The Wnt signaling pathway provides key information during development of vertebrates and invertebrates, and mutations in this pathway lead to various forms of cancer. Wnt binding to its receptor causes the stabilization and nuclear localization of β-catenin. Nuclear β-catenin then functions to activate transcription in conjunction with the transcription factor TCF. A recent report has challenged this basic precept of the Wnt signaling field, arguing that the nuclear localization of β-catenin may be unrelated to its function and that β-catenin functions at the plasma membrane to activate this signaling pathway. Here we present evidence that the pathway in fact does depend on the nuclear localization of β-catenin. We reexamine the functionality of various truncations of β-catenin and find that only the most severe truncations are true signaling-null mutations. Further, we define a signaling-null condition and use it to show that membrane-tethered β-catenin is insufficient to activate transcription. We also define two novel loss-of-function mutations that are not truncations, but are missense point mutations that retain protein stability. These alleles allow us to show that the membrane-bound form of activated β-catenin does indeed depend on the endogenous protein. Further, this activity is dependent on the presence of the C-terminus-specific negative regulator Chibby. Our data clearly show that nuclear localization of β-catenin is in fact necessary for Wnt pathway activation.

Introduction The Wnt signal transduction pathway has been studied extensively in both vertebrate and invertebrate systems. The  Drosophila  ortholog  wingless  ( wg ) is a segment polarity gene that defines posterior cell fates in each of the larval segments (for a review of the various functions of Wg, see  Wodarz and Nusse 1998 ). The pathway is activated when the extracellular ligand Wg binds to the transmembrane receptors Frizzled and Arrow. These in turn activate Disheveled (Dsh), which inactivates a complex composed of Axin, adenomatous polyposis coli (APC), and Zeste-white 3 (Zw3) (the  Drosophila  homolog of glycogen synthase kinase [GSK3β]). This complex is responsible for the retention of Armadillo (Arm) in the cytoplasm, for its phosphorylation, and thus for its targeting for ubiquitination and destruction. When the complex is inactivated by Dsh, the intracellular levels of Arm increase, and Arm enters the nucleus, where in combination with the transcription factor TCF/Pangolin, it activates the transcription of genes such as cyclin D and c- myc  ( Wodarz and Nusse 1998 ). We have argued that Axin plays a key role in the Wnt signaling process, functioning both as an anchor for Arm and a scaffold for the degradation complex. Wnt signaling results in a visible reduction in Axin levels, and mutations in Axin cause a relocalization of Arm to the nucleus ( Tolwinski and Wieschaus 2001 ;  Tolwinski et al. 2003 ). The nuclear import and export of Arm are not clearly understood (for a review, see  Henderson and Fagotto 2002 ), but Arm can cross the nuclear membrane by interacting with the nuclear pore complex directly. Once in the nucleus, Arm interacts with a variety of nuclear factors, in particular the transcription factor TCF/LEF ( Behrens et al. 1996 ;  Molenaar et al. 1996 ;  Brunner et al. 1997 ;  van de Wetering et al. 1997 ). The β-catenin–TCF complex releases repression and activates transcription ( Cavallo et al. 1998 ). A recent study has challenged this view and has questioned the importance of nuclear localization of Arm protein ( Chan and Struhl 2002 ). These authors' conclusions were based primarily on the observation that a membrane-tethered, stabilized form of Arm (Arm ΔArm ) causes activation of the Wnt pathway without entering the nucleus. However, this is not the first time that the controversy about the location of Arm/β-catenin function has arisen. Previously, a group working with amphibian embryos had found that membrane-tethered plakoglobin, a close relative of β-catenin, can activate Wnt signaling ( Merriam et al. 1997 ). Another group showed, however, that expression of membrane-tethered forms of β-catenin leads to the nuclear localization of endogenous β-catenin ( Miller and Moon 1997 ). When the endogenous Arm/β-catenin gene was mutated, the activity of membrane-tethered forms was lost ( Cox et al. 1999 b). These experiments illustrate the importance of following the activity of the endogenous allele in evaluating the activity of membrane-tethered forms. Previously, we had expressed the same membrane-tethered form used by  Chan and Struhl (2002 ) in embryos with various endogenous  arm  mutations and had concluded that it functions by titrating Axin to the membrane, releasing the endogenous Arm protein and allowing it to move freely into the nucleus ( Tolwinski and Wieschaus 2001 ). These experiments are difficult, because none of the cell-viable alleles are absolute genetic nulls, as Arm plays essential roles in both Wnt signaling and cell adhesion. In this study, we reexamine Arm function using three classes of previously described  arm  alleles. We find that by manipulating their levels and localizations, many alleles believed to be signaling nulls can still activate transcription. When the cell-adhesive defects of the most severe class of alleles are rescued, however, the mutant protein still fails to signal, allowing us to assay the activity of membrane-tethered Arm in a true signaling-null background. We find that nuclear localization is necessary for pathway activation and that exclusively membrane-bound forms of Arm are insufficient for this. We use two novel missense mutations in  arm  to assess the nuclear activity of Arm and confirm that negative regulation by the transcriptional regulator Chibby (Cby) is required for patterning.
Introduction
The Wnt signal transduction pathway has been studied extensively in both vertebrate and invertebrate systems. The  Drosophila  ortholog  wingless  ( wg ) is a segment polarity gene that defines posterior cell fates in each of the larval segments (for a review of the various functions of Wg, see  Wodarz and Nusse 1998 ). The pathway is activated when the extracellular ligand Wg binds to the transmembrane receptors Frizzled and Arrow. These in turn activate Disheveled (Dsh), which inactivates a complex composed of Axin, adenomatous polyposis coli (APC), and Zeste-white 3 (Zw3) (the  Drosophila  homolog of glycogen synthase kinase [GSK3β]). This complex is responsible for the retention of Armadillo (Arm) in the cytoplasm, for its phosphorylation, and thus for its targeting for ubiquitination and destruction. When the complex is inactivated by Dsh, the intracellular levels of Arm increase, and Arm enters the nucleus, where in combination with the transcription factor TCF/Pangolin, it activates the transcription of genes such as cyclin D and c- myc  ( Wodarz and Nusse 1998 ).DrosophilawinglesswgDrosophilamycWe have argued that Axin plays a key role in the Wnt signaling process, functioning both as an anchor for Arm and a scaffold for the degradation complex. Wnt signaling results in a visible reduction in Axin levels, and mutations in Axin cause a relocalization of Arm to the nucleus ( Tolwinski and Wieschaus 2001 ;  Tolwinski et al. 2003 ). The nuclear import and export of Arm are not clearly understood (for a review, see  Henderson and Fagotto 2002 ), but Arm can cross the nuclear membrane by interacting with the nuclear pore complex directly. Once in the nucleus, Arm interacts with a variety of nuclear factors, in particular the transcription factor TCF/LEF ( Behrens et al. 1996 ;  Molenaar et al. 1996 ;  Brunner et al. 1997 ;  van de Wetering et al. 1997 ). The β-catenin–TCF complex releases repression and activates transcription ( Cavallo et al. 1998 ).A recent study has challenged this view and has questioned the importance of nuclear localization of Arm protein ( Chan and Struhl 2002 ). These authors' conclusions were based primarily on the observation that a membrane-tethered, stabilized form of Arm (Arm ΔArm ) causes activation of the Wnt pathway without entering the nucleus. However, this is not the first time that the controversy about the location of Arm/β-catenin function has arisen. Previously, a group working with amphibian embryos had found that membrane-tethered plakoglobin, a close relative of β-catenin, can activate Wnt signaling ( Merriam et al. 1997 ). Another group showed, however, that expression of membrane-tethered forms of β-catenin leads to the nuclear localization of endogenous β-catenin ( Miller and Moon 1997 ). When the endogenous Arm/β-catenin gene was mutated, the activity of membrane-tethered forms was lost ( Cox et al. 1999 b). These experiments illustrate the importance of following the activity of the endogenous allele in evaluating the activity of membrane-tethered forms. Previously, we had expressed the same membrane-tethered form used by  Chan and Struhl (2002 ) in embryos with various endogenous  arm  mutations and had concluded that it functions by titrating Axin to the membrane, releasing the endogenous Arm protein and allowing it to move freely into the nucleus ( Tolwinski and Wieschaus 2001 ). These experiments are difficult, because none of the cell-viable alleles are absolute genetic nulls, as Arm plays essential roles in both Wnt signaling and cell adhesion.armIn this study, we reexamine Arm function using three classes of previously described  arm  alleles. We find that by manipulating their levels and localizations, many alleles believed to be signaling nulls can still activate transcription. When the cell-adhesive defects of the most severe class of alleles are rescued, however, the mutant protein still fails to signal, allowing us to assay the activity of membrane-tethered Arm in a true signaling-null background. We find that nuclear localization is necessary for pathway activation and that exclusively membrane-bound forms of Arm are insufficient for this. We use two novel missense mutations in  arm  to assess the nuclear activity of Arm and confirm that negative regulation by the transcriptional regulator Chibby (Cby) is required for patterning.armarmResults Membrane-Tethered Arm Is Dependent upon the Endogenous  arm  Allele The original mutants in the  arm  gene were classified into three groups based upon their phenotypes and the position of stop codons that result in truncated proteins. The “weak” class has the smallest truncations and is represented by  arm XM19 . In germline clones (where maternal and zygotic contribution of protein is removed;  Chou and Perrimon 1992 ), its phenotype is identical to loss-of-function  wg  mutations ( Figure 1 B;  Peifer and Wieschaus 1990 ). The “medium” class, represented here by  arm O43A01 , shows defects in adhesion as well as transcription. Here germline clones give embryos that fail to differentiate an intact cuticle ( Figure 1 C;  Tolwinski and Wieschaus 2001 ). The “strong” class ( arm XK22 ) does not allow proper progression through oogenesis and germline clones do not make eggs ( Figure 1 D;  Peifer et al. 1993 ).  