Wnt pathway activation by ADP-ribosylation: APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation

Wnt/β-catenin signal transduction directs metazoan development and is deregulated in numerous human congenital disorders and cancers. In the absence of Wnt stimulation, a multi-protein 'destruction complex', assembled by the scaffold protein Axin, targets the key transcriptional activator β-catenin for proteolysis. Axin is maintained at very low levels that limit destruction complex activity, a property that is currently being exploited in the development of novel therapeutics for Wnt-driven cancers. This study used an in vivo approach in Drosophila to determine how tightly basal Axin levels must be controlled for Wnt/Wingless pathway activation, and how Axin stability is regulated. For nearly all Wingless-driven developmental processes, a three- to four-fold increase in Axin was found to be insufficient to inhibit signaling, setting a lower-limit for the threshold level of Axin in the majority of in vivo contexts. Further, both the tumor suppressor Adenomatous polyposis coli (APC) and the ADP-ribose polymerase Tankyrase (Tnks) were found to have evolutionarily conserved roles in maintaining basal Axin levels below this in vivo threshold, and separable domains were defined in Axin that are important for APC- or Tnks-dependent destabilization. Together, these findings reveal that both APC and Tnks maintain basal Axin levels below a critical in vivo threshold to promote robust pathway activation following Wnt stimulation (Yang, 2016).

The Wnt/β-catenin signal transduction pathway directs fundamental processes during metazoan development and tissue homeostasis, whereas deregulation of Wnt signalling underlies numerous congenital disorders and carcinomas. Two multimeric protein complexes with opposing functions -- the cytoplasmic destruction complex and the plasma membrane-associated signalosome -- control the stability of the transcriptional co-factor β-catenin to coordinate the state of Wnt pathway activation. In the absence of Wnt stimulation, β-catenin is targeted for proteasomal degradation by the destruction complex, which includes the two tumour suppressors: Axin and Adenomatous polyposis coli (APC), and two kinases: casein kinase α (CK1α) and glycogen synthase kinase 3 (GSK3). Engagement of Wnt with its transmembrane receptors, Frizzled and low-density lipoprotein receptor-related protein 5/6 (herein LRP6), induces rapid LRP6 phosphorylation, recruitment of Axin to phospho-LRP6, and assembly of the signalosome, which includes two other Axin-associated components, GSK3 and Dishevelled (Dvl). Signalosome assembly results in the inhibition of β-catenin proteolysis; consequently stabilized β-catenin promotes the transcriptional regulation of Wnt pathway target genes (Yang, 2016).

As a key component in both the destruction complex and the signalosome, Axin is tightly regulated. Under basal conditions, Axin is maintained at very low levels, and serves as the concentration-limiting scaffold for assembly of the destruction complex. Following Wnt exposure, the rapid association of phospho-Axin with phospho-LRP6 triggers Axin dephosphorylation, inducing a conformational change that inhibits Axin's interaction with both the destruction and signalosome complexes. Axin is subsequently degraded; however, Axin proteolysis occurs several hours after Wnt exposure, and thus does not regulate Axin's essential role during the initial activation of the Wnt pathway (Yang, 2016).

The mechanisms that rapidly reprogram Axin from inhibitory to stimulatory roles following Wnt exposure remain uncertain. In current models, Wnt stimulation induces Axin's dissociation from the destruction complex, thereby promoting its interaction with the signalosome. As Wnt stimulation induces Axin dephosphorylation, decreased phosphorylation was postulated to facilitate the dissociation of Axin from the destruction complex; however, recent work revealed that the interaction of Axin with LRP6 precedes Axin dephosphorylation, and that dephosphorylation serves to inhibit, rather than enhance this interaction (Kim, 2013) Furthermore, some findings have challenged prevailing models, providing evidence that Axin's interaction with the destruction complex is not diminished upon Wnt stimulation. Thus, whereas the rapid switch in Axin function following Wnt stimulation is essential for the activation of signalling, the underlying mechanisms remain uncertain (Yang, 2016).

During investigation of this critical process, an unanticipated role was discovered for the ADP-ribose polymerase Tankyrase (Tnks) in the reprogramming of Axin activity following Wnt exposure. As Tnks-mediated ADP-ribosylation is known to target Axin for proteolysis, small molecule Tnks inhibitors have become lead candidates for development in the therapeutic targeting of Wnt-driven cancers. This study identified a novel mechanism through which Tnks regulates Axin: by promoting Axin's central role in rapid Wnt pathway activation. Wnt stimulation was found to modulate Axin levels biphasically in both Drosophila and human cells. Unexpectedly, Axin is rapidly stabilized following Wnt stimulation, before its ultimate proteolysis hours later. In an evolutionarily conserved process, the ADP-ribosylated pool of Axin is preferentially increased immediately following Wnt exposure. ADP-ribosylation enhances Axin's association with phospho-LRP6, providing a mechanistic basis for the rapid switch in Axin function following Wnt stimulation. These results thus indicate that Tnks inhibition not only increases basal Axin levels, but also impedes the Wnt-dependent interaction between Axin and LRP6, suggesting a basis for the potency of Tnks inhibitors in Wnt-driven cancers. Thus, Tnks not only targets Axin for proteolysis independently of Wnt stimulation, but also promotes Axin's central role in Wnt pathway activation, which may be relevant to the context-dependent activation of Wnt signalling and the treatment of Wnt-driven cancers with Tnks inhibitors (Yang, 2016).

Wnt exposure induces biphasic regulation in the level of Axin, and a large increase in the level of ADP-ribosylated Axin immediately after stimulation. ADP-ribosylation enhances the interaction of Axin with phospho-LRP6, and promotes the activation of Wnt signalling. These findings lead to three major revisions of the current model for the role of Tnks in the activation of the Wnt pathway. First, Tnks serves bifunctional roles under basal conditions and after stimulation, revealing a remarkable economy and coordination of pathway components. Second, the results provide a mechanistic basis for the rapid reprogramming of Axin function in response to Wnt stimulation, and thereby reveal an unanticipated role for Tnks in this process. These findings suggest that Wnt exposure either rapidly increases the ADP-ribosylation of Axin or inhibits the targeting of ADP-ribosylated Axin for proteasomal degradation, through mechanisms yet to be elucidated. Finally, pharmacologic inactivation of Tnks was shown to diminish the interaction of Axin with LRP6, revealing a previously unknown mechanism through which small molecule Tnks inhibitors disrupt Wnt signalling, distinct from their known role in stabilizing the destruction complex inhibitors (Yang, 2016).

In the absence of Wnt stimulation, the concentration-limiting levels of Axin regulate its scaffold function in the destruction complex. As components of the destruction complex participate in other signalling pathways, the low levels of Axin were proposed to maintain modularity of the Wnt pathway. The new findings indicate that Axin levels are not only regulated in the absence of Wnt, but also regulated biphasically following Wnt stimulation. This sequential modulation of Axin divides activation of the pathway into an early, fast phase and a delayed long-term phase. During embryogenesis, the earliest expression of Wg triggers the rapid appearance of Axin in segmental stripes, which is a novel hallmark for the initial activation of the pathway. The findings reveal that Wnt exposure induces a rapid increase in the total level of Axin, and importantly, a preferential increase in the level of the ADP-ribosylated Axin. The early Axin stripes are absent in Tnks null mutant embryos and are also absent when the Tnks binding domain in Axin is deleted. Therefore, it is proposed that Axin ADP-ribosylation contributes to Axin stabilization and to the rapid response to Wg stimulation (Yang, 2016).

It is postulated that the initial increase in levels of ADP-ribosylated Axin jump-starts the response to Wnt stimulation by enhancing the Axin-LRP6 interaction, whereas the subsequent decrease in Axin levels prolongs the duration of signalling by reducing destruction complex assembly. Thus, Wnt stimulation induces rapid increases in the levels of not only cytoplasmic β-catenin, but also ADP-ribosylated Axin. Previous work that coupled mathematical modelling with experimental analysis revealed that several Wnt signalling systems were responsive to the relative change in β-catenin levels, rather than their absolute value. This dependence was proposed to impart robustness and resistance to noise and cellular variation. The current data raise the possibility that a similar principle applies to changes in Axin levels on the Axin-LRP6 interaction, as the marked increase in ADP-ribosylated Axin levels following Wnt stimulation is evolutionarily conserved. Thus, the relative change in levels of ADP-ribosylated Axin may promote signalling following Wnt exposure by facilitating the fold change in β-catenin levels (Yang, 2016).

The current findings have relevance for the context-specific in vivo roles of Tnks in Wnt signalling suggested in previous studies. Tnks inhibition disrupts Wnt signalling in a number of cultured cell lines, but in vivo studies in several model organisms suggested that the requirement for Tnks in promoting Wnt signalling is restricted to specific cell types or developmental stages. In mice, functional redundancy exists between the two Tnks homologues, such that Tnks single mutants are viable and fertile, whereas double mutants display embryonic lethality without overt Wnt-related phenotypes. However, a missense mutation in the TBD of Axin2 that is predicted to disrupt ADP-ribosylation resulted in either activating or inhibiting effects on Wnt signalling that were dependent on developmental stage. Tnks inhibitors resulted in the same paradoxical effects, suggesting complex roles in mouse embryonic development. Analogously, treatment of fish with Tnks inhibitors resulted in no observed defects in Wnt-mediated processes during development; however, the regeneration of injured fins in adults, a process that requires Wnt signalling, was disrupted (Yang, 2016).

Similarly, the finding that Drosophila Tnks null mutants are viable (Wang, 2016a; Wang, 2016b; Feng, 2014) was unexpected, as Tnks is highly evolutionarily conserved, and no other Tnks homologues exist in fly genomes. Nonetheless, the current studies reveal that a less than twofold increase in Axin levels uncovers the importance of Tnks in promoting Wg signalling during embryogenesis. Therefore, it is postulated that Tnks loss can be compensated during development unless Axin levels are increased, but that the inhibition of Wg signalling resulting from Tnks inactivation cannot be attributed solely to increased Axin levels. Furthermore, Drosophila Tnks is essential for Wg target gene activation in the adult intestine, and exclusively within regions of the gradient where Wg is present at relatively low concentration. Thus, the context-specific roles of Tnks observed in different model organisms may reflect the mechanisms described herein, which reveal that the Wnt-induced association of Axin with LRP6 occurs even in the absence of Axin ADP-ribosylation, but is markedly enhanced in its presence. It is postulated that by enhancing this interaction, Tnks-dependent ADP-ribosylation of Axin serves to amplify the initial response to Wnt stimulation, and thus is essential in a subset of in vivo contexts (Yang, 2016).

The recent discovery that Tnks enhances signalling in Wnt-driven cancers has raised the possibility that Tnks inhibitors will offer a promising new therapeutic option. Indeed, preclinical studies have supported this possibility. Tnks inhibitors were thought previously to disrupt Wnt signalling solely by increasing the basal levels of Axin, and thus by increasing destruction complex activity. However, the current findings indicate that the degree to which the basal level of Axin increases following Tnks inactivation is not sufficient to disrupt Wnt signalling in some in vivo contexts. Instead, the results reveal that Tnks inhibition simultaneously disrupts signalling at two critical and functionally distinct steps: by promoting activity of the destruction complex and by diminishing an important step in signalosome assembly: the Wnt-induced interaction between LRP6 and Axin. On the basis of these findings, it is proposed that the efficacy of Tnks inhibitors results from their combined action at both of these steps, providing a rationale for their use in the treatment of a broad range of Wnt-driven cancers. Therefore, these results suggest that in contrast with the current focus on tumours in which attenuation of the destruction complex aberrantly activates Wnt signalling (such as those lacking APC), the preclinical testing of Tnks inhibitors could be expanded to include cancers that are dependent on pathway activation by Wnt stimulation. These include the colorectal, gastric, ovarian and pancreatic cancers that harbour inactivating mutations in RNF43, a negative Wnt feedback regulator that promotes degradation of the Wnt co-receptors Frizzled and LRP6 (Yang, 2016).

Protein Interactions

An examination was performed to see whether Drosophila Axin (Axn) produced by in vitro translation could interact with fragments of Armadillo fused to glutathione S-transferase (GST). Axn specifically interacted with the Armadillo repeat domain of Arm (amino acids 140 to 713), but not with its NH2-terminal (amino acids 1 to 139) or its COOH-terminal (amino acids 714 to 843) domain. Pull-down assays with a series of deletion fragments of Axn showed that a fragment of Axn containing amino acids 459 to 538 binds to Arm. This region corresponds to the beta-catenin-binding domain of Axin and conductin/Axil and contains a small segment (amino acids 494 to 525) that is highly conserved, suggesting that it may function in binding to Arm (Hamada, 1999).

Mammalian Axin family members interact not only with beta-catenin but also with GSK-3beta and APC. In line with these findings, it has been found that the RGS domain of Axn interacts with a fragment of D-APC (amino acids 757 to 1270). This region of D-APC corresponds to the region of APC that interacts with beta-catenin, conductin, and Axin. However, Axn does not bind to Zeste white-3/Shaggy (ZW3/Sgg; Drosophila GSK-3beta) (Hamada, 1999).

Immunoprecipitation of either Arm or Zw3 co-precipitates the endogenous Axn protein from embryo lysates. Immunoprecipitation of Axn also precipitates Zw3 protein; however, no Arm could be detected in Axn immunoprecipitates from embryo lysates. To confirm that Arm co-immunoprecipitates with Axn, Axn was overexpressed under the control of the heat-shock promoter in Schneider 2 cells. Upon induction of the Axn transgene, significantly more Zw3 and Arm co-immunoprecipitate with Axn, thus demonstrating that Axn forms a complex or complexes with Zw3 and Arm (Willert, 1999b).

The protein-serine kinase Shaggy(Zeste-white3) [Sgg(Zw3)] is the Drosophila homolog of mammalian glycogen synthase kinase-3 and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. Sgg(Zw3) is a putative component of the Wingless (Wg) pathway; epistasis analyses suggest that Sgg(Zw3) function is repressed by Wg signaling. The biochemical consequences of Wg signaling with respect to the Sgg(Zw3) protein kinase has been investigated in two types of Drosophila cell lines and in embryos. Sgg(Zw3) activity is inhibited following exposure of cells to Wg protein and by expression of downstream components of Wg signaling, Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation of Sgg(Zw3) is accompanied by serine phosphorylation. The level of Sgg(Zw3) activity regulates the stability of Armadillo protein and modulates the level of phosphorylation of Drosophila Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and provide a model for dissecting the molecular interactions between the signaling proteins (Ruel, 1999).

Drosophila Armadillo plays two distinct roles during development. It is a component of adherens junctions, and functions as a transcriptional activator in response to Wingless signaling. In the current model, Wingless signal causes stabilization of cytoplasmic Armadillo allowing it to enter the nucleus where it can activate transcription. However, the mechanism of nuclear import and export remains to be elucidated. Two gain-of-function alleles of Armadillo are shown to activate Wingless signaling by different mechanisms. The S10 allele localizes to the nucleus, where it activates transcription. In contrast, the DeltaArm allele localizes to the plasma membrane, and forces endogenous Arm into the nucleus. Therefore, DeltaArm is dependent on the presence of a functional endogenous allele of arm to activate transcription. DeltaArm may function by titrating Axin protein to the membrane, suggesting that Axin acts as a cytoplasmic anchor keeping Arm out of the nucleus. In axin mutants, Arm is localized to the nuclei. Nuclear retention is dependent on dTCF/Pangolin. This suggests that cellular distribution of Arm is controlled by an anchoring system, where various nuclear and cytoplasmic binding partners determine its localization (Tolwinski, 2001).

