Protein Phosphatase 2A (PP2A) has a heterotrimeric-subunit structure,consisting of a core dimer of ∼36 kDa catalytic and ∼65 kDa scaffold subunits complexed to a third variable regulatory subunit. Several studies have implicated PP2A in Wg/Wnt signaling. However, reports on the precise nature of PP2A role in Wg/Wnt pathway in different organisms are conflicting. We show that twins (tws), which codes for the B/PR55 regulatory subunit of PP2A in Drosophila, is a positive regulator of Wg/Wnt signaling. In tws- wing discs both short- and long-range targets of Wingless morphogen are downregulated. Analyses of tws- mitotic clones suggest that requirement of Tws in Wingless pathway is cell-autonomous. Epistatic genetic studies indicate that Tws functions downstream of Dishevelled and upstream of Sgg and Armadillo. Our results suggest that Tws is required for the stabilization of Armadillo/β-catenin in response to Wg/Wnt signaling. Interestingly,overexpression of, otherwise normal, Tws protein induce dominant-negative phenotypes. The conflicting reports on the role of PP2A in Wg/Wnt signaling could be due to the dominant-negative effect caused by the overexpression of one of the subunits.

Wnt signaling is one of the important signal transduction pathways regulating several events during growth and development and is also implicated in a variety of cancers (reviewed by Polakis, 2000). Stabilization of β-catenin, a highly oncogenic protein, is the key event in the transduction of the Wnt signal (reviewed by Polakis, 1999). In the absence of Wnt signal, APC recruits β-catenin to Axin/GSK3β complex. The N-terminal region of β-catenin contains a series of serine and threonine residues, which are phosphorylated first by Casein Kinase I(Liu et al., 2002) and subsequently by GSK3β. FWD1 or βTrCP, members of the F-box protein family, binds to phosphorylated β-catenin resulting in its degradation by the ubiquitin and proteasome pathways(Marikawa and Elinson, 1998; Winston et al., 1999). Binding of Wnt to its cell-surface receptor of the Frizzled family causes the activation of Dsh, a cytoplasmic protein, which is then recruited to the cell membrane. Activated Dsh antagonizes GSK3β activity, to stabilize and elevate the levels of cytoplasmic β-catenin. Subsequently, β-catenin complexes with TCF/LEF to transduce Wg/Wnt signaling. Thus, repression of the activity of GSK-3β appears to be central to the regulation of Wnt signal transduction pathway.

The ability of Axin to bind Protein Phosphatase 2A (PP2A)(Willert et al, 1999; Hsu et al., 1999) suggests that this phosphatase can interact with the Axin-APC-GSK3β-catenin complex. It might modulate the effect of the kinase on one or more substrates,thereby affecting Wnt signaling. However, studies involving the inhibition of PP2A by okadaic acid in mammalian cell lines are conflicting. Seeling et al.(Seeling et al., 1999) and Li et al. (Li et al., 2001)report that PP2A is a negative regulator of Wnt signaling, while studies by Willert et al. (Willert et al.,1999) suggest that PP2A is a positive regulator. The predominant form of PP2A in cells has a heterotrimeric-subunit structure, consisting of a core dimer of ∼36 kDa catalytic (C) and ∼65 kDa scaffold (A) subunits complexed to a third variable regulatory (B) subunit. Knockout mouse embryos lacking the catalytic subunit of PP2A show decreased levels of β-catenin,suggesting its positive role in Wnt signaling upstream of β-catenin(Gotz et al., 2000).

Relatively large number of B or B-related polypeptides are known, many of which are present in multiple isoforms (reviewed by Janssens and Goris, 2001). They express in tissue- and temporal-specific manner during development. Reports also indicate that they may target the PP2A catalytic complex to intracellular sites such as microtubules(Sontag et al., 1995) or the nucleus (McCright et al.,1996). These features of the PP2A regulatory subunits suggest that it is the B subunit that defines the substrate specificity of a given PP2A heterotrimeric complex and its physiological role. Thus, identification of the B subunit(s) that interacts with components of Wnt pathway will elucidate the mechanism by which PP2A regulates Wnt signaling. Indeed, evidence is available for the involvement of a regulatory subunit in the Wnt pathway. For example,overexpression of B56 in mammalian cells or in Xenopusembryos/explants results in the reduction of β-catenin levels suggesting a negative role upstream of β-catenin(Seeling et al., 1999; Li et al., 2001). Overexpression of B′/PR61 inhibits β-catenin-induced axis duplication in Xenopus embryos, suggesting that this subunit is also a negative regulator of Wnt signaling(Ratcliffe et al., 2000). These reports do not reconcile with the findings from mouse knockout experiments that the catalytic subunit positively regulates Wnt signaling(Gotz et al., 2000) as well as to a recent report that shows loss of Wnt signaling in embryos depleted with PR/B56ϵ transcripts (Yang et al.,2003). However, none of the above studies combine both loss- and gain-of-function approaches to examine the role of a given subunit, which may help in better understanding the role of PP2A in Wnt signaling.

We have earlier reported that overexpression of human APC (hAPC) in Drosophila downregulates Wingless (Wg; Drosophila homologue of Wnt) signaling (Bhandari and Shashidhara, 2001). We had observed that deficiency mutation Df(3R)by62 enhanced hAPC-induced eye phenotypes, but not its overlapping deficiency Df(3R)by10. Thus, the locus/loci that enhances hAPC-induced phenotypes was mapped to 85E11-85F16, the cytological location of tws, which encodes Drosophila homologue of B (of type PR55)regulatory subunit of PP2A. We have examined a possible requirement of Tws in Wg signal transduction. We have employed both loss- and gain-of-function genetic studies to determine the precise role of Tws in Wg signaling. Results outlined in this report suggest that tws is a positive regulator of Wg signaling. Tws is required for the stabilization of Armadillo (Arm; Drosophila homologue of β-catenin) in response to Wg signaling. Its primary role in Wg pathway appears to be inhibition of Shaggy (Sgg; Drosophila homologue of GSK3β) activity.

Fly stocks and generation of UAS-Tws

Recombinant chromosomes and combinations of GAL4 drivers, UAS lines,different mutations and/or markers were by standard genetic techniques. FLP-FRT method (Xu and Rubin,1993) was used for generating mitotic clones of twsP, which was first recombined with P[FRT]82 πMyc. Clones were generated with the help of hsFLP using either forked,arm-lacZ or Ubi-GFP as clonal markers. Germline clones of P[FRT]82 twsP were generated using P[FRT]82 ovoD as described in Chou and Perrimon(Chou and Perrimon, 1996).

