Copyright � 2009 by the Genetics Society of AmericaDOI: 10.1534/genetics.108.098236

Note

Membrane Bound Axin Is Sufficient for Wingless Signalingin Drosophila Embryos

Nicholas S. Tolwinski1

Program in Developmental Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10065

Manuscript received November 2, 2008Accepted for publication January 2, 2009

ABSTRACT

The Wingless signaling pathway controls various developmental processes in both vertebrates andinvertebrates. Here I probe the requirement for nuclear localization of APC2 and Axin in the Wg signaltransduction pathway during embryonic development of Drosophila melanogaster. I find that nuclearlocalization of APC2 appears to be required, but Axin can block signaling when tethered to themembrane. These results support the model where Axin regulates Armadillo localization and activity inthe cytoplasm.

THE Wnt/Wingless (Wg) signaling pathway playsessential roles in the development of animals.

Aspects of signaling regulate cell proliferation, differen-tiation, polarity, and survival. Importantly, Wg pathwaycomponents have been found to affect stem cell main-tenance and tumor progression. The basic step in signaltransduction through this pathway is the regulation ofArmadillo/b-catenin (Arm/b-cat) levels. In the absenceof extracellular ligand, a degradation complex consist-ing of the scaffold proteins Axin and APC and the kinasesCKI and Zw3 (Shaggy, GSK3) forms. This complexmediates the phosphorylation of Arm, tagging it forproteasome-mediated degradation. Upon ligand bind-ing, the receptor complex of Frizzled (Fz) and Arrow(LRP5/6, Arr) activates Disheveled (Dsh), which in turninhibits the degradation complex leading to increasedArm protein levels. Arm in turn translocates to thenucleus where it activates transcription in conjunctionwith the transcription factor TCF (Logan and Nusse

2004).Previous results suggested a model where ligand-

mediated receptor activation recruited Axin to themembrane where it bound Arr in a larger complex withDsh and Fz. It was further shown that Axin was the rate-limiting component, and its levels were regulatedthrough proteasomal degradation in a signal-dependentmanner (Lee et al. 2003; Tolwinski et al. 2003;

Tolwinski and Wieschaus 2004b). Other approachesshowed that the membrane localization of the degrada-tion complex was a key step in signaling as GSK3 and CKIpotentiated signaling at the membrane by phosporylat-ing LRP (Davidson et al. 2005; Zeng et al. 2005). Other,somewhat contradictory findings were that Axin wasrequired in the nucleus for pathway activation (Cong

and Varmus 2004; Wiechens et al. 2004). Anotherimportant finding was that APC, a component of thedegradation complex, was shown to function in nuclearexport of Arm (Rosin-Arbesfeld et al. 2000), and inopposing transcriptional activation by the Arm/TCFcomplex (Sierra et al. 2006).

Here I report that membrane-tethered Axin is suffi-cient for the proper transduction of Wg signal in thepatterning of the Drosophila embryo. In contrast,membrane-tethered APC2 does not rescue signaling.

MATERIALS AND METHODS

Alleles used: axinS044230 (Hamada et al. 1999), apc1Q8 (Ahmed

et al. 1998), apc2d40 (McCartney et al. 1999), and zw3M11-1

(Perrimon and Smouse 1989). Please see Flybase for details ofalleles.

Crosses for the third chromosome (Ch.) mutants:

(Ch. 3-left arm) da-GAL4, (Ch. 3-right arm) FRT 82B, axinS044230/TM3

(3L) da-GAL4, (3R) FRT 82B, apc1Q8, apc2d40/TM3(3L) da-GAL4, (3R) FRT 82B, apc1Q8, apc2d40, axinS044230/TM3.