Evidence is provided for the titration model, but focus is on potential cytoplasmic anchors that retain ß-catenin/Arm in the cytoplasm. Endogenous Arm accumulates in the nucleus in response to expression of DeltaArm, and the underlying mechanism appears to be independent of protein levels. DeltaArm functions downstream of zw3, and does not increase endogenous protein levels appreciably. These results point to a mechanism by which DeltaArm affects some component of the cytoplasmic retention machinery. axin may be this component, since its mutation leads to nuclear Arm accumulation, and its overexpression prevents it. Axin appears to be amenable to a titration model, because its function is highly dose dependent. Only maternal mutation of axin leads to a naked cuticle with a partial rescue by a paternal copy. Zygotic mutation doesn’t produce an embryonic phenotype. Overexpression leads to a wg phenotype only if expressed very early. Observations in tissue culture show that Axin is localized to the cytoplasmic membrane and the cytoplasm, but is excluded from the nucleus. Also, mutant forms of Arm lacking repeats that are required for Axin binding localize to the nucleus. Therefore, a model is favored in which DeltaArm directly titrates out Axin, leading to nuclear localization of endogenous Arm. DeltaArm retains arm repeats 3 through 8, shown to be required for Axin binding, and may sequester Axin away from endogenous Arm. This suggests a dual role for Axin, both as a scaffold for degradation and as a component of the cytoplasmic retention machinery (Tolwinski, 2001).

Studies have found that ß-catenin import is constitutive. They suggest a system of cytoplasmic and nuclear anchors that control the flow of ß-catenin into and out of the nucleus. However, prevention of import by cytoplasmic anchoring may be the regulated step, since export is probably controlled by APC. In resting cells, ß-catenin is observed mostly at the cell membrane, therefore it seems likely that localization of ß-catenin to this compartment prevents it from entering the nucleus. Axin has been observed to localize to the plasma membrane, as well as the cytoplasm, and is thus well positioned to function as an anchor. A strong nuclear localization of Arm is observed in experiments where no Axin protein is present. In contrast, overexpressed Axin prevents the nuclear accumulation of Arm normally associated with DeltaArm expression (Tolwinski, 2001).

A model is favored where the dynamic import and export of Arm is controlled by binding partners in the cytoplasm and the nucleus. Axin is involved in cytoplasmic anchoring, and dTCF/Pan is involved in nuclear retention. Arm retained in the cytoplasm is degraded unless it enters adherens junctions. In response to Wg, degradation stops, and Arm accumulates in the cytoplasm bound to Axin. Some Arm enters the nucleus where it binds dTCF/Pan. An equilibrium is reached as a result of active import and export, and inactive degradation. This is the situation in Arm stripes where diffuse staining throughout the cell is observed. However, the existence of anchoring offers a second level of signaling control that could induce a rapid and concentrated nuclear accumulation of Arm with no change in levels. Specific nuclear accumulation has been observed in Xenopus and sea urchin. Though levels were not measured, the striking lack of cytoplasmic ß-catenin is suggestive of a lack of cytoplasmic anchoring. Another response of this type may be what is observed in the epithelial to mesenchyme transition. Here, ILK is overexpressed in epithelial cells resulting in very high nuclear accumulation of ß-catenin without an increase in levels, suggesting the possibility of inhibition of cytoplasmic anchoring (Tolwinski, 2001).

Recently, two studies have suggested that APC is involved in the nuclear export of Arm/ß-catenin. APC contains a nuclear export signal (NES) which is required for efficient export of ß-catenin from the nucleus. Combining this result with the current data, it is proposed that there are at least two levels of control of Arm/ß-catenin localization involving cytoplasmic anchoring and active export. APC may play a role in preventing Arm/ß-catenin from accumulating in the nucleus due to dTCF binding. Both controls must be overcome to accumulate enough Arm/ß-catenin to activate transcription (Tolwinski, 2001).

Activation of the Wnt signaling cascade provides key signals during development and in disease. By designing a Wnt receptor with ligand-independent signaling activity, evidence is provided that physical proximity of Arrow (LRP) to the Wnt receptor Frizzled-2 triggers the intracellular signaling cascade. A branch of the Wnt pathway has been uncovered in which Armadillo activity is regulated concomitantly with the levels of Axin protein. The intracellular pathway bypasses Gsk3ß/Zw3, the kinase normally required for controlling ß-catenin/Armadillo levels, suggesting that modulated degradation of Armadillo is not required for Wnt signaling. It is proposed that Arrow (LRP) recruits Axin to the membrane, and that this interaction leads to Axin degradation. As a consequence, Armadillo is no longer bound by Axin, resulting in nuclear signaling by Armadillo (Tolwinski, 2003).

The data argue for a different regulatory mechanism of Wg signal transduction, proceeding through the inhibition of the protein Axin, rather than through the inhibition of Zw3/GSK3β. Axin has been identified in both vertebrates and invertebrates as a negative component of the pathway. Later work established Axin as a critical scaffold protein required for the assembly and function of the degradation complex. This complex functions in the destruction of Arm/β-catenin by bringing the kinase Zw3 and Arm into close proximity, leading to the phosphorylation of Arm, and thereby targeting it to the proteasome for degradation. For efficient Arm degradation, both Axin and APC must be present in the complex. How Wg input controls activity of the degradation complex has never been properly established, although most models have focused on the inhibition of the kinase Zw3. It is also unclear whether Arm degradation always plays a central role in converting Wnt input into transcriptional responses. In sea urchins and mammals, the most obvious response to Wnt signaling is a relocalization of Arm protein from the cytoplasm to the nucleus; it has been shown that both Axin and APC have a profound effect on Arm localization that cannot be explained by their interaction with Zw3 or the degradation complex alone (Tolwinski, 2003).

Evidence is presented that the Wg signal can be transmitted through a posttranslational regulation of Axin accumulation. Despite uniform transcription of Axin using the UAS/GAL4 system, Axin accumulates to different levels in different cells across each parasegment. Cells with lower steady-state levels of Axin are those exposed to Wg input, and this was strictly dependent on Wg. Loss of Wg causes excess accumulation of Axin, whereas uniform Wg expression (and therefore signaling) lowers total Axin levels. The phenomena observed in embryos parallel earlier reports showing that Axin accumulation is affected by Wnt signaling in tissue culture cells. GSK3β phosphorylation of Axin leads to its stabilization. However, the actual role that phosphorylation plays appears to be more complex, since further work contradicted this finding. In the current experiments, the phosphorylation state of Axin was not examined in cells responding to Wg (those with low Axin levels), nor in those not exposed to Wg (high Axin levels). Therefore, whether modification may inactivate Axin or whether modification leads to removal of Axin by degradation cannot be distinguished. It was found, however, that Zw3 kinase activity is not necessary for the reduction in Axin accumulation that is observed; the Axin striping pattern is maintained in embryos that lack Zw3 function. These results argue for a link between Wg signaling and Axin accumulation that is independent of the Zw3-mediated degradation complex (Tolwinski, 2003).

Although Zw3 does not appear to be required for Axin degradation, the more upstream component, Arrow, appears to be important for this mode of Wg signal transduction. The cytoplasmic domain of Arrow interacts with Axin in the yeast two-hybrid system, an interaction also identified for one of the mammalian LRPs, mLRP5, whose rapid binding of mAxin is ligand stimulated. Binding data for Arrow are largely in agreement with the mammalian study, except that no contribution is found of the Zw3 binding region of Axin in binding of Arrow bait. Interestingly, full-length Axin fails to interact significantly with the Arrow C terminus in yeast and all the Axin clones isolated in the library screen lack sequences N-terminal to position 353. This finding suggests that an inhibitory domain is present in Axin, N-terminal to the Zw3 binding domain, and that this inhibitory domain prevents Axin from binding Arrow. It is possible that the Wnt signal necessary for the mouse Axin interaction with LRP5 induces a conformational change in Axin that removes, modifies, or otherwise displaces the inhibitory domain. In contrast, Armadillo bait significantly binds both full-length and truncated Axin. These data taken together with the demonstration in Drosophila that signaling leads to loss in Axin striping and a lowered steady-state level of Axin, suggest that the Arrow/LRP5 interaction with Axin induces a change in activity and/or stability of Axin (Tolwinski, 2003).

The prevailing view on Wnt reception states that Arrow/LRP5,6 function as coreceptors together with Frizzled proteins. It is well established that Frizzled proteins bind Wnt ligands and that this interaction is essential for Wnt signal transduction. Initial work on LRP6 extended this model in suggesting that Wnt provides a bridging function in assembling a complex of Frizzleds and Arrow, at least for the particular combination mFz8/mWnt-1/mLRP6. However, biochemically, such complex formation has also not always been confirmed. In addition, the functional significance for signaling of the observed ternary complex has not been demonstrated in vivo. Therefore an experiment was designed that tested whether, in vivo, physical proximity of Arrow and Frizzled-2 is sufficient for signaling. In fact, it was found that Frizzled-2 can initiate ligand-independent signal transduction. The constitutive activity of the Fz2-Arr[intra]chimeric protein is significant, since only expression of the fusion protein but not expression of the individual components (Fz2 and Arr[intra]) activate signal transduction. It is inferred that association of Frizzled2 with the Arrow C terminus is indeed a key step in signal initiation in vivo, and that the proximity afforded here by protein fusion also occurs during normal signaling. The Fz2-Arr[intra] chimera is uncoupled from the need for ligand to trigger the intracellular signal transduction cascade. Therefore, whether the Arrow extracellular domain participates in a true 'reception' complex with Fz2 in Wg binding cannot be addressed. Nevertheless, Arrow, or at least its C terminus, likely interacts intimately with Fz2 during signal initiation at the membrane. In addition, activation of the pathway by the Fz2-Arr[intra] chimera proceeds through canonical pathway components, most notably requiring Disheveled, a result consistent with the finding that Dsh functions downstream of Arrow. In cultured vertebrate cells, one report has suggested that in some circumstances, LRP6 can induce Wnt signal transduction independently of Disheveled. In contrast, the experiment of overexpressing biologically active Arrow cytoplasmic sequences in the form of the Fz2-Arr[intra] chimera revealed a strict Dsh dependence, suggesting instead an obligate role for the Dsh protein at signal initiation by Arrow, and by extension, presumably by vertebrate LRP5,6 (Tolwinski, 2003).

Though the Fz2-Arr[intra] chimera clearly signals, it is not as active as a Wg-stimulated endogenous receptor complex. Presumably, and not surprisingly, the protein fusion will present a distorted topology to cofactors required in the signal initiation complex, and therefore is not optimally configured for initiating signal transduction. This may explain why the chimera retains some measure of reliance on endogenous Arrow, as is apparent from a reduced level of signaling in its absence (Tolwinski, 2003).

In summary, Arrow and the Frizzled family of Wnt receptors function in a protein complex that triggers the intracellular signaling cascade. By binding to and causing a reduction in steady-state levels of Axin, Arrow provides a pivotal link between the receptor complex on the cell surface and the downstream events that control Arm activity. One consequence of Axin degradation may reflect its role as a scaffold for Zw3-mediated degradation of Arm. However, because zw3- embryos still respond to Wg input though they fail to degrade Arm, regulation of the degradation complex cannot be the only target of Wg signaling. A Zw3-independent branch in the Wg pathway is proposed, one that might regulate the release of Armadillo from Axin, resulting in nuclear accumulation and signaling (Tolwinski, 2003).

Subcellular localization of Axin depends on Dishevelled

Wnt signaling causes changes in gene transcription that are pivotal for normal and malignant development. A key effector of the canonical Wnt pathway is ß-catenin, or Drosophila Armadillo. In the absence of Wnt ligand, ß-catenin is phosphorylated by the Axin complex, which earmarks it for rapid degradation by the ubiquitin system. Axin acts as a scaffold in this complex, to assemble ß-catenin substrate and kinases (casein kinase I [CKI] and glycogen synthase kinase 3ß [GSK3]). The Adenomatous polyposis coli (APC) tumor suppressor also binds to the Axin complex, thereby promoting the degradation of ß-catenin. In Wnt signaling, this complex is inhibited; as a consequence, ß-catenin accumulates and binds to TCF proteins to stimulate the transcription of Wnt target genes. Wnt-induced inhibition of the Axin complex depends on Dishevelled (Dsh), a cytoplasmic protein that can bind to Axin, but the mechanism of this inhibition is not understood. This study shows that Wingless signaling causes a striking relocation of Drosophila Axin from the cytoplasm to the plasma membrane. This relocation depends on Dsh. It may permit the subsequent inactivation of the Axin complex by Wingless signaling (Cliffe, 2003).

To study the subcellular distribution of Drosophila Axin, this protein was tagged with green fluorescent protein (GFP) and it was expressed throughout the embryo with the GAL4 system. This results in mutant embryonic cuticles with a 'denticle lawn' phenotype that mimics loss of Wingless signaling. Axin-GFP produces the same phenotype as untagged Axin; this finding indicates that the tagged protein is fully functional. This phenotype reflects a loss of Armadillo, which in turn causes premature loss of expression of Wingless target genes, including wingless itself, in midembryogenesis. Similarly, overexpression of Axin proteins in mammalian cells reduces their β-catenin levels. Thus, elevated Axin levels in the Drosophila embryo counteract the normal Wingless response in cells that are stimulated by Wingless ('+Wg cells') (Cliffe, 2003).

The subcellular distribution of Axin-GFP was studied at late embryonic stages, i.e., in epidermal cells that are no longer stimulated by Wingless ('-Wg cells'). In these -Wg cells, conspicuous green dots are seen throughout the cytoplasm. Similar dots have been observed in vertebrate cells expressing tagged Axin; these dots are associated with vesicles. Interestingly, most of the Axin-GFP dots coincide with dots of E-APC staining. E-APC is the main APC protein expressed in the Drosophila embryonic epidermis; many of the E-APC dots accumulate in apicolateral regions along the plasma membrane. This can be seen in young embryos that have just begun to express Axin-GFP, but, in older embryos in which Axin-GFP has accumulated to high levels, E-APC is largely delocalized from the plasma membrane and is recruited into the cytoplasmic Axin-GFP dots, presumably by direct binding. It is likely that these dots represent the Axin destruction complex. Thus, in -Wg cells, this complex appears to be located predominantly in the cytoplasm, where it actively promotes the degradation of Armadillo (Cliffe, 2003).

Next, Axin-GFP was expressed in embryos without APC function, i.e., in embryos that express a mutant E-APC protein (N175K) and lack the second APC protein (dAPC) that acts redundantly with E-APC. These APC double mutants show very few Axin-GFP dots, and the green fluorescence appears mostly diffuse or grainy. Indeed, the staining of the mutant N175K protein itself appears grainy and is much less dotty than the staining of wild-type E-APC. The few remaining dots colocalize with Axin-GFP dots. Thus, E-APC is required for the formation of the Axin-GFP dots, indicating that the N175K mutant cannot promote Axin complex formation (Cliffe, 2003).

