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Negative Regulation of Wingless Signaling by D-Axin, a Drosophila Homolog of Axin

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Science  12 Mar 1999:
Vol. 283, Issue 5408, pp. 1739-1742
DOI: 10.1126/science.283.5408.1739

Abstract

Wnt/Wingless directs many cell fates during development. Wnt/Wingless signaling increases the amount of β-catenin/Armadillo, which in turn activates gene transcription. Here theDrosophila protein D-Axin was shown to interact with Armadillo and D-APC. Mutation of d-axin resulted in the accumulation of cytoplasmic Armadillo and one of the Wingless target gene products, Distal-less. Ectopic expression of d-axininhibited Wingless signaling. Hence, D-Axin negatively regulates Wingless signaling by down-regulating the level of Armadillo. These results establish the importance of the Axin family of proteins in Wnt/Wingless signaling in Drosophila.

The Wnt/Wingless (Wg) signal-transduction pathway is involved in cell-cell signaling in many developmental processes (1). Wnt/Wg signaling promotes the stabilization of β-catenin/Armadillo (Arm, a Drosophilahomolog of β-catenin) by negatively regulating the activity of glycogen synthase kinase–3β (GSK-3β). β-Catenin/Arm binds to transcription factors of the LEF/TCF family and thereby modulates expression of Wnt/Wg-responsive genes (2). The colorectal tumor-suppressor gene product APC induces down-regulation of β-catenin, and mutation of APC results in the accumulation of the latter (3). Mutations that activate β-catenin have also been detected in some tumors with intact APC (4). Therefore, regulation of the level of β-catenin is critical for Wnt/Wg signaling during development and tumorigenesis. Here, we show that the Drosophila protein D-Axin interacts with Arm and D-APC and is a negative regulator of Wg signaling.

To identify Arm-interacting proteins, we performed a yeast two-hybrid screen of a Drosophila embryo cDNA library using the Armadillo repeat domain of Arm as target and identified a protein that we designate D-Axin (5). Sequence analysis of thed-axin cDNA showed that it encodes a protein of 743 amino acids. A region near its NH2- terminus (amino acids 51 to 171) shows similarity to the regulator of G protein signaling (RGS) domain, whereas its COOH-terminus contains a region (amino acids 687 to 734) homologous to a conserved sequence near the NH2-terminus of Dishevelled (Dsh) (Fig. 1B). Thus, D-Axin has a domain structure very similar to that of proteins of the mammalian Axin family, Axin and conductin/Axil (6–9), suggesting that D-Axin is aDrosophila homolog of the Axin family of proteins.

Figure 1

Structures of the d-axingene and its protein product and physical interaction of D-Axin with Arm and D-APC. (A) Genomic structure of thed-axin locus. The triangle indicates the location of thel(3)S044230 P insertion. The insertion site is 135 base pairs downstream of the d-axin transcription start site. White boxes indicate untranslated regions of the d-axincDNA. Black boxes indicate coding regions. E, Eco RI site. (B) Schematic representation of the D-Axin protein. Thed-axin sequence has been deposited in GenBank (accession number AF086811). (C) D-Axin binds to the Armadillo repeat domain of Arm. In vitro–translated 35S-labeled D-Axin (IVT D-Axin) was incubated with purified GST or GST-Arm fusion proteins (N-ter, amino acids 1 to 139; Arm, the Armadillo repeat domain, amino acids 140 to 713; C-ter, amino acids 714 to 843) immobilized to glutathione-Sepharose. The bound proteins were analyzed by SDS-PAGE and autoradiography. (D) Arm binds to amino acids 459 to 538 of D-Axin. The in vitro–translated 35S-labeled Armadillo repeat domain of Arm (amino acids 140 to 713) was incubated with GST– or GST–D-Axin–Sepharose. (E) D-Axin interacts with D-APC, but not with ZW3/Sgg. In vitro–translated35S-labeled D-Axin was incubated with GST-, GST–D-APC (amino acids 757 to 1270)–, GST-Arm (the Armadillo repeat domain)–, or GST-ZW3/Sgg (full-length)–Sepharose.

We examined whether D-Axin produced by in vitro translation could interact with fragments of Arm fused to glutathione S-transferase (GST) (10). D-Axin specifically interacted with the Armadillo repeat domain of Arm (amino acids 140 to 713), but not with its NH2-terminal (amino acids 1 to 139) or its COOH-terminal (amino acids 714 to 843) domain (Fig. 1C). Pull-down assays with a series of deletion fragments of D-Axin showed that a fragment of D-Axin containing amino acids 459 to 538 bound to Arm (Fig. 1D). This region corresponds to the β-catenin–binding domain of Axin and conductin/Axil and contains a small segment (amino acids 494 to 525) that is highly conserved, suggesting that it may function in binding to Arm.