Full-length (2.5 kb) tws cDNA clone (LD12394; http://www.fruitfly.org)was subcloned into the P-element vector, pCaSpeR-UAS(Brand and Perrimon, 1993). The construct was first sequenced to ensure that no mutations have been introduced during cloning. All the transgenic flies (29 independent lines) generated from this construct were crossed to various GAL4 drivers to express Tws in different tissues. All transgenic lines showed similar phenotypes, although they differed in the severity of the phenotypes. UAS-Tws23 gave the strongest effect as a single insertion, which was used in all the experiments reported here.

Other UAS lines used were, UAS-armS2 and UAS-armS10(Pai et al., 1997),UAS-Flu-ΔArm (Zecca et al.,1996), UAS-Dsh (Neumann and Cohen, 1996), UAS-DN-TCF/pan (van der Wetering et al., 1997),UAS-APC/FL (expressing full-length human APC) and UAS-hAPC/CBD (expressing theβ-catenin binding domain of human APC)(Bhandari and Shashidhara,2001), UAS-Sgg (Steitz et al.,1998), UAS-DNGSK-3β (dominant negative form of Xenopus Sgg/GSK-3β) (Jia et al., 2002) and UAS-Cadi5 (intracellular domain of Drosophila E-cadherin) (Sanson et al., 1996). GAL4 strains used were en-GAL4(Brand and Perrimon, 1993), vg-GAL4 (Simmonds et al.,1995), hs-GAL4 (Bloomington stock list; originally developed by Andrea Brand) and 405-GAL4 (expressed only in the differentiating neurons of wing and eye imaginal discs) (Bhandari and Shashidhara, 2001).

Histology

X-gal staining and immunohistochemical staining were essentially as described (Ghysen and O'Kane,1989; Patel et al.,1989). The lacZ reporter gene constructs used were vg-DV enhancer-lacZ and vg-quadrant enhancer-lacZ(Kim et al., 1996). FITC-labeled actin-Phalloidin was purchased from Molecular Probes. The primary antibodies used were, monoclonal anti-Ac(Skeath and Carroll, 1991),anti-Arm (Riggleman et al.,1990), anti β-galactosidase (Sigma, St Louis, MO), anti-Ci(Motzny and Holmgren, 1995),anti-Cut (Blochlinger et al.,1993), anti-Dll (Vachon et al., 1992), anti-Fas3 (Patel et al., 1987), anti-Myc-tag (purchased from Sigma, St. Louis, MO),anti-Sca (Mlodzik et al.,1990) and anti-Wg (Brook and Cohen, 1996) and polyclonal anti-Arm(Ruel et al., 1999),β-galactosidase (raised by A. Khar, CCMB, India), anti-Tws(Gomes et al., 1993), anti-Vg(Williams et al., 1993). Anti-Ac, Anti-Arm, anti-Fas3, anti-Sca and anti-Wg were obtained from the Development Studies Hybridoma Bank, University of Iowa, USA. Fluorescence images were obtained either on a Zeiss Axiocam digital camera, on Meridian Ultima confocal microscope or on Zeiss LSM/Meta. Control and experimental images were digitized always at identical fluorescence microscope and camera settings. The adult appendages were processed for microscopy as described before (Shashidhara et al.,1999).

Comparative analysis of tws alleles

twsP and tws60, the two homozygous lethal alleles of tws, were isolated by Uemura et al.(Uemura et al., 1993). Genetically twsP is stronger of the two and shows no or very low levels of Tws expression (Shiomi et al., 1994) (Fig. 1C). Both the alleles exhibit mirror-symmetric pattern duplication in wing imaginal discs (Uemura et al.,1993) (Fig. 1E). aar1, a homozygous viable allele, shows characteristic mitotic (abnormal anaphase) defects (Gomes et al., 1993), but has no wing disc phenotype. aar1 hemizygous larvae, i.e. over the deficiency Df(3R)by62, which uncovers tws, showed wing disc duplications (data not shown). As all the three alleles showed pattern duplication in wing discs, their phenotypes do not seem to be allele-specific. We have used the stronger two alleles (twsP and tws60) in our studies reported here. Both the alleles are recessive; the heterozygous flies do not show any phenotype.

Fig. 1.

Effect of tws mutation on Wg, DV-Vg and Ct expression patterns.(A-C) Tws expression levels (detected by anti-Tws antibody staining) in wild-type (A), twsP/+ (B) and twsP/twsP (C) wing discs. Homozygous mutants barely have any gene product. (D,E) Wild-type (D) and twsP/twsP (E) wing discs stained for Ci, which is expressed only in the anterior compartment. Note complete mirror-symmetry duplication in twsP/twsP disc as reported by Uemura et al. (Uemura et al., 1993).(F,G) Wild-type (F) and twsP/twsP (G)wing discs stained for Wg. (H,I) Higher magnification images of wild-type (H)and twsP/twsP (I) wing discs showing Wg expression in the DV boundary in more detail. Note that Wg levels in the DV boundary of twsP/twsP discs are marginally lower, while the expression pattern itself is unchanged compared with wild type. (J,K) Wild-type (J) and twsP/twsP (K) wing discs stained for vg-BE. There is no change in the expression pattern of vg-BE between normal and mutant discs. (L,M) Wild type (L) and twsP/twsP (M) wing discs stained for Ct. Note reduction in Ct expression levels in the mutant disc.

Fig. 1.

Effect of tws mutation on Wg, DV-Vg and Ct expression patterns.(A-C) Tws expression levels (detected by anti-Tws antibody staining) in wild-type (A), twsP/+ (B) and twsP/twsP (C) wing discs. Homozygous mutants barely have any gene product. (D,E) Wild-type (D) and twsP/twsP (E) wing discs stained for Ci, which is expressed only in the anterior compartment. Note complete mirror-symmetry duplication in twsP/twsP disc as reported by Uemura et al. (Uemura et al., 1993).(F,G) Wild-type (F) and twsP/twsP (G)wing discs stained for Wg. (H,I) Higher magnification images of wild-type (H)and twsP/twsP (I) wing discs showing Wg expression in the DV boundary in more detail. Note that Wg levels in the DV boundary of twsP/twsP discs are marginally lower, while the expression pattern itself is unchanged compared with wild type. (J,K) Wild-type (J) and twsP/twsP (K) wing discs stained for vg-BE. There is no change in the expression pattern of vg-BE between normal and mutant discs. (L,M) Wild type (L) and twsP/twsP (M) wing discs stained for Ct. Note reduction in Ct expression levels in the mutant disc.

Wg signaling is compromised in tws mutant discs

Both twsP and tws60 alleles survive up to early pupal stages, probably owing to maternal contribution of the gene product in the embryo. We made attempts to generate germline clones (see Materials and methods) to remove maternal contribution of tws. Embryos not carrying any maternal product of tws die prior to segmentation (data not shown). We, therefore, restricted our analyses using wing development, which is also a convenient assay system to study Wg pathway.