The N175K mutant bears a missense mutation in a surface residue of its Armadillo repeat domain, and its loss of function is due to its inability to associate with the plasma membrane. This results in naked cuticles, the hallmark of ubiquitous Wingless activation. Intriguingly, the N175K mutant is a fully stable protein that retains its Axin binding site. It binds to Axin as efficiently as wild-type E-APC in vitro. Thus, the inability of the N175K mutant protein to associate with the plasma membrane appears to be the sole reason for its failure to promote Axin complex assembly (Cliffe, 2003).

Expression of Axin-GFP in the APC double mutant embryos restores their mutant phenotype partially toward normal. Thus, Axin-GFP is less active in these mutants; this finding confirms that Axin function depends on APC. This dependence is strong but not absolute, and it is likely to reflect the role of APC in promoting Axin complex assembly. Moreover, overexpression of Axin-GFP compensates to some extent for the loss of APC. This parallels the results in APC mutant cancer cells in which overexpressed Axin proteins can bypass the function of APC; this finding suggested that APC has a regulatory role with regard to Axin. This regulatory role could be to target Axin to a specific subcellular location: one would expect APC-mediated targeting to be less critical at elevated levels of Axin expression (Cliffe, 2003).

Axin-GFP expression was examined next in the epidermis of 3- to 6-hr-old embryos; at this stage, stripes of +Wg cells alternate with stripes of -Wg cells. As in older embryos, conspicuous dots of Axin-GFP are scattered throughout the cytoplasm of -Wg cells. Strikingly, in +Wg cells, these dots are associated almost exclusively with apicolateral regions of the plasma membrane. This is observed neither in the epidermis of older embryos that lack Wingless expression nor in wingless mutants. Conversely, coexpression of Wingless with Axin-GFP causes a relocation of virtually all Axin-GFP dots to the plasma membrane and also restores the membrane-associated staining of E-APC in older embryos. Thus, Wingless signaling is both necessary and sufficient for relocation of the Axin-GFP dots to the plasma membrane. Notably, a FRET signal between Axin and LRP-5 has been observed in Wnt-stimulated mammalian cells; this result suggested a Wnt-induced recruitment of Axin to the plasma membrane. This result is the first direct demonstration that Wnt signaling triggers a relocation of Axin to the plasma membrane (Cliffe, 2003).

Axin-GFP levels were examined by Western blot analysis to confirm that Axin-GFP is expressed at moderate levels as an intact full-length fusion protein. Coexpression with Wingless does not change these levels of Axin-GFP, although this analysis can only detect a maximal reduction to 50%. The exposure of these embryos to ubiquitous Wingless was 0-8 hr, so the inability to detect a decrease in Axin-GFP levels in response to Wingless is not inconsistent with the previously determined half-life of tagged mammalian Axin of 4 hr under Wnt signaling conditions. Under these experimental conditions, the main effect of Wingless signaling is clearly a relocation of Axin to the plasma membrane rather than a destabilization of Axin (Cliffe, 2003).

It was asked whether relocation of Axin-GFP to the plasma membrane might be sufficient for its inactivation. If so, overexpressed Wingless should block the excessive activity of Axin-GFP. This is only partly true: some restoration of naked cuticle (predominantly along the midline) is seen in embryos coexpressing Wingless and Axin-GFP compared to embryos expressing Axin-GFP alone. Thus, a component upstream of Axin but downstream of Wingless may be limiting in the inactivation of Axin. The relocation of Axin to the plasma membrane may be a necessary first step toward its inactivation (Cliffe, 2003).

To identify further components of the Wingless pathway that are required for this relocation, Axin-GFP was examined in various mutants. In sgg mutants, there are no significant changes in the subcellular distribution of the Axin-GFP dots, and their relocation to the plasma membrane in +Wg cells appears normal. Likewise, the few residual GFP-Axin in +Wg cells of APC double mutants are associated with the plasma membrane. Thus, neither GSK3 nor APC are required for relocation of Axin-GFP to the plasma membrane. Interestingly however, none of the Axin-GFP dots are associated with the plasma membrane in dsh mutants; Wingless is still expressed in these mutants at this stage). This is the case even if Wingless is coexpressed with Axin-GFP in these mutants. Thus, Dsh is the most downstream-acting component of the Wnt pathway that is required for the relocation of Axin-GFP to the plasma membrane (Cliffe, 2003).

It was asked whether overexpressed Dsh may mediate additional relocation. In wing discs, GFP-Dsh is associated with apicolateral regions of the plasma membrane whether or not Wingless signals. In the embryo, GFP-Dsh is expressed very weakly, and it can only be detected in late stages when Wingless has ceased to signal in the epidermis. In these -Wg cells, GFP-Dsh is detectable in the cytoplasm throughout the embryo and forms occasional dots; it is also weakly associated with the plasma membrane. Notably, overexpressed Dsh causes additional relocation of GFP-Axin from the cytoplasm to the plasma membrane. Most embryos show wide zones of membrane-associated Axin-GFP spanning +Wg cells that alternate with narrow zones of cytoplasmic Axin-GFP dots coincinding with -Wg cells, but, occasionally, the membrane relocation is seen throughout the embryo. This suggests that the additional Dsh-mediated relocation depends on low levels of Wingless signaling (Cliffe, 2003).

Overexpressed Dsh results in a partially naked cuticle, with only small denticles remaining. Thus, Dsh may inhibit endogenous Axin by relocating it to the plasma membrane. Consistent with this, limited restoration of naked cuticle is seen if Axin-GFP is coexpressed with Dsh, and an abundance of small denticles are seen that are sparse in wg mutants or in cuticles expressing Axin-GFP alone. As in the case of Wingless, this suppression of the activity of Axin-GFP is mild, suggesting that the putative limiting component is upstream of Dsh. This component may be Arrow, given that Arrow can bind to Axin and that an interaction between these two components in mammalian cells is induced by Wnt signaling (Cliffe, 2003).

Membrane bound forms of activated Armadillo ('Arm*', i.e., forms lacking their N termini) show significantly more signaling activity than Arm* without a membrane-targeting domain; this finding led to the suggestion that Armadillo exerts its signaling function in the cytoplasm rather than in the nucleus. However, overexpression of membrane-targeted Arm* causes a dramatic relocation of Axin-GFP, and of E-APC, to the plasma membrane throughout the embryonic epidermis, presumably by direct binding. This mimics the Wingless-induced membrane relocation of Axin-GFP, except that the membrane-targeted Arm* relocates Axin-GFP and E-APC to the entire lateral membrane where it itself is localized. No such relocation is seen under conditions of ubiquitous high levels of untargeted Arm*. The striking relocation of Axin-GFP to the plasma membrane by the membrane-targeted Arm* may cause its inactivation even in cells that are only weakly stimulated by Wingless; thus, this finding provides an alternative explanation for the increased activity of membrane bound Armadillo (Cliffe, 2003).

This work provides evidence that the assembly of Axin complex in the cytoplasm depends on a membrane-targeting function of E-APC. This function may also affect targeting to internal membranes, or vesicles, suggesting that the Axin complex may be associated with vesicles. In support of this, overexpressed Axin is associated with vesicles in Xenopus embryos. Furthermore, Dsh (which is required for the Wingless-induced membrane relocation of Axin) is also associated with vesicles, and to some extent with the plasma membrane, in vertebrate and Drosophila cells. Indeed, Axin and Dsh colocalize after overexpression in vertebrate cells. Notably, the DIX domain of the mammalian Dsh protein Dvl-2 contains a phospholipid binding motif that is conserved in the DIX domain of Axin, and targeting of Dvl-2 to vesicles by this motif is essential for its function in controlling the degradation of β-catenin (Cliffe, 2003).

Therefore, a possible model is that the Axin complex and Dsh are associated with the same vesicles, which may be recycling endocytic vesicles. Dsh may target these vesicles constitutively to the plasma membrane, where the Axin complex can interact potentially with Wnt receptors. This complex may be retained at the plasma membrane as a result of a Wnt-induced interaction between Axin and LRP/Arrow, and this retention may allow its subsequent inactivation. It is noted that LRPs are thought to recycle to the plasma membrane through endocytic vesicles, like their rapidly recycling LDL receptor relative. Recycling vesicles may thus provide a platform for APC-mediated assembly of the Axin complex and may convey this complex to the plasma membrane for inactivation by Wnt receptors (Cliffe, 2003).

Axin and the Axin/Arrow-binding protein DCAP mediate glucose-glycogen metabolism

Axin was found as a negative regulator of the canonical Wnt pathway. Human LRP5 was originally found as a candidate gene of insulin dependent diabetes mellitus (IDDM), but its Drosophila homolog, Arrow, works as a co-receptor of the canonical Wnt signal. A previous paper described Drosophila Axin (Daxin)-binding SH3 protein, DCAP, a homolog of mammalian CAV family protein (Yamazaki, 2002). Among the subtypes, DCAPL3 shows significant homology with CAP, an essential component of glucose transport in insulin signal. Further binding assay revealed that DCAP binds to not only Axin but also Arrow, and Axin binds to not only GSK3beta but also Arrow. However, overexpression and RNAi experiments of DCAP do not affect the canonical Wnt pathway. As DCAP is expressed predominantly in insulin-target organs, and as RNAi of DCAP disrupts the pattern of endogenous glycogen accumulation in late stage embryos, it is suggested that DCAP is also involved in glucose transport. Moreover, early stage embryos lacking maternal Axin show significant delay of initial glycogen decomposition, and RNAi of Axin in S2 cells revealed quite increase of endogenous glycogen level as well as GSK3beta. These results suggest that Axin and DCAP mediate glucose-glycogen metabolism in embryo. In addition, the interaction among Axin, Arrow, and DCAP implies a possible cross-talk between Wnt signal and insulin signal (Yamazaki, 2003).

This study shows that DCAP binds to not only Axin but also the cytoplasmic tail of Arrow through SH3 domains. Usually, SH3 proteins mediate protein-protein interaction in signal transduction and often link different pathways for cross talking of signals. Therefore, it is possible that DCAP can connect Wnt signaling molecules to other pathways. Although it could not be proven that DCAP participates in the canonical Wnt pathway, it was shown that DCAP is also a functional homolog of mammalian CAP in insulin signal and controls proper localization of endogenous glycogen in late stage embryos. These results suggest that the glucose transport by DCAP can be affected by Wnt signaling molecules (Yamazaki, 2003).

In the case of Axin, it binds to GSK3β, a glucose-glycogen metabolism-modifying enzyme, and also binds to DCAP, one of the components for insulin-dependent glucose transport. Novel glycogen phenotypes were found in D-axin null mutants and DCAP-RNAi embryos. In addition, RNAi of Axin in S2 increases the level of endogenous glycogen. These findings mean that Axin controls glycogen level as well as GSK3β, and this function is thought to be another important function of Axin as well as inhibiting the canonical Wnt signal (Yamazaki, 2003).

The binding between DCAP and Arrow is also quite intriguing. Arrow is a coreceptor of the canonical Wnt pathway in Drosophila. However, its human homolog LRP5 was originally identified as a candidate gene for IDDM4. Axin binds to not only GSK3β and DCAP but also Arrow/LRP5. Therefore, it is also possible that Axin, Arrow, GSK3β, and DCAP work together in glucose-glycogen metabolism or insulin signal (Yamazaki, 2003).

Although LRP5 is well studied in the canonical Wnt pathway, the function in insulin signal still needs to be investigated further. In a previous report, LRP5 was shown to be expressed in β cells of pancreatic Langerhans islets. The disruption of LRP5 can inhibit glucose uptake into β cells and cause Insulin-Dependent Diabetes Mellitus (IDDM), because β cells would misjudge blood glucose as low levels and would not release insulin. Mammalian CAP is also supposed to interact with LRP5, so this interaction may give significant insights in IDDM (Yamazaki, 2003).

Moreover, recent works revealed that Drosophila insulin signal controls cell size and number in late stage embryo during development. It is after the initial canonical Wnt signal but the same period as DCAP expression. This means that insulin signal has another function other than controlling blood glucose level. Therefore, it is considered that Wnt signal and insulin signal molecules interact each other for development other than the canonical Wnt pathway or controlling blood glucose (Yamazaki, 2003).

Until now, several cross talks have been reported in Wnt signal. As GSK3β and Arrow are already known to be multifunctional molecules, it is quite reasonable that Axin is another bifunctional molecule both in Wnt signal and glucose-glycogen metabolism. Taking together these data, it is also reasonable to think that Wnt signal has a cross-talk with insulin signal. However, the role of Axin in the initial glycogen decomposition is still unclear. It is also unknown why DCAP has five different spliced forms. To study these questions will give significant insights into the signaling network of development (Yamazaki, 2003).

Signals across the plasma membrane to activate the Β-catenin pathway by forming oligomers containing its receptors, Frizzled and LRP: Axin interacts with the intracellular domain of LRP/Arrow

Wnt-induced signaling via ß-catenin plays crucial roles in animal development and tumorigenesis. Both a seven-transmembrane protein in the Frizzled family and a single transmembrane protein in the LRP family (LDL-receptor-related protein 5/6 or Arrow) are essential for efficiently transducing a signal from Wnt, an extracellular ligand, to an intracellular pathway that stabilizes ß-catenin by interfering with its rate of destruction. However, the molecular mechanism by which these two types of membrane receptors synergize to transmit the Wnt signal is not known. Mutant and chimeric forms of Frizzled, LRP and Wnt proteins, small inhibitory RNAs, and assays for ß-catenin-mediated signaling and protein localization in Drosophila S2 cells and mammalian 293 cells were used to study transmission of a Wnt signal across the plasma membrane. The findings are consistent with a mechanism by which Wnt protein binds to the extracellular domains of both LRP and Frizzled receptors, forming membrane-associated hetero-oligomers that interact with both Disheveled (via the intracellular portions of Frizzled) and Axin (via the intracellular domain of LRP). This model takes into account several observations reported here: the identification of intracellular residues of Frizzled required for ß-catenin signaling and for recruitment of Dvl to the plasma membrane; evidence that Wnt3A binds to the ectodomains of LRP and Frizzled, and demonstrations that a requirement for Wnt ligand can be abrogated by chimeric receptors that allow formation of Frizzled-LRP hetero-oligomers. In addition, the ß-catenin signaling mediated by ectopic expression of LRP is not dependent on Disheveled or Wnt, but can also be augmented by oligomerization of LRP receptors (Cong, 2004).