These lines were crossed to FRT 82B, OvoD males resultingin females that made only maternally mutant eggs of which50% have da-GAL4. This is because the recombination only

1Address for correspondence: Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 423, New York, NY 10065.E-mail: tolwinsn@mskcc.org

Genetics 181: 1169–1173 (March 2009)

occurs on 3R (Xu and Rubin 1993; Chou and Perrimon

1996). The resulting females were crossed to males:

(Ch. 2) UAS-axin; (Ch. 3) mutant*/TM3 balancer(Ch. 2) UAS-myr-axin; (Ch. 3) mutant*/TM3 balancer(Ch. 2) UAS-apc2; (Ch. 3) mutant*/TM3 balancer(Ch. 2) UAS-myr-apc2; (Ch. 3) mutant*/TM3 balancer.

Crosses for the first or X chromosome mutants:

y, zw3M11-1 FRT 101/FM6 (Ch. 1); arm-GAL4 (Ch. 2).

This line was crossed to OvoD, FRT101 males resulting infemales that made only maternally mutant eggs of which 50%have arm-GAL4. These females were crossed to homozygousUAS transgenes as above. In all cases, the GAL4/UAS systemwas used to express transgenes (Brand and Perrimon 1993).

Genotypes were identified unambiguously as follows. Thedaughterless-GAL4 insertion is located on the left arm ofchromosome 3. Therefore, when germline clones are madefor the right arm of the third chromosome, all embryos arematernally mutant but only 50% express GAL4. Similarly forCh. 1, the driver assorts independently of the mutant chro-mosome as it is on Ch. 2. Maternal and zygotic (M/Z) mutantsfor zw3, axin and the triple mutant are completely naked,whereas maternal (M) only mutants retain a small number ofdistinct denticles making them readily identifiable. Also, theUAS transgene was zygotically crossed in as homozygous.Combining these facts, 50% of embryos express the transgene,and I observe that 50% show an effect depending on thetransgene used irrespective of whether they are zygoticallymutant (see numbers in Table 1). The remaining 50% can beclassified according to phenotype into half M/Z and half Mdepending on whether small numbers of denticles are presentor absent. Having these two classes each present at 25% showsthat the embryonic patterning defects are mostly due to loss ofthe maternal contribution of these genes rather than on thezygotic contribution, an effect also observed in Hamada et al.(1999) and Peterson-Nedry et al. (2008).

The apc double mutant differs only in that, although M/Zmutants are completely naked similar to the others discussedabove, the M only embryos are fully, paternally rescued andhatch. Still, as in the above case, 50% express the transgene,and therefore it is simply a matter of counting the four classesthat result (Ahmed et al. 2002). Embryos were collected at 25�before being dechorionated and mounted in Hoyer’s media.The results of one representative experiment that was re-peated multiple times are quantified in Table 1.

Molecular Biology: Myristoylated constructs were made byadding a sequence identical to the NH2 terminus of src(MGNKCCSKRQGTMAGNI) to the NH2 teminus of both axinand apc2 by PCR. This sequence has proven to be very effectivefor membrane targeting of arm (Zecca et al. 1996; Tolwinski

and Wieschaus 2001; Tolwinskiand Wieschaus 2004a). ThePCR products were then transferred by Gateway cloning (In-vitrogen) into a pUASt with COOH-terminal 3XFLAG tag vector(http://www.ciwemb.edu/labs/murphy/Gateway%20vectors.

html), and injected by standard methods. I used full-lengthaxin constructs kindly provided by Karl Willert and Roel Nusse(Willert et al. 1999) and UAS-apc2 constructs that were kindlyprovided by Mariann Bienz.

Antibody stainings were performed as previously described(Tolwinski and Wieschaus 2001) using mouse monoclonalFLAG M2 (Sigma) and rabbit polyclonal Arm (Riggleman

et al. 1990).

RESULTS AND DISCUSSION

Mutations in axin result in constitutive activation ofthe Wg pathway, or the complete absence of patterningof the ventral embryonic epidermis—the naked pheno-type (Hamada et al. 1999; Willert et al. 1999). In con-trast, overexpression of axin leads to complete lack ofWg signaling and loss of patterning or the uniformdenticle—the wg phenotype (Hamada et al. 1999;Willert et al. 1999). In axin mutant embryos, I assayedthe ability of axin that was tethered to the membrane torescue the naked phenotype. As shown in Figure 1B, thismembrane-bound form can rescue patterning to someextent. In contrast, an unmodified axin, leads to com-plete loss of patterning and the wg phenotype Figure 1A.