Cox et al. (1999 b) showed that the junctional defects of the “medium” alleles can be circumvented by coexpression of a membrane-tethered full-length form of Arm (Arm S18 ) ( Figure 2 ). We have confirmed their findings and extended them to the “strong” allele during oogenesis. We show that uniform expression of Arm S18  allows  arm XK22 germ cells to produce normal eggs and rescues the adhesive defects of both  arm XK22  and  arm O43A01  embryos. The membrane-tethered form does not, however, rescue the signaling defects associated with either of these alleles and the embryos show typical  wg  phenotypes (see  Figure 1 G and  1 H). Figure 1 Arm ΔArm  Requires Endogenous Arm Endogenous allele indicated at top; ectopically expressed transgenes indicated at left. (A) The wild-type cuticle of a  Drosophila  embryo. (B) The  arm XM19  “weak” allele phenotype, similar to  wg  mutations in which the entire cuticle is covered with denticles. (C) The  arm O43A01  “medium” allele phenotype shows disintegrated embryos in which cells delaminate owing to an inability to form adherens junctions. (D)  arm XK22  “strong” allele does not produce embryos, owing to an oogenesis defect. (E) A wild-type embryo expressing Arm S18  shows a wild-type cuticle. (F)  arm XM19  mutant expressing Arm S18  is rescued to a wild-type cuticle. (G)  arm O43A01  mutant expressing Arm S18  shows rescued adhesion, but a  wg  mutant signaling phenotype. (H)  arm XK22  mutant expressing Arm S18  also shows rescued adhesion, as well as a  wg  mutant signaling phenotype. (I) Coexpression of Arm ΔArm  and Arm S18  in wild-type embryos leads to naked cuticle or the uniform Wg active phenotype. (J) Coexpression of Arm ΔArm  and Arm S18  leads to naked cuticle or the uniform Wg active phenotype in an  arm XM19  mutant background. (K) Coexpression of Arm ΔArm  and Arm S18  in  arm O43A01  mutant embryos leads to naked cuticle or the uniform Wg active phenotype. (L) However, coexpression of Arm ΔArm  and Arm S18  in “strong” mutant  arm XK22  background shifts embryos back to the  wg  mutant phenotype. Expression of the membrane-tethered, stabilized form of Arm (Arm ΔArm ) leads to uniform activation of signaling in all cells. This effect is independent of whether the cell is exposed to Wg signal or not, because Arm ΔArm  functions independently of Wg ligand. The membrane-tethered, unstabilized form of Arm (Arm S18 ) leads to pathway activation only in cells that receive Wg signal, because this form of Arm is still subject to Wg-dependent phosphorylation and phosphorylation-dependent degradation. Figure 2 Structure of Arm Protein and Alleles Arm protein consists of three regions. The N-terminus is required for transactivation, for phosphorylation-based and proteasome-mediated degradation, and for α-catenin binding. The central repeats region is a superhelical structure that contains the binding sites for most of Arm's binding partners, including APC, TCF, Cadherin, and Axin. The C-terminus is required for Cby and Teashirt binding and transactivation. The  arm F1a  mutation causes an arginine-to-histidine change within repeat six. The  arm LM134  mutation causes a serine-to-phenylalanine change in repeat five. The “weak” allele  arm XM19  removes the entire C-terminus. The “medium” allele  arm O43A01  causes early termination within repeat nine. The “strong” allele  arm XK22  causes early termination within repeat six. The Arm ΔArm  transgene consists of the entire repeats region and C-terminus fused to an HA tag and myristoylation sequence at the N-terminus under GAL4/UAS control. The Arm S10  transgene contains a small deletion in the N-terminus, which removes the four phosphorylation sites necessary for degradation and is under GAL4/UAS control. The Arm S8  transgene contains a deletion of approximately a third of the C-terminus and is under endogenous promoter control. The Arm S18  transgene contains the entire Arm sequence fused to the CAAX myristoylation sequence of Ras and is under endogenous promoter control. The UAS–Arm XM19  is the equivalent of the  arm XM19  allele in deletion, but is fused to an N-terminal HA tag and is under GAL4/UAS control. Expression of Arm S18  has no effect on the cuticle of wild-type embryos (compare  Figure 1 E to  1 A), but it does rescue the signaling defects of  arm  alleles, like  arm XM19 , that have only short C-terminal truncations ( Cox et al. 1999 b; see  Figure 1 F). These alleles normally show very low levels of protein ( Peifer and Wieschaus 1990 ), and  Cox et al. (1999 b) postulated that expression of a membrane-tethered Arm might “free up” the endogenous mutant protein, allowing the “weak” allele to signal. The low levels of  arm XM19  may reflect degradation of nonsense mRNAs triggered by the premature stop codon in this mutant (reviewed in  Wagner and Lykke-Andersen 2002 ). To eliminate this degradation, we expressed a cDNA version of the  arm XM19  allele under GAL4/UAS control ( Brand and Perrimon 1993 ) in embryos mutant for  arm XM19  ( Figure 3 B). To avoid the possibility of overexpression artifacts, we also expressed a smaller C-terminal deletion from the endogenous promoter (Arm S8 ;  Orsulic and Peifer 1996 ). In both experiments, the truncated protein from the transgene accumulated to levels approximating those observed in wild-type ( Figure 3 G and  3 H) and in the characteristic striped pattern indicative of response to the Wg signal ( Peifer and Wieschaus 1990 ). The truncated protein rescued the  arm XM19  phenotype to a wild-type cuticle pattern and allowed hatching ( Figure 3 C). When combined with a mutation in the kinase  zw3 , Arm S8  causes the cuticles of these embryos to appear uniformly naked (compare  Figure 3 E to  3 D), as would be expected since the Arm S8  protein is expressed to high uniform levels throughout the epidermis when Zw3 is removed ( Figure 3 I). These experiments argue that the C-terminus is not essential for signaling or transcriptional activation of Wnt targets required for cuticle patterning. However, as we do not obtain adult flies containing exclusively the truncated alleles, it is very likely that the C-terminus is not entirely expendable and must have important functions later in development. Figure 3 C-Terminally Truncated Arm Can Signal If Its Levels Are Increased (A)  arm XM19  shows a  wg  mutant phenotype. (B) Expression of GAL4/UAS-driven Arm XM19  protein in  arm XM19  mutant background rescues this to a wild-type pattern. (C) The same is true of expression of an endogenous promoter-driven truncation Arm S8 . (D) Removal of Zw3 has no effect on  arm XM19  cuticle pattern. (E) However, when Arm S8  is introduced into  arm XM19 , zw3  mutants, the cuticle is naked. (F) Wild-type embryo is shown for comparison. (G–I) Arm stainings reveal that expression of UAS–Arm XM19  (stained for the HA tag [G]) and Arm S8  (stained for Arm [H]) is present in stripes corresponding to Wg striping, whereas removal of Zw3, along with Arm S8  expression, leads to uniform and high levels of Arm throughout the epidermis (I). Null Allele Background Proves That Arm ΔArm  Cannot Signal on Its Own The fact that  arm XM19  is able to signal when expressed at normal levels invalidates its use in tests for a direct activity of membrane-tethered Arm in Wnt signaling ( Chan and Struhl 2002 ). Therefore, expression of Arm ΔArm  in a “weak” allele background cannot address whether membrane-tethered Arm activates transcription without ever entering the nucleus, since a membrane-untethered, signaling-competent form of Arm is also present. To directly address whether the Arm ΔArm  transgene can transmit Wg signal on its own, we turned to the “strong” and “medium” alleles. Although Arm S18  is not sufficient to restore signaling to these alleles, it raises the possibility that stronger expression of stabilized, membrane-tethered Arm (Arm ΔArm ) might reveal some signaling capacity of those alleles as well. Experiments of this kind have been difficult with Arm ΔArm , given that it lacks the α-catenin-binding site and fails to rescue the junctional defect in “medium” and “strong” endogenous  arm  allele backgrounds. We have found that by expressing both Arm ΔArm  and Arm S18 , we can recover intact embryos in all backgrounds tested. We find that “medium” and “weak” alleles can be induced to activate transcription, but the “strong”  arm  allele cannot (see  Figure 1 J– 1 L), consistent with the position of the “medium” alleles in the hypomorphic allelic series. These findings demonstrate that Arm ΔArm  is dependent upon the endogenous form of  arm , as it cannot activate transcription in the “strong” allele background. Loss-of-Function Missense Mutations When Arm ΔArm  is expressed in a wild-type embryo, it strongly activates Wg signaling ( Figure 4 C;  Chan and Struhl 2002 ).  Chan and Struhl (2002 ) suggest that this is because this membrane-tethered form of Arm can signal on its own. The results presented above argue, on the other hand, that it does so by stabilizing the endogenous protein. To further test this, we asked whether expression of Arm ΔArm  can induce Wg signaling when endogenous Arm is replaced by signaling-deficient Arm. We turned to two novel missense mutations where the rest of the  arm  coding region remains intact. Because these alleles do not produce truncations through stop codons, they are immune to nonsense mRNA degradation ( Wagner and Lykke-Andersen 2002 ). Both mutations result in amino acid substitutions close to repeat seven, a key hinge region postulated to be important in binding of TCF ( Huber et al. 1997 ;  Graham et al. 2000 ). Both mutants retain the phosphorylation sites required for degradation and therefore accumulate in stripes in response to Wg signal ( Figure 5 I and  5 J). They supply apparent wild-type junctional activity and accumulate to high levels in all cells when the kinase responsible for the degradation signal (Zw3) is removed ( Figure 5 K and  5 L). The primary phenotype of these alleles is a loss or reduction of Wnt transcriptional responses ( Figure 5 A and  5 B). The  arm F1a  allele produced a partial loss-of-function phenotype, and germline clone embryos show some residual naked cuticle.  