What is the mechanism by which Frizzled transduces a Wnt signal? Mutations that disrupt the signaling activity of Frizzled also affect the ability of Frizzled to induce membrane translocation of Dvl and reduce physical interaction between Frizzled and Dvl, suggesting that a physical interaction between Frizzled and Dvl is required for the signaling activity of Frizzled. It is proposed that Frizzled might function as a docking site for Dvl in ß-catenin signaling. The results are consistent with the finding that the Lys-Thr-x-x-x-Trp motif at the C-terminal tail of Frizzled is not only required for activating ß-catenin signaling, but also for inducing Dvl membrane translocation. The PDZ domain of Dvl has been shown to directly bind to a peptide of C-terminal region of Frizzled containing the Lys-Thr-x-x-x-Trp motif, and this peptide can inhibit Wnt/ß-catenin signaling in Xenopus. However, the binding is relatively weak (Kd~10 microM). The current results suggest that multiple regions of Frizzled might be involved in the binding with Dvl and could increase the binding affinity (Cong, 2004).

The same structural elements may be required for Frizzled to function in both the planar polarity and the ß-catenin pathways, since membrane translocation of Dvl has been implicated in planar polarity signaling, and residues essential for the activity of Frizzled in ß-catenin signaling are also important for Frizzled-induced translocation of Dvl to the plasma membrane. It is possible that other proteins in the Frizzled-Dvl complex, such as LRP in ß-catenin signaling and Flamingo in planar polarity signaling, determine the signaling consequences of interaction between Frizzled and Dvl (Cong, 2004).

What is the role of LRP in transmitting the Wnt signal and what is the function of its extracellular domain of LRP for receiving the Wnt signal? An in vitro binding assay has suggested that Wnt1 is able to bind to the extracellular domain of LRP, but analogous binding was not observed in studies with Wg protein. Results from in vitro binding assays need to be treated cautiously, as the concentrations of ligands and receptors in these assays could be significantly higher than in physiological situations, and certain components normally involved in formation of the receptor complex could be missing in these assays. Therefore, functional data are necessary to address the significance of potential binding between Wnt and LRP. The extracellular domain of LRP can be functionally replaced by the extracellular domain of Frizzled, suggesting a physiological role for a direct, or indirect, interaction of Wnt with the extracellular domain of LRP (Cong, 2004).

LRP can also transmit a signal via ß-catenin without a requirement for Wnt. Advantage was taken of two commonly used inducible oligomerization strategies to demonstrate that oligomerization of LRP6 increases its signaling activity and its interaction with Axin. Interestingly, it has been shown that the second cysteine-rich domain of DKK2 stimulates ß-catenin signaling via LRP independently of Dvl. Further experiments are needed to determine whether this DKK2 fragment activates LRP by altering the oligomerization status of LRP (Cong, 2004).

Why is it necessary and sufficient to bring LRP and Frizzled into proximity for transducing the Wnt signal? RNA interference studies have indicated that signaling by overexpressed LRP is strictly Dvl independent, and Dvl becomes important once Wnt and Frizzled are involved. Axin is known to interact with the C terminus of LRP, and Dvl can interact with Frizzled. Presumably, once overexpressed, a high concentration of membrane LRP is able to bring endogenous Axin to the plasma membrane, based solely on its affinity with Axin, so that Axin might be inactivated or degraded. This would explain why the signaling activity of ectopically expressed LRP is Dvl independent. Under normal physiological conditions, Frizzled and Dvl might be required to translocate Axin to the membrane LRP upon Wnt signaling. Dvl might function as a molecular chaperone to deliver Axin to the Frizzled-LRP complex, based on its affinity with both Frizzled and Axin. In addition, Frizzled and Dvl might also enhance the binding affinity between LRP and Axin through promoting phosphorylation of LRP. Therefore, the Wnt-Frizzled-LRP complex might serve as a high-affinity docking site for Axin. This model is also in agreement with the recent finding that Wnt induces translocation of Axin to the membrane in a Dvl-dependent manner. Consistent with its proposed role as a shuttle, Dvl is associated with intracellular vesicles, and interacts with both actin stress fibers and microtubules (Cong, 2004).

It is proposed that Wnt stimulates the ß-catenin pathway by relocating Axin to the plasma membrane and inactivating Axin. It is still not clear whether Wnt-induced Axin membrane translocation is a prerequisite for dissociation of the ß-catenin degradation complex. Furthermore, it is unknown whether the only function of Dvl is to facilitate the transport of Axin to the plasma membrane. It is possible that Dvl also brings certain factors to Axin upon Wnt signaling and promotes inactivation of the Axin complex. Indeed, it has been suggested that in response to Wnt, Dvl can recruit Frat/GBP, a strong inhibitor of GSK3, to the Axin-GSK3-ß-catenin complex, although a requirement for Frat/GBP in Wnt signaling has not been established genetically. Furthermore, it is unclear whether inhibition of GSK3 normally plays a major role in Wnt signaling, although dominant-negative mutants of GSK3 can activate ß-catenin signaling. It has been shown that Wnt induces dephosphorylation of Axin, which might reflect inhibition of GSK3 or dissociation of the Axin-GSK3 complex. Dephosphorylated Axin appears to be less stable and binds ß-catenin less efficiently. It is currently unknown how membrane translocation of Axin is coupled to dephosphorylation and destabilization of Axin. More work will be necessary to illustrate fully the molecular mechanism by which Wnt induces the stabilization of ß-catenin (Cong, 2004).

Notch synergizes with axin to regulate the activity of armadillo in Drosophila

Cell fate decisions require the integration of various signalling inputs at the level of transcription and signal transduction. Wnt and Notch signalling are two important signalling systems that operate in concert in a variety of systems in vertebrates and invertebrates. There is evidence that the Notch receptor can modulate Wnt signalling and that its target is the activity and levels of Armadillo/β-catenin. This function of Notch has been characterized in relation to Axin, a key element in the regulation of Wnt signalling that acts as a scaffold for the Shaggy/GSK3beta-dependent phosphorylation of Armadillo/beta-catenin. While Notch can regulate ectopic Wingless signalling caused by loss of function of Shaggy, it can only partially regulate the ectopic Wnt signalling induced by the loss of Axin function. The same interactions are observed in tissue culture cells where a synergy is observed in between Axin and Notch in the regulation of Armadillo/β-catenin. These results provide evidence for a function of Axin in the regulation of Armadillo that is different from its role as a scaffold for GSK3β (Hayward, 2006).

The trimeric G protein Go inflicts a double impact on axin in the Wnt/frizzled signaling pathway

The Wnt/Frizzled signaling pathway plays crucial roles in animal development and is deregulated in many cases of carcinogenesis. Frizzled proteins initiating the intracellular signaling are typical G protein-coupled receptors and rely on the trimeric G protein Go for Wnt transduction in Drosophila. However, the mode of action of Go and its interplay with other transducers of the pathway such as Dishevelled and Axin remained unclear. This study shows that the alpha-subunit of Go directly acts on Axin, the multidomain protein playing a negative role in the Wnt signaling. G alpha o physically binds Axin and re-localizes it to the plasma membrane. Furthermore, G alpha o suppresses Axin's inhibitory action on the Wnt pathway in Drosophila wing development. The interaction of G alpha o with Axin critically depends on the RGS domain of the latter. Additionally, the betagamma-component of Go (see Gβ13F) can directly bind and recruit Dishevelled from cytoplasm to the plasma membrane, where activated Dishevelled can act on the DIX domain of Axin. Thus, the two components of the trimeric Go protein mediate a double-direct and indirect-impact on different regions of Axin, which likely serves to ensure a robust inhibition of this protein and transduction of the Wnt signal (Egger-Adam, 2009).

This study has demonstrated that Gαo can physically bind the RGS domain of Axin and recruit it to the plasma membrane, the action likely leading to the destabilization of the Axin-based β-catenin destruction complex and propagation of the Wnt signal inside the cell. In support of this idea, this study has shown that Gαo can suppress the Wnt loss-of-function phenotypes induced by Axin over-expression in wing imaginal discs. This rescue critically depends on the presence of the RGS domain, reiterating the crucial role of this domain for the interaction with Gαo. While the GTP-bound form of Gαo is unable to change the phenotypes of the AxinΔRGS expression, the GDP-bound forms of Gαo even dramatically enhance these phenotypes. It is hypothesized that this enhancement is due to sequestration of the Gβγ heterodimer by the GDP-forms of Gαo. It was also shown that Gβγ can directly bind and recruit Dsh from the cytoplasm to the plasma membrane, thus possibly contributing to the propagation of the Wnt signal (Egger-Adam, 2009).

The RGS domain of Axin, responsible for the interaction with Gαo, is important for the full range of Axin activity in wing imaginal discs. Indeed, over-expression of the ΔRGS form of Axin only partially suppresses Wnt signaling in this tissue. The RGS domain of Axin is known to bind APC, another component of the β-catenin-destruction complex. The inability of AxinΔRGS to directly interact with APC is the likely reason for the reduced activity of this construct in Drosophila wings and in vertebrates. The Gαo and Gαq proteins were shown to dissociate the Axin-based destruction complexes in mammalian cells. It is proposed that in Drosophila, Gαo leads to a similar dissociation of the destruction complex through direct binding to the RGS domain of Axin, which recruits Axin to the plasma membrane and probably displaces APC from Axin (Egger-Adam, 2009).

In vitro, the purified RGS domain of Axin binds equally well both the GDP- and the GTP-loaded forms of Gαo. It also lacks the GTPase-activating protein (GAP) activity towards Gαo, typical for other RGS domains. These data agree with the absence of some of the conserved residues required for the GAP action in Axin RGS. Thus, biochemically Axin binds Gαo regardless of its nucleotide form. However, in vivo the GDP- and the GTP-loaded forms of Gαo behave differently towards Axin. Only Gαo[GTP] is capable of recruiting Axin-GFP to the plasma membrane in the salivary glands. Similarly, Gαo[GTP] is much more potent in rescuing the Axin full-length over-expression effects in wing imaginal discs and adult wings. This seeming contradiction is explained by the fact that in vivo the GDP-loaded forms of Gαo bind the βγ-subunits, recreating the trimeric Go complexes. Indeed, over-expressed, the wild-type Gαo was shown to compete with other Gα proteins for the βγ-subunits. Only the Gαo[GTP] form can stay free and thus exert its activities on Axin in full (Egger-Adam, 2009).

In contrast, the wild-type Gαo also possesses a capacity of over-activating the Wnt pathway in wing imaginal discs, and can to a certain degree rescue the phenotypes of Axin over-expression in this tissue. This contrasts with its inability to recruit Axin-GFP to the plasma membrane in salivary glands. These differences between the two tissues correlate with the degree of Wnt signal transduction. Indeed, the Wnt pathway is highly active in the wing imaginal discs, and Gαo can further enhance the pathway relying on the activity of Fz receptors. In contrast, in larval salivary glands the Wnt pathway is silent, which is illustrated by the cytoplasmic localization of Dsh in this tissue, expected to be plasma membrane localized when the pathway is on. It thus seems probable that in the salivary glands Gαo, forming trimeric Go complexes with Gβγ, fails to be further converted into the monomeric form due to the absence of the Wnt/Fz activity. In contrast, wing imaginal discs provide enough Wnt/Fz activity to activate endogenous as well as exogenous Go, which can then recruit Axin and thus propagate the signal (Egger-Adam, 2009).

The ability of the GDP-bound forms of Gαo to bind to the βγ-subunits is the likely reason for the aggravation of the AxinΔRGS phenotype induced by Gαo. This form, even upon conversion to the GTP-bound state by the action of the Wnt/Fz complexes, can no longer bind the RGS-lacking Axin and suppress Axin's negative action on the Wnt signal transduction. However, it can bind Gβγ. It is proposed that Gβγ plays, in addition to Gαo-GTP, a positive role in the Wnt signal transduction through its ability to bind and recruit Dsh to the plasma membrane. Over-expression of Gαo reduces the amounts of free Gβγ, reducing the efficiency of Dsh re-localization. It is proposed that when the endogenous full-length Axin is present, over-expression of Gαo has the overall stimulating effect on the Wnt signaling in wing discs due to increased generation of Gαo-GTP, which binds and antagonizes Axin. It is only in the artificial situation of over-expression of AxinΔRGS that the other, negative, effect of Gαo can be revealed. To prove that Gαo aggravates the AxinΔRGS phenotypes due to sequestration of Gβγ, the mutant Gαo[GDP] protein unable to charge with GTP but still capable to bind Gβγ was ested, and this form was found to be similar to Gαo in enhancing the AxinΔRGS phenotypes (Egger-Adam, 2009).

Direct experiments were performed testing the involvement of Gβγ in Wnt signaling. In accordance with predictions, down-regulation of Gβγ results in a clear reduction of the Wnt signaling in Drosophila wings and wing discs, affecting the short-range target genes of the Wnt pathway. As over-expression of Gβ alone leads to trapping Dsh in the cytoplasm, such over-expression also produces drastic dominant effects on Wnt signaling in wing discs. Unfortunately, it was not possible to confirm that Dsh was trapped in the cytoplasm of the epithelial cells of such discs due to the low resolution of the Dsh staining obtained in these thin columnar cells. Additionally, not only localization but also abundance of the components of the Wnt pathway are known to change in cells with high levels of Fz activation as part of the feedback regulation. Thus, interpretation of Dsh localization in wing imaginal discs upon perturbations of the Wnt pathway will be difficult. Instead, analysis of a tissue where the Wnt pathway is endogenously silent, such as salivary glands, allows analysis of the direct influence of the subunits of the trimeric Go complex on cellular localization of the components of the Wnt pathway. This analysis led to the identification of the plasma membrane re-localization of Axin by Gαo and of Dsh by Gβγ as such direct cellular responses. These primary responses are probably then utilized in the physiological context as the basis to build positive and negative feedbacks for the final outcome of Wnt signal propagation (Egger-Adam, 2009).

While the numerous data indicate that Gβγ is necessary for the proper activation of the Wnt pathway, probably through plasma membrane re-localization of Dsh, it was not possible to over-activate the Wnt pathway by over-expression of Gβ and Gγ together. Instead, the pathway was down-regulated, although to a weaker extent than that seen by over-expression of Gβ alone. This observation is not easy to reconcile with the other data. One possible explanation is that in the wing discs, unlike the salivary glands, co-overexpression of Gγ might be insufficient to attract the complete pool of Gβ to the plasma membrane, and significant amounts of Gβ may still remain cytoplasmic and retain Dsh. Along these lines, co-overexpression of Gγ shows a partial 'rescue' of the phenotypes induced by Gβ over-expression. Another possible explanation involves the notion of the negative feedback regulation in the Wnt cascade. Proteosomal degradation of Dsh during Wnt signal transduction has been demonstrated. A recent work has shown that targeted plasma membrane localization of Dsh by the Wnt activation or by the Gβγ subunits also destines it for the lysosomal degradation in vertebrate cells. Thus, the activity of Gβγ in the Wnt signaling may be multistep: the initial recruitment of Dsh from the cytosol may serve to activate the pathway, but the persistent membrane localization will lead to Dsh degradation. While Gβ RNAi targeting shows that the Gβγ complex is necessary for the proper Wnt signaling, activation of such a negative feedback loop may underlie the phenotypes observed upon the persistent over-expression of Gβγ. In this scenario, Gβγ will be added to the growing list of regulators of the Wnt pathway, which have both positive and negative activities in this signaling (Egger-Adam, 2009).