There are two genes that encode apc in Drosophila,apc 1 and 2. They are largely redundant, and only doublemutants lead to strong pathway activation in embryos(Ahmed et al. 2002; McCartney et al. 2006). In apc1 apc2double mutants, I assayed the ability of membrane-tethered apc2 to block signaling activation. As shown inFigure 1H, there was no real effect on the patterning ofthe cuticle. In contrast, expression of untethered apc2rescued to a wild-type cuticle pattern (Figure 1G). Ifurther used the apc1 apc2 double mutants to test theactivity of both membrane-tethered and -untetheredaxin. Interestingly, expression of axin abolished signal-ing brought about by loss of both apc genes leading toa completely denticle-covered cuticle (Figure 1E). Thetethered axin, however, was unable to overcome the lossof apc and signaling was not blocked (naked phenotype,Figure 1F).

To further assay the activity of the membrane-tetheredand -untethered apc2 and axin, I expressed all four in atriple mutant for axin apc1 apc2. In the triple mutant,only untethered Axin was able to block signaling (Figure1I), as both membrane-tethered axin and apc2 and theuntethered apc2 failed to restore patterning (Figure 1,

TABLE 1

Summary of phenotypes shown in Figure 1 along with number of embryos scored

UAS-Axin UAS-myrAxin UAS-APC2 UAS-myrAPC2

axin Wg 50% (n ¼ 54) Wg 50% (n ¼ 96) Naked 100% (n ¼ 53) Naked 100% (n ¼ 113)apc 1, 2 Wg 50% (n ¼ 89) Naked 50% (n ¼ 85) Wild-type 75% (n ¼ 41) Naked 50% (n ¼ 36)axin, apc 1, 2 Wg 50% (n ¼ 79) Naked 100% (n ¼ 100) Naked 100% (n ¼ 84) Naked 100% (n ¼ 109)zw3 Wg 50% (n ¼ 52) Weak rescue 50% (n ¼ 64) Naked 100% (n ¼ 90) Naked 100% (n ¼ 78)

1170 N. S. Tolwinski

J–L). This finding suggests that in the absence of APC,Axin at the membrane is insufficient to block signaling,perhaps because APC is not available to export Arm fromthe nucleus. Arm that is made in the cytoplasm maytherefore elude the membrane bound Axin and activatesignaling when APC isn’t present to export it out of thenucleus.

The triple mutant could only be rescued by theexpression of untethered axin, and further, expressionboth apc2 and tethered apc2 failed to rescue thesignaling activation brought about by the loss of axin

alone (Figure 1, C and D). These results taken togetherimply that in the complete absence of Axin, signalingcannot be blocked by APC in any form. This is supportedby the fact that in zw3 mutant embryos expression ofuntethered axin blocked signaling, and tethered axincould do so minimally (Figure 1, M and N). Expressionof either form of apc2 had no effect in zw3 mutants(Figure 1, O and P). All these combinations takentogether suggest that only wild-type Axin can blocksignaling in all the other destruction complex mutants.Membrane bound Axin could only rescue axin mutants