arm LM134  produces a stronger phenotype comparable to a loss of  wg  function, although it may not be a signaling null (see below). Figure 4 Expression of Arm ΔArm  Leads to the Nuclear Localization of Endogenous Arm Protein (A) Wild-type Arm protein appears in stripes that correspond to cells responding to Wg signaling. (B) Expression of Arm ΔArm  in an  arm F1a  background leads to the nuclear localization of endogenous Arm. (C and D) Dark-field images reveal that expression of both Arm ΔArm  and Arm S10  leads to similar naked cuticle phenotypes. (E) An anti-Arm Western blot showing a faster-migrating band, which correlates with endogenous Arm's being active, and a slower-migrating band, which correlates with Arm's being inactive. Figure 5 ΔArm Functions through Endogenous Arm (A) Embryonic cuticle of  arm F1a  mutant showing a weak loss-of-function phenotype. (B) Cuticle of  arm LM134  mutant embryo showing a strong loss-of-signaling phenotype. (C) Embryo mutant for  arm F1a  expressing Arm ΔArm  showing relatively normal segment polarity. (D)  arm LM134  mutant expressing Arm ΔArm  also shows segment polarity. (E and F) Both alleles in combination with a null  zw3  allele and expressing Arm ΔArm  show a complete lack of denticles. (G and H) Both alleles expressing the activated but nontethered form of stabilized Arm, Arm S10 , show the naked cuticle phenotype. (I–L) In both missense alleles, the mutant protein is expressed in stripes (I and J), corresponding with Wg expression (data not shown), which is abolished when the key degradation kinase Zw3 is removed (K and L). We asked whether these signaling-deficient alleles could block the cell fate transformation and Wnt target activation observed when Arm ΔArm  is expressed in wild-type epidermis. If Arm ΔArm  functions independently of the endogenous protein, then all cells should assume the naked cell fate. However, this does not occur ( Figure 5 C and  5 D). Instead, both point mutants produce a cuticle pattern with periodic denticle belts and regions of intervening naked cuticle. This periodicity may reflect the fact that  arm F1a  and  arm LM134  can still be controlled by Wg even when Arm ΔArm  is expressed. This periodicity is, in fact, abolished when Zw3 activity is removed from such embryos (i.e., in triply mutant  zw3, arm F1a ; Arm ΔArm  embryos). Under these conditions, all cells in the cuticle take on the naked cell fate ( Figure 5 E and  5 F). Since Arm ΔArm  lacks the N-terminal sites that respond to Zw3, the sensitivity of the double-mutant phenotype confirms that the pattern of the double mutant is dependent on the endogenous Arm protein. The behavior of membrane-tethered Arm ΔArm  contrasts with that of other stabilized forms of Arm that would be predicted to move more freely between the cytoplasm and the nucleus. Arm S10 , for example, contains a small N-terminal deletion that blocks Zw3 phosphorylation, but preserves binding sites for various nuclear proteins (see  Figure 2 ;  Pai et al. 1997 ). Arm S10  is not membrane-tethered, but the cell fate transformations it produces are identical to those produced by Arm ΔArm  (compare  Figure 4 C and  4 D). They do not, however, depend on the endogenous allele and are still observed in an  arm F1a  or  arm LM134  germline clone background ( Figure 5 G and  5 H). Arm ΔArm  Causes Nuclear Localization and Mobility Shift of Endogenous Arm All of our experiments argue that Arm ΔArm  produces its effect on transcription by activating the endogenous alleles. To investigate the mechanism that underlies this effect, we looked at the in situ localization of the endogenous Arm protein and its migration pattern on Western blots. Expression of Arm ΔArm  is sufficient to drive both wild-type and the point mutant forms of Arm into nuclei (see  Figure 4 A and  4 B;  Miller and Moon 1997 ;  Tolwinski and Wieschaus 2001 ). Generally, the most obvious feature observed upon removal of any of the negative factors of the Wg pathway is the rapid accumulation of Arm in cells. However, another feature is the phosphorylation state of the Arm protein.  Peifer et al. (1994 a) found that a fast-migrating band of Arm corresponds with active Wg signaling and that a slower-migrating band corresponds with Wg's being off. Therefore, it is the unphosphorylated band that corresponds with signaling. Here we show that, on Western blots, endogenous Arm protein responds to Arm ΔArm  expression in much the same way that it does to the removal of negative components of the pathway such as Axin and APC1 and APC2 (see  Figure 4 E). We see a downshift of the protein, which is directly opposite to what is seen when a positive component of the pathway is removed (Dsh or Wg; see  Figure 4 E). Wild-type embryos show the expected intermediate phenotype, as they have both active and inactive forms of Arm protein (see  Figure 4 E). The observed shift is most likely the result of phosphorylation ( Peifer et al. 1994 a), though we do not address this directly in this study. The C-Terminus of Arm Is Necessary for Cby-Mediated Repression Although the missense mutations we have used in our studies produce (on average) weaker phenotypes, they are more effective at blocking the cell-fate transformation induced by Arm ΔArm  than the “medium” C-terminal truncation mutants (compare  Figure 1 K with  Figure 5 C and  5 D). The comparison is somewhat indirect, owing to the necessity of expressing Arm S18  in the “medium”  arm  allele background in order to get intact embryos. However, we find that expression of Arm S18  in an  arm F1a  background has no visible effect on the cuticle (data not shown). Therefore, the activity of C-terminally truncated  arm  alleles in response to ΔArm expression suggests that, under certain conditions, removal of the C-terminus may actually enhance the transcriptional activity of Arm. One possibility is suggested by the recent discovery of Cby ( Takemaru et al. 2003 ), a nuclear negative regulator of the Wg pathway that binds to the C-terminus of Arm. To test whether nuclear Cby affected the transformation produced by Arm ΔArm , we used RNA interference (RNAi) to reduce Cby levels in  arm F1a  embryos with and without Arm ΔArm . In the absence of Arm ΔArm , i.e., in embryos where most Arm F1a  protein is cytoplasmic, Cby RNAi has no effect ( Figure 6 D). However, when Arm ΔArm  is present, lowering Cby levels leads to increased naked cuticle characteristic of Wnt pathway activation (compare  Figure 6 B to  6 C). We propose that Cby's effect on  arm F1a  protein is dependent on Arm ΔArm  relocalizing Arm to the nucleus. Figure 6 Relief of C-Terminal Repression through the Elimination of Cby Leads to Uniform Activation of Signaling (A) A wild-type cuticle shown for comparison. (B) Expression of Arm ΔArm  in the  arm F1a  background. (C) Expression of a Cby RNAi construct along with Arm ΔArm  in the  arm F1a  background. (D) Expression of a Cby RNAi construct in an  arm F1a  background.
Results
Membrane-Tethered Arm Is Dependent upon the Endogenous  arm  Allele The original mutants in the  arm  gene were classified into three groups based upon their phenotypes and the position of stop codons that result in truncated proteins. The “weak” class has the smallest truncations and is represented by  arm XM19 . In germline clones (where maternal and zygotic contribution of protein is removed;  Chou and Perrimon 1992 ), its phenotype is identical to loss-of-function  wg  mutations ( Figure 1 B;  Peifer and Wieschaus 1990 ). The “medium” class, represented here by  arm O43A01 , shows defects in adhesion as well as transcription. Here germline clones give embryos that fail to differentiate an intact cuticle ( Figure 1 C;  Tolwinski and Wieschaus 2001 ). The “strong” class ( arm XK22 ) does not allow proper progression through oogenesis and germline clones do not make eggs ( Figure 1 D;  Peifer et al. 1993 ).  Cox et al. (1999 b) showed that the junctional defects of the “medium” alleles can be circumvented by coexpression of a membrane-tethered full-length form of Arm (Arm S18 ) ( Figure 2 ). We have confirmed their findings and extended them to the “strong” allele during oogenesis. We show that uniform expression of Arm S18  allows  arm XK22 germ cells to produce normal eggs and rescues the adhesive defects of both  arm XK22  and  arm O43A01  embryos. The membrane-tethered form does not, however, rescue the signaling defects associated with either of these alleles and the embryos show typical  wg  phenotypes (see  Figure 1 G and  1 H). Figure 1 Arm ΔArm  Requires Endogenous Arm Endogenous allele indicated at top; ectopically expressed transgenes indicated at left. (A) The wild-type cuticle of a  Drosophila  embryo. (B) The  arm XM19  “weak” allele phenotype, similar to  wg  mutations in which the entire cuticle is covered with denticles. (C) The  arm O43A01  “medium” allele phenotype shows disintegrated embryos in which cells delaminate owing to an inability to form adherens junctions. (D)  arm XK22  “strong” allele does not produce embryos, owing to an oogenesis defect. (E) A wild-type embryo expressing Arm S18  shows a wild-type cuticle. (F)  arm XM19  mutant expressing Arm S18  is rescued to a wild-type cuticle. (G)  arm O43A01  mutant expressing Arm S18  shows rescued adhesion, but a  wg  mutant signaling phenotype. (H)  arm XK22  mutant expressing Arm S18  also shows rescued adhesion, as well as a  wg  mutant signaling phenotype. (I) Coexpression of Arm ΔArm  and Arm S18  in wild-type embryos leads to naked cuticle or the uniform Wg active phenotype. (J) Coexpression of Arm ΔArm  and Arm S18  leads to naked cuticle or the uniform Wg active phenotype in an  arm XM19  mutant background. (K) Coexpression of Arm ΔArm  and Arm S18  in  arm O43A01  mutant embryos leads to naked cuticle or the uniform Wg active phenotype. (L) However, coexpression of Arm ΔArm  and Arm