A model ia favored whereby Gβγ-induced plasma membrane re-localization of Dsh serves as an initial positive impact to activate the Wnt signal propagation. If this is correct, what may be the immediate consequences of the Gβγ-induced plasma membrane recruitment of Dsh? This scaffolding protein is known to become hyper-phosphorylated upon plasma membrane localization, which correlates with its activity in the Wnt signal transduction. Dsh is known to directly bind Axin through the DIX domain heterodimerization. Although a direct interaction of Gβγ with Axin's protein phosphatase 2A-binding region (N-terminal to the DIX domain) has recently been demonstrated in mammalian cells, no ability was found of Gβγ to re-localize or directly bind Drosophila Axin. Overall, the data and the above considerations lead to the proposal of the following model of the action of the trimeric Go protein in the Drosophila Wnt/Fz pathway (Egger-Adam, 2009).

The trimeric Go protein is a direct target of the activated Fz receptors. Wnt ligand binding to Fz activates the guanine nucleotide exchange activity of Fz towards Go. This in turn dissociates the trimeric Go complex into Gαo-GTP and Gβγ. It is proposed that both these components of the trimeric complex have the initial positive activity in Wnt signal propagation. Gαo-GTP directly binds to the RGS domain of Axin, recruiting Axin to the plasma membrane and dissociating the Axin-based β-catenin destruction complex. In contrast, Gβγ recruits and contributes to activation of Dsh, which then can bind the DIX domain of Axin and thus also promote dissociation of the destruction complex. These two branches of G protein–mediated signal propagation converge on the Axin complex to cooperatively ensure its efficient inhibition. Such a double effect on Axin emanating from the trimeric Go complex may serve to ensure a robust activation of the Wnt signaling (Egger-Adam, 2009).

Modulation of the ligand-independent traffic of Notch by Axin and Apc contributes to the activation of Armadillo in Drosophila

There is increasing evidence for close functional interactions between Wnt and Notch signalling. In many instances, these are mediated by convergence of the signalling events on common transcriptional targets, but there are other instances that cannot be accounted for in this manner. Studies in Drosophila have revealed that an activated form of Armadillo, the effector of Wnt signalling, interacts with, and is modulated by, the Notch receptor. Specifically, the ligand-independent traffic of Notch serves to set up a threshold for the amount of this form of Armadillo and therefore for Wnt signalling. In the current model of Wnt signalling, a complex assembled around Axin and Apc allows GSK3 (Shaggy) to phosphorylate Armadillo and target it for degradation. However, genetic experiments suggest that the loss of function of any of these three elements does not have the same effect as elevating the activity of β-catenin. This study shows that Axin and Apc, but not GSK3, modulate the ligand-independent traffic of Notch. This finding helps to explain unexpected differences in the phenotypes obtained by different ways of activating Armadillo function and provides further support for the notion that Wnt and Notch signalling form a single functional module (Muñ-Descalzo, 2011).

Cells expressing ArmS10, a form of Arm that is insensitive to phosphorylation by GSK3, do not overgrow and remain integrated in the epithelium. Clones of cells mutant for Axin, a central element of the Arm destruction complex, exhibit very high levels of Arm, some of which can be found in the nucleus, and exhibit overgrowths and round edges suggestive of defects in cellular recognition. These phenotypes are related to, but distinct from, those caused by expression of ArmS10 and support the contention that Axin exerts controls on the activity of Arm that are additional to those mediated through its role as a scaffold for GSK3. The effects of Axin loss of function are reminiscent of those caused by expression of ArmS10 in cells with compromised Notch function. Since these effects are caused by the loss of the ligand-independent traffic of Notch, this study tested whether Axin exerts some effect on the traffic of Notch (Muñ-Descalzo, 2011).

Clones of cells mutant for Axin did not show alterations in ligand-dependent Notch signalling, although they exhibited a mild but reproducible increase in Notch protein on the apical side, and overexpression of Axin reduced the amount of Notch present at the cell surface. These observations suggest that Axin regulates the amount of Notch at the cell surface. To test whether this control is exerted by targeting the endocytosis and traffic of Notch, label and chase experiments were performed with Notch. Under the experimental conditions and focusing the analysis in the pouch of the wing imaginal disc, labelled Notch disappeared from the cell surface within 10 minutes of the chase and could be found in punctate intracellular structures, presumably vesicles associated with endocytic traffic. Performing the same assay in the absence of Axin revealed that the endocytosis and traffic of Notch is impaired in Axin mutant cells, and after 30 minutes a substantial amount of Notch could still be detected on the cell surface. This suggests that Axin is involved in, or can influence, the traffic of Notch. Performing the same experiment in discs overexpressing Axin, a decrease was observed in the amount of Notch over time. Altogether, these results suggest that Axin contributes to the removal of Notch from the cell surface and to targeting it for degradation (Muñ-Descalzo, 2011).

Regulation of the activity of Arm by Notch is mediated by its ligand-independent traffic as shown by the activity of chimeric receptors in which the extracellular domain of Notch has been substituted by the extracellular domain of CD8 (CeN) or Torso (TN; Tor - FlyBase). Since Wingless signalling promotes the traffic and degradation of these receptors and cells lacking Axin have elevated levels of Wnt signalling, this study examined what would happen to the stability of CeN in this situation. Surprisingly, the levels of CeN remained largely unchanged in clones of cells mutant for Axin, suggesting that in the absence of Axin, despite high levels of Wnt signalling, CeN cannot be degraded . This could be because Axin is required for the degradation of CeN or because this degradation is dependent on Wnt and Dsh but not on Axin. A contribution of Axin is favoured by the observations that overexpression of Axin reduces, and Axin loss of function increases, Notch levels (Muñ-Descalzo, 2011).

A functional relationship between Axin and Notch is also highlighted by the observation that, in tissue culture, simultaneous reductions of Notch and Axin induce very high levels of Arm activity. However, in vivo, simultaneous loss of both Notch and Axin leads to a suppression of the growth induced by the loss of Axin alone, a phenotype that is associated with extensive cell death and perhaps reflects a synergy of the roles of each protein in apoptosis. For this reason, to test the synergy between the two proteins in determining Arm activity in vivo, a NotchRNAi construct was expressed that reduces, but does not abolish, Notch function in clones of cells mutant for Axin. Under these conditions, there is no apoptosis and larger outgrowths than those promoted by the loss of Axin alone were observed. These phenotypes indicate a synergistic effect of the mutations and suggest that Axin is involved in the modulation of Notch while it traffics through the cell (Muñ-Descalzo, 2011).

Apc, a second element of the Arm destruction complex, is encoded in Drosophila by Apc1 (Apc - FlyBase) and Apc2, which play redundant roles in the regulation of Wnt signalling. In order to test whether Apc is also involved in the traffic of Notch, clones of cells mutant for Apc1 and Apc2 were generated in wing imaginal discs and the traffic of Notch was assessed. In these clones, cells exhibited very similar phenotypes to those of Axin mutants in terms of growth, overall shape and levels of Arm. In addition, they exhibited altered traffic of Notch. However, instead of being clearly localised in vesicles or in the cell membranes, as in the case of Axin mutant cells, Notch protein appeared as a 'fuzzy' stain throughout the cytoplasm of the Apc1/2 mutant cells that was not associated with any subcellular structure. Axin and Apc have been shown to play functionally related, but distinct, roles in the regulation of Arm/δ-catenin and these differences might extend to their effects on Notch (Muñ-Descalzo, 2011).

The function of Axin and Apc is to provide a scaffold for the phosphorylation of Arm/β-catenin by Sgg/GSK3. Since, in mammalian systems, GSK3 has been shown to phosphorylate Notch and there are reports of interactions between Notch and Sgg in Drosophila, tests were performed to see whether Sgg has an effect on the traffic of Notch. Clones of cells mutant for sgg displayed elevated levels of Arm but no discernible effects on the endocytosis and traffic of Notch. This is consistent with the observation that Sgg is not required for the effects of Notch on Wnt signalling (Muñ-Descalzo, 2011).

In addition to their interactions with Wnt signalling, Axin and Apc display interactions with other signalling pathways and, in the case of Apc, with the cytoskeleton. These additional interactions might contribute to the differences between the effects of activated Arm and the loss of function of Axin and Apc. Notwithstanding this, the results reveal a function of Axin and Apc in the traffic of Notch. Previous studies have shown that compromising the traffic of Notch elevates the activity of an activated form of Arm. In Axin or Apc1,Apc2 mutant clones, in addition to the elevation of active Arm, the traffic of Notch is compromised and probably contributes to the increase in Arm activity. In this situation, the levels and activity of Arm would be higher than those resulting from the expression of an activated form of Arm alone. There is evidence that Axin functions in the regulation of Arm activity in a manner that is independent of its role as a scaffold for GSK3. Some of these effects could be mediated through its role in the endocytosis and traffic of Notch, which also could traffic with a GSK3-independent form of Arm (Muñ-Descalzo, 2011).

These results underscore the inadequacy of the notion that Wnt signalling flows through a linear pathway to target the destruction complex and promote β-catenin transcriptional activity. Although this framework helps to explain some of the effects associated with Wnt signalling, it is inconsistent with the observation that, in many instances, changes in the concentration of Arm/β-catenin are insufficient to promote transcriptional activity. While the axis Wnt-Dsh-Axin/Apc-β-catenin is the backbone of Wnt signalling, it is clear that there are additional elements that are not simply modulatory add-ons. In this regard, the interactions between Wnt and Notch signalling are a recurrent theme in developmental biology and disease and might not reflect a simple functional convergence in specific processes at the transcriptional level. The results presented in this study reinforce the notion that Wnt and Notch configure a molecular device (Wntch), in which the mutual control of their activities serves to regulate the assignation of cell fates with the effect of Notch providing a buffer to fluctuations in the resting levels of Arm (Muñ-Descalzo, 2011).



In order to study the Axin protein in embryos, antibodies were raised. Endogenous Axn protein is only detected with the Axin antibodies by immunoprecipitation followed by immunoblotting (IP-western). Two antibodies directed to distinct regions of Axin recognize the same set of bands with apparent molecular weight of 95 kDa, strongly suggesting that these antibodies detect the Axn protein. Furthermore, overexpressed Axn migrates at the identical position. At present, the difference in Axn giving rise to the two bands is not known. It is possible that various phosphorylation states generate the two species of Axn protein, as is the case for mouse Axin (Willert, 1999a). Alternatively, the two separately migrating Axn species could be the A1 and A2 forms of Axn. These forms of Axn consist of amino acid differences between two independent Axin isolates of Willert (1999b) and the previously published Axn sequence (Hamada, 1999). Form A2 includes the six amino acids SRSGSS while Form A1 does not (Willert, 1999b).

The time course of endogenous Axn expression in the fly embryo was examined. Immunofluorescence staining of whole embryos with the Axn antibodies produces weak staining. Thus, the Axn protein may be present at very low to undetectable levels or the Axn specific antibodies do not react efficiently in fluorescence assays. By IP-western, Axn protein was detectable at low levels during the earliest timepoints (0- to 1-hour old embryos), suggesting that some Axn protein is maternally contributed. At later timepoints, Axn protein accumulates to higher levels until it reaches a plateau at about 3-4 hours after egg laying (Willert, 1999b).

Effects of Mutation or Deletion

Wingless is critical for patterning and cell fate determination in embryonic segmentation. Although embryos that are zygotically mutant for Axn appear to have almost normal segment patterning, embryos devoid of both maternal and zygotic Axn gene products are completely naked, lacking all denticles on the ventral cuticle. Embryos that lack the maternal Axn product but have received one paternal wild-type copy of the gene have some denticles on the ventral cuticle, suggesting that the zygotic Axn product can partially rescue the Axn maternal deficiency. These phenotypes are similar to those of embryos derived from homozygous zw3/sgg female germ lines and to those of embryos ubiquitously expressing the wild-type Wg or constitutively active Arm. Thus, Wg signaling is constitutively activated in embryos lacking maternal Axn (Hamada, 1999).

Wg is required for the organization of wing blade development, especially for specification of the wing margin structure. Clones of Axn mutant cells marked with a yellow mutation produce ectopic marginal bristles cell autonomously. Wg also plays an essential role in organizing leg structures; ectopic activation of Wg signaling induces supernumerary outgrowth on the dorsal side of normal legs. The Axn clone also produces a supernumerary leg from the dorsal side of the normal leg. Furthermore, Wg signaling is required for the formation of sternites in the ventral side of the adult abdomen, and its ectopic activation results in the appearance of ectopic sternite structures. The same phenotype is observed in an abdomen containing Axn clones. During wing disc development, Wg signaling is induced along the dorsoventral compartment boundary in the wing imaginal disc. Arm accumulates in the cytoplasm, associates with its partner, Pangolin/DTcf, and activates expression of target genes such as Distal-less (Dll). In Axn clones, the levels of Arm are markedly enhanced in a strictly cell-autonomous manner. In addition, Arm is localized predominantly in the cytoplasm and nuclei in the Axn mutant clones, in contrast to the membrane localization observed in wild-type cells. The levels of Dll expression are also elevated in the Axn clones in a cell-autonomous manner. These results suggest that Axn negatively regulates Wg signaling by down-regulating intracellular levels of Arm and that this regulatory mechanism is essential for Wg signaling (Hamada, 1999).

To further examine the function of Axn, the Axn gene was ectopically expressed using the GAL4/UAS system. In contrast to the phenotypes observed with the Axn mutant clones, ectopic expression of Axn induces notches in the wing, generation of a supernumerary leg from the ventral side of the normal leg, and loss of the sternite structure in the abdomen. In addition, when Axn is expressed in the posterior compartment under the control of engrailed-GAL4, Dll expression is severely repressed in the posterior region of the dorsoventral compartment border. Thus, ectopic expression of Axn exerts an inhibitory effect on Wg signaling (Hamada, 1999).

It is concluded that Axn is required in vivo for the negative regulation of Wg signaling. Of particular interest is the finding that the levels of cytoplasmic Arm are highly and uniformly elevated wherever Axn clones are located in the wing discs. For example, the accumulation of Arm in Axn clones is observed not only around the region where Wg is secreted but also in the region where Wg is not supposed to reach. Together with the fact that Axn is ubiquitously expressed, these findings suggest that Wg activity is not required for the effect of Axn. It is speculated that the Axin family of proteins functions to establish a threshold to prevent premature signaling events caused by Wg/Wnt and to restrict areas that are capable of responding to Wg/Wnt (Hamada, 1999).

naked cuticle (nkd) is an embryonic lethal recessive zygotic mutation that produces multiple segmentation defects, the most prominent of which is the replacement of denticles by excess naked cuticle. This phenotype is also seen in embryos exposed to excess Wg, as well as in embryos lacking both maternal and zygotic contributions from any of three genes that antagonize Wg: zeste-white3/glycogen synthase kinase 3beta (zw3/gsk3beta), D-axin and D-Apc2. In nkd embryos, hh and en transcripts initiate normally but accumulate in broad stripes, including cells further from the source of Wg, which suggests that these cells are hypersensitive to Wg. Next, a stripe of new wg transcription appears just posterior to the expanded Hh/En stripe. This extra wg stripe requires both wg and hh activity and is required for the excess naked cuticle seen in nkd mutants. The death of cells producing Hh/En contributes to the marked shortening of nkd mutant cuticles (Zeng, 2000).