Growth and patterning during fly wing development are mediated by signaling from its dorsoventral (DV) organizer. Interactions between dorsal and ventral cells of the wing pouch set up the organizer by activating Notch (N) in the DV boundary (Diaz-Benjumea and Cohen,1993; Diaz-Benjumea and Cohen,1995; Williams et al.,1994; Irvine and Wieschaus,1994; Kim et al.,1996; de Celis et al.,1996). N, in turn, activates Wingless (Wg), Cut (Ct) and Vestigial(Vg) in the DV boundary (Couso et al.,1995; Kim et al.,1995; Rulifson and Blair,1995; Kim et al.,1996; Neumann and Cohen,1996). Wg is known to diffuse to non-DV cells from the DV boundary and acts as a morphogen (Zecca et al.,1996; Neumann and Cohen,1997). First, it collaborates with N to activate Cut (Ct) in a cell-autonomous manner (de Celis and Bray,1997). Highest levels of secreted Wg are required to activate Achaete (Ac) and Scabrous (Sca) expression in the sensory mother cells (SMC)along the DV boundary (Zecca et al.,1996; Neumann and Cohen,1997), whereas moderate levels are enough to activate Distal-less(Dll) and low-levels to activate Vg(Neumann and Cohen, 1997). Thus, Vg is expressed in both DV and non-DV cells. Two different enhancers regulate Vg expression in DV and non-DV cells(Kim et al., 1996). They are vg-boundary enhancer (vg-BE) and vg-quadrant enhancer (vg-QE).

Anti-Wg antibody staining of twsP mutant discs revealed that the levels of Wg are marginally reduced, but the pattern of expression is unchanged (Fig. 1G,I). However, vg-BE expression remained robust(Fig. 1K), suggesting that N pathway is not affected in tws mutant discs. We then examined a number of targets of Wg pathway. Ct expression, which is dependent on both N and Wg signaling, is normally seen as a continuous narrow line along the entire DV boundary (Fig. 1L). In tws mutant wing discs, its expression is irregular and discontinuous (Fig. 1M). We observed partial to complete loss of Ac(Fig. 2B) and Sca(Fig. 2D) expression,particularly in the presumptive margin SMCs, and moderate to severe reduction in Dll and vg-QE expression (Fig. 2F,H). Downregulation of Ac, Sca, Dll and vg-QE suggests that Wg signaling is down regulated in twsP wing discs. Observed downregulation of Wg expression could be due to interference in its autoregulation.

Fig. 2.

Downregulation of targets of Wg signaling in twsP/twsP wing discs. Wild-type wing discs (A,C,E,G) and twsP/twsP wing discs (B,D,F,H) showing the expression patterns of Ac (A,B), Sca (C,D), Dll(E,F) and vg-QE (G,H). Note that, compared with their wild-type counterparts, levels of all the targets of Wg are lower in twsP background. Control and experimental images were digitized at identical fluorescence and camera settings.

Fig. 2.

Downregulation of targets of Wg signaling in twsP/twsP wing discs. Wild-type wing discs (A,C,E,G) and twsP/twsP wing discs (B,D,F,H) showing the expression patterns of Ac (A,B), Sca (C,D), Dll(E,F) and vg-QE (G,H). Note that, compared with their wild-type counterparts, levels of all the targets of Wg are lower in twsP background. Control and experimental images were digitized at identical fluorescence and camera settings.

Uemura et al. (Uemura et al.,1993) have reported normal expression of Sca in margin SMCs in tws mutant background. They used the lacZ enhancer-trap insertion strain ScaryXho38 in tws60 discs,while we examined endogenous levels of Sca protein in twsPmutant discs. Therefore, the apparent discrepancy is probably due to the differences in the genetic backgrounds (twsP is stronger than tws60) and assays (lacZ versus antibody staining) used.

Clonal analysis of twsP allele

Serrated wing margin phenotypes are characteristics of loss of Wg(hypomorphic wg mutants)(Phillips and Whittle, 1993)or loss of its upstream regulators such as N and Ser(Neumann and Cohen, 1996). Downregulation of targets of Wg such as Ac and Sca in twsPwing discs predict serrated anterior margin in adult wings. As twsP mutants die in late larval/early pupal stages, we employed a clonal analysis approach to examine the requirement of twsgene product in adult wing margin formation. We observed higher frequency of mitotic clones when FLP expression was induced at 48-72 hours AEL. Consistent with our prediction on downregulation of Wg signaling in twsP discs, a large number of adult flies with twsP/twsP clones showed serrated wing margin phenotype (n=27; Fig. 3A,B). Interestingly, the frequency of the clones observed in imaginal discs was far higher than the number of clones observed in pharate adult/adult flies. Consistent with this observation, imaginal discs showed large clusters of tws+/tws+ cells with small or no associated twsP/twsP spots(Fig. 3C). Because we also observed larger twsP/twsP clones(Fig. 3D), it is likely that they grow slowly and subsequently eliminated by neighboring cells.

Fig. 3.

Loss-of-function clones of twsP affect wing margin.(A,B) Cuticle of phenotypes displayed by the mitotic clones of twsP. We observed characteristic serrated-margin phenotypes in both anterior (A) and posterior (B) compartments. (C,D) Wing discs with twsP/twsP clones. tws+/tws+ cells are marked with GFP. Note large clusters of tws+/tws+ cells with smaller or no associated twsP/twsP spots (C). Occasionally larger clusters of twsP/twsP cells were also seen (D). (E-H) Wing discs with twsP/twsP clones stained for Wg (E),Ct (F), Dll (G) and vg-QE (H). Arm-lacZ (red) was used to mark twsP/twsP clones in E-G, and GFP was used in H. For all the discs, the viability of twsP/twsP clones were confirmed by DAPI staining (data not shown). We did not observe any change in Wg expression levels, while Ct showed marginal reduction in its levels. Partial to complete loss of Dll and vg-QE was observed in twsP/twsP clones.

Fig. 3.

Loss-of-function clones of twsP affect wing margin.(A,B) Cuticle of phenotypes displayed by the mitotic clones of twsP. We observed characteristic serrated-margin phenotypes in both anterior (A) and posterior (B) compartments. (C,D) Wing discs with twsP/twsP clones. tws+/tws+ cells are marked with GFP. Note large clusters of tws+/tws+ cells with smaller or no associated twsP/twsP spots (C). Occasionally larger clusters of twsP/twsP cells were also seen (D). (E-H) Wing discs with twsP/twsP clones stained for Wg (E),Ct (F), Dll (G) and vg-QE (H). Arm-lacZ (red) was used to mark twsP/twsP clones in E-G, and GFP was used in H. For all the discs, the viability of twsP/twsP clones were confirmed by DAPI staining (data not shown). We did not observe any change in Wg expression levels, while Ct showed marginal reduction in its levels. Partial to complete loss of Dll and vg-QE was observed in twsP/twsP clones.

