenin, which has escaped tagging by GSK3 for ubiquitin-proteasome degradation. Because this complex enters endosomal trafficking and becomes degraded, this non-canonical, non-transcriptional function of Notch would be important to buffer activated Arm/β-catenin in order to keep low levels of spontaneous Wnt activity in the system. This antagonistic interaction of Notch and Wnt was proposed as an example of how biological systems decrease their own noise. This is in agreement with the results shown in this study during early Xenopus development (Acosta, 2011).

ß-catenin and early mammalian development

The oocyte to embryo transition in metazoans depends on maternal proteins and transcripts to ensure the successful initiation of development, and the correct and timely activation of the embryonic genome. The maternal gene encoding the cell adhesion molecule E-cadherin was conditionally eliminated and the ß-catenin gene was partially eliminated from the mouse oocyte. Oocytes lacking E-cadherin, or expressing a truncated allele of ß-catenin without the N-terminal part of the protein, give rise to embryos whose blastomeres do not adhere. Blastomere adhesion is restored after translation of protein from the wild-type paternal alleles: at the morula stage in embryos lacking maternal E-cadherin, and at the late four-cell stage in embryos expressing truncated ß-catenin. This suggests that adhesion per se is not essential in the early cleavage stage embryos; that embryos develop normally if compaction does not occur until the morula stage, and that the zona pellucida suffices to maintain blastomere proximity. Although maternal E-cadherin is not essential for the completion of the oocyte-to-embryo transition, absence of wild-type ß-catenin in oocytes does statistically compromise developmental success rates. This developmental deficit is alleviated by the simultaneous absence of maternal E-cadherin, suggesting that E-cadherin regulates nuclear ß-catenin availability during embryonic genome activation (de Vries, 2004).

Culture of embryonic stem (ES) cells at high density inhibits both ß-catenin signaling and neural differentiation. ES cell density does not influence ß-catenin expression, but a greater proportion of ß-catenin is targeted for degradation in high-density cultures. Moreover, in high-density cultures, ß-catenin is preferentially localized to the membrane further reducing ß-catenin signaling. Increasing ß-catenin signaling by treatment with Wnt3a-conditioned medium, by overexpression of ß-catenin, or by overexpression of a dominant-negative form of E-cadherin promotes neurogenesis. Furthermore, ß-catenin signaling is sufficient to induce neurogenesis in high-density cultures even in the absence of retinoic acid (RA), although RA potentiates the effects of ß-catenin. By contrast, RA does not induce neurogenesis in high-density cultures in the absence of ß-catenin signaling. Truncation of the armadillo domain of ß-catenin, but not the C terminus or the N terminus, eliminates its proneural effects. The proneural effects of ß-catenin reflect enhanced lineage commitment rather than proliferation of neural progenitor cells. Neurons induced by ß-catenin overexpression either alone or in association with RA express the caudal neuronal marker Hoxc4. However, RA treatment inhibits the ß-catenin-mediated generation of tyrosine hydroxylase-positive neurons, suggesting that not all of the effects of RA are dependent upon ß-catenin signaling. These observations suggest that ß-catenin signaling promotes neural lineage commitment by ES cells, and that ß-catenin signaling may be a necessary co-factor for RA-mediated neuronal differentiation. Further, enhancement of ß-catenin signaling with RA treatment significantly increases the numbers of neurons generated from ES cells, thus suggesting a method for obtaining large numbers of neural species for possible use in ES cell transplantation (Otero, 2004).