Recently, we and others have shown that proteins of the mammalian Axin family interact not only with β-catenin but also with GSK-3β and APC (6–9). In line with these findings, we found that the RGS domain of D-Axin interacts with a fragment of D-APC (amino acids 757 to 1270) (Fig. 1E). This region of D-APC corresponds to the region of APC that interacts with β-catenin, conductin, and Axin (7, 11, 12). However, D-Axin did not bind to Zeste white-3/Shaggy (ZW3/Sgg, Drosophila GSK-3β) (Fig. 1E).

The d-axin gene was mapped to the 99D1-7 region of the third chromosome, and its genomic organization was determined (Fig. 1A) (13). Whole-mount in situ hybridization to embryos and imaginal discs and Northern (RNA) blotting with the coding region of the d-axin cDNA as a probe revealed that d-axintranscripts are provided by maternal contribution and expressed ubiquitously throughout all developmental stages (11). To analyze the biological function of D-Axin in vivo, we examined a series of lethal lines that have mutations in the 99D1-7 region and found one line, l(3)S044230, that contains a P-element inserted into the 5′-untranslated region of the first exon of d-axin (Fig. 1A) (14). Although l(3)S044230 homozygous animals died during the first larval instar stage, normal adults were produced upon precise excision of the P-element. In addition, ubiquitous expression of the d-axin cDNA with an inducible heat shock GAL4 system rescued 5 to 10% of l(3)S044230homozygous animals, allowing them to survive until the late third larval instar or pupal stage (15). Furthermore, no signal was observed when d-axin cDNA probe was hybridized in situ to l(3)S044230 homozygous embryos (11). Thus, l(3)S044230 is considered to be a loss-of-function allele of the d-axin locus.

Wg is critical for patterning and cell fate determination in embryonic segmentation (1). Although embryos that are zygotically mutant for d-axin appeared to have almost normal segment patterning (Fig. 2B), embryos devoid of both maternal and zygotic d-axin gene products were completely naked, lacking all denticles on the ventral cuticle (Fig. 2C) (16). Embryos that lack the maternald-axin product but have received one paternal wild-type copy of the gene had some denticles on the ventral cuticle, suggesting that the zygotic d-axin product can partially rescue thed-axin maternal deficiency (Fig. 2D). These phenotypes are similar to those of embryos derived from homozygous zw3/sggfemale germ lines and to those of embryos ubiquitously expressing the wild-type Wg or constitutively active Arm (17). Thus, Wg signaling is constitutively activated in embryos lacking maternal d-axin.

Figure 2

Ventral cuticle phenotype of d-axinembryos. (A) Cuticle of a wild-type embryo. Each segment produces denticles in the anterior region and naked cuticle in the posterior region. (B) Cuticle of an embryo zygotically mutant for d-axin does not exhibit any major defects. (C) Cuticle of an embryo lacking both maternal and zygoticd-axin is entirely naked. Filzkorper is present. (D) Cuticle of an embryo that lacks maternald-axin but has a wild-type paternal copy of the gene has occasional patches of denticles (indicated by the arrow). All embryos are shown with anterior oriented to the left.

Wg is required for the organization of wing blade development, especially for specification of the wing margin structure (18). We found that clones of d-axin mutant cells marked with a yellow mutation produced ectopic marginal bristles cell autonomously (Fig. 3, A and B) (19). Wg also plays an essential role in organizing leg structures; ectopic activation of Wg signaling induces supernumerary outgrowth on the dorsal side of normal legs (18,20). The d-axin clone also produced a supernumerary leg from the dorsal side of the normal leg (Fig. 3C). Furthermore, Wg signaling is required for the formation of sternites in the ventral side of the adult abdomen, and its ectopic activation results in the appearance of ectopic sternite structures (21). The same phenotype was observed in an abdomen containing d-axinclones (Fig. 3D). During wing disc development, Wg signaling is induced along the dorsoventral compartment boundary in the wing imaginal disc. Arm accumulates in the cytoplasm, associates with its partner, Pangolin/DTcf, and activates expression of target genes such asDistal-less (Dll) (1,2, 22). In these d-axin clones, the levels of Arm were markedly enhanced in a strictly cell-autonomous manner (Fig. 3, F and G) (19, 23). In addition, Arm was localized predominantly in the cytoplasm and nuclei in thed-axin mutant clones, in contrast to the membrane localization observed in wild-type cells (Fig. 3G). The levels ofDll expression were also elevated in the d-axinclones in a cell-autonomous manner (Fig. 3H). These results suggest that D-Axin negatively regulates Wg signaling by down-regulating intracellular levels of Arm and that this regulatory mechanism is essential for Wg signaling.