In viable tws- mitotic clones (confirmed by DAPI staining), we did not observe any change in the levels of Wg expression(Fig. 3E), whereas we observed marginal reduction in Ct expression levels(Fig. 3F). Furthermore, we observed significant reduction in both Dll(Fig. 3G) and vg-QE(Fig. 3H) expression in tws- mitotic clones. Observation of particular significance here is the cell-autonomous reduction in Dll and vg-QE expression. This supports our earlier conclusion that Wg signal transduction pathway, and not the expression of Wg ligand, is compromised in tws- cells. The observed reduction in Wg levels in twsP/twsP wing discs(Fig. 1G,I) is therefore attributed to interference in its autoregulation.

Arm is not stabilized in tws mutant wing discs

We then examined the levels of Arm, the key regulator of Wg signaling. In addition to being an effector of Wg signaling, Arm binds to intracellular domain of E-cadherin and participates in modulating the cell-adhesion properties of the cell. Stabilization of cytoplasmic Arm is a key step in the transduction of Wg signaling. Thus, although Arm is present in all the cells,cytoplasmic levels of Arm, which transduces Wg signaling, are higher only in cells wherein Wg signaling is active(Peifer et al., 1994). In wing discs, cells immediately adjacent to the DV boundary show higher levels of Arm than non-DV cells (Jiang and Struhl,1998; Collins and Treisman,2000; Bhandari and Shashidhara,2001; Mohit et al.,2003) (Fig. 4A). Unlike in wild-type discs, Arm levels are lower in the DV boundary of all tws homozygous mutant discs examined (n>40; Fig. 4B). In twsP/twsP wing discs, peripodial and disc proper cells are often in the same focal plane, suggesting that these discs are flatter than the wild-type discs(Fig. 4B). We therefore examined if loss of Arm in tws- background is due to changed disc morphology. Noncytoplasmic Arm (Cadherin-bound form) is normally localized to the apical/subapical surface of wing epithelial cells. However,there was no difference between wild-type and twsP/twsP discs in the intensities of Arm in adherens junctions of cells around the DV boundary(Fig. 4B). In addition to Arm,we used actin-phalloidin to mark the apical surface and Fas3 to mark the basolateral surface. Confocal examination of tws homozygous mutant discs stained with these markers suggested normal apicobasal polarity of disc cells (data not shown). In addition, normally observed intense staining of actin-phalloidin in the cells adjacent to the DV boundary(Fig. 4D) was not affected in tws- wing discs (Fig. 4F), indicating similar cell densities around the DV boundary of wild-type and mutant wing discs. Thus, observed reduction in Arm levels in tws- discs is not due to loss of apicobasal polarity, or to changes in cell morphology or to a decrease in the density of cells in and around the DV boundary. Finally, clonal removal of twsPfrom the cells abutting the DV boundary caused cell-autonomous downregulation of Arm to the levels normally seen in distant non-DV cells(Fig. 4G). Therefore, reduction in Arm in tws- discs is probably due to lowering of the levels of cytoplasmic Arm.

Fig. 4.

Arm is not stabilized in the DV boundary of twsP/twsP wing discs. (A) Wild-type wing disc stained with anti-Arm (red) and anti-Wg (green) antibodies. Note higher levels of Arm in cells adjacent to Wg-expressing DV boundary compared with distant non-DV cells. (B) twsP/twsP wing disc stained with anti-Arm (red) and anti-Wg (green) antibodies. Note reduction in the levels of Arm in cells adjacent to the Wg-expressing DV boundary. Also seen in B are large peripodial cells. In wild-type wing discs, they normally are not in the same focal plane as disc cells. A′ and B′ show x-z sections of the cells around the DV boundary of wild type (A′) and twsP/twsP(B′) wing discs. Although Arm is localized apically, owing to its stabilization in the cytoplasm of DV cells, it is seen all along the cross-section of the cells. However, in mutant cells, Arm is localized only to the apical adherens junctions. (C,D) Wild-type wing disc double-stained (but shown in single channels) with anti-Arm (C) and FITC-conjugated actin-phalloidin (D). Note that both Arm and Actin-phalloidin show intense staining in the cells surrounding the DV boundary (arrows in C,D). (E,F) twsP/twsP wing disc double-stained(shown in single channels) with anti-Arm (E) and FITC-conjugated actin-phalloidin (F). Note reduced levels of Arm in the DV boundary (arrow in E). However, actin-phalloidin staining is as intense as in the wild-type disc(arrow in F). (G) hsFLP; P[FRT]82 twsP/P[FRT]82 arm-lacZ wing disc stained for Arm (green). twsP/twsP mitotic clones are marked with arm-lacZ (red), which is shown in single channel in G′. G″ is a merge of G and G′. A large clone abutting the DV boundary is shown. Note that levels of Arm are lower in twsP/twsP cells compared with neighboring tws+ cells. The viability of twsP/twsP cells were confirmed by DAPI staining (data not shown). (H,I) vg-GAL4; UAS-armS2 (H)and vg-GAL4; UAS-armS2twsP/twsP (I) wing discs stained for Myc tag, which marks expression of only the overexpressed Arm. Note that Myc staining in I is significantly lower than in H, suggesting enhanced degradation of Arm in twsP/twsP discs.(J,K) UAS-armS10;vg-GAL4 (J) and UAS-armS10;vg-GAL4; twsP/twsP (K) wing discs stained for Myc tag. Levels of Myc in K are comparable with those in J,suggesting that twsP/twsP has no effect on degradation-resistant form of Arm.

Fig. 4.