Many components of the Wnt/ ß-catenin signaling pathway are expressed during mouse pre-implantation embryo development, suggesting that this pathway may control cell proliferation and differentiation at this time. No evidence is found for a functional activity of this pathway in cleavage-stage embryos using the Wnt-reporter line, BAT-gal. To further probe the activity of this pathway, ß-catenin signaling was activated by mating a zona pellucida3-cre (Zp3-cre) transgenic mouse line with a mouse line containing an exon3-floxed ß-catenin allele. The result is expression of a stabilized form of ß-catenin, resistant to degradation by the GSK3 ß-mediated proteasome pathway, expressed in the developing oocyte and in each cell of the resulting embryos. Nuclear localization and signaling function of ß-catenin were not observed in cleavage-stage embryos derived from these oocytes. These results indicate that in pre-implantation embryos, molecular mechanisms independent of the GSK3 ß-mediated ubiquitination and proteasome degradation pathway inhibit the nuclear function of ß-catenin. Although the mutant blastocysts initially developed normally, they then exhibited a specific phenotype in the embryonic ectoderm layer of early post-implantation embryos. A nuclear function of ß-catenin in the mutant epiblast is shown that leads to activation of Wnt/ ß-catenin target genes. As a consequence, cells of the embryonic ectoderm change their fate, resulting in a premature epithelial-mesenchymal transition (Kemler, 2004).

Differential gene regulation integrated in time and space drives developmental programs during embryogenesis. To understand how the program of gastrulation is regulated by Wnt/ß-catenin signaling, genome-wide expression profiling of conditional ß-catenin mutant embryos was performed. Known Wnt/ß-catenin target genes, known components of other signaling pathways, as well as a number of uncharacterized genes were downregulated in these mutants. To further narrow down the set of differentially expressed genes, whole-mount in situ screening was used to associate gene expression with putative domains of Wnt activity. Several potential novel target genes were identified by this means and two, Grsf1 and Fragilis2, were functionally analyzed by RNA interference (RNAi) in completely embryonic stem (ES) cell-derived embryos. The gene encoding the RNA-binding factor Grsf1 is important for axial elongation, mid/hindbrain development and axial mesoderm specification, and Fragilis2, encoding a transmembrane protein, regulates epithelialization of the somites and paraxial mesoderm formation. Intriguingly, the knock-down phenotypes recapitulate several aspects of Wnt pathway mutants, suggesting that these genes are components of the downstream Wnt response. This functional genomic approach allows the rapid identification of functionally important components of embryonic development from large datasets of putative targets (Lickert, 2005).

The observed Grsf1 knock-down phenotypes remarkably recapitulate distinct aspects of the CKO mutant phenotype and other Wnt pathway mutants, suggesting that Grsf1 is a crucial mediator of the Wnt/ß-catenin signaling cascade. The lack of T expression in the anterior primitive streak of Grsf1 knock-down embryos is comparable to lack of T expression in Wnt3a mutants, offering an explanation for the axis truncation in both mutants. The normal expression of the Wnt/ß-catenin target genes, Cdx1 and Grsf1, in Grsf1 knock-down embryos suggests that Grsf1 acts downstream of the Wnt/ß-catenin signaling pathway selectively on target mRNAs and is not involved in signal transduction, e.g., by stabilizing components of the pathway. This might also be the case for mid/hindbrain development, where Grsf1 is necessary for maintaining Fgf8 and Gbx2 expression, two factors important for the establishment of the mid/hindbrain boundary. The comparison of putative mRNA targets of the RNA-binding factor Grsf1 with all the deregulated genes from the ß-catenin target gene screen revealed several potentially coregulated transcripts, which might explain similarities in the Grsf1 and CKO mutant phenotypes (Lickert, 2005).

Fragilis2 is expressed in the primitive streak, including the base of the allantois, where the PGCs are localized at late gastrulation stage, and in the paraxial and lateral mesoderm, as well as in the first forming somites at E8.5. Studies in the immune system suggest a role for Fragilis2 (human orthologs Leu13/9-27/IFITM1) as part of a transmembrane multiprotein signaling complex implicated in inhibition of cell proliferation and homotypic cell adhesion. Histological analysis of Fragilis2-silenced embryos revealed a defect in epithelialization of the somites, consistent with a function in homotypic cell adhesion. Additionally, marker gene analysis revealed that Fragilis2 knock-down embryos show reduced expression of PAPC, a gene implicated in somite epithelialization, and reduced expression of the paraxial mesoderm markers T and Tbx6 at tailbud stage. These phenotypes are very similar to the paraxial mesoderm and somite segmentation defects seen in several different Wnt mutants, thus it seems likely that Fragilis2 is a crucial downstream mediator of the Wnt/ß-catenin signaling cascade in these processes, mediating homotypic cell adhesion (Lickert, 2005).