The posteriorly expressed signaling molecules Hedgehog and Decapentaplegic drive photoreceptor differentiation in the Drosophila eye disc, while at the anterior lateral margins Wingless expression blocks ectopic differentiation. Mutations in axin prevent photoreceptor differentiation and leads to tissue overgrowth; both these effects are due to ectopic activation of the Wingless pathway. In addition, ectopic Wingless signaling causes posterior cells to take on an anterior identity, reorienting the direction of morphogenetic furrow progression in neighboring wild-type cells. Signaling by Dpp and Hh normally blocks the posterior expression of anterior markers such as Eyeless. Wingless signaling is not required to maintain anterior Eyeless expression and in combination with Dpp signaling can promote Ey downregulation, suggesting that additional molecules contribute to anterior identity. Along the dorsoventral axis of the eye disc, Wingless signaling is sufficient to promote dorsal expression of the Iroquois gene mirror, even in the absence of the upstream factor pannier. However, Wingless signaling does not lead to ventral mirror expression, implying the existence of ventral repressors (Lee, 2001).

Two characteristics distinguish anterior from posterior behavior in the eye disc: growth occurs in the anterior, with the exception of the second mitotic wave, and differentiation occurs in the posterior. Wg signaling regulates both of these properties. Wg signaling promotes the growth of eye disc cells. Loss of axin causes dramatic overgrowth and outgrowth of cells in the eye disc, and this phenotype is due only to excessive Wg pathway activity, since it can be blocked by a dominant negative form of dTCF/Pangolin. The strength of the phenotype may reflect higher levels of Wg signaling than are induced by loss of sgg; perhaps Axin contributes to retaining Arm in the cytoplasm, in addition to promoting its phosphorylation. Vertebrate Axin has been shown to associate with mitogen-activated protein kinase kinase kinase 1 and activate the c-jun N-terminal kinase (JNK) pathway. However, JNK signaling does not appear to be essential for the growth or differentiation of cells in the Drosophila eye disc, and it does not contribute to the axin mutant phenotype in the eye. The ability of Wg signaling to promote overgrowth in the eye disc is consistent with the reduction in the size of the eye disc caused by loss of Wg signaling (Lee, 2001).

Loss of axin function at the posterior margin results in outgrowths from the disc, over-riding the normal control of organ size. axin mutant clones also form smooth borders with surrounding cells, suggesting that their ability to adhere to wild-type cells is decreased. Growth control requires the formation of normal junctions between cells, so it is possible that the outgrowth results from this loss of adhesion. Because the posterior margin is the site of dpp expression prior to initiation, the outgrowth observed could also require Dpp signaling; in the leg disc, overlap between dpp and wg promotes the extension of a proximal-distal axis. However, punt;axin double mutant clones show a similar degree of overgrowth, suggesting that Dpp signaling does not contribute to this (Lee, 2001).

Early transplantation experiments and other studies have suggested that the region of the eye disc anterior to the morphogenetic furrow contains intrinsic positional information; the subsequent finding that Hh is essential for furrow movement has been taken to mean that all this information originates posterior to the furrow. The observations presented here challenge this view by showing that Wg signaling can generate a source of anterior positional information that appears to attract the morphogenetic furrow toward itself. Cells mutant for axin autonomously express ey and other markers for the region anterior to the furrow, including hth, tsh and mirr. Cells adjacent to an axin mutant clone show a reorientation of the pattern of Atonal expression and cells at the internal border of the clone express Hairless, which is likely to be activated by Dpp signaling from adjacent wild-type cells. This suggests that axin mutant cells produce a non-autonomous signal that maintains nearby cells in an Ato-expressing state; since wg-lacZ expression is activated in axin mutant clones, the signal could itself be Wg (Lee, 2001).

In addition to providing anterior information to the eye disc, Wg acts early in development to define its dorsal domain. Dorsal wg expression is controlled by pnr, and ectopic eye formation caused by loss of pnr. This can be blocked by restoring wg, suggesting that wg is a downstream effector of pnr. In addition, Wg signaling is necessary to maintain the expression of mirr and can induce ectopic mirr expression along the ventral margin. axin mutation has been used to clarify the relationships between pnr, wg and mirr. Small clones of cells mutant for pnr maintain mirr expression, showing that pnr does not have a direct effect on mirr, but acts through one or more non-autonomous factors. This is consistent with the expression of pnr in a smaller domain than mirr. Restoring Wg signaling to pnr mutant eye discs, by making pnr;axin double mutant clones in a Minute background, allows the expression of mirr; thus, no other factor downstream of pnr can be essential for mirr expression (Lee, 2001).

hh was expressed dorsally in early eye discs and activation of the Hh pathway in ventral ptc clones leads to ectopic mirr expression. The results are consistent with two possible roles for Hh. Dorsal hh expression could be independent of pnr and contribute to mirr activation in the absence of both pnr and axin. The dorsal domain of hh expression in the eye disc is indeed not stably established until the second larval instar, while wg and pnr are expressed dorsally from late embryonic stages. Alternatively, Hh could act downstream of pnr but upstream of wg to activate mirr. In support of this hypothesis, anterior ventral ptc clones activate mirr non-autonomously, and have also been shown to activate wg expression. Because ventral axin clones do not activate mirr expression, this mechanism would imply that Hh activates factors in addition to wg that allow ventral expression of mirr (Lee, 2001).

The secreted glycoprotein Wingless (Wg) acts through a conserved signaling pathway to regulate expression of Wingless pathway nuclear targets. Wg signaling causes nuclear translocation of Armadillo, the fly ß-catenin, which then complexes with the DNA-binding protein TCF (Pangolin), enabling it to activate transcription. Though many nuclear factors have been implicated in modulating TCF/Armadillo activity, their importance remains poorly understood. A ubiquitously expressed protein, Pygopus, is required for Wg signaling throughout Drosophila development. Pygopus contains a PHD finger at its C terminus, a motif often found in chromatin remodeling factors. Overexpression of pygopus also blocks the pathway, consistent with the protein acting in a complex. The pygopus mutant phenotype is highly, though not exclusively, specific for Wg signaling. Epistasis experiments indicate that Pygopus acts downstream of Armadillo nuclear import, consistent with the nuclear location of heterologously expressed protein. These data argue strongly that Pygopus is a new core component of the Wg signaling pathway that acts downstream or at the level of TCF (Parker, 2002).

If pygo is a core component of Wg signaling in the fly, where does it act in the pathway? This question was approached using epistasis analysis. Initially, this was achieved via overexpression. In the absence of Wnt signaling, ß-catenin (and by extension Arm) is believed to be phosphorylated at serine and threonine residues at its N terminus via the GSK3ß/Axin/APC complex. If these residues are deleted or substituted, ß-catenin becomes resistant to degradation. In flies, these mutant forms of Arm (Arm*) activate Wg signaling independently of Wg. When placed under the control of the GMR promoter, Arm* causes a small eye phenotype similar to that of GMR-wg. Co-expression of pygo severely suppresses this phenotype. This strongly suggests that pygo overexpression blocks Wg signaling downstream of Wg-induced Arm stabilization (Parker, 2002).

To examine the position of pygo in the pathway using loss-of-function genetics, Axin;pygo double mutants were created. In Axin mutants, the signaling pathway is constitutively activated because of stabilization of Arm. As found in vertebrate systems, Axin functions in a complex with Sgg to phosphorylate Arm. The Wg target gene Senseless (Sens) was used as a readout in wing imaginal discs. In pygo clones, Sens expression adjacent to the dorsal/ventral Wg stripe is lost. In Axin clones, Sens is activated, no matter where in the presumptive wing blade the clones are located, since loss of Axin constitutively activates Wg signaling. In Axin;pygo double mutant clones, Sens expression is always lost. Thus, pygo acts downstream of Axin in this assay (Parker, 2002).

Epitasis analysis was also performed in the eye. At the beginning of the third larval instar, a wave of apical constriction of the columnar epithelial cells, called the morphogenetic furrow (MF) sweeps across the presumptive eye from the posterior to the anterior. Behind the MF, ordered clusters of photoreceptors develop. When Wg signaling is activated in the primordial eye, such as in Axin mutant clones, no photoreceptors are specified. Thus, the eye offers another test of whether pygo is epistatic to Axin (Parker, 2002).

Photoreceptor development, as judged by Elav staining, appears normal in pygo mutant cells. Even at higher magnification, no detectable difference was observed in the photoreceptor clusters between pygo-positive and pygo mutant cells. Clones that lacked Axin lack any evidence of photoreceptor development. This dramatic phenotype is completely rescued in Axin;pygo double mutant clones, clearly demonstrating that pygo is epistatic to (acts downstream of) Axin. This is consistent with the overexpression studies that suggest pygo acts downstream of Arm stabilization (Parker, 2002).

When Wg signaling is activated, Arm is stabilized and translocates to the nucleus. In Drosophila, it has proved very difficult to detect nuclear Arm, even in cells receiving high levels of endogenous Wg. However, Axin maternal and zygotic mutant embryos display high levels of nuclear Arm. Because attempts to make Axin;pygo germline clones were unsuccessful, clones in the wing disc were generated to investigate Arm levels and localization. In clones of cells lacking pygo, Arm is present at low levels at the cell periphery, consistent with its role in adherence junctions. In Axin clones, Arm protein levels are greatly increased in both the nucleus and cytoplasm. Axin;pygo double mutant clones also have high levels of cytosolic and nuclear Arm, though the nuclear levels of Arm appear slightly less than in Axin clones. These data are interpreted to mean that Arm is still stabilized in the absence of pygo (as would be expected if pygo acts downstream of Axin) and that, for the most part, pygo is not required for Arm nuclear import (Parker, 2002).

These experiments indicate that pygo acts downstream of Axin, an activated form of Arm and Arm nuclear import. Consistent with this, a tagged form of Pygo is nuclear. Taken together these data strongly suggests that Pygo acts in the nucleus, probably at the transcriptional level (Parker, 2002).

How pygo influences transcription of Wg target genes in the nucleus could occur in several ways. Simple explanations include the possibility that pygo could simply be required for the interaction of Arm with TCF, or for TCF to bind to DNA. However, the fact that Arm still accumulates to high levels in the nuclei of Axin;pygo mutant cells may indicate that the Arm/TCF/DNA complex still forms in the absence of pygo. It has been shown that expression of a dominant-negative version of TCF (which lacks the Arm-binding domain but retains its ability to bind DNA) prevents Arm nuclear accumulation. This supports the idea that TCF acts as a nuclear tether for stabilized Arm. Using this line of reasoning, Arm is still found in the nuclei of Axin;pygo mutant cells because it is still bound by TCF, which is still localized properly on the DNA. It should be noted a subtle reduction in nuclear Arm accumulation is seen in Axin;pygo versus Axin mutant cells. However, the small difference suggests that this effect may be indirect (Parker, 2002).

Drosophila dishevelled (dsh) functions in two pathways: it is necessary to transduce Wingless (Wg) signaling and it is required in planar cell polarity. To learn more about how Dsh can discriminate between these functions, genetic screens were performed to isolate additional dsh alleles and the potential role of protein phosphorylation was examined by site-directed mutagenesis. Two alleles were identified with point mutations in the Dsh DEP (Dishevelled, Egl-10, Pleckstrin) domain that specifically disrupt planar polarity signaling. When positioned in the structure of the DEP domain, these mutations are located close to each other and to a previously identified planar polarity mutation. In addition to the requirement for the DEP domain, it was found that a cluster of potential phosphorylation sites in a binding domain for the protein kinase PAR-1 is also essential for planar polarity signaling. To identify regions of dsh that are necessary for Wg signaling, a screen was carried out for mutations that modified a GMR-GAL4;UAS-dsh overexpression phenotype in the eye. Many alleles of the transgene containing missense mutations were recovered, including mutations in the DIX (Dishevelled, Axin) domain and in the DEP domain, the latter group mapping separately from the planar polarity mutations. In addition, several transgenes had mutations within a domain containing a consensus sequence for an SH3-binding protein. Second-site-suppressing mutations were recovered in axin, mapping at a region that may specifically interact with overexpressed Dsh (Penton, 2002). In addition to mutations in the UAS-Dsh transgene itself, the UAS-dsh misexpression screen yielded second-site modifiers on the third and fourth chromosome. Modifiers on the first and second chromosome could not be recovered due to the strategy of the screen. Five of the second-site modifiers map near axin and were indeed found to contain mutations within the axin gene. They behave as dominant suppressors of Dsh misexpression phenotypes in both the wing and eye but do not modify Wg or DFz2 misexpression phenotypes. In addition, these alleles are homozygous viable and have no phenotype when they are recombined away from UAS-dsh. Since Axin normally suppresses Wg signaling, and null axin alleles do not interact with UAS-dsh, it is inferred that these alleles specifically suppress overexpressed forms of Dsh but do not affect Dsh that is regulated by Wg signaling. This would imply that overexpressed Dsh works through a mechanism that is different from Dsh when activated by Wg. For example, overexpressed Dsh may interact with Axin through binding to a domain that is different from the Axin domain that interacts with Wg-activated Dsh (Penton, 2002).

The Wingless protein plays an important part in regional specification of imaginal structures in Drosophila, including defining the region of the eye-antennal disc that will become retina. Wingless signaling establishes the border between the retina and adjacent head structures by inhibiting the expression of the eye specification genes eyes absent, sine oculis and dachshund. Ectopic Wingless signaling leads to the repression of these genes and the loss of eyes, whereas loss of Wingless signaling has the opposite effects. Wingless expression in the anterior of wild-type discs is complementary to that of these eye specification genes. Contrary to previous reports, it has been found that under conditions of excess Wingless signaling, eye tissue is transformed not only into head cuticle but also into a variety of inappropriate structures (Baonza, 2002).

In order to analyse the effect of ectopic activation of the Wingless pathway during the development of the eye-antennal imaginal disc, clones either mutant for the negative regulator of Wingless signaling, Axin, or expressing an activated form of Armadillo (Arm*) were induced. The loss of eye identity caused by the ectopic activation of Wingless, suggests a possible function for Wingless in the regulation of the eye selector genes. The top of the genetic hierarchy involved in eye specification appears to be the Pax6 homolog, Eyeless. In the third instar eye disc the expression of Eyeless is restricted to the region anterior to the furrow and, despite the Wingless-induced inhibition of eye development, the expression of Eyeless in this region is not affected by axin- clones. This lack of an effect anterior to the furrow, despite the overgrowth and abnormal Distal-less expression in the same region, implies that misregulation of Eyeless is not the primary cause of the transformations caused by ectopic Wingless activity (Baonza, 2002).

Downstream of Eyeless (although feedback relationships makes the epistatic relationship complex) are other transcription factors required for eye specification, including Eyes absent, Sine oculis and Dachshund. A phenotype similar to axin- clones of excess proliferation and consequent overgrowth is caused by loss of Eyes absent and Sine oculis. Moreover, as in axin- clones, clones mutant for sine oculis ectopically express Eyeless in the region posterior to the furrow. The similar mutant phenotypes shown by the loss of function of these genes and the ectopic activation of Wingless signaling make them good candidates to be regulated by the Wingless pathway (Baonza, 2002).