Arm is not stabilized in the DV boundary of twsP/twsP wing discs. (A) Wild-type wing disc stained with anti-Arm (red) and anti-Wg (green) antibodies. Note higher levels of Arm in cells adjacent to Wg-expressing DV boundary compared with distant non-DV cells. (B) twsP/twsP wing disc stained with anti-Arm (red) and anti-Wg (green) antibodies. Note reduction in the levels of Arm in cells adjacent to the Wg-expressing DV boundary. Also seen in B are large peripodial cells. In wild-type wing discs, they normally are not in the same focal plane as disc cells. A′ and B′ show x-z sections of the cells around the DV boundary of wild type (A′) and twsP/twsP(B′) wing discs. Although Arm is localized apically, owing to its stabilization in the cytoplasm of DV cells, it is seen all along the cross-section of the cells. However, in mutant cells, Arm is localized only to the apical adherens junctions. (C,D) Wild-type wing disc double-stained (but shown in single channels) with anti-Arm (C) and FITC-conjugated actin-phalloidin (D). Note that both Arm and Actin-phalloidin show intense staining in the cells surrounding the DV boundary (arrows in C,D). (E,F) twsP/twsP wing disc double-stained(shown in single channels) with anti-Arm (E) and FITC-conjugated actin-phalloidin (F). Note reduced levels of Arm in the DV boundary (arrow in E). However, actin-phalloidin staining is as intense as in the wild-type disc(arrow in F). (G) hsFLP; P[FRT]82 twsP/P[FRT]82 arm-lacZ wing disc stained for Arm (green). twsP/twsP mitotic clones are marked with arm-lacZ (red), which is shown in single channel in G′. G″ is a merge of G and G′. A large clone abutting the DV boundary is shown. Note that levels of Arm are lower in twsP/twsP cells compared with neighboring tws+ cells. The viability of twsP/twsP cells were confirmed by DAPI staining (data not shown). (H,I) vg-GAL4; UAS-armS2 (H)and vg-GAL4; UAS-armS2twsP/twsP (I) wing discs stained for Myc tag, which marks expression of only the overexpressed Arm. Note that Myc staining in I is significantly lower than in H, suggesting enhanced degradation of Arm in twsP/twsP discs.(J,K) UAS-armS10;vg-GAL4 (J) and UAS-armS10;vg-GAL4; twsP/twsP (K) wing discs stained for Myc tag. Levels of Myc in K are comparable with those in J,suggesting that twsP/twsP has no effect on degradation-resistant form of Arm.

To further test if stabilization of cytoplasmic Arm is compromised in twsP discs, we used Myc-tagged degradation-resistant and degradation-sensitive forms of Arm (armS10 and armS2)(Pai et al, 1997). armS2 functions as wild-type protein and is susceptible to the degradation machinery (Pai et al,1997). Furthermore, similar to endogenous Arm, armS2 is stabilized only in Wg signaling cells, e.g. in the wing disc DV boundary(Mohit et al., 2003). Thus,relative levels of armS2 in the DV boundary of wing discs can be used as an estimate of the relative efficiency of Arm-degradation machinery. Anti-Myc antibody staining of discs expressing armS2 showed accumulation of Myc tag in the DV cells of wild-type discs(Fig. 4H), but not in twsP/twsP discs(Fig. 4I). This suggests enhanced-degradation of Arm in tws- discs. armS10 has an internal deletion of residues 43-87 at the N terminus, which removes residues that are normally phosphorylated by Sgg, thus making Arm degradation resistant (Pai et al, 1997). In contrast to the effect of tws mutation on endogenous Arm and overexpressed armS2, the levels of degradation-resistant form of Arm was not affected by twsPmutation (Fig. 4K). These results suggest that Tws functions upstream of Arm and probably mediates stabilization of Arm levels in the cytoplasm in response to Wg signaling.

Tws functions downstream of Dsh and upstream of Sgg

We further examined if twsP modifies phenotypes induced by the overexpression of known positive and negative effectors of Wg signaling. Positive regulators such as Dsh and activated Arm and negative regulators such as Sgg, dominant-negative TCF/pan (DN-TCF/pan), intracellular domain of Cadherin (Cadintra) were overexpressed in wild-type and in twsP heterozygous backgrounds(Table 1). Severity of wing margin phenotypes (supernumerary bristles; Fig. 5A) induced by ectopic Dsh was significantly reduced when it was expressed in twsPheterozygous background (Fig. 5B). Conversely, wing-to-notum transformations induced by ectopic Sgg (Fig. 5C) were significantly enhanced when expressed in twsP heterozygous background (Fig. 5D). Interestingly, phenotypes generated by the ectopic expression of degradation-resistant forms of Arm (UAS-ΔArm or UAS-armS10crossed to vg-GAL4) were not even marginally affected by twsP (data not shown).

Table 1.

twsPsuppresses phenotypes caused by gain of Wg and enhances those caused by loss of Wg

Construct or gene overexpressed (vg-GAL4 mediated)Status of tws mutation
+/+ (n≥40 in all cases)twsP/+ (n≥30 in all cases)
Positive regulators of Wg signaling   
Dsh Tufts of ectopic bristles Supressed (very few bristles seen) 
Activated Arm Tufts of ectopic bristles Unaffected (tufts of ectopic bristles) 
Negative regulators of Wg signaling   
hAPC/FL No phenotype Enhanced (mild serrated margin) 
Sgg/GSK3 Reduced wings, serrated margins and 30% flies show partial wing to notum transformation Enhanced (complete wing to notum transformation and the frequency increased to 87%) 
DN-TCF/pan Reduced wings and serrated margins (normally seen in 30% of flies) Enhanced [severe wing-margin and also wing to notum transformation (100% of flies)] 
Cadintra Serrated wing margin Enhanced (increase in both the number and severity of serrated margins) 
Construct or gene overexpressed (vg-GAL4 mediated)Status of tws mutation
+/+ (n≥40 in all cases)twsP/+ (n≥30 in all cases)
Positive regulators of Wg signaling   
Dsh Tufts of ectopic bristles Supressed (very few bristles seen) 
Activated Arm Tufts of ectopic bristles Unaffected (tufts of ectopic bristles) 
Negative regulators of Wg signaling   
hAPC/FL No phenotype Enhanced (mild serrated margin) 
Sgg/GSK3 Reduced wings, serrated margins and 30% flies show partial wing to notum transformation Enhanced (complete wing to notum transformation and the frequency increased to 87%) 
DN-TCF/pan Reduced wings and serrated margins (normally seen in 30% of flies) Enhanced [severe wing-margin and also wing to notum transformation (100% of flies)] 
Cadintra Serrated wing margin Enhanced (increase in both the number and severity of serrated margins) 

Modification of phenotypes induced by various positive and negative regulators of Wg signaling in twsP/+ background. twsP does not affect phenotypes induced by armS10, suggesting that Tws may function upstream of Arm. However,none of the hetero-alleleic combinations such as (1) spdhl2/+; twsP/+,(2) wgP/+; twsP/+,(3) spdfg/spdfg; twsP/+, (4) sggM111/+; twsP/+and (5) armH4/+; twsP/+ showed any dominant interaction.

Fig. 5.