Figure 1.—Expression of membrane-tethered axin blocks Wg signaling activation, in contrast to membrane-tethered apc2which does not. (A) axinS044230 germline clone or maternally and zygoticaly (M/Z) embryos expressing a wild-type form of axinunder daGAL4/UAS control show a wg phenotype or ectopic denticles. (B) axinS044230 mutant embryos expressing axin with anNH2-terminal myristoylation site or UAS-myr-axin also blocks Wg signaling pathway activation. (C and D) Neither expressionof apc2 nor expression of myr-apc2 in axinS044230 mutant embryos has an effect, as shown by embryos completely devoid of denticlesor the naked phenotype. (E) apc1Q8 apc2d40 double mutants are naked, but expression of axin blocks this and leads to the wg phe-notype. (F) Expression of myr-axin has no effect on the naked phenotype of apc1Q8 apc2d40 double mutants. (G) However, expres-sion of apc2 restores wild-type patterning, but (H) expression of myr-apc2 cannot restore patterning. (I) Triple mutant embryos foraxinS044230 apc1Q8 apc2d40 loose three of the components of the Arm destruction complex, and therefore have a very strong Wg gain-of-function phenotype, which can be blocked by expression of axin. Expression of ( J) myr-axin has no effect in triple mutants, nordoes expression of apc2 (K), or myr-apc2 (L). (M) Mutations in the kinase zw3 lead to strong activation of Wg signaling, which canbe blocked by expression of axin. However, expression of myr-axin (N) has a very weak rescuing effect, and apc2 (O) and myr-apc2(P) have no effect on patterning in zw3 mutants.

Note 1171

and to a much lesser extent zw3 mutants. Expressionof apc2 only rescued apc mutants, whereas membranebound apc2 could not rescue any of the destructioncomplex mutants or combinations.

Therefore, as predicted from the mutant studiesabove, expression of myr-axin in otherwise wild-type em-bryos is sufficient to block Wg signaling (Figure 2A), butmyr-apc2 has minor effects (Figure 2B). Both constructsappear to be localized primarily to the membrane(Figure 2, C–H), but unfortunately I cannot excludethe possibility that our membrane-tethering system is atsome low level leaky. However, on the basis of the factthat I do not observe either APC or Axin in the nucleus,that the constructs are functionally distinct, and myprevious observations of the efficacy of this membrane-tethering sequence (Tolwinski and Wieschaus 2004a),it is likely that the function of these alleles is largely at themembrane.

Overall, these results suggest that the different mem-bers of the degradation complex perform different rolesin signal transduction. Specifically, since UAS-axin blockssignaling in apc and zw3 mutants it appears to be epistaticto the other destruction complex components. From abiochemical perspective, Axin is thought to be the rate-limiting component as it appears to be present at�5000fold lower levels in extracts from Xenopus oocytes (Lee

et al. 2003), and its levels can be modulated in responseto Wg signaling (Tolwinski et al. 2003). A recent study,

however, showed that in fly eye development APC1 alsoexists at threshold levels and that this is required forproper graded responses to Wg signal (Benchabane

et al. 2008). This is unlikely to be true for embryonic Wgsignaling, as loss of APC1 has no effect on embryogenesisand appears to mainly affect eye development (Ahmed

et al. 1998).An important finding shown here is that Axin when

tethered to the membrane cannot block signaling in theabsence of APC. This suggests that either APC is re-quired for nuclear export when Axin is absent from thecytoplasm and nucleus or Axin is required in the cyto-plasm and not just the membrane for its Arm anchoringrole (Tolwinskiand Wieschaus 2001; Krieghoff et al.2006). However, our finding raises a major conundrumin that expression of Axin alone can rescue the loss ofZw3. As phosphorylation of Arm by Zw3 is required forArm proteasome-mediated degradation, loss of Zw3leads to very high levels of Arm (Siegfried et al. 1994).In contrast, Zw3 phosphorylation has the opposite effecton Axin, stabilizing its levels and preventing its degra-dation (Yamamoto et al. 1999; Lee et al. 2003). There-fore, it is unclear how unregulated levels of Arm proteincan be blocked from entering the nucleus by Axinexpression unless the expression of Axin is at enor-mously high levels. This is unlikely since Axin levelsshould be lower when Zw3 is absent, leading to theconclusion that there must be another pathway that may

