perspective

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Armadillo/β-catenin signals in the nucleus –proof beyond a reasonable doubt?

Mariann Bienz and Hans Clevers

Wnt signalling results in transcriptional stimulation of genes controlling normal and malignantdevelopment. A key effector of the canonical Wnt pathway is β-catenin (also known as Drosophilamelanogaster Armadillo (Arm)), thought to function as a nuclear co-activator of TCF transcriptionfactors. This has been challenged by unexpected observations of membrane-bound Arm/β-cateninsignalling activity. Plausible explanations allow these observations to be reconciled with the largebody of evidence supporting a nuclear function of Arm/β-catenin.

Cells have two pools of Arm/β-catenin— a membrane-associated pool ofstable proteins that function in cellu-

lar adhesion and a soluble pool of highlyunstable protein that transduces the canon-ical Wnt signal1,2. The former binds to E-cadherin and α-catenin, thus linking thetwo proteins within adherens junctions,whereas the latter binds to Axin and theadenomatous polyposis coli (APC) tumoursuppressor (Fig. 1) to be targeted for protea-somal destruction. After Wnt signalling, sol-uble Arm/β-catenin becomes stabilized andis detectable in the nucleus. Here, it binds toTCF to co-activate the transcription of Wnttarget genes3–8. This co-activator function ofArm/β-catenin involves the recruitment ofthe CBP/p300 histone acetyltransferase9,10

and the Brahma–Brg-1 chromatin remodel-ling complex11 (Fig. 1).

This view — that Arm/β-catenin signalsin the nucleus by forming a transcriptionfactor complex with TCF — has recentlybeen challenged by evidence fromDrosophila, mainly on the basis of signallingactivity of membrane-anchored forms ofArm12. The Wnt research field has alreadyexperienced a previous challenge of a simi-lar kind five years ago, when a membrane-anchored form of plakoglobin (a relative ofβ-catenin) was shown to provide signallingactivity in Xenopus laevis embryos13.However, an explanation for this unex-pected signalling activity in the cytoplasmwas soon found14, as it was demonstratedthat overexpressed membrane-anchoredβ-catenin competes with endogenousβ-catenin for its binding partners, such asE-cadherin and APC (Fig. 1), antagonisingWnt signalling. This competition results inthe stabilisation and nuclear translocationof endogenous β-catenin. Plakoglobincompetes in the same way, so its apparentsignalling activity is probably indirect andmediated by endogenous β-catenin14. Thesame results were later obtained in mam-malian cells transfected with a differentmembrane-anchored β-catenin construct,

the transcriptional activity of which wasalso mediated by stabilisation and nucleartranslocation of endogenous β-catenin15.

These experiments in Xenopus and cul-tured mammalian cells were thus compli-cated by the presence of wild-type β-catenin. To circumvent this, two parallelseries of experiments were carried out inDrosophila, in which wild-type Arm can beremoved using genetic manipulation16–18.Two different forms of membrane-boundArm were tested to determine whether theycould rescue different Arm mutants (unlessotherwise specified, the term ‘mutant’means ‘mutant in the germ-line andzygote’). One form was anchored with atransmembrane domain (identical to theform used by Miller and Moon; ‘TM’(ref. 14)), the other tethered to the mem-brane by a CAAX box (‘MT’), resulting inlipid modification. Hypomorphic mutantswere used, as oogenesis is disrupted if Armis completely removed, precluding theassessment of signalling in the progeny19.

Cox et al.16,17 first tested TM- and MT-Arm in armXM19 mutants expressing an Armmutant (XM19 protein) lacking the car-boxyl terminus. XM19 protein retains itsadhesion function, but on its own has littleor no signalling activity20–22. Surprisingly,the signalling defects in this mutant aresubstantially rescued by the membrane-bound Arm — however, they are also res-cued by the overexpression of a XM19-likeC-terminal truncation of Arm that is notmembrane-bound17. It was also demon-strated that when MT-Arm produces sig-nalling activity in armXM19 mutants, it doesit the ‘Miller and Moon’ way, namely bychasing endogenous Arm protein into thenucleus18.