Tws functions downstream of Dsh to inhibit Sgg activity. (A,B) vg-GAL4/UAS-Dsh (A) and vg-GAL4/+;UAS-Dsh/twsP (B) wing blades. Inset in A shows expression pattern of vg-GAL4. Dsh-induced phenotype (ectopic bristles along the posterior margin) is suppressed by one copy of twsPmutation. (C) vg-GAL4;UAS-Sgg fly showing underdeveloped wing blades owing to loss of Wg signaling. (D) vg-GAL4/+;UAS-Sgg/twsP fly showing enhanced phenotype. Note total loss of wings and wing-to-notum transformation (shown at higher magnification in D′), a characteristic loss-of-wg phenotype. (E,F) vg-GAL4/+; UAS-Dsh twsP/twsP(E) and vg-GAL4/UASDNGSK-3β; twsP/twsP (F) wing discs stained for Dll expression. Overexpression of dominant-negative Sgg/GSK3β, but not Dsh, causes the rescue of twsP/twsPdiscs at the level of Dll expression (refer to Fig. 2F for Dll expression in twsP/twsP discs). (G) vg-GAL4/UAS-APC/CBD wing disc stained for Arm. Note very high levels of Arm in the DV boundary (compare with Fig. 4C) because of its sequestration by APC. In these cells, APC sequesters Arm, because Sgg (thereby the degradation machinery) is inhibited by Wg. (H) vg-GAL4/UAS-hAPC/CBD; twsP/twsP wing disc stained for Arm. In tws mutant background, APC fails to sequester Arm, suggesting that Sgg is active in all cells including the DV boundary.

Fig. 5.

Tws functions downstream of Dsh to inhibit Sgg activity. (A,B) vg-GAL4/UAS-Dsh (A) and vg-GAL4/+;UAS-Dsh/twsP (B) wing blades. Inset in A shows expression pattern of vg-GAL4. Dsh-induced phenotype (ectopic bristles along the posterior margin) is suppressed by one copy of twsPmutation. (C) vg-GAL4;UAS-Sgg fly showing underdeveloped wing blades owing to loss of Wg signaling. (D) vg-GAL4/+;UAS-Sgg/twsP fly showing enhanced phenotype. Note total loss of wings and wing-to-notum transformation (shown at higher magnification in D′), a characteristic loss-of-wg phenotype. (E,F) vg-GAL4/+; UAS-Dsh twsP/twsP(E) and vg-GAL4/UASDNGSK-3β; twsP/twsP (F) wing discs stained for Dll expression. Overexpression of dominant-negative Sgg/GSK3β, but not Dsh, causes the rescue of twsP/twsPdiscs at the level of Dll expression (refer to Fig. 2F for Dll expression in twsP/twsP discs). (G) vg-GAL4/UAS-APC/CBD wing disc stained for Arm. Note very high levels of Arm in the DV boundary (compare with Fig. 4C) because of its sequestration by APC. In these cells, APC sequesters Arm, because Sgg (thereby the degradation machinery) is inhibited by Wg. (H) vg-GAL4/UAS-hAPC/CBD; twsP/twsP wing disc stained for Arm. In tws mutant background, APC fails to sequester Arm, suggesting that Sgg is active in all cells including the DV boundary.

We then examined the effect of overexpression of Dsh and a dominant-negative form of Sgg/GSK-3β (DN-GSK3β) in homozygous twsP wing discs. We overexpressed Dsh and DNGSK3β in the wing disc DV boundary using vg-GAL4 driver. As the requirement of Dsh, Sgg and Tws is cell autonomous, we examined the effect only at the levels of Dll expression. Overexpression of Dsh in twsP/twsP wing discs did not affect the levels of Dll (Fig. 5E),which remained at the levels normally seen in mutant discs (compare with Fig. 2F). By contrast,overexpression of DN-GSK3β caused significant rescue of wing discs at the level of Dll expression (Fig. 5F). These results suggest that Tws normally functions downstream of Dsh and upstream of Sgg.

In both Drosophila and in mammalian cells, APC binds to Arm/β-catenin even when Wg/Wnt is active(Papkoff et al., 1996; Bhandari and Shashidhara,2001). In those cells, wherein Sgg is inactivated, APC sequesters Arm/β-catenin rather than recruiting it to degradation machinery. For example, overexpression of human colon cancer gene APC in wing discs sequesters Arm only in DV cells (Bhandari and Shashidhara, 2001; Mohit et al., 2003) (Fig. 5G). In other cells, overexpressed APC participates in the Arm-degradation machinery and hence no change in Arm expression is observed. Because only unphosphorylated Arm/β-catenin is sequestered and not the phosphorylated form (Munemitsu et al.,1996), the amount of Arm sequestered by overexpressed APC would provide a relative estimate of Sgg activity. Overexpressed APC in twsP/twsP background did not sequester Arm (Fig. 5H), indicating that in this genetic background Sgg is active in DV cells. Thus, the normal function of tws gene appears to be inactivation of Sgg in response to Wg signal.

Ectopic Tws induces gain-of-Wg-function phenotypes

Results described above show that Tws functions as a positive regulator of Wg signaling, which is consistent with the phenotypes observed in mice lacking the catalytic subunit of PP2A. Most of the observations against this, wherein PP2A is shown as a negative regulator of Wnt/Wg signaling comes from studies involving overexpression of the regulatory subunit. We therefore analyzed the effect of ectopic expression of Tws in the context of Wg signaling.

We generated transgenic flies that enable overexpression of Tws using GAL4-UAS system. We first tested if the transgene expresses normal protein. We expressed Tws using hs-GAL4 driver in homozygous twsP and tws60 backgrounds and examined if UAS-Tws could rescue the mutations. Temperature-shift experiments, wherein the developing animals were kept at temperatures between 25°C and 28°C for varying lengths of time, did not show any rescue of twsP mutant phenotypes. We also tried a number of heat-shock regimes (1 hour pulse at 37°C), but with no success. However, low levels of expression of UAS-Tws driven by the basal activity of the heat-shock promoter at 25°C was sufficient to rescue late larval/early pupal lethal phenotype of tws60. Rescued pharate adults showed distinctive adult structures(Fig. 6B,C, n=20), but were unable to eclose. Significantly, wing blades of rescued pharate adults showed normal wing margin, suggesting recovery of Wingless signaling. No wing disc showed pattern duplication (n=28), suggesting rescue of both Wingless-dependent and independent developmental events. Inability to rescue twsP, the stronger of the two alleles, suggests that survival of homozygous twsP larvae may require higher levels of tws gene product. Indeed, expression of UAS-Tws with the help of a stronger driver (en-GAL4) resulted in the rescue of twsP mutant flies at the levels of both pattern duplication in discs (data not shown) and adult wing blade(Fig. 6D). Close observation of the cuticle in the rescued pharate adults indicated that the rescue was limited to posterior compartment (data not shown), which reflects the fact that Tws was expressed only in that compartment.

Fig. 6.