S18  in “strong” mutant  arm XK22  background shifts embryos back to the  wg  mutant phenotype. Expression of the membrane-tethered, stabilized form of Arm (Arm ΔArm ) leads to uniform activation of signaling in all cells. This effect is independent of whether the cell is exposed to Wg signal or not, because Arm ΔArm  functions independently of Wg ligand. The membrane-tethered, unstabilized form of Arm (Arm S18 ) leads to pathway activation only in cells that receive Wg signal, because this form of Arm is still subject to Wg-dependent phosphorylation and phosphorylation-dependent degradation. Figure 2 Structure of Arm Protein and Alleles Arm protein consists of three regions. The N-terminus is required for transactivation, for phosphorylation-based and proteasome-mediated degradation, and for α-catenin binding. The central repeats region is a superhelical structure that contains the binding sites for most of Arm's binding partners, including APC, TCF, Cadherin, and Axin. The C-terminus is required for Cby and Teashirt binding and transactivation. The  arm F1a  mutation causes an arginine-to-histidine change within repeat six. The  arm LM134  mutation causes a serine-to-phenylalanine change in repeat five. The “weak” allele  arm XM19  removes the entire C-terminus. The “medium” allele  arm O43A01  causes early termination within repeat nine. The “strong” allele  arm XK22  causes early termination within repeat six. The Arm ΔArm  transgene consists of the entire repeats region and C-terminus fused to an HA tag and myristoylation sequence at the N-terminus under GAL4/UAS control. The Arm S10  transgene contains a small deletion in the N-terminus, which removes the four phosphorylation sites necessary for degradation and is under GAL4/UAS control. The Arm S8  transgene contains a deletion of approximately a third of the C-terminus and is under endogenous promoter control. The Arm S18  transgene contains the entire Arm sequence fused to the CAAX myristoylation sequence of Ras and is under endogenous promoter control. The UAS–Arm XM19  is the equivalent of the  arm XM19  allele in deletion, but is fused to an N-terminal HA tag and is under GAL4/UAS control. Expression of Arm S18  has no effect on the cuticle of wild-type embryos (compare  Figure 1 E to  1 A), but it does rescue the signaling defects of  arm  alleles, like  arm XM19 , that have only short C-terminal truncations ( Cox et al. 1999 b; see  Figure 1 F). These alleles normally show very low levels of protein ( Peifer and Wieschaus 1990 ), and  Cox et al. (1999 b) postulated that expression of a membrane-tethered Arm might “free up” the endogenous mutant protein, allowing the “weak” allele to signal. The low levels of  arm XM19  may reflect degradation of nonsense mRNAs triggered by the premature stop codon in this mutant (reviewed in  Wagner and Lykke-Andersen 2002 ). To eliminate this degradation, we expressed a cDNA version of the  arm XM19  allele under GAL4/UAS control ( Brand and Perrimon 1993 ) in embryos mutant for  arm XM19  ( Figure 3 B). To avoid the possibility of overexpression artifacts, we also expressed a smaller C-terminal deletion from the endogenous promoter (Arm S8 ;  Orsulic and Peifer 1996 ). In both experiments, the truncated protein from the transgene accumulated to levels approximating those observed in wild-type ( Figure 3 G and  3 H) and in the characteristic striped pattern indicative of response to the Wg signal ( Peifer and Wieschaus 1990 ). The truncated protein rescued the  arm XM19  phenotype to a wild-type cuticle pattern and allowed hatching ( Figure 3 C). When combined with a mutation in the kinase  zw3 , Arm S8  causes the cuticles of these embryos to appear uniformly naked (compare  Figure 3 E to  3 D), as would be expected since the Arm S8  protein is expressed to high uniform levels throughout the epidermis when Zw3 is removed ( Figure 3 I). These experiments argue that the C-terminus is not essential for signaling or transcriptional activation of Wnt targets required for cuticle patterning. However, as we do not obtain adult flies containing exclusively the truncated alleles, it is very likely that the C-terminus is not entirely expendable and must have important functions later in development. Figure 3 C-Terminally Truncated Arm Can Signal If Its Levels Are Increased (A)  arm XM19  shows a  wg  mutant phenotype. (B) Expression of GAL4/UAS-driven Arm XM19  protein in  arm XM19  mutant background rescues this to a wild-type pattern. (C) The same is true of expression of an endogenous promoter-driven truncation Arm S8 . (D) Removal of Zw3 has no effect on  arm XM19  cuticle pattern. (E) However, when Arm S8  is introduced into  arm XM19 , zw3  mutants, the cuticle is naked. (F) Wild-type embryo is shown for comparison. (G–I) Arm stainings reveal that expression of UAS–Arm XM19  (stained for the HA tag [G]) and Arm S8  (stained for Arm [H]) is present in stripes corresponding to Wg striping, whereas removal of Zw3, along with Arm S8  expression, leads to uniform and high levels of Arm throughout the epidermis (I).
Membrane-Tethered Arm Is Dependent upon the Endogenous  arm  Allele
armThe original mutants in the  arm  gene were classified into three groups based upon their phenotypes and the position of stop codons that result in truncated proteins. The “weak” class has the smallest truncations and is represented by  arm XM19 . In germline clones (where maternal and zygotic contribution of protein is removed;  Chou and Perrimon 1992 ), its phenotype is identical to loss-of-function  wg  mutations ( Figure 1 B;  Peifer and Wieschaus 1990 ). The “medium” class, represented here by  arm O43A01 , shows defects in adhesion as well as transcription. Here germline clones give embryos that fail to differentiate an intact cuticle ( Figure 1 C;  Tolwinski and Wieschaus 2001 ). The “strong” class ( arm XK22 ) does not allow proper progression through oogenesis and germline clones do not make eggs ( Figure 1 D;  Peifer et al. 1993 ).  Cox et al. (1999 b) showed that the junctional defects of the “medium” alleles can be circumvented by coexpression of a membrane-tethered full-length form of Arm (Arm S18 ) ( Figure 2 ). We have confirmed their findings and extended them to the “strong” allele during oogenesis. We show that uniform expression of Arm S18  allows  arm XK22 germ cells to produce normal eggs and rescues the adhesive defects of both  arm XK22  and  arm O43A01  embryos. The membrane-tethered form does not, however, rescue the signaling defects associated with either of these alleles and the embryos show typical  wg  phenotypes (see  Figure 1 G and  1 H).armarm XM19wgarm O43A01arm XK22arm XK22arm XK22arm O43A01wgExpression of Arm S18  has no effect on the cuticle of wild-type embryos (compare  Figure 1 E to  1 A), but it does rescue the signaling defects of  arm  alleles, like  arm XM19 , that have only short C-terminal truncations ( Cox et al. 1999 b; see  Figure 1 F). These alleles normally show very low levels of protein ( Peifer and Wieschaus 1990 ), and  Cox et al. (1999 b) postulated that expression of a membrane-tethered Arm might “free up” the endogenous mutant protein, allowing the “weak” allele to signal. The low levels of  arm XM19  may reflect degradation of nonsense mRNAs triggered by the premature stop codon in this mutant (reviewed in  Wagner and Lykke-Andersen 2002 ). To eliminate this degradation, we expressed a cDNA version of the  arm XM19  allele under GAL4/UAS control ( Brand and Perrimon 1993 ) in embryos mutant for  arm XM19  ( Figure 3 B). To avoid the possibility of overexpression artifacts, we also expressed a smaller C-terminal deletion from the endogenous promoter (Arm S8 ;  Orsulic and Peifer 1996 ). In both experiments, the truncated protein from the transgene accumulated to levels approximating those observed in wild-type ( Figure 3 G and  3 H) and in the characteristic striped pattern indicative of response to the Wg signal ( Peifer and Wieschaus 1990 ). The truncated protein rescued the  arm XM19  phenotype to a wild-type cuticle pattern and allowed hatching ( Figure 3 C). When combined with a mutation in the kinase  zw3 , Arm S8  causes the cuticles of these embryos to appear uniformly naked (compare  Figure 3 E to  3 D), as would be expected since the Arm S8  protein is expressed to high uniform levels throughout the epidermis when Zw3 is removed ( Figure 3 I). These experiments argue that the C-terminus is not essential for signaling or transcriptional activation of Wnt targets required for cuticle patterning. However, as we do not obtain adult flies containing exclusively the truncated alleles, it is very likely that the C-terminus is not entirely expendable and must have important functions later in development.armarm XM19arm XM19arm XM19arm XM19arm XM19zw3Null Allele Background Proves That Arm ΔArm  Cannot Signal on Its Own The fact that  arm XM19  is able to signal when expressed at normal levels invalidates its use in tests for a direct activity of membrane-tethered Arm in Wnt signaling ( Chan and Struhl 2002 ). Therefore, expression of Arm ΔArm  in a “weak” allele background cannot address whether membrane-tethered Arm activates transcription without ever entering the nucleus, since a membrane-untethered, signaling-competent form of Arm is also present. To directly address whether the Arm ΔArm  transgene can transmit Wg signal on its own, we turned to the “strong” and “medium” alleles. Although Arm S18  is not sufficient to restore signaling to these alleles, it raises the possibility that stronger expression of stabilized, membrane-tethered Arm (Arm ΔArm ) might reveal some signaling capacity of those alleles as well. Experiments of this kind have been difficult with Arm ΔArm , given that it lacks the α-catenin-binding site and fails to rescue the junctional defect in “medium” and “strong” endogenous  arm  allele backgrounds. We have found that by expressing both Arm ΔArm  and Arm S18 , we can recover intact embryos in all backgrounds tested. We find that “medium” and “weak” alleles can be induced to activate transcription, but the “strong”  arm  allele cannot (see  Figure 1 J– 1 L), consistent with the position of the “medium” alleles in the hypomorphic allelic series. These findings demonstrate that Arm ΔArm  is dependent upon the endogenous form of  arm , as it cannot activate transcription in the “strong” allele background.