Wnt signaling has been implicated in stem cell (SC) biology, but little is known about how stabilized ß-catenin functions within native SC niches. This was addressed by defining the impact of ß-catenin stabilization on maintenance, proliferation, and lineage commitment of multipotent follicle SCs when in their native niche and in culture. Gain of function mutations and inducible loss of function mutations were employed to demonstrate that ß-catenin stabilization is essential for promoting the transition between SC quiescence and conversion to proliferating transit amplifying (TA) progeny. Purified SCs isolated directly from wild-type and elevated ß-catenin follicles were transcriptionally profiled in both resting and activated states to uncover the discrete set of genes whose expression in native SCs is dependent upon ß-catenin stabilization. Finally, the underlying mechanism was addressed; in the SC niche, Wnt signaling and ß-catenin stabilization transiently activates Lef1/Tcf complexes and promote their binding to target genes that promote TA cell conversion and proliferation to form the activated cells of the newly developing hair follicle. These changes precede subsequent Wnt signals that impact on the TA progeny to specify the differentiation lineages of the follicle (Lowry, 2005).

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).

Modulation of β-catenin function maintains mouse epiblast stem cell and human embryonic stem cell self-renewal

Wnt/beta-catenin signalling has a variety of roles in regulating stem cell fates. Its specific role in mouse epiblast stem cell self-renewal, however, remains poorly understood. This study shows that Wnt/β-catenin functions in both self-renewal and differentiation in mouse epiblast stem cells. Stabilization and nuclear translocation of β-catenin and its subsequent binding to T-cell factors induces differentiation. Conversely, retention of stabilized β-catenin in the cytoplasm maintains self-renewal. Cytoplasmic retention of β-catenin is effected by stabilization of Axin2, a downstream target of β-catenin, or by genetic modifications to β-catenin that prevent its nuclear translocation. This study also found that human embryonic stem cell and mouse epiblast stem cell fates are regulated by β-catenin through similar mechanisms. The results elucidate a new role for β-catenin in stem cell self-renewal that is independent of its transcriptional activity and will have broad implications in understanding the molecular regulation of stem cell fate (Kim, 2013).

This study demonstrates that Wnt/β-catenin signalling can promote self-renewal or differentiation of mouse EpiSCs and human ESCs. The retention of stabilized β-catenin in the cytoplasm maintains mouse EpiSC and human ESC self-renewal, whereas nuclear translocation of β-catenin and subsequent binding to TCFs induces differentiation. The finding that cytoplasmic and nuclear β-catenin pools are both involved in regulating cell fates might provide a rational explanation for some of the diverse and sometimes opposite effects of Wnt/β-catenin observed in different contexts. More importantly, this study reveals a new functional avenue of the canonical Wnt/β-catenin pathway, which current dogma depicts as being functionally defined by nuclear translocation of β-catenin and its subsequent binding to TCFs (Kim, 2013).

The gene regulatory effects of Wnt/β-catenin signalling are initiated upon the binding of β-catenin to TCFs in the nucleus. So how is cytoplasmic β-catenin involved in regulating cell fates? One possible model of regulation is suggested by the interaction of cytoplasmic β-catenin with cadherin, α-catenin and actin filaments, as these interactions have been shown to have multiple and important roles in regulating cellular organization, cell adhesion and signal transduction from cell surface to the nucleus. Another possibility is that cytoplasmic β-catenin might hold negative regulators of self-renewal in the cytoplasm, preventing them from entering the nucleus and activating or suppressing transcription of their target genes. In this scenario, cytoplasmic β-catenin would promote stem cell self-renewal by alleviating the self-renewal suppression effect of these negative regulators. This might be the case in mouse EpiSCs and human ESCs in which the persistence of β-catenin in the cytoplasm is associated with self-renewal (Kim, 2013).