Rescue of tws mutant phenotypes by UAS-Tws. (A) tws60/tws60 pupa. Homozygous twsP and tws60 mutants die at early pupal stages and do not show any signs of appendage development. (B,C) Ventral(B) and dorsal (C) views of two different tws60/tws60 pharate adults rescued by the expression of UAS-Tws using hs-GAL4 driver. Note normal development of all appendages. Eye in of the pharate adult in B is white because of prolonged preservation of the sample in ethanol prior to photography. (D) Partial rescue of twsP/twsP flies by en-GAL4 mediated UAS-Tws expression. Note that development of all appendages is partially rescued as Tws is expressed only in the posterior compartment. Overexpression of Tws in tws60/tws60background induces gain-of-Wg phenotype. (E,F) Wild-type wing blades showing posterior hinge (E) and anterior margin (F) at higher magnification. (G)UAS-armS10; vg-GAL4 wing blade with tufts of ectopic bristles (arrow) at the posterior hinge. (H) 405-GAL4/UAS-armS10wing blade with anterior margin-specific bristles in ectopic positions(arrow). Inset shows 405-GAL4 expression pattern. (I,J) Similar phenotypes(arrows) are observed when Tws is overexpressed in tws60/tws60 background using hs-GAL4.

Fig. 6.

Rescue of tws mutant phenotypes by UAS-Tws. (A) tws60/tws60 pupa. Homozygous twsP and tws60 mutants die at early pupal stages and do not show any signs of appendage development. (B,C) Ventral(B) and dorsal (C) views of two different tws60/tws60 pharate adults rescued by the expression of UAS-Tws using hs-GAL4 driver. Note normal development of all appendages. Eye in of the pharate adult in B is white because of prolonged preservation of the sample in ethanol prior to photography. (D) Partial rescue of twsP/twsP flies by en-GAL4 mediated UAS-Tws expression. Note that development of all appendages is partially rescued as Tws is expressed only in the posterior compartment. Overexpression of Tws in tws60/tws60background induces gain-of-Wg phenotype. (E,F) Wild-type wing blades showing posterior hinge (E) and anterior margin (F) at higher magnification. (G)UAS-armS10; vg-GAL4 wing blade with tufts of ectopic bristles (arrow) at the posterior hinge. (H) 405-GAL4/UAS-armS10wing blade with anterior margin-specific bristles in ectopic positions(arrow). Inset shows 405-GAL4 expression pattern. (I,J) Similar phenotypes(arrows) are observed when Tws is overexpressed in tws60/tws60 background using hs-GAL4.

Activation of Wg pathway by overexpressing Wg or other positive regulators like Dsh, stable/activated form of Arm etc., in the wing blade results in bristle formation at ectopic positions(Phillips and Whittle, 1993; Rulifson et al., 1996)(Fig. 6G,H). As the heat shock promoter causes ubiquitous expression of the GAL4, and if tws indeed is a positive regulator of Wg signaling, ectopic bristles are likely to appear in the wing blades of rescued flies. Consistent with this hypothesis,scattered ectopic bristles were present within the wing blades of rescued flies (Fig. 6I,J).

Overexpression of Tws in the wild-type background functions as dominant negative

Results described in the previous section clearly indicate that UAS-Tws construct we employed in our study functions like wild-type Tws. It rescued tws mutation and also showed gain-of-Wg signaling. However, when Tws was overexpressed in wild-type background, we observed downregulation of Wg signaling. Overexpression of Tws specifically in the DV boundary using vg-GAL4 was enough to cause downregulation of Ct expression in the DV boundary (Fig. 7A), Sca expression in the presumptive margin SMCs(Fig. 7B), and Dll(Fig. 7C) and Vg(Fig. 7E) expression in non-DV cells. Arm levels along the DV boundary were also downregulated in wing discs overexpressing Tws using vg-GAL4(Fig. 7G). Adult flies displayed characteristic serrated wing margin(Fig. 7H), which is similar to the phenotype observed in wing blades carrying twsPmitotic clones. In addition, we observed enhanced notching of wing margin when Tws was overexpressed in wgP/+ background (data not shown). These results suggest that ectopic Tws in wild-type background functions as dominant-negative that recreates the tws mutant phenotypes both at the molecular levels in discs and adult phenotypic levels. This is further supported by the observation that co-expression of degradation resistant form of Arm (armS10) with Tws was able to suppress wing margin phenotype (Fig. 7I). However, degradation sensitive form of Arm (armS2) did not show any effect on Tws-induced phenotypes (data not shown). This suggests that, similar to loss of tws, overexpressed Tws causes downregulation of Wg signaling by interfering with an event downstream of Dsh and upstream of Arm.

Fig. 7.

Dominant-negative phenotypes caused by the overexpression of Tws in wild-type background. (A-C and E) vg-GAL4/UAS-Tws wing discs stained with Ct (A), Sca (B), Dll (C) and Vg (E) antibodies. (D) Wild-type wing disc stained with anti-Vg antibodies. Tws overexpression caused the downregulation of both short-range (Ct expression in the DV boundary and Sca expression in the presumptive margin SMCs; arrow in B) and long-range (Dll and Vg) targets of Wg signaling. Refer to Fig. 1L, Fig. 2C and Fig. 2E, for wild-type expression patterns of Ct, Sca and Dll, respectively. (F-G) Wild-type (F) and vg-GAL4/UAS-Tws (G) wing discs stained with Arm. Note overexpression of Tws causes reduction in cytoplasmic Arm levels. (H) vg-GAL4/UAS-Tws wing blade showing serrated posterior margin. (I)UAS-armS10; vg-GAL4/UAS-Tws wing blade. Note suppression of Tws-induced serrated margin phenotype. This wing blade is indistinguishable from UAS-armS10; vg-GAL4 wing blades (data not shown).

Fig. 7.

Dominant-negative phenotypes caused by the overexpression of Tws in wild-type background. (A-C and E) vg-GAL4/UAS-Tws wing discs stained with Ct (A), Sca (B), Dll (C) and Vg (E) antibodies. (D) Wild-type wing disc stained with anti-Vg antibodies. Tws overexpression caused the downregulation of both short-range (Ct expression in the DV boundary and Sca expression in the presumptive margin SMCs; arrow in B) and long-range (Dll and Vg) targets of Wg signaling. Refer to Fig. 1L, Fig. 2C and Fig. 2E, for wild-type expression patterns of Ct, Sca and Dll, respectively. (F-G) Wild-type (F) and vg-GAL4/UAS-Tws (G) wing discs stained with Arm. Note overexpression of Tws causes reduction in cytoplasmic Arm levels. (H) vg-GAL4/UAS-Tws wing blade showing serrated posterior margin. (I)UAS-armS10; vg-GAL4/UAS-Tws wing blade. Note suppression of Tws-induced serrated margin phenotype. This wing blade is indistinguishable from UAS-armS10; vg-GAL4 wing blades (data not shown).

The Wg/Wnt signal transduction pathway plays an important role in a variety of developmental and differentiation processes in many organisms (reviewed by Wodarz and Nusse, 1998) and is aberrantly activated in majority of human colorectal cancers as well as in other tumor types (reviewed by Bienz and Clevers, 2000; Peifer and Polakis, 2000; Polakis,2000). Newly emerging information implicates Wnt pathway in regulating angiogenesis (Ishikawa et al,2001; Wright et al,1999), adipogenesis (Ross et al, 2000) and B-lymphocyte proliferation(Reya et al, 2000). Thus,understanding the regulation of Wg/Wnt signaling would not only help us to understand developmental processes, but also to understand the onset and progression of various diseases.