These results show conclusively thatXM19 protein retains residual signallingactivity through which overexpressed TM-and MT-Arm protein can function16–18.They make a strong case, as did Miller andMoon, that this indirect action is caused bycompetition of exogenous protein with

negative regulators of the Wnt pathway,such as Axin and APC. Molecular evidencesupports the hypothesis that XM19 proteinretains signalling activity — this truncationretains the last two Arm repeats, and thuspart of the Brg-1 and part of the mainCBP/p300-binding site9–11 (Fig. 1). In addi-tion, the amino terminus of β-catenin con-tains a second transactivation domain15 (aswell as the C-terminal transactivationdomain8), which is also retained in theXM19 protein. Finally, this truncationretains the interaction domain with theLegless–Pygopus (Lgs–Pygo) complex, twonew components of the Wnt signallingpathway required for the transcriptionalactivity of Arm and β-catenin23–26.

However, the residual signalling activityof XM19 protein meant that these experi-ments did not test the signalling ability ofmembrane-bound Arm. Cox et al.16 thusturned to another allele, armXP33, whichretains just enough adhesion function toallow oogenesis to occur, although only a feweggs mature19,20,27,28. This allele thus offersthe closest approximation to a null back-ground available (see also below). WhenTM-Arm is overexpressed in these mutants,it rescues their early embryonic adhesiondefects, but not the signalling defects (thepossibility of contamination can be ruledout, as the rescued armXP33 mutants are qual-itatively distinct from zygotically mutantarmXP33 embryos)16. This experiment pro-vided strong support for the idea that mem-brane-bound Arm does not signal, a conclu-sion also reached by Tolwinski andWieschaus, who found that MT-Arm lackedany activity when overexpressed in arm043A01

mutants18 (expressing a truncation similar tothe XP33 protein; Fig. 1). These authors fur-ther demonstrated the importance of theAxin–APC complex in anchoring Arm in thecytoplasm, in addition to promoting itsdestruction. Together, these results provide acompelling case for the requirement of Armto be able to transit from the plasma mem-brane to signal effectively.

© 2003 Nature Publishing Group

perspective

Chan and Struhl have now re-examinedthis issue12. They generated 29 differentArm constructs, including a TM-Arm, MT-Arm and one targeted to the nucleus (‘NLS(nuclear localization sequence (NLS)-Arm’), and tested these in wild-type andarm mutant cells of two different tissues.They demonstrate that overexpression ofTM- and MT-Arm produces signallingactivity in wild-type cells and rescues armmutants, although NLS-Arm has neitheractivity. They conclude from this, and fromthe additional supportive experimentsbelow, that Arm exerts its signalling func-tion in the cytoplasm rather than in thenucleus.

In the light of the previous work14–18,this is rather puzzling, and one’s instinctiveresponse is to wonder whether Chan andStruhl can rule out the ‘Miller and Moon’explanation; that is, that their membrane-bound Arm functions indirectly throughstabilising endogenous Arm. This ultimate-ly boils down to the question of whether thearm mutants that were used lack any resid-ual signalling function. Chan and Struhl domost of their experiments in armXM19

mutants that retain residual signallingactivity revealed in the presence of overex-pressed Arm16–18, as discussed above. Thus,the ability of their membraned-bound Armto rescue this mutant is not surprising. Thekey experiment in their paper12 is based ona more severe allele , arm043A01, whose prod-uct is also deficient in cellular adhesion18. Intheir hands, membrane-bound Arm canprovides some rescuing activity in thismutant12, but there are two caveats with thisresult. First, it conflicts with very similarexperiments done by Tolwinski and