Figure 2.—Expression of mem-brane-tethered axin blocks Wgsignaling in the epidermal pat-terning of Drosophila embryos.(A) daGAL4.UAS-myr-Axin em-bryos show a loss of naked cu-ticle phenotype. (B) daGAL4.UAS-myr-APC2 embryos show mainlynormal patterning although occa-sional extra denticles can beobserved (arrow). (C) Immuno-fluorescence with FLAG antibod-ies shows that myr-Axin appearsto be localized to the membrane.(D) Arm staining which is mainlymembrane localized is shown forcomparison as well as an overlay(E). (F) Immunofluorescencewith FLAG antibodies shows thatmyr-APC2 also appears to be lo-calized to the membrane. (G)Arm staining is shown for com-parison as well as an overlay (H).Bar, 20 mm.

1172 N. S. Tolwinski

target Arm for degradation, or the Arm protein presentunder these conditions somehow lacks activity or activa-tion by an as yet unidentified component or components.

Finally, although these experiments may raise morenew questions than they answer, it is important to notethat they support much of what is believed to be themajor form of pathway activation. Upon Wg binding tothe receptor Fz, a complex involving Fz, Dsh, Arr, andthe destruction complex forms. At this point Dshsomehow inactivates the phosphorylation of Arm andallows it to enter the nucleus where it is involved intranscription. APC’s role appears to be twofold, as it isinvolved in the destruction complex and in the export ofArm from the nucleus. That this robust complex formsat the plasma membrane seems certain (Cliffe et al.2003; Davidson et al. 2005; Zeng et al. 2005; Peterson-Nedry et al. 2008), but some unanswered questionsremain such as how does Dsh inactivate the destructioncomplex and whether there is an as yet uncharacterizedactivation step for Arm.

LITERATURE CITED

Ahmed, Y., S. Hayashi, A. Levine and E. Wieschaus, 1998 Reg-ulation of armadillo by a Drosophila APC inhibits neuronal ap-optosis during retinal development. Cell 93: 1171–1182.

Ahmed, Y., A. Nouri and E. Wieschaus, 2002 Drosophila Apc1 andApc2 regulate Wingless transduction throughout development.Development 129: 1751–1762.

Benchabane, H., E. G. Hughes, C. M. Takacs, J. R. Baird andY. Ahmed, 2008 Adenomatous polyposis coli is present nearthe minimal level required for accurate graded responses tothe Wingless morphogen. Development 135: 963–971.

Brand, A. H., and N. Perrimon, 1993 Targeted gene expression asa means of altering cell fates and generating dominant pheno-types. Development 118: 401–415.

Chou, T. B., and N. Perrimon, 1996 The autosomal FLP-DFS tech-nique for generating germline mosaics in Drosophila melanogaster.Genetics 144: 1673–1679.

Cliffe, A., F. Hamada and M. Bienz, 2003 A role of Dishevelled inrelocating Axin to the plasma membrane during wingless signal-ing. Curr. Biol. 13: 960–966.

Cong, F., and H. Varmus, 2004 Nuclear-cytoplasmic shuttling of Ax-in regulates subcellular localization of beta-catenin. Proc. Natl.Acad. Sci. USA 101: 2882–2887.

Davidson, G., W. Wu, J. Shen, J. Bilic, U. Fenger et al., 2005 Caseinkinase 1 gamma couples Wnt receptor activation to cytoplasmicsignal transduction. Nature 438: 867–872.

Hamada, F., Y. Tomoyasu, Y. Takatsu, M. Nakamura, S. Nagai et al.,1999 Negative regulation of Wingless signaling by D-axin, aDrosophila homolog of axin. Science 283: 1739–1742.

Krieghoff, E., J. Behrens and B. Mayr, 2006 Nucleo-cytoplasmicdistribution of beta-catenin is regulated by retention. J. CellSci. 119: 1453–1463.

Lee, E., A. Salic, R. Kruger, R. Heinrich and M. W. Kirschner,2003 The roles of APC and Axin derived from experimentaland theoretical analysis of the Wnt pathway. PLoS Biol. 1: E10.