Similar to many other signal transduction pathways, changes in phosphorylation-dephosphorylation status of Wg/Wnt pathway components are key to its regulation. Although the role of PP2A as a dephosphorylating agent is established by studies on Pp2a- mice, there has not been a consensus on (1) whether PP2A positively or negatively regulates Wg/Wnt signaling, (2) does PP2A function downstream or upstream (or both) ofβ-catenin and (3) which regulatory subunit of PP2A functions to modify Wg/Wnt signaling.

tws is a positive regulator of Wg/Wnt signaling

Results of our studies described in this report show that Drosophila homologue of B/PR55 subunit of PP2A is involved in modifying Wg signaling. We have observed partial to complete downregulation of short- (Ct and Sca) and long-range (Dll and Vg) targets of Wg pathway in tws- background. The downregulation of Wg signaling in wing discs was reflected in adult phenotypes, such as serrated wing margin in mitotic clones of tws. We have also observed that loss-of-Wg phenotypes (induced by the overexpression of DN-TCF/pan or Sgg or Cadintra) are enhanced in tws heterozygous mutant background. In addition, mutation in tws suppressed the phenotypes induced by Dsh, a positive component of Wg signaling. Finally, some of the phenotypes induced by the overexpression of Tws are characteristic of gain-of-Wg phenotypes. These results suggest that Tws functions as a positive regulator of Wg signaling.

We have further shown that overexpression of, otherwise normal, Tws protein induce dominant-negative phenotypes. The dominant-negative phenotype is unlikely to be neomorphic or antimorphic, as UAS-Tws rescued twsalleles (at the levels of both Wingless-dependent and independent developmental events) and also induced gain-of-Wg phenotypes. The dominant-negative phenotype is probably due to imbalance in the relative amounts of the three subunits in the heterotrimeric complex, proper formation of which is obligatory for PP2A function. Thus, the conflicting reports on the role of PP2A in Wnt signaling could be due to the dominant negative effect caused by the overexpression of one of the subunits.

Mechanism of Tws function

In tws mutant background, cytoplasmic Arm levels are downregulated. Even the overexpressed Arm was degraded in tws- background. Furthermore, loss of tws had no effect on the degradation-resistant form of Arm, which suggests that Tws functions upstream of Arm to mediate Wg signaling. We could not confirm these results directly by western blotting, as only a very small fraction (such as DV cells) of wing disc shows changes in Arm levels in response to Wg signaling. Nevertheless, results presented in this report suggest that stabilization of the cytoplasmic form of Arm by Wg signaling is dependent on Tws function.

A dominant-negative form of Sgg/GSK-3β was able to rescue tws- phenotype at the level of Dll expression. However,overexpression of Dsh failed to rescue Dll expression in tws- discs, suggesting that Tws functions downstream of Dsh and upstream of Sgg to stabilize cytoplasmic Arm in response to Wg signaling. Preliminary results presented here suggest that function of Tws in Wg pathway is inactivation of Sgg. Normally, overexpressed APC sequesters Arm only in those cells in which Sgg activity is downregulated(Bhandari and Shashidhara,2001). In other cells, APC participates in Arm-degradation machinery. In tws- wing discs, overexpressed APC failed to sequester Arm in DV cells, suggesting that loss of tws results in upregulation of Sgg activity. However, recently, Yang et al.(Yang et al., 2003) have reported that PR/B56ϵ functions upstream of Dsh to regulate Wnt signaling in Xenopus embryos. The PR/B56ϵ homologue in Drosophilais widerborst (with 80% identity at the protein level), which is involved in the determination of planar cell polarity(Hannus et al., 2002). widerborst is also known to be functional upstream of Dsh, but not in the canonical Wg/Wnt pathway. Although Tws homologues in other organisms have not been well characterized, our studies are consistent with a role for PP2A in dephosphorylation of Axin (Willert et al., 1999).

The next question is on the substrate of PP2A function in Wg pathway. In mammalian cells, Axin is dephosphorylated in response to Wnt signaling(Willert et al., 1999). Furthermore, dephosphorylated Axin binds β-catenin less efficiently than the phosphorylated form. Thus, dephosphorylation of Axin would freeβ-catenin from the degradation machinery(Willert et al., 1999). Thus,Tws may function by inhibiting the activity of Axin, which acts a scaffold protein to bring Sgg and Arm to close proximity. Further biochemical work is in progress to determine phosphorylated status of Arm in tws- background and to determine if Tws directly binds to Sgg or Axin or both.

Role of tws in other signal transduction pathways

The current study was mainly concerned with the role of Tws in Wg signaling. tws alleles were initially isolated based on two different kinds of phenotypes, both of which are not related to Wg signaling. The catalytic subunit of PP2A in Drosophila is encoded by microtubule star (mts). Loss-of-function of either mts causes overcondensation of chromatin and blocks the cell division at mitosis, between prophase and anaphase (Snaith et al.,1996). Similar phenotypes are reported for the aar1 allele of tws(Gomes et al., 1993). These phenotypes reflect the interaction between Tws and cyclinB/cdc2, which are required for G2-M transition (Minshull et al., 1996).

The pattern duplication in twsP is always limited to posterior compartment and is mainly due to loss of En, which in turn causes ectopic Dpp expression (Uemura et al.,1993). Vn/EGFR signaling is known to specify notum by antagonizing wing development and by activating notum-specifying genes(Baonza et al., 2000; Wang et al., 2000; Pallavi and Shashidhara,2003). Thus, loss of EGFR signaling would lead to notum-to-wing transformation. Furthermore, Vn/EGFR is required for the maintenance of En in the posterior compartment, defect in which would lead to pattern duplications of the kind seen in twsP background. Thus, it is likely that Tws is also functional in EGFR signal transduction pathway. Consistent with this, tws alleles are known to interact with Rasalleles and enhance eye phenotypes of the latter (Wassarman, 1996).

We thank Ann-Mari Voi for help in generating UAS-Tws and S. Cohen for helpful discussions. We thank K. Basler, S. Carroll, S. Cohen, D. Glover, J. Jiang, R. Nagaraj, E. Sanchez-Herrero, T. Uemura, the Bloomington Stock Center and the Developmental Studies Hybridoma Bank for fly stocks and antibodies;and members of the laboratory for technical help, advice and discussions. R.B. was a recipient of Development Traveling and UNESCO-MCBN short-term fellowships to visit EMBL. This work was supported by a grant to L.S.S. from Indian Council of Medical Research (Government of India).

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