Null Allele Background Proves That Arm ΔArm  Cannot Signal on Its Own
The fact that  arm XM19  is able to signal when expressed at normal levels invalidates its use in tests for a direct activity of membrane-tethered Arm in Wnt signaling ( Chan and Struhl 2002 ). Therefore, expression of Arm ΔArm  in a “weak” allele background cannot address whether membrane-tethered Arm activates transcription without ever entering the nucleus, since a membrane-untethered, signaling-competent form of Arm is also present. To directly address whether the Arm ΔArm  transgene can transmit Wg signal on its own, we turned to the “strong” and “medium” alleles. Although Arm S18  is not sufficient to restore signaling to these alleles, it raises the possibility that stronger expression of stabilized, membrane-tethered Arm (Arm ΔArm ) might reveal some signaling capacity of those alleles as well. Experiments of this kind have been difficult with Arm ΔArm , given that it lacks the α-catenin-binding site and fails to rescue the junctional defect in “medium” and “strong” endogenous  arm  allele backgrounds. We have found that by expressing both Arm ΔArm  and Arm S18 , we can recover intact embryos in all backgrounds tested. We find that “medium” and “weak” alleles can be induced to activate transcription, but the “strong”  arm  allele cannot (see  Figure 1 J– 1 L), consistent with the position of the “medium” alleles in the hypomorphic allelic series. These findings demonstrate that Arm ΔArm  is dependent upon the endogenous form of  arm , as it cannot activate transcription in the “strong” allele background.arm XM19armarmarmLoss-of-Function Missense Mutations When Arm ΔArm  is expressed in a wild-type embryo, it strongly activates Wg signaling ( Figure 4 C;  Chan and Struhl 2002 ).  Chan and Struhl (2002 ) suggest that this is because this membrane-tethered form of Arm can signal on its own. The results presented above argue, on the other hand, that it does so by stabilizing the endogenous protein. To further test this, we asked whether expression of Arm ΔArm  can induce Wg signaling when endogenous Arm is replaced by signaling-deficient Arm. We turned to two novel missense mutations where the rest of the  arm  coding region remains intact. Because these alleles do not produce truncations through stop codons, they are immune to nonsense mRNA degradation ( Wagner and Lykke-Andersen 2002 ). Both mutations result in amino acid substitutions close to repeat seven, a key hinge region postulated to be important in binding of TCF ( Huber et al. 1997 ;  Graham et al. 2000 ). Both mutants retain the phosphorylation sites required for degradation and therefore accumulate in stripes in response to Wg signal ( Figure 5 I and  5 J). They supply apparent wild-type junctional activity and accumulate to high levels in all cells when the kinase responsible for the degradation signal (Zw3) is removed ( Figure 5 K and  5 L). The primary phenotype of these alleles is a loss or reduction of Wnt transcriptional responses ( Figure 5 A and  5 B). The  arm F1a  allele produced a partial loss-of-function phenotype, and germline clone embryos show some residual naked cuticle.  arm LM134  produces a stronger phenotype comparable to a loss of  wg  function, although it may not be a signaling null (see below). Figure 4 Expression of Arm ΔArm  Leads to the Nuclear Localization of Endogenous Arm Protein (A) Wild-type Arm protein appears in stripes that correspond to cells responding to Wg signaling. (B) Expression of Arm ΔArm  in an  arm F1a  background leads to the nuclear localization of endogenous Arm. (C and D) Dark-field images reveal that expression of both Arm ΔArm  and Arm S10  leads to similar naked cuticle phenotypes. (E) An anti-Arm Western blot showing a faster-migrating band, which correlates with endogenous Arm's being active, and a slower-migrating band, which correlates with Arm's being inactive. Figure 5 ΔArm Functions through Endogenous Arm (A) Embryonic cuticle of  arm F1a  mutant showing a weak loss-of-function phenotype. (B) Cuticle of  arm LM134  mutant embryo showing a strong loss-of-signaling phenotype. (C) Embryo mutant for  arm F1a  expressing Arm ΔArm  showing relatively normal segment polarity. (D)  arm LM134  mutant expressing Arm ΔArm  also shows segment polarity. (E and F) Both alleles in combination with a null  zw3  allele and expressing Arm ΔArm  show a complete lack of denticles. (G and H) Both alleles expressing the activated but nontethered form of stabilized Arm, Arm S10 , show the naked cuticle phenotype. (I–L) In both missense alleles, the mutant protein is expressed in stripes (I and J), corresponding with Wg expression (data not shown), which is abolished when the key degradation kinase Zw3 is removed (K and L). We asked whether these signaling-deficient alleles could block the cell fate transformation and Wnt target activation observed when Arm ΔArm  is expressed in wild-type epidermis. If Arm ΔArm  functions independently of the endogenous protein, then all cells should assume the naked cell fate. However, this does not occur ( Figure 5 C and  5 D). Instead, both point mutants produce a cuticle pattern with periodic denticle belts and regions of intervening naked cuticle. This periodicity may reflect the fact that  arm F1a  and  arm LM134  can still be controlled by Wg even when Arm ΔArm  is expressed. This periodicity is, in fact, abolished when Zw3 activity is removed from such embryos (i.e., in triply mutant  zw3, arm F1a ; Arm ΔArm  embryos). Under these conditions, all cells in the cuticle take on the naked cell fate ( Figure 5 E and  5 F). Since Arm ΔArm  lacks the N-terminal sites that respond to Zw3, the sensitivity of the double-mutant phenotype confirms that the pattern of the double mutant is dependent on the endogenous Arm protein. The behavior of membrane-tethered Arm ΔArm  contrasts with that of other stabilized forms of Arm that would be predicted to move more freely between the cytoplasm and the nucleus. Arm S10 , for example, contains a small N-terminal deletion that blocks Zw3 phosphorylation, but preserves binding sites for various nuclear proteins (see  Figure 2 ;  Pai et al. 1997 ). Arm S10  is not membrane-tethered, but the cell fate transformations it produces are identical to those produced by Arm ΔArm  (compare  Figure 4 C and  4 D). They do not, however, depend on the endogenous allele and are still observed in an  arm F1a  or  arm LM134  germline clone background ( Figure 5 G and  5 H).
Loss-of-Function Missense Mutations
When Arm ΔArm  is expressed in a wild-type embryo, it strongly activates Wg signaling ( Figure 4 C;  Chan and Struhl 2002 ).  Chan and Struhl (2002 ) suggest that this is because this membrane-tethered form of Arm can signal on its own. The results presented above argue, on the other hand, that it does so by stabilizing the endogenous protein. To further test this, we asked whether expression of Arm ΔArm  can induce Wg signaling when endogenous Arm is replaced by signaling-deficient Arm. We turned to two novel missense mutations where the rest of the  arm  coding region remains intact. Because these alleles do not produce truncations through stop codons, they are immune to nonsense mRNA degradation ( Wagner and Lykke-Andersen 2002 ). Both mutations result in amino acid substitutions close to repeat seven, a key hinge region postulated to be important in binding of TCF ( Huber et al. 1997 ;  Graham et al. 2000 ). Both mutants retain the phosphorylation sites required for degradation and therefore accumulate in stripes in response to Wg signal ( Figure 5 I and  5 J). They supply apparent wild-type junctional activity and accumulate to high levels in all cells when the kinase responsible for the degradation signal (Zw3) is removed ( Figure 5 K and  5 L). The primary phenotype of these alleles is a loss or reduction of Wnt transcriptional responses ( Figure 5 A and  5 B). The  arm F1a  allele produced a partial loss-of-function phenotype, and germline clone embryos show some residual naked cuticle.  arm LM134  produces a stronger phenotype comparable to a loss of  wg  function, although it may not be a signaling null (see below).armarm F1aarm LM134wgWe asked whether these signaling-deficient alleles could block the cell fate transformation and Wnt target activation observed when Arm ΔArm  is expressed in wild-type epidermis. If Arm ΔArm  functions independently of the endogenous protein, then all cells should assume the naked cell fate. However, this does not occur ( Figure 5 C and  5 D). Instead, both point mutants produce a cuticle pattern with periodic denticle belts and regions of intervening naked cuticle. This periodicity may reflect the fact that  arm F1a  and  arm LM134  can still be controlled by Wg even when Arm ΔArm  is expressed. This periodicity is, in fact, abolished when Zw3 activity is removed from such embryos (i.e., in triply mutant  zw3, arm F1a ; Arm ΔArm  embryos). Under these conditions, all cells in the cuticle take on the naked cell fate ( Figure 5 E and  5 F). Since Arm ΔArm  lacks the N-terminal sites that respond to Zw3, the sensitivity of the double-mutant phenotype confirms that the pattern of the double mutant is dependent on the endogenous Arm protein.arm F1aarm LM134zw3, arm F1a ;The behavior of membrane-tethered Arm ΔArm  contrasts with that of other stabilized forms of Arm that would be predicted to move more freely between the cytoplasm and the nucleus. Arm S10 , for example, contains a small N-terminal deletion that blocks Zw3 phosphorylation, but preserves binding sites for various nuclear proteins (see  Figure 2 ;  Pai et al. 1997 ). Arm S10  is not membrane-tethered, but the cell fate transformations it produces are identical to those produced by Arm ΔArm  (compare  Figure 4 C and  4 D). They do not, however, depend on the endogenous allele and are still observed in an  arm F1a  or  arm LM134  germline clone background ( Figure 5 G and  5 H).arm F1aarm LM134Arm ΔArm  Causes Nuclear Localization and Mobility Shift of Endogenous Arm All of our experiments argue that Arm ΔArm  produces its effect on transcription by activating the endogenous alleles. To investigate the mechanism that underlies this effect, we looked at the in situ localization of the endogenous Arm protein and its migration pattern on Western blots. Expression of Arm ΔArm  is sufficient to drive both wild-type and the point mutant forms of Arm into nuclei (see  Figure 4 A and  4 B;  Miller and Moon 1997 ;  Tolwinski and Wieschaus 2001 ). Generally, the most obvious feature observed upon removal of any of the negative factors of the Wg pathway is the rapid accumulation of Arm in cells. However, another feature is the phosphorylation state of the Arm protein.  Peifer et al. (1994 a) found that a fast-migrating band of Arm corresponds with active Wg signaling and that a slower-migrating band corresponds with Wg's being off. Therefore, it is the unphosphorylated band that corresponds with signaling. Here we show that, on Western blots, endogenous Arm protein responds to Arm ΔArm  expression in much the same way that it does to the removal of negative components of the pathway such as Axin and APC1 and APC2 (see  Figure 4 E). We see a downshift of the protein, which is directly opposite to what is seen when a positive component of the pathway is removed (Dsh or Wg; see  Figure 4 E). Wild-type embryos show the expected intermediate phenotype, as they have both active and inactive forms of Arm protein (see  Figure 4 E). The observed shift is most likely the result of phosphorylation ( Peifer et al. 1994 a), though we do not address this directly in this study.