Logan, C. Y., and R. Nusse, 2004 The Wnt signaling pathway indevelopment and disease. Annu. Rev. Cell Dev. Biol. 20: 781–810.

McCartney, B. M., H. A. Dierick, C. Kirkpatrick, M. M. Moline, A.Baas et al., 1999 Drosophila APC2 is a cytoskeletally associatedprotein that regulates wingless signaling in the embryonic epider-mis. J. Cell Biol. 146: 1303–1318.

McCartney, B. M., M. H. Price, R. L. Webb, M. A. Hayden, L. M.Holot et al., 2006 Testing hypotheses for the functions ofAPC family proteins using null and truncation alleles in Drosoph-ila. Development 133: 2407–2418.

Perrimon, N., and D. Smouse, 1989 Multiple functions of a Dro-sophila homeotic gene, zeste-white 3, during segmentation andneurogenesis. Dev. Biol. 135: 287–305.

Peterson-Nedry, W., N. Erdeniz, S. Kremer, J. Yu, S. Baig-Lewis

et al., 2008 Unexpectedly robust assembly of the Axin destruc-tion complex regulates Wnt/Wg signaling in Drosophila as re-vealed by analysis in vivo. Dev. Biol. 320: 226–241.

Riggleman, B., P. Schedl and E. Wieschaus, 1990 Spatial expres-sion of the Drosophila segment polarity gene armadillo is post-transcriptionally regulated by wingless. Cell 63: 549–560.

Rosin-Arbesfeld, R., F. Townsley and M. Bienz, 2000 The APC tu-mour suppressor has a nuclear export function. Nature 406:1009–1012.

Siegfried, E., E. L. Wilder and N. Perrimon, 1994 Components ofwingless signalling in Drosophila. Nature 367: 76–80.

Sierra, J., T. Yoshida, C. A. Joazeiro and K. A. Jones, 2006 TheAPC tumor suppressor counteracts beta-catenin activation andH3K4 methylation at Wnt target genes. Genes Dev. 20: 586–600.

Tolwinski, N. S., and E. Wieschaus, 2001 Armadillo nuclear im-port is regulated by cytoplasmic anchor Axin and nuclear anchordTCF/Pan. Development 128: 2107–2117.

Tolwinski, N. S., M. Wehrli, A. Rives, N. Erdeniz, S. DiNardo et al.,2003 Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity. Dev.Cell 4: 407–418.

Tolwinski, N. S., and E. Wieschaus, 2004a A nuclear function forarmadillo/beta-catenin. PLoS Biol. 2: E95.

Tolwinski, N. S., and E. Wieschaus, 2004b Rethinking WNTsignal-ing. Trends Genet. 20: 177–181.

Wiechens, N., K. Heinle, L. Englmeier, A. Schohl and F. Fagotto,2004 Nucleo-cytoplasmic shuttling of Axin, a negative regulatorof the Wnt-beta-catenin pathway. J. Biol. Chem. 279: 5263–5267.

Willert, K., C. Y. Logan, A. Arora, M. Fish and R. Nusse, 1999 ADrosophila Axin homolog, Daxin, inhibits Wnt signaling. Devel-opment 126: 4165–4173.

Xu, T., and G. M. Rubin, 1993 Analysis of genetic mosaics in devel-oping and adult Drosophila tissues. Development 117: 1223–1237.

Yamamoto, H., S. Kishida, M. Kishida, S. Ikeda, S. Takada et al.,1999 Phosphorylation of axin, a Wnt signal negative regulator,by glycogen synthase kinase-3beta regulates its stability. J. Biol.Chem. 274: 10681–10684.

Zecca, M., K. Basler and G. Struhl, 1996 Direct and long-rangeaction of a wingless morphogen gradient. Cell 87: 833–844.

Zeng, X., K. Tamai, B. Doble, S. Li, H. Huang et al., 2005 A dual-kinase mechanism for Wnt co-receptor phosphorylation and ac-tivation. Nature 438: 873–877.

Communicating editor: T. Schupbach

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