Wieschaus who used the same MT-Arm asChan and Struhl, but found it lacked anyactivity when overexpressed in arm043A01

mutants18. Indeed, when Chan and Struhlused the same overexpression conditions,their result is arguably the same — no sig-nificant signalling activity12. Second, theonly convincing activity of MT-Arm detect-ed by Chan and Struhl in these mutants isafter overexpression with the paired GAL4driver12, one of the earliest and strongestembryonic drivers known. As discussed, itis hard to exclude that this activity does notreflect a slight residual function of endoge-nous 043A01 protein, which is revealedunder these overexpression conditions.Although 043A01 and XP33 protein aresimilar C-terminal truncations (Fig. 1), thelatter is unusually unstable and thus barelydetectable (although other truncations,even shorter ones, are readily detectable)20.Thus, armXP33 would have been the allele ofchoice, as it provides the most stringentloss-of-function conditions for functionaltests of overexpressed Arm constructs(recall that MT-Arm did not exhibit anysignalling activity in armXP33 mutantembryos16). Regrettably, Chan and Struhlwere unable to generate mutant embryoswith this allele.

The failure of Chan and Struhl’s NLS-Arm to provide signalling function12 alsodisagrees with previous results that demon-strate signalling and transcriptional activityof NLS-β-catenin in Xenopus embryos andtransfected cells14,15. Furthermore, it con-tradicts work demonstrating that NLS-Armprovides rescuing and ectopic signallingactivity in armXM19 mutant Drosophilaembryos, arguing that targetting Arm to the

nucleus promotes its signalling activity16.One possible explanation for this discrepan-cy may be that the NLS was attached differ-ently — each of the three signalling-compe-tent constructs have an amino-terminalNLS14–16, whereas the signalling-deficientconstruct has a C-terminal NLS12. Theobserved nuclear localization of the latter12

does not necessarily mean that this con-struct is functional, and one might like tosee an independent test of its potential fortranscriptional activation. The same appliesto the Arm construct in which the C termi-nus was substituted by the VP16 transacti-vation domain, which did not produce anyactivity12, but no test was carried out for itsbasic functionality. Finally, Chan andStruhl’s Arm fusion to the repressor domainof Engrailed (and a GAL4 DNA-bindingdomain) did provide rescue activity. Thiscontrasts with a similar fusion (in whichthe C terminus of β-catenin was substitutedwith the Engrailed repressor domain),which had a a severe dominant-negativeeffect in Xenopus embryos29.

The key observations of Chan andStruhl directly contradict results from pre-vious analyses14–18,29. Despite this, are theauthors providing a plausible new model ofhow Arm may exert signalling function inthe cytoplasm? They offer two possiblemodels. First, they consider the possibilityof TCF being a transcriptional repressorthat Arm needs to export from the nucleusfor Wnt target genes to be transcribed. Thisidea is supported by their observation thatthe C terminus of Arm could be substitutedfunctionally by the C terminus ofDrosophila α-importin (predicted to medi-ate nuclear export), but not by a mutant Cterminus with two mutated glutamateresidues that are conserved between Armand α-importin12. However, these tworesidues are neither conserved in other β-catenins, nor have they been shown to berequired for nuclear export ofα-importin30. Therefore it remains unclearwhy the functional substitution was suc-cessful. Chan and Struhl also provide evi-dence that dTCF is exported in response toWingless (Wg) signalling along theprospective wing margin12. However, thisdoes not seem to be the case in the embryowhere dTCF appears to be in the nucleus,independently of whether or not Wg sig-nals24. Furthermore, in Xenopus and mam-malian cells, TCF is localized to the nucleus,regardless of Wnt signalling and of the sub-cellular location of β-catenin.