Arm ΔArm  Causes Nuclear Localization and Mobility Shift of Endogenous Arm
All of our experiments argue that Arm ΔArm  produces its effect on transcription by activating the endogenous alleles. To investigate the mechanism that underlies this effect, we looked at the in situ localization of the endogenous Arm protein and its migration pattern on Western blots. Expression of Arm ΔArm  is sufficient to drive both wild-type and the point mutant forms of Arm into nuclei (see  Figure 4 A and  4 B;  Miller and Moon 1997 ;  Tolwinski and Wieschaus 2001 ).Generally, the most obvious feature observed upon removal of any of the negative factors of the Wg pathway is the rapid accumulation of Arm in cells. However, another feature is the phosphorylation state of the Arm protein.  Peifer et al. (1994 a) found that a fast-migrating band of Arm corresponds with active Wg signaling and that a slower-migrating band corresponds with Wg's being off. Therefore, it is the unphosphorylated band that corresponds with signaling. Here we show that, on Western blots, endogenous Arm protein responds to Arm ΔArm  expression in much the same way that it does to the removal of negative components of the pathway such as Axin and APC1 and APC2 (see  Figure 4 E). We see a downshift of the protein, which is directly opposite to what is seen when a positive component of the pathway is removed (Dsh or Wg; see  Figure 4 E). Wild-type embryos show the expected intermediate phenotype, as they have both active and inactive forms of Arm protein (see  Figure 4 E). The observed shift is most likely the result of phosphorylation ( Peifer et al. 1994 a), though we do not address this directly in this study.The C-Terminus of Arm Is Necessary for Cby-Mediated Repression Although the missense mutations we have used in our studies produce (on average) weaker phenotypes, they are more effective at blocking the cell-fate transformation induced by Arm ΔArm  than the “medium” C-terminal truncation mutants (compare  Figure 1 K with  Figure 5 C and  5 D). The comparison is somewhat indirect, owing to the necessity of expressing Arm S18  in the “medium”  arm  allele background in order to get intact embryos. However, we find that expression of Arm S18  in an  arm F1a  background has no visible effect on the cuticle (data not shown). Therefore, the activity of C-terminally truncated  arm  alleles in response to ΔArm expression suggests that, under certain conditions, removal of the C-terminus may actually enhance the transcriptional activity of Arm. One possibility is suggested by the recent discovery of Cby ( Takemaru et al. 2003 ), a nuclear negative regulator of the Wg pathway that binds to the C-terminus of Arm. To test whether nuclear Cby affected the transformation produced by Arm ΔArm , we used RNA interference (RNAi) to reduce Cby levels in  arm F1a  embryos with and without Arm ΔArm . In the absence of Arm ΔArm , i.e., in embryos where most Arm F1a  protein is cytoplasmic, Cby RNAi has no effect ( Figure 6 D). However, when Arm ΔArm  is present, lowering Cby levels leads to increased naked cuticle characteristic of Wnt pathway activation (compare  Figure 6 B to  6 C). We propose that Cby's effect on  arm F1a  protein is dependent on Arm ΔArm  relocalizing Arm to the nucleus. Figure 6 Relief of C-Terminal Repression through the Elimination of Cby Leads to Uniform Activation of Signaling (A) A wild-type cuticle shown for comparison. (B) Expression of Arm ΔArm  in the  arm F1a  background. (C) Expression of a Cby RNAi construct along with Arm ΔArm  in the  arm F1a  background. (D) Expression of a Cby RNAi construct in an  arm F1a  background.
The C-Terminus of Arm Is Necessary for Cby-Mediated Repression
Although the missense mutations we have used in our studies produce (on average) weaker phenotypes, they are more effective at blocking the cell-fate transformation induced by Arm ΔArm  than the “medium” C-terminal truncation mutants (compare  Figure 1 K with  Figure 5 C and  5 D). The comparison is somewhat indirect, owing to the necessity of expressing Arm S18  in the “medium”  arm  allele background in order to get intact embryos. However, we find that expression of Arm S18  in an  arm F1a  background has no visible effect on the cuticle (data not shown). Therefore, the activity of C-terminally truncated  arm  alleles in response to ΔArm expression suggests that, under certain conditions, removal of the C-terminus may actually enhance the transcriptional activity of Arm. One possibility is suggested by the recent discovery of Cby ( Takemaru et al. 2003 ), a nuclear negative regulator of the Wg pathway that binds to the C-terminus of Arm. To test whether nuclear Cby affected the transformation produced by Arm ΔArm , we used RNA interference (RNAi) to reduce Cby levels in  arm F1a  embryos with and without Arm ΔArm . In the absence of Arm ΔArm , i.e., in embryos where most Arm F1a  protein is cytoplasmic, Cby RNAi has no effect ( Figure 6 D). However, when Arm ΔArm  is present, lowering Cby levels leads to increased naked cuticle characteristic of Wnt pathway activation (compare  Figure 6 B to  6 C). We propose that Cby's effect on  arm F1a  protein is dependent on Arm ΔArm  relocalizing Arm to the nucleus.armarm F1aarmarm F1aarm F1aDiscussion In this study we offer genetic proof that the nuclear localization of Arm is important for the activation of the pathway. The dissenting view ( Chan and Struhl 2002 ) relied on C-terminal truncations that we have shown retain their ability to signal if their levels are increased. These alleles also appear to bypass the normal nuclear regulation by Cby. We show that full-length loss-of-function forms of Arm provide a novel way of assessing the activity of the pathway. Finally, we show that in an approximate signaling-null condition, Arm ΔArm  cannot activate transcription on its own. Based on these findings, we propose that membrane-tethered Arm, whether wild-type or activated, cannot activate transcription on its own. It does, however, have a profound effect on the endogenous form, forcing both “weak” and “medium” alleles to translocate to the nucleus and activate transcription. Our findings extend and build upon the original nuclear localization of Arm model ( Miller and Moon 1997 ;  Cox et al. 1999 b). Further support for the nuclear localization of Arm model has recently been provided by the publication of a study that uses tissue culture experiments to show that nuclear localization of Arm is required ( Cong et al. 2003 ). Our results also point to an unexpected feature of Arm, namely that the C-terminus, although it has been shown to supply transcriptional activation ( Hsu et al. 1998 ), does not appear to be required for Wnt activation.  Cox et al. (1999 a) studied this aspect of Arm function and found that a C-terminally truncated form of Arm can significantly rescue the signaling defects of  arm  mutants, but is not as good as the wild-type form at transcriptional activation. Further, given that  arm  mutant flies expressing the transgene that lacks the C-terminus do not survive to adulthood, the C-terminus may not be entirely expendable. This may point to the requirement for Cby-based repression or Teashirt-mediated activation at a later stage of development, as both these proteins function by binding the C-terminus of Arm ( Gallet et al. 1999 ;  Takemaru et al. 2003 ). However, taken together with the finding that an N-terminally truncated Arm sent to the nucleus fails to activate transcription ( Chan and Struhl 2002 ), it appears that it is the N-terminus that is most important for the nuclear transactivation and chromatin remodeling functions ascribed to β-catenin ( Hsu et al. 1998 ;  Hecht and Kemler 2000 ;  Takemaru and Moon 2000 ;  Barker et al. 2001 ;  Tutter et al. 2001 ;  Bienz and Clevers 2003 ). We have previously shown that the “medium”  arm  mutant ( arm O43A01 , which creates a stop codon eliminating repeats 10 through 12 and the entire C-terminus) does not signal in the presence of uniform Arm ΔArm  ( Tolwinski and Wieschaus 2001 ).  Chan and Struhl (2002 ) found that  arm O43A01  embryos expressing high levels of Arm ΔArm  from the paired GAL4 driver were able to activate Wnt targets. But since neither Arm ΔArm  nor  arm O43A01  can provide junctional Arm activity, the abnormalities of these embryos make these experiments difficult to interpret. As an alternative, we used a membrane-tethered but otherwise wild-type form of Arm (Arm S18 ), which we expressed in  arm O43A01  mutant embryos (see  Figure 1 G). The Arm S18  allele rescues the junctional defects, but does not allow signaling. Similar results have been obtained with another “medium” allele,  arm XP33  ( Cox et al. 1999 b). However, when combined with Arm ΔArm  and Arm S18 ,  arm O43A01  can now be clearly seen to activate naked cell fates. It thus appears that even the “medium” alleles of  arm  actually do retain some ability to function when Arm ΔArm  is present. This is not observed in the larger truncations (“strong” alleles), consistent with the “medium” alleles retaining the TCF-binding region ( Graham et al. 2000 ). The question now becomes what is Arm ΔArm  doing at the membrane that causes such drastic change in the signaling kinetics of the pathway. We have previously argued that Arm ΔArm  may function by titrating the cytoplasmic anchoring activity of Axin and by therefore allowing rapid enrichment of Arm in the nucleus. We have in fact observed such an enrichment and have shown that it is counteracted by increasing the level of Axin ( Tolwinski and Wieschaus 2001 ). Further work has pointed to the importance of controlling Axin stability in pathway activation ( Salic et al. 2000 ;  Mao et al. 2001 ;  Lee et al. 2003 ;  Tolwinski et al. 2003 ). Expression of large quantities of a stabilized, membrane-tethered form of Arm might also remove additional cytoplasmic inhibitory factors, preventing them from interacting with nontethered Arm. In turn, even lower-level or lower-activity alleles will now be able to activate transcription, simply owing to the complete lack of inhibiting factors. The missense mutations described here provide a glimpse of the in vivo activity of Arm protein. Structural studies of β-catenin found that although the central repeat region forms a uniformly repeating super helix, one α-helix was missing from repeat seven. The missing helix might allow a local flexibility in the structure and led the authors to define this region as a potential hinge ( Huber et al. 1997 ). Further crystallographic analysis concluded that this region was important for TCF binding ( Graham et al. 2000 ). Both our point mutations cluster around this repeat and would probably lead to structural consequences for this hinge. The apparent specificity of these alleles for the transcriptional response to Wnt signaling provides in vivo evidence that the postulated hinge may be very important for that aspect of Arm protein function.