The expression patterns of Eyes absent, Sine oculis and Dachshund in axin- and/or arm* mutant clones were examined in third instar eye discs. At this stage, Dachshund is expressed at high levels on either side of the morphogenetic furrow, whereas Eyes absent and Sine oculis are expressed in all the cells of the eye primordium. In order to produce large patches of mutant tissue, the Minute technique was used. In axin- M+ clones the expression of Eyes absent in front of the furrow is always autonomously eliminated. This effect is not only seen in large clones that touch the eye margin but also in small internal clones. Identical results were obtained with Sine oculis and Dachshund: their expression was autonomously lost from anterior axin- M+ clones. Consistent with these results, in arm*-expressing clones Eyes absent, Dachshund and sine oculis (detected with a lacZ reporter construct) are similarly autonomously eliminated. It is therefore concluded that Wingless signaling represses the expression of the eye selector genes eyes absent, dachshund and sine oculis anterior to the morphogenetic furrow. Posterior to the furrow, however, some clones express high levels of Eyes absent, and Dachshund. This effect is always associated with overgrowth, and this expression is restricted to only some cells in these clones (Baonza, 2002).

Identifying the signals involved in maintaining stem cells is critical to understanding stem cell biology and to using stem cells in future regenerative medicine. In the Drosophila ovary, Hedgehog is the only known signal for maintaining somatic stem cells (SSCs). Wingless (Wg) signaling is also essential for SSC maintenance in the Drosophila ovary. Wg is expressed in terminal filament and cap cells, a few cells away from SSCs. Downregulation of Wg signaling in SSCs through removal of positive regulators of Wg signaling, dishevelled and armadillo, results in rapid SSC loss. Constitutive Wg signaling in SSCs through the removal of its negative regulators, Axin and shaggy, also causes SSC loss. Also, constitutive wg signaling causes over-proliferation and abnormal differentiation of somatic follicle cells. This work demonstrates that wg signaling regulates SSC maintenance and that its constitutive signaling influences follicle cell proliferation and differentiation. In mammals, constitutive ß-catenin causes over-proliferation and abnormal differentiation of skin cells, resulting in skin cancer formation. Possibly, mechanisms regulating proliferation and differentiation of epithelial cells, including epithelial stem cells, are conserved from Drosophila to man (Song, 2003).

Wg produced from terminal filament and cap cells may reach SSCs at a distance of a few cells by either diffusion or active transport, and then Wg directly controls SSC maintenance. Furthermore, correct intermediate levels of wg signaling seem to be important for maintaining SSCs in the Drosophila ovary. Reduction of wg signaling in SSCs by removal of positive regulators such as arm and dsh causes rapid SSC loss, as does constitutive wg signaling in SSCs by removal of negative regulators such as Axn and sgg. wg signaling maintains SSCs through several possible mechanisms: (1) wg signaling could be required for SSC self-renewal and/or survival; (2) it could maintain the association of SSCs with IGS cells, and/or (3) both mechanisms could work simultaneously. DE-cadherin-mediated cell adhesion has been shown to be important for keeping SSCs in their niche; it also shares arm as a common component with wg signaling. wg signaling is known to regulate levels of arm, which are also important for DE-cadherin-mediated cell adhesion. Thus, it is possible that wg signaling regulates cell adhesion between SSCs and their niches. In addition, arm mutant clonal analysis strongly argues that wg signaling must also directly regulate SSC self-renewal and/or survival. arm2 mutant SSC clones are lost very quickly over time in comparison with wild-type SSC clones, and the arm2 mutation primarily affects wg signaling but does not disrupt DE-cadherin-mediated cell adhesion. Therefore, wg signaling controls SSC maintenance through regulating SSC self-renewal/survival and/or cell adhesion between SSCs and their niche cells. The temperature-sensitive allele of wg gives very mild phenotypes in follicle cell production, however, removal of wg downstream components has a dramatic impact on SSC maintenance. In Drosophila, there are six other wg-related genes. This raises an interesting possibility that other wg-like molecules could also be involved in regulating SSC maintenance (Song, 2003).

In addition to wg signaling, hh signaling is also essential for SSC maintenance and proliferation. Hyperactive hh signaling causes follicle cell over-proliferation and abnormal differentiation of follicle cells. Disrupting hh signaling in SSCs by removing the function of hh downstream components such as Smoothened and Cubitus interruptus results in rapid SSC loss. Similarly, reduction or elimination of wg signaling also causes rapid SSC loss. Removal of patched, a negative regulator of the hh pathway, stabilizes SSCs. However, SSCs mutant for negative regulators for the wg pathway, sgg and Axn, are destabilized. All the evidence indicates that wg and hh may use different mechanisms to regulate SSCs in the Drosophila ovary (Song, 2003).

Constitutive wg signaling increases the division rates of early follicle cell progenitors in the germarium. When Fz2, dsh and activated arm are over-expressed, extra follicle cells accumulate in the ovarioles, suggesting that hyper-activation of wg signaling causes over-proliferation of follicle cells. Furthermore, sgg or Axn mutations cause over-proliferation of follicle cells, resulting in the formation of extra follicle cells that accumulate outside egg chambers. These cells are not mitotically active and usually assume some stalk cell characteristics. These results suggest that production of extra follicle cells by excessive wg signaling is because of higher mitotic activities of progenitors and/or SSCs in the germarium. It is important to note that sgg mutations are more potent than Axn in stimulating the proliferation of follicle cell progenitors. The different potencies may be because of differences in how these mutations affect wg signaling. Alternatively, because sgg negatively regulates hh signaling, sgg could be involved in negatively regulating both hh and wg signaling in the ovary. It has been demonstrated that excessive hh signaling causes extra follicle cells to accumulate outside egg chambers. Therefore, it might be probable that sgg is involved in regulating both hh and wg signaling pathways in follicle cells of the Drosophila ovary (Song, 2003).

This study also demonstrates that constitutive wg signaling disrupts the normal differentiation of somatic follicle cells. Mutant Axn or sgg follicle cells in and outside the germarium express higher levels of Hts in their membranes and tend to accumulate between egg chambers. In ovarioles that contain a majority of mutant follicle cells, germline cysts fail to undergo normal morphological changes necessary for proper encapsulation by follicle cells, although they are wild type, suggesting that the mutant follicle cells are defective in their interactions with germ cells. Although some of them are recruited to egg chambers, these mutant follicle cells have abnormal morphologies (e.g. smaller and irregular sizes). Huli tai shao is present not only on spectrosomes in GSCs, cystoblasts and fusomes in early germline cysts, but also on the membranes of somatic follicle cells. The abnormal follicle cell phenotype may be because of abnormal levels of Hts, which may prevent follicle cells from shape changes and growth. The extra mutant follicle cells accumulating outside egg chambers express Lamin C and do not divide similar to stalk cells. However, unlike stalk cells, they express high levels of Fas3. Similar to the mutant follicle cells in the germarium, the mutant follicle cells that are recruited to egg chambers also express high levels of Hts. Unlike the follicle cells in the germarium, the cells fail to express high levels of Fas3. These results indicate that constitutive wg signaling in follicle cells disrupts proper follicle cell differentiation (Song, 2003).

Cellular interaction between the proximal and distal domains of the limb plays key roles in proximal-distal patterning. In Drosophila, these domains are established in the embryonic leg imaginal disc as a proximal domain expressing escargot, surrounding the Distal-less expressing distal domain in a circular pattern. The leg imaginal disc is derived from the limb primordium that also gives rise to the wing imaginal disc. Essential roles of Wingless in patterning the leg imaginal disc are described. (1) Wingless signaling is essential for the recruitment of dorsal-proximal, distal, and ventral-proximal leg cells. Wingless requirement in the proximal leg domain appears to be unique to the embryo, since it has previously been shown that Wingless signal transduction is not active in the proximal leg domain in larvae. (2) Downregulation of Wingless signaling in wing disc is essential for its development, suggesting that Wg activity must be downregulated to separate wing and leg discs. In addition, evidence is provided that Dll restricts expression of a proximal leg-specific gene expression. It is proposed that those embryo-specific functions of Wingless signaling reflect its multiple roles in restricting competence of ectodermal cells to adopt the fate of thoracic appendages (Kubota, 2003).

To confirm whether Wg signaling is required cell autonomously for leg disc development, the dominant-negative forms of Drosophila TCF (DTCFdeltaN) or Drosophila axin (Daxin) were expressed in the limb primordia. The Dll-Gal4 driver, which is turned on in the limb primordium at stage 11 and continues to be active in leg and wing discs, was used. In Dll-GAL4 embryos carrying UAS-DTCFdeltaN or UAS-Daxin, the overall size of leg discs was reduced. Expression of Esg was preferentially reduced in the dorsal side. The drastic reduction of Dll mRNA and protein in distal leg cells in the armH8.6 mutants as well as in DTCFdeltaN- and Daxin-expressing embryos demonstrate that Wg signaling is required for both proximal and distal leg cells. In arm mutants, Hth-expressing cells expand to the distal domain. This observation suggests that, upon loss of Wg signaling, prospective leg disc cells lose their identity and adopt the fate of trunk ectoderm. However, disc-specific reduction of Wg signaling does not affect wing disc formation, although the Dll-Gal4 driver is active in the wing primordium. It is concluded that late function of Wg signaling promotes formation of the leg disc with a higher requirement in the proximal domain, but is dispensable for wing disc formation (Kubota, 2003).

Steep differences in wingless signaling trigger Myc-independent competitive cell interactions

Wnt signaling is a key regulator of development that is often associated with cancer. Wingless, a Drosophila Wnt homolog, has been reported to be a survival factor in wing imaginal discs. However, it was found that prospective wing cells survive in the absence of Wingless as long as they are not surrounded by Wingless-responding cells. Moreover, local autonomous overactivation of Wg signaling (as a result of a mutation in APC or axin) leads to the elimination of surrounding normal cells. Therefore, relative differences in Wingless signaling lead to competitive cell interactions. This process does not involve Myc, a well-established cell competition factor. It does, however, require Notum, a conserved secreted feedback inhibitor of Wnt signaling. It is suggested that Notum could amplify local differences in Wingless signaling, thus serving as an early trigger of Wg signaling-dependent competition (Vincent, 2011).

One conclusion from this work is that Wg signaling is not intrinsically required for wing cell survival and that, instead, competitive cell interactions triggered by local differences in Wingless signal transduction influence survival decisions. Such local differences can arise between clones that either cannot transduce the signal (e.g., fz fz2 or arrow mutant) or overactivate signaling (e.g., axin or APC mutant). In both cases, the low signaling cells are eliminated. It has been suggested that other forms of cell competition could be relevant to cancer. Moreover, mutations in axin and APC are found in a variety of cancers. Therefore, it is conceivable that humans precancerous APC or axin mutant cells could acquire a competitive advantage that enables them to clear surrounding normal tissue, thus contributing to tissue colonization. As this study has shown, this is not mediated by local differences in the activity of Myc, a key regulator of ribosomal activity and a well-established factor of cell competition. In fact, the competitive nature of axin mutant cells was boosted by experimentally increasing their relative content of functional ribosomes. By analogy, in humans, loss of axin (or APC) and increased translational potential are two features that could have additive effects in boosting early tumor progression and enabling tumors to overcome preexisting barriers to tissue growth (Vincent, 2011).

Although the cell biological basis of Wg signaling-induced competition remains to be elucidated, this study has identified one important mediator, the secreted phospholipase encoded by notum. notum knockdown prevents axin mutant cells from taking over the wing pouch even though these cells are themselves insensitive to Notum activity. Therefore, the overgrowth of axin mutant cells is not solely an autonomous consequence of overactive Wg signaling. As a result of high signaling activity, axin mutant cells secrete Notum, which inhibits signaling in neighboring wildtype cells. Thus, an initial signaling difference is amplified and then transduced into downstream events that lead to the elimination of normal cells, which is required for axin mutant cells to overgrow and take over the tissue (Vincent, 2011).

Wnt signaling cross-talks with JH signaling by suppressing Met and gce expression

Juvenile hormone (JH) plays key roles in controlling insect growth and metamorphosis. However, relatively little is known about the JH signaling pathways. Until recent years, increasing evidence has suggested that JH modulates the action of 20-hydroxyecdysone (20E) by regulating expression of broad (br), a 20E early response gene, through Met/Gce and Kr-h1. To identify other genes involved in JH signaling, a novel Drosophila genetic screen was designed to isolate mutations that derepress JH-mediated br suppression at early larval stages. It was found that mutations in three Wnt signaling negative regulators in Drosophila, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), caused precocious br expression, which could not be blocked by exogenous juvenile hormone analogs (JHA). A similar phenotype was observed when armadillo (arm), the mediator of Wnt signaling, was overexpressed. qRT-PCR revealed that Met, gce and Kr-h1expression are suppressed in the Axn, slmb and nkd mutants as well as in arm gain-of-function larvae. Furthermore, ectopic expression of gce restored Kr-h1 expression but not Met expression in the arm gain-of-function larvae. Taken together, it is concluded that Wnt signaling cross-talks with JH signaling by suppressing transcription of Met and gce, genes that encode for putative JH receptors. The reduced JH activity further induces down-regulation of Kr-h1expression and eventually derepresses br expression in the Drosophila early larval stages (Abdou, 2011).

JH transduces its signal through Methoprene-tolerant (Met), Germ cell-expressed (Gce) and Krüppel-homolog 1 (Kr-h1) and the p160/SRC/NCoA-like molecule (Taiman in Drosophila and FISC in Aedes). The Drosophila Met and gce genes encode two functionally redundant bHLH-PAS protein family members, which have been proposed to be components of the elusive JH receptor. Both Met and gce mutants are viable and resistant to JH analogs (JHA) as well as to natural JH III. However, Met-gce double mutants are prepupal lethal and phenocopies CA-ablation flies. The Met protein binds JH III with high affinity. In Tribolium, suppression of Met activity by injecting double-stranded (ds) Met RNA causes precocious metamorphosis. Kr-h1 is considered as a JH signaling component working downstream of Met. In both Drosophila and Tribolium, Kruppel-homolog1 (Kr-h1) mRNA exhibits high levels during the embryonic stage and is continuously expressed in the larvae; then, it disappears during pupal and adult development. Kr-h1 expression can be induced in the abdominal integument by exogenous JH analog (JHA) at pupariation. Suppression of Kr-h1 by dsRNA in the early larval instars of Tribolium causes precocious br expression and premature metamorphosis after one succeeding instar. Thus, Kr-h1 is necessary for JH to maintain the larval state during a molt by suppressing br expression. Studies in Aedes, Drosophila and Tribolium have demonstrated that the p160/SRC/NCoA-like molecule is also required for JH to induce expression of Kr-h1 and other JH response genes. For example, Aedes FISC forms a functional complex with Met on the JH response element in the presence of JH and directly activates transcription of JH target genes (Abdou, 2011 and references therein).