Figure 3

Constitutive activation of Wg signaling ind-axin mutant clones. (A) A wing carryingd-axin mutant clones. Wing-margin structures are ectopically produced by the d-axin clones. Cells belonging to the clones are marked with the yellow mutation. (B) A close-up view of the ectopic wing-margin bristles in (A) (marked by the rectangle). (C) A leg carrying the d-axin clones. The supernumerary leg (indicated by the arrow) branches out from the dorsal surface of the normal leg. (D) Ventral surface of an abdomen carrying the d-axin clones. Ectopic bristles are formed in the pleura by the d-axin clones (indicated by the arrow). (E to G) A wild-type wing disc (E) and a wing disc carrying the d-axin clones (F and G) stained with anti-Arm (red). The d-axin clones are marked by the absence of green Myc-GFP expression (indicated by arrows). In the wild-type wing disc, Arm is ubiquitously expressed at low levels in the wing disc and accumulates preferentially at the cell membrane (E). In thed-axin clones, high levels of Arm accumulate in a cell-autonomous manner (F and G). Single-channel view indicates that Arm in the d-axin clones accumulates preferentially in the cytoplasm and nuclei, whereas Arm in normal cells is localized to the cell membrane (G). (H) A wing disc carrying thed-axin clones stained with anti-Dll (red). Thed-axin clones are marked as in (F) and (G) by the absence of green Myc-GFP expression (indicated by the arrow). In the wild-type wing disc, Dll is expressed along the dorsoventral compartment border (Fig. 4D). In the d-axin clones, Dll is ectopically expressed at high levels in a cell-autonomous manner.

To further examine the function of D-Axin, we ectopically expressed thed-axin gene using the GAL4/UAS system (15). In contrast to the phenotypes observed with the d-axin mutant clones (Fig. 3, A to D), ectopic expression of d-axin induced notches in the wing (Fig. 4A), generation of a supernumerary leg from the ventral side of the normal leg (Fig. 4B), and loss of the sternite structure in the abdomen (Fig. 4C). In addition, whend-axin was expressed in the posterior compartment under the control of engrailed-GAL4, Dll expression was severely repressed in the posterior region of the dorsoventral compartment border (Fig. 4, D and E). Thus, ectopic expression ofd-axin exerts an inhibitory effect on Wg signaling.

Figure 4

Ectopic overexpression ofd-axin causes inhibition of Wg signaling. (A) A wing of a 71B-GAL4:UAS–d-axin fly. Notches are indicated by the arrowhead. (B) A leg of atsh-GAL4:UAS–d-axin fly. The supernumerary leg (indicated by the arrowhead) branches out from the ventral surface of the normal leg. Compare with the leg carrying the d-axin clones in Fig. 3C. (C) Sternites of a tsh-GAL4:UAS–d-axin fly. Sternite bristles are missing (indicated by the arrowhead), and the entire ventral surface is composed of pleura. Compare with the abdomen carrying the d-axin clones in Fig. 3D. (Dand E) A wild-type wing disc (D) and a wing disc of an engrailed-GAL4:UAS–d-axin fly (E) stained with anti-Dll (red). Dll expression is repressed in the posterior region of the dorsoventral compartment border where d-axinis ectopically expressed by the engrailed-GAL4 expression system (indicated by the arrowhead). Wing discs are shown with anterior oriented to the left.

Using genetic analysis in Drosophila, we have demonstrated that D-Axin is required in vivo for the negative regulation of Wg signaling. Of particular interest is the finding that the levels of cytoplasmic Arm are highly and uniformly elevated whereverd-axin clones are located in the wing discs (Fig. 3). For example, the accumulation of Arm in d-axin clones was observed not only around the region where Wg is secreted (indicated by the arrow in Fig. 3F) but also in the region where Wg is not supposed to reach (indicated by the arrowhead in Fig. 3F) (22). Together with the fact that d-axin is ubiquitously expressed, these findings suggest that Wg activity is not required for the effect of D-Axin. We speculate that the Axin family of proteins functions to establish a threshold to prevent premature signaling events caused by Wg/Wnt and to restrict areas that are capable of responding to Wg/Wnt.

  • * To whom correspondence should be addressed. E-mail: akiyama{at}imcbns.iam.u-tokyo.ac.jp

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