Discussion
In this study we offer genetic proof that the nuclear localization of Arm is important for the activation of the pathway. The dissenting view ( Chan and Struhl 2002 ) relied on C-terminal truncations that we have shown retain their ability to signal if their levels are increased. These alleles also appear to bypass the normal nuclear regulation by Cby. We show that full-length loss-of-function forms of Arm provide a novel way of assessing the activity of the pathway. Finally, we show that in an approximate signaling-null condition, Arm ΔArm  cannot activate transcription on its own. Based on these findings, we propose that membrane-tethered Arm, whether wild-type or activated, cannot activate transcription on its own. It does, however, have a profound effect on the endogenous form, forcing both “weak” and “medium” alleles to translocate to the nucleus and activate transcription. Our findings extend and build upon the original nuclear localization of Arm model ( Miller and Moon 1997 ;  Cox et al. 1999 b). Further support for the nuclear localization of Arm model has recently been provided by the publication of a study that uses tissue culture experiments to show that nuclear localization of Arm is required ( Cong et al. 2003 ).Our results also point to an unexpected feature of Arm, namely that the C-terminus, although it has been shown to supply transcriptional activation ( Hsu et al. 1998 ), does not appear to be required for Wnt activation.  Cox et al. (1999 a) studied this aspect of Arm function and found that a C-terminally truncated form of Arm can significantly rescue the signaling defects of  arm  mutants, but is not as good as the wild-type form at transcriptional activation. Further, given that  arm  mutant flies expressing the transgene that lacks the C-terminus do not survive to adulthood, the C-terminus may not be entirely expendable. This may point to the requirement for Cby-based repression or Teashirt-mediated activation at a later stage of development, as both these proteins function by binding the C-terminus of Arm ( Gallet et al. 1999 ;  Takemaru et al. 2003 ). However, taken together with the finding that an N-terminally truncated Arm sent to the nucleus fails to activate transcription ( Chan and Struhl 2002 ), it appears that it is the N-terminus that is most important for the nuclear transactivation and chromatin remodeling functions ascribed to β-catenin ( Hsu et al. 1998 ;  Hecht and Kemler 2000 ;  Takemaru and Moon 2000 ;  Barker et al. 2001 ;  Tutter et al. 2001 ;  Bienz and Clevers 2003 ).armarmWe have previously shown that the “medium”  arm  mutant ( arm O43A01 , which creates a stop codon eliminating repeats 10 through 12 and the entire C-terminus) does not signal in the presence of uniform Arm ΔArm  ( Tolwinski and Wieschaus 2001 ).  Chan and Struhl (2002 ) found that  arm O43A01  embryos expressing high levels of Arm ΔArm  from the paired GAL4 driver were able to activate Wnt targets. But since neither Arm ΔArm  nor  arm O43A01  can provide junctional Arm activity, the abnormalities of these embryos make these experiments difficult to interpret. As an alternative, we used a membrane-tethered but otherwise wild-type form of Arm (Arm S18 ), which we expressed in  arm O43A01  mutant embryos (see  Figure 1 G). The Arm S18  allele rescues the junctional defects, but does not allow signaling. Similar results have been obtained with another “medium” allele,  arm XP33  ( Cox et al. 1999 b). However, when combined with Arm ΔArm  and Arm S18 ,  arm O43A01  can now be clearly seen to activate naked cell fates. It thus appears that even the “medium” alleles of  arm  actually do retain some ability to function when Arm ΔArm  is present. This is not observed in the larger truncations (“strong” alleles), consistent with the “medium” alleles retaining the TCF-binding region ( Graham et al. 2000 ).armarm O43A01arm O43A01arm O43A01arm O43A01arm XP33arm O43A01armThe question now becomes what is Arm ΔArm  doing at the membrane that causes such drastic change in the signaling kinetics of the pathway. We have previously argued that Arm ΔArm  may function by titrating the cytoplasmic anchoring activity of Axin and by therefore allowing rapid enrichment of Arm in the nucleus. We have in fact observed such an enrichment and have shown that it is counteracted by increasing the level of Axin ( Tolwinski and Wieschaus 2001 ). Further work has pointed to the importance of controlling Axin stability in pathway activation ( Salic et al. 2000 ;  Mao et al. 2001 ;  Lee et al. 2003 ;  Tolwinski et al. 2003 ). Expression of large quantities of a stabilized, membrane-tethered form of Arm might also remove additional cytoplasmic inhibitory factors, preventing them from interacting with nontethered Arm. In turn, even lower-level or lower-activity alleles will now be able to activate transcription, simply owing to the complete lack of inhibiting factors.The missense mutations described here provide a glimpse of the in vivo activity of Arm protein. Structural studies of β-catenin found that although the central repeat region forms a uniformly repeating super helix, one α-helix was missing from repeat seven. The missing helix might allow a local flexibility in the structure and led the authors to define this region as a potential hinge ( Huber et al. 1997 ). Further crystallographic analysis concluded that this region was important for TCF binding ( Graham et al. 2000 ). Both our point mutations cluster around this repeat and would probably lead to structural consequences for this hinge. The apparent specificity of these alleles for the transcriptional response to Wnt signaling provides in vivo evidence that the postulated hinge may be very important for that aspect of Arm protein function.Note  As the final version of this paper was being prepared, the paper by  Chan and Struhl (2002 ) was retracted.
Note
As the final version of this paper was being prepared, the paper by  Chan and Struhl (2002 ) was retracted.Materials and Methods Fly Strains The wild-type strain used was Oregon R. See Flybase ( http://flybase.bio.indiana.edu ) for details on mutants used. Hypomorphic mutants of  arm  are as follows:  arm LM134  TCC to TTC at nucleotide 2776,  arm F1a  CGC to CAC at nucleotide 2990,  arm XM19  stop codon at nucleotide 3850,  arm O43A01  stop codon at nucleotide 3404,  arm XP33  stop codon at nucleotide 3466,  arm XK22  stop codon at nucleotide 3013. Other alleles used were  axin S044230  ,  zw3 M11–1 ,  dsh V26 ,  apc1 Q8 ,  apc2 d40 , and  wg IG22 . Crosses and Expression of UAS Constructs
 arm  mutants As Arm and many other  Drosophila  proteins are contributed maternally, to fully evaluate the function of a mutant protein, one needs to make embryos maternally and zygotically mutant. Therefore, maternally mutant eggs were generated by the dominant female sterile technique ( Chou and Perrimon 1992 ). For all expression experiments, the Arm–GAL4 driver was used. All X-chromosome mutants use FRT 101. The  arm  mutants used were as follows:
 arm F1a  zw3 M11–1  (maternal)/Y (zygotic)
 arm LM134  zw3 M11–1  (maternal)/Y (zygotic)
 arm F1a  (maternal)/Y (zygotic)
 arm LM134  (maternal)/Y (zygotic)
 arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm F1a  zw3 M11–1  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm LM134  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm LM134  zw3 M11–1  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm O43A01  (maternal)/Y (zygotic)
 arm O43A01  (maternal)/Y (zygotic); Arm S18  (zygotic)
 arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–WIZ–Cby, UAS–ΔArm (zygotic)
 arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–WIZ–Cby (zygotic)
 arm XK22  (maternal)/Y (zygotic); Arm S18  (maternal)
 arm XK22  (maternal)/Y (zygotic); MAT–GAL4/UAS–ΔArm (zygotic); Arm S18  (maternal) UAS transgenes and GAL4 driver lines Previously published transgenes used were UAS–Arm*[S10], a small deletion of the phosphorylation sites ( Pai et al. 1996 ); the Arm–GAL4 driver ( Sanson et al. 1996 ); Arm S18 , a mirystoylated, membrane-tethered full-length Arm ( Cox et al. 1999 b); and UAS–WIZ–Cby for RNAi ( Takemaru et al. 2003 ). ΔArm is a pUAST transgene that deletes the first 128 amino acids, including the GSK3β and CKII phosphorylation sites, the CBP acetylation site, the α-catenin-binding domain, and a transactivation domain ( Zecca et al. 1996 ). Antibodies and Immunofluorescence  Embryos were treated and stained as described previously ( Tolwinski and Wieschaus 2001 ), except that they were fixed with heptane/4% formaldehyde in phosphate buffer (0.1 M NaPO 4  [pH 7.4]). The antibodies used were anti-Arm (monoclonal antibody [mAb] N2 7A1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, United States), rabbit anti-Arm ( Peifer et al. 1994 b), rabbit anti-c-Myc (Santa Cruz Biotechnology, Santa Cruz, California, United States), and anti-Sexlethal (mAb M-14; Developmental Studies Hybridoma Bank). Staining, detection, and image processing were as described previously ( Tolwinski and Wieschaus 2001 ). Though not shown, the Sexlethal antibody was used to sex embryos. This allows for the identification of male embryos laid by germline clone mothers, which are hemizygous and therefore maternally and zygotically mutant for X-chromosome genes. Western Blotting Embryos were lysed in extract buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 10% glycerol; Complete Mini Protease, Sigma, St. Louis, Missouri, United States), and the extracts were separated by 7.5% SDS-PAGE and were blotted as described elsewhere ( Peifer et al. 1994 a). Maternally and zygotically mutant embryos were hand-selected using standard GFP balancers ( http://flybase.bio.indiana.edu ).
Materials and Methods
Fly Strains The wild-type strain used was Oregon R. See Flybase ( http://flybase.bio.indiana.edu ) for details on mutants used. Hypomorphic mutants of  arm  are as follows:  arm LM134  TCC to TTC at nucleotide 2776,  arm F1a  CGC to CAC at nucleotide 2990,  arm XM19  stop codon at nucleotide 3850,  arm O43A01  stop codon at nucleotide 3404,  arm XP33  stop codon at nucleotide 3466,  arm XK22  stop codon at nucleotide 3013. Other alleles used were  axin S044230  ,  zw3 M11–1 ,  dsh V26 ,  apc1 Q8 ,  apc2 d40 , and  wg IG22 .