In an attempt to isolate other genes involving JH signaling, a novel genetic screen was conducted, and mutations in were identified in three Wnt signaling component genes, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), induced precocious br expression, which was similar to a loss of JH activity. The evolutionarily conserved Wnt signaling pathway controls numerous developmental processes. The key mediator of the Drosophila Wnt pathway is Armadillo (Arm, the homolog of vertebrate β-catenin). When the Wnt signaling ligand, Wingless (Wg), is absent, the destruction complex is active and phosphorylates Arm, earmarking it for degradation. Upon Wg stimulation, the destruction complex is inactivated; as a result, unphosphorylated Arm accumulates in the cytosol and is targeted to the nucleus to stimulate transcription of Wnt target genes. Many players in the Wnt signaling pathway negatively regulate its activity. For example, Axin (Axn) is one of the main components of the destruction complex. Supernumerary limbs (Slmb) recognizes phosphorylated Arm and targets it for polyubiqitination and proteasomal destruction. Naked cuticle (Nkd) antagonizes Wnt signaling by inhibiting nuclear import of Arm. The current investigations reveal that the high activity of Wnt signaling in the Axn, slmb, and nkd mutants suppresses the transcription of Met and gce, genes encoding for putative JH receptors, thus linking Wnt signaling to JH signaling and insect metamorphosis for the first time (Abdou, 2011).

The 'status quo' action of JH in controlling insect metamorphosis is conserved in hemimetabous and most holometabous insects. However, the larval-pupal transition in higher Diptera, such as Drosophila, has largely lost its dependence on JH. For instance, in most insects, the addition of JH in larvae at the last instar causes the formation of supernumerary larvae. However, exogenous JH does not prevent pupariation and pupation in Drosophila, and instead disrupts the development of only the adult abdominal cuticle and some internal tissues. The molecular mechanisms underlying these differential responses to JH are not clear (Abdou, 2011).

Broad is a JH-dependent regulator that specifies pupal development and mediates the 'status quo' action of JH. In the relatively basal holometabolous insects, such as beetles and moths, JH is both necessary and sufficient to repress br expression during all of the larval stages. These studies revealed that JH is also required during the early larval stages in the more derived groups of the holometabolous insects, such as Drosophila, but it is not sufficient to repress br expression at the late 3rd instar. During the early larval stages, overexpression of the JH-degradative enzyme JHE, reduction of JH biosynthesis or disruption of the JH signaling always causes precocious br expression in the fat body. However, exogenous JHA treatment can not repress br expression in the fat body of late 3rd instar larvae. The molecular mechanism underlying the developmental stage-specific responses of the br gene to JH signaling remains to be clarified (Abdou, 2011).

As knowledge of signal transduction increases, the next step is to understand how individual signaling pathways integrate into the broader signaling networks that regulate fundamental biological processes. In vertebrates, Wnt signaling has been found to interact with different hormone signaling pathways to mediate various developmental events. For example, the Wnt/beta-catenin signaling pathway interacts with thyroid hormones in the terminal differentiation of growth plate chondrocytes and interacts with estrogen to regulate early gene expression in response to mechanical strain in osteoblastic cells. In insects, both Wnt and JH signaling are important regulatory pathways, each controlling a wide range of biological processes. This study reports that the Wnt signaling pathway interacts with JH in regulating insect development. During the Drosophila early larval stages, elevated Wnt signaling activity in the Axn, slmb, nkd mutants and arm-GAL4/UAS-armS10 flies represses Met and gce expression, which down-regulates Kr-h1 and causes precocious br expression in the fat body. Ectopic expression of UAS-gce in the arm-GAL4/UAS-armS10 larvae is sufficient for restoring Kr-h1 expression and then repressing br expression (Abdou, 2011).

Arm is a co-activator that interacts with Drosophila TCF homolog Pangolin (Pan), a Wnt-response element-binding protein, to stimulate expression of Wnt signaling target genes. In the absence of nuclear Arm, Pan interacts with Groucho, a co-repressor, to repress transcription of Wingless-responsive genes. Upon the presence of nuclear Arm, it binds to Pan, converting it into a transcriptional activator to promote the transcription of Wingless-responsive genes. It is proposed that Wnt signaling indirectly suppresses Met and gce expression by activating an unknown transcriptional repressor (Abdou, 2011).

JH signaling is well known to be a systemic factor that decides juvenile versus adult commitment. Wg is a morphogen that tissue-autonomously promotes proliferation and patterning during organogenesis. The current studies show that ectopically activating Wg signaling, either by mutations of negative regulators or by the ectopic expression of Arm, results in br derepression via loss of Met and Gce. How and why does the localized Wg signaling regulate the global JH signaling during insect development? It is hypothesized that though JH signaling activity is globally controlled by JH titer in the hemolymph, distinct tissues may response to JH with different sensitivity, which could be regulated by Wnt signaling-mediated Met and gce expression. Actually, it was found that precocious br expression is detectible in the fat body but not midgut of the Axn mutant 2nd instar larvae. This is one line of evidence to support that Wnt signaling regulates Met and gce expression in a tissue-specific manner (Abdou, 2011).

APC2 and Axin promote mitotic fidelity by facilitating centrosome separation and cytoskeletal regulation

To ensure the accurate transmission of genetic material, chromosome segregation must occur with extremely high fidelity. Segregation errors lead to chromosomal instability (CIN), with deleterious consequences. Mutations in the tumor suppressor adenomatous polyposis coli (APC) initiate most colon cancers and have also been suggested to promote disease progression through increased CIN, but the mechanistic role of APC in preventing CIN remains controversial. Using fly embryos as a model, this study investigated the role of APC proteins in CIN. The findings suggest that APC2 loss leads to increased rates of chromosome segregation error. This occurs through a cascade of events beginning with incomplete centrosome separation leading to failure to inhibit formation of ectopic cleavage furrows, which result in mitotic defects and DNA damage. Several hypotheses related to the mechanism of action of APC2 were tested, revealing that APC2 functions at the embryonic cortex with several protein partners, including Axin, to promote mitotic fidelity. These in vivo data demonstrate that APC2 protects genome stability by modulating mitotic fidelity through regulation of the cytoskeleton (Poulton, 2013).

This study has used Drosophila embryos to explore how APC proteins regulate mitotic fidelity in vivo. The findings corroborate studies in mammalian cells suggesting that APC promotes genomic stability through its cytoskeletal functions. Mammalian APC may also promote genomic stability by regulating Wnt signaling, but the current data indicate that the role of APC2 in preventing CIN in syncytial embryos does not involve this. Furthermore, although APC2 mutants have increased mitotic defects, most mitoses proceed without error. Thus, APC2 is not a central part of the mitotic apparatus in vivo, which contrasts with studies suggesting that APC proteins are key mitotic regulators. Instead, the data suggest that APC2 ensures high-fidelity mitosis. Although this might reflect functional differences between human and fly APCs, it may also suggest that cultured cells represent a sensitized situation with an elevated mitotic error rate, in which removal of fidelity regulators such as APC has a greater impact; consistent with this, cultured cells are prone to karyotypic anomalies. It is important to note that the precise series of events triggered by APC2 loss in syncytial fly embryos are likely to be specific to that system, with APC playing diverse cytoskeletal roles at different times and places, both within the same animal and between species (Poulton, 2013).

The small but significant increase in mitotic failure in APC2 mutants prompted an analysis backwards from nuclear removal, with the idea that the primary defect might be significantly more frequent, but might not always trigger nuclear removal. Mechanistically, the data are consistent with a model wherein APC2 promotes high-fidelity chromosome segregation through a primary role in facilitating centrosome separation. In APC2 mutants, centrosome separation defects are highly elevated, with 12% of nuclei having separation reduced more than two standard deviations from the WT mean. Although this did not have apparent effects on spindle structure, it had an unexpected consequence. APC2 mutants had a dramatic increase in ectopic cleavage furrows, the likelihood of which was highly correlated with reduced centrosome separation. The possible causal connection between incomplete centrosome separation and ectopic furrowing was further supported by an analysis of EB1 knockdown, as EB1 acts independently of APC2 in syncytial embryo. Although it is contended that the similar effects of Eb1-RNAi and APC2 loss on centrosome separation and ectopic furrowing support the role of these phenotypes in downstream chromosome mis-segregation and nuclear fallout, it should be noted that EB1 appears to play a direct role in chromosome segregation. Thus, it is possible that this shared defect in chromosome segregation is responsible for nuclear fallout in both mutants, irrespective of their similarities in centrosome separation defects and ectopic furrows (Poulton, 2013).

Ectopic furrows form where cleavage furrows would form in standard mitotic divisions. Indeed, formation of normal cytokinetic furrows is prevented in syncytial divisions by preventing Rho activation in the spindle midzone; although centralspindlin proteins localize there, RhoGEF2 does not, and artificial elevation of Rho activity triggers ectopic furrows. The data suggest how ectopic furrows are normally prevented in early embryos. They are consistent with the hypothesis that proper centrosome separation leads to furrow formation around the dividing 'cell', rather than over the metaphase plate where it would form in normal cytokinesis. When centrosome separation is reduced, the ectopic RhoGEF2 recruitment observed at the spindle midzone might be sufficient to trigger ectopic furrowing. The model suggests that failure to fully separate centrosomes and subsequent formation of ectopic furrows then leads to mitotic defects. Nuclei and associated mitotic spindles were found to be able to be physically displaced by ingressing ectopic furrows, perhaps disrupting chromosome segregation. This provides strong selective pressure for the existing mechanism preventing ectopic furrowing (Poulton, 2013).

How then does this trigger nuclear removal? Nuclear removal can be initiated by DNA damage, monitored by the DNA damage sensor CHK2. Strikingly, APC2 mutants had more chromosome segregation errors than wild type, many of which were followed by nuclear removal. Furthermore, nuclei undergoing fallout accumulated the DNA damage marker γH2Av. These data are consistent with the hypothesis that in APC2 mutants, chromosome mis-segregation generates DNA damage, thus triggering CHK2-mediated nuclear removal. Indeed, nuclear removal was blocked in chk2;APC2 double mutants. Recent studies in mammalian cells indicate that chromosome mis-segregation can cause DNA damage; intriguingly, one suggests cytokinetic furrow ingression on lagging chromosomes damages DNA. Given the significant increase in lagging chromosomes and the ectopic cleavage furrows over spindles in APC2 mutants, it is tempting to speculate that a similar mechanism for generating DNA damage might be involved (Poulton, 2013).

Of course, APC2 loss also might lead to chromosome segregation errors by other mechanisms. For example, centrosome separation defects are sufficient to generate merotelic microtubule attachments, thus leading to chromosome mis-segregation. The rapidity of syncytial divisions may make early embryos particularly sensitive to this type of error. Furthermore, in syncytial embryos the shared cytoplasm might make it difficult to alter timing of the metaphase-anaphase transition locally at an affected nucleus to allow repair of microtubule attachment errors. Together, these data suggest that although APC2 loss may lead to chromosome mis-segregation and nuclear fallout by multiple means, most if not all of these pathways begin with defects in centrosome separation. It is thought that these findings on the role of APC2 in mitotic fidelity in syncytial embryos may extend to other cell types, as fly neural stem cells lacking APC proteins have a significantly longer mitotic cycle, suggesting mitotic defects. It will be interesting to see whether human APC promotes mitotic fidelity, at least in part, by regulating centrosome positioning; consistent with this, human APC facilitates centrosome movement in migrating neurons by stabilizing microtubule interactions with cortical actin Furthermore, in human colon cancer cells, merlin (neurofibromin 2), ezrin and APC2 govern centrosome and spindle positioning by regulating astral microtubule attachment to cortical actin, suggesting a conserved role for APC in positioning centrosomes and spindles. These findings may also help in understanding better the role of APC mutations in cancer. In most colon cancers, one APC allele bears a premature stop codon in the mid-region, truncating the protein. A fly mutant (APC2d40) that mimics these truncations has increased nuclear fallout, like APC2 null embryos. APC2d40 mutants were also found to have reduced centrosome separation. This suggests that at least some aspects of the model describing the mechanistic role of APC2 in promoting mitotic fidelity may apply to colon tumors with truncated APC (Poulton, 2013).

The data also test key aspects of models describing where and how APC2 regulates the cytoskeleton. These analyses suggest that APC2 acts at the embryonic cortex, where it binds βcat. These findings, together with the role of APC2 in centrosome separation, led to revision of a previous model. It is proposed that APC2 facilitates stable interaction of astral microtubules with cortical actin to promote centrosome separation. This model is consistent with numerous studies indicating that cortical microtubule attachment helps mediate centrosome separation by generating pulling forces on astral microtubules through cortical dynein. It is now important to explore the effects of APC2 on microtubule dynamics directly (Poulton, 2013).

These analyses of APC2 domain-deleted mutants revealed two additional mechanistic insights. First, the Arm repeats of APC2 are necessary for syncytial cytoskeletal function, suggesting that they bind an important partner. Based on the model that APC2 facilitates microtubule-cortex interactions, one attractive candidate is KAP3. Second, the SAMP repeats, which allow APC2 to bind the destruction complex scaffold Axin, are also required for the cytoskeletal role of APC2. Furthermore, Axin mutants, like APC2 mutants, had reduced centrosome separation, and increased ectopic furrows and nuclear fallout. These data suggest that in syncytial embryos, APC2 acts as part of a multiprotein complex sharing many components with the Wnt-regulatory destruction complex. However, comparing APC2 mutants rescuing mitotic fidelity (this study) or Wnt signaling strongly suggests that in syncytial embryos this complex has a distinct role from its Wnt-regulatory function. It will be important to determine the role of Axin in this complex, as this study found it is not required to localize APC2 to the cortex, unlike the destruction complex component GSK3 (Poulton, 2013).

These in vivo studies indicate that APC helps to ensure mitotic fidelity via cytoskeletal regulation, but show that, at least in syncytial fly embryos, this contribution is relatively subtle. Furthermore, the data suggest that embryos possess mechanisms to help compensate for defects caused by APC2 loss: although centrosomal separation defects affect 12% of nuclei, only 7% have ectopic furrows and only 2% experience chromosome segregation defects or nuclear fallout. It will be important to identify mechanisms buffering the effects of APC2 loss and thus reducing mitotic defects (Poulton, 2013).

Although the precise role that APC2 plays in syncytial embryos and the cascade of consequences of APC2 loss are likely to be confined to that system, it is hypothesized that APC proteins play analogous roles in other tissues, subtly regulating the cytoskeleton to promote mitotic fidelity. This is consistent with observations in cultured mammalian cells, which suggest that APC loss exerts diverse effects on the cytoskeleton, reducing mitotic fidelity. Such roles for APC proteins in mitotic fidelity and the possible mechanisms compensating for APC loss may have interesting implications for cancer progression. In colon polyps and early adenomas, prior to accumulation of additional mutations present in carcinomas, cells prone to massive mitotic defects would probably be eliminated by surveillance processes, such as DNA damage checkpoints, whereas cells with subtle perturbations of mitotic fidelity like those caused by APC2 loss might persist. Occasional mitotic errors may then help induce genomic instability spurring tumor progression. Furthermore, APC mutation might sensitize cells to checkpoint loss, as cytoskeletal defects caused by APC loss may be less effectively buffered, thereby elevating CIN. This provides a clear, testable model for how APC mutation contributes to tumor initiation and progression (Poulton, 2013).


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Axin: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 21 November 2016

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