The role of β-catenin in human ESC self-renewal has been controversial. It has been suggested that activation of β-catenin by Wnt ligands or GSK3 inhibitors can promote human ESC self-renewal. Other studies showed that Wnt/β-catenin signalling is dispensable for human ESC self-renewal, and that its activation predominantly induces differentiation. The finding that activation of β-catenin can promote human ESC self-renewal or differentiation, and that the respective outcome is dictated by whether β-catenin translocates into the nucleus, provides a rational explanation for earlier, seemingly paradoxical results. This study also found that FGF2 and CHIR/IWR-1 act synergistically to promote human ESC self-renewal. This is noteworthy because in mouse ESCs, LIF and CHIR/PD can also independently promote self-renewal, yet there is a synergistic effect when the two are combined. Understanding how these different pathways work independently or synergistically to maintain stem cell self-renewal will advance efforts to better control stem cell fate, which is critical to the future of regenerative medicine (Kim, 2013).

ß-catenin and the non-canonical WNT pathway in mammals

The Wnt signalling pathway (see Habas and Dawid Dishevelled and Wnt signaling: is the nucleus the final frontier?) regulates many developmental processes through a complex of beta-catenin and the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of high-mobility-group transcription factors. Wnt stabilizes cytosolic beta-catenin, which then binds to TCF and activates gene transcription. This signalling cascade is conserved in vertebrates, Drosophila and C. elegans. In C. elegans, the proteins MOM-4 and LIT-1 regulate Wnt signalling to polarize responding cells during embryogenesis. MOM-4 and LIT-1 are homologous to TAK1 (a kinase activated by transforming growth factor-beta) mitogen-activated protein-kinase-kinase kinase (MAP3K) and MAP kinase (MAPK)-related NEMO-like kinase (NLK), respectively, in mammalian cells. These results raise the possibility that TAK1 and NLK are also involved in Wnt signalling in mammalian cells. This study shows that TAK1 activation stimulates NLK activity and downregulates transcriptional activation mediated by beta-catenin and TCF. Injection of NLK suppresses the induction of axis duplication by microinjected beta-catenin in Xenopus embryos. NLK phosphorylates TCF/LEF factors and inhibits the interaction of the beta-catenin-TCF complex with DNA. Thus, the TAK1-NLK-MAPK-like pathway negatively regulates the Wnt signalling pathway (Ishitani, 1999).

The Wnt/beta-catenin signaling pathway regulates many developmental processes by modulating gene expression. Wnt signaling induces the stabilization of cytosolic beta-catenin, which then associates with lymphoid enhancer factor and T-cell factor (LEF-1/TCF) to form a transcription complex that activates Wnt target genes. A specific mitogen-activated protein (MAP) kinase pathway involving the MAP kinase kinase kinase TAK1 and MAP kinase-related Nemo-like kinase (NLK) suppresses Wnt signaling. This study investigated the relationships among NLK, beta-catenin, and LEF-1/TCF. It was found that NLK interacts directly with LEF-1/TCF and indirectly with beta-catenin via LEF-1/TCF to form a complex. NLK phosphorylates LEF-1/TCF on two serine/threonine residues located in its central region. Mutation of both residues to alanine enhanced LEF-1 transcriptional activity and rendered it resistant to inhibition by NLK. Phosphorylation of TCF-4 by NLK inhibited DNA binding by the beta-catenin-TCF-4 complex. However, this inhibition was abrogated when a mutant form of TCF-4 was used in which both threonines were replaced with valines. These results suggest that NLK phosphorylation on these sites contributes to the down-regulation of LEF-1/TCF transcriptional activity (Ishitani, 2003b; full text of article).

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