Fly Strains
The wild-type strain used was Oregon R. See Flybase ( http://flybase.bio.indiana.edu ) for details on mutants used. Hypomorphic mutants of  arm  are as follows:  arm LM134  TCC to TTC at nucleotide 2776,  arm F1a  CGC to CAC at nucleotide 2990,  arm XM19  stop codon at nucleotide 3850,  arm O43A01  stop codon at nucleotide 3404,  arm XP33  stop codon at nucleotide 3466,  arm XK22  stop codon at nucleotide 3013. Other alleles used were  axin S044230  ,  zw3 M11–1 ,  dsh V26 ,  apc1 Q8 ,  apc2 d40 , and  wg IG22 .armarm LM134arm F1aarm XM19arm O43A01arm XP33arm XK22axin S044230zw3 M11–1dsh V26apc1 Q8apc2 d40wg IG22Crosses and Expression of UAS Constructs
 arm  mutants As Arm and many other  Drosophila  proteins are contributed maternally, to fully evaluate the function of a mutant protein, one needs to make embryos maternally and zygotically mutant. Therefore, maternally mutant eggs were generated by the dominant female sterile technique ( Chou and Perrimon 1992 ). For all expression experiments, the Arm–GAL4 driver was used. All X-chromosome mutants use FRT 101. The  arm  mutants used were as follows:
 arm F1a  zw3 M11–1  (maternal)/Y (zygotic)
 arm LM134  zw3 M11–1  (maternal)/Y (zygotic)
 arm F1a  (maternal)/Y (zygotic)
 arm LM134  (maternal)/Y (zygotic)
 arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm F1a  zw3 M11–1  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm LM134  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm LM134  zw3 M11–1  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm O43A01  (maternal)/Y (zygotic)
 arm O43A01  (maternal)/Y (zygotic); Arm S18  (zygotic)
 arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–WIZ–Cby, UAS–ΔArm (zygotic)
 arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–WIZ–Cby (zygotic)
 arm XK22  (maternal)/Y (zygotic); Arm S18  (maternal)
 arm XK22  (maternal)/Y (zygotic); MAT–GAL4/UAS–ΔArm (zygotic); Arm S18  (maternal) UAS transgenes and GAL4 driver lines Previously published transgenes used were UAS–Arm*[S10], a small deletion of the phosphorylation sites ( Pai et al. 1996 ); the Arm–GAL4 driver ( Sanson et al. 1996 ); Arm S18 , a mirystoylated, membrane-tethered full-length Arm ( Cox et al. 1999 b); and UAS–WIZ–Cby for RNAi ( Takemaru et al. 2003 ). ΔArm is a pUAST transgene that deletes the first 128 amino acids, including the GSK3β and CKII phosphorylation sites, the CBP acetylation site, the α-catenin-binding domain, and a transactivation domain ( Zecca et al. 1996 ).
Crosses and Expression of UAS Constructs
arm  mutants As Arm and many other  Drosophila  proteins are contributed maternally, to fully evaluate the function of a mutant protein, one needs to make embryos maternally and zygotically mutant. Therefore, maternally mutant eggs were generated by the dominant female sterile technique ( Chou and Perrimon 1992 ). For all expression experiments, the Arm–GAL4 driver was used. All X-chromosome mutants use FRT 101. The  arm  mutants used were as follows:
 arm F1a  zw3 M11–1  (maternal)/Y (zygotic)
 arm LM134  zw3 M11–1  (maternal)/Y (zygotic)
 arm F1a  (maternal)/Y (zygotic)
 arm LM134  (maternal)/Y (zygotic)
 arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm F1a  zw3 M11–1  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm LM134  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm LM134  zw3 M11–1  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)
 arm O43A01  (maternal)/Y (zygotic)
 arm O43A01  (maternal)/Y (zygotic); Arm S18  (zygotic)
 arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–WIZ–Cby, UAS–ΔArm (zygotic)
 arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–WIZ–Cby (zygotic)
 arm XK22  (maternal)/Y (zygotic); Arm S18  (maternal)
 arm XK22  (maternal)/Y (zygotic); MAT–GAL4/UAS–ΔArm (zygotic); Arm S18  (maternal)
arm  mutants
armAs Arm and many other  Drosophila  proteins are contributed maternally, to fully evaluate the function of a mutant protein, one needs to make embryos maternally and zygotically mutant. Therefore, maternally mutant eggs were generated by the dominant female sterile technique ( Chou and Perrimon 1992 ). For all expression experiments, the Arm–GAL4 driver was used. All X-chromosome mutants use FRT 101. The  arm  mutants used were as follows:Drosophilaarmarm F1a  zw3 M11–1  (maternal)/Y (zygotic)arm F1a  zw3 M11–1arm LM134  zw3 M11–1  (maternal)/Y (zygotic)arm LM134  zw3 M11–1arm F1a  (maternal)/Y (zygotic)arm F1aarm LM134  (maternal)/Y (zygotic)arm LM134arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)arm F1aarm F1a  zw3 M11–1  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)arm F1a  zw3 M11–1arm LM134  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)arm LM134arm LM134  zw3 M11–1  (maternal)/Y (zygotic); Arm–GAL4/UAS–ΔArm (zygotic)arm LM134  zw3 M11–1arm O43A01  (maternal)/Y (zygotic)arm O43A01arm O43A01  (maternal)/Y (zygotic); Arm S18  (zygotic)arm O43A01arm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–WIZ–Cby, UAS–ΔArm (zygotic)arm F1aarm F1a  (maternal)/Y (zygotic); Arm–GAL4/UAS–WIZ–Cby (zygotic)arm F1aarm XK22  (maternal)/Y (zygotic); Arm S18  (maternal)arm XK22arm XK22  (maternal)/Y (zygotic); MAT–GAL4/UAS–ΔArm (zygotic); Arm S18  (maternal)arm XK22UAS transgenes and GAL4 driver lines Previously published transgenes used were UAS–Arm*[S10], a small deletion of the phosphorylation sites ( Pai et al. 1996 ); the Arm–GAL4 driver ( Sanson et al. 1996 ); Arm S18 , a mirystoylated, membrane-tethered full-length Arm ( Cox et al. 1999 b); and UAS–WIZ–Cby for RNAi ( Takemaru et al. 2003 ). ΔArm is a pUAST transgene that deletes the first 128 amino acids, including the GSK3β and CKII phosphorylation sites, the CBP acetylation site, the α-catenin-binding domain, and a transactivation domain ( Zecca et al. 1996 ).
UAS transgenes and GAL4 driver lines
Previously published transgenes used were UAS–Arm*[S10], a small deletion of the phosphorylation sites ( Pai et al. 1996 ); the Arm–GAL4 driver ( Sanson et al. 1996 ); Arm S18 , a mirystoylated, membrane-tethered full-length Arm ( Cox et al. 1999 b); and UAS–WIZ–Cby for RNAi ( Takemaru et al. 2003 ). ΔArm is a pUAST transgene that deletes the first 128 amino acids, including the GSK3β and CKII phosphorylation sites, the CBP acetylation site, the α-catenin-binding domain, and a transactivation domain ( Zecca et al. 1996 ).Antibodies and Immunofluorescence  Embryos were treated and stained as described previously ( Tolwinski and Wieschaus 2001 ), except that they were fixed with heptane/4% formaldehyde in phosphate buffer (0.1 M NaPO 4  [pH 7.4]). The antibodies used were anti-Arm (monoclonal antibody [mAb] N2 7A1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, United States), rabbit anti-Arm ( Peifer et al. 1994 b), rabbit anti-c-Myc (Santa Cruz Biotechnology, Santa Cruz, California, United States), and anti-Sexlethal (mAb M-14; Developmental Studies Hybridoma Bank). Staining, detection, and image processing were as described previously ( Tolwinski and Wieschaus 2001 ). Though not shown, the Sexlethal antibody was used to sex embryos. This allows for the identification of male embryos laid by germline clone mothers, which are hemizygous and therefore maternally and zygotically mutant for X-chromosome genes.
Antibodies and Immunofluorescence
Embryos were treated and stained as described previously ( Tolwinski and Wieschaus 2001 ), except that they were fixed with heptane/4% formaldehyde in phosphate buffer (0.1 M NaPO 4  [pH 7.4]). The antibodies used were anti-Arm (monoclonal antibody [mAb] N2 7A1; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa, United States), rabbit anti-Arm ( Peifer et al. 1994 b), rabbit anti-c-Myc (Santa Cruz Biotechnology, Santa Cruz, California, United States), and anti-Sexlethal (mAb M-14; Developmental Studies Hybridoma Bank). Staining, detection, and image processing were as described previously ( Tolwinski and Wieschaus 2001 ). Though not shown, the Sexlethal antibody was used to sex embryos. This allows for the identification of male embryos laid by germline clone mothers, which are hemizygous and therefore maternally and zygotically mutant for X-chromosome genes.Western Blotting Embryos were lysed in extract buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 10% glycerol; Complete Mini Protease, Sigma, St. Louis, Missouri, United States), and the extracts were separated by 7.5% SDS-PAGE and were blotted as described elsewhere ( Peifer et al. 1994 a). Maternally and zygotically mutant embryos were hand-selected using standard GFP balancers ( http://flybase.bio.indiana.edu ).
Western Blotting
Embryos were lysed in extract buffer (50 mM Tris [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 10% glycerol; Complete Mini Protease, Sigma, St. Louis, Missouri, United States), and the extracts were separated by 7.5% SDS-PAGE and were blotted as described elsewhere ( Peifer et al. 1994 a). Maternally and zygotically mutant embryos were hand-selected using standard GFP balancers ( http://flybase.bio.indiana.edu ).Supporting Information
 Accession Numbers
 The GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession numbers for the genes and alleles discussed in this paper are  apc1 Q8  (U77947),  apc2 d40  (AF091430),  arm  (X54468),  axin S044230  (AF086811),  dsh V26  (U02491),  wg IG22  (NM 164746), and  zw3 M11–1  (X54005).
Supporting Information
Accession NumbersAccession NumbersThe GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/ ) accession numbers for the genes and alleles discussed in this paper are  apc1 Q8  (U77947),  apc2 d40  (AF091430),  arm  (X54468),  axin S044230  (AF086811),  dsh V26  (U02491),  wg IG22  (NM 164746), and  zw3 M11–1  (X54005).apc1 Q8apc2 d40armaxin S044230dsh V26wg IG22zw3 M11–1