Although TCF proteins can function asrepressors, Arm/β-catenin and as an anti-repressor (for example, see refs 7, 31–34),there is also strong evidence that TCF pre-dominantly mediates transcriptional acti-vation35. For example, Chan and Struhldemonstrated that a dTCF construct fusedto the VP16 transcriptional activator pro-vides signalling function in armXM19

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N–TAD C-TAD

CBP/p300

Lgs–Pygo

E-Cadherin

Brg-1

TCF

Axin, APC

α-cat

armxp33 armxm19

Figure 1 ββ-catenin/Arm and its binding partners. The primary structure of ββ-catenin/Arm is depicted(roughly to scale), with its highly conserved Arm repeat domain (12 repeats boxed). Its N-terminalGSK3 and CKI phosphorylation sites (small arrows) are required for proteasome-mediated destruc-tion, and its C-terminal domain is required for signalling. The interaction domains with its bindingpartners are bracketed (predominantly cytoplasmic proteins above; nuclear proteins below); the twotranscriptional activation domains (TAD) are also indicated. Large arrows mark truncation end pointsof a signalling-null (XP33) and a signalling-defective mutant (XM19) of Arm (the 043A01 truncation issimilar to XP33, though its precise lesion has not been published). For references, see text.

© 2003 Nature Publishing Group

mutants12. Perhaps the strongest evidencefor the activator function of TCFs comesfrom loss-of-function analysis — the phe-notypes of TCF loss in Drosophila6,8 andmammals36–39 resemble loss of Wnt sig-nalling (that is, loss of activating compo-nents of the pathway) . If TCF factors func-tioned predominantly as repressors, onewould expect their loss to produce mutantphenotypes mimicking loss of negative Wntregulators (for example, glycogen synthasekinase 3β (GSK3β), Axin and APC), but thelatter tend to be the opposite of the TCFmutant phenotypes21,22,40–44. Taken togeth-er, this means that the ‘exporting TCFrepressor’ model is unlikely to provide ageneral explanation as to how Arm/β-catenin functions. In addition, as TM-Armdoes not seem to gain access to the nucleus,it would have to deplete the nucleus of TCFrepressor through cytoplasmic retention,and this retention would be dependant oncontinuous nuclear–cytoplasmic shuttlingof TCF. This shuttling would have to behighly active to allow a rapid response to

Wnt signalling — however, the TCF repres-sor is thought to be complexed withGroucho and chromatin in the absence ofWnt signalling32–34.

Chan and Struhl propose a secondmodel, in which Arm activates TCF in thecytoplasm. As they do not make any specif-ic suggestion for the possible nature of thisactivation mechanism, this model is harderto refute. The cytoplasmic activation modeldoes not seem plausible to us for a numberof reasons that present an argument for ajoint nuclear function of β-catenin and TCF.First, a version of TCF, in which the β-catenin-binding domain has been replacedby the C-terminal transcriptional activationdomain of β-catenin or by a heterologoustransactivation domain is localized to thenucleus and mimics Wnt signalling inXenopus embryos and in mammaliancells15,45. This supports the hypothesis thatthe essential function of β-catenin is to serveas a transcriptional co-activator for TCF inthe nucleus. Second, all known TCF proteinsseem to be exclusively nuclear15,24,36,46,47

(Fig. 2a). Indeed, overexpression of TCFproteins in Xenopus or mammalian cellstypically causes efficient translocation ofβ-catenin into the nucleus3–5. Third, theknown co-activators of β-catenin/Arm,CBP/p300 and Brahma/Brg-1, are nuclearfactors that modify histones and remodelchromatin, respectively (see above). Fourth,Pygo and Lgs, which are required for thetranscriptional activity of Arm/β-cateninare also nuclear23–26 (Fig. 2b). Indeed, p300,Brg-1 and Pygo have all been detected in acomplex with β-catenin/Arm in vivo9,11,24,26.Furthermore, a stable quaternary complexbetween β-catenin and the interactiondomains of p300, TCF-4 and cognate DNAcan be demonstrated in vitro48, indicatingthat β-catenin can bind simultaneously toDNA-bound TCF and to the p300 co-activator. Fifth, in a cell-free transcriptionsystem based on reconstituted chromatintemplates, β-catenin facilitates chromatinaccess and transactivation dependant onits CBP/p300-binding domain49.Individually, none of these reasons ruleout the possibility that one or more ofthese nuclear factors need to be activatedin the cytoplasm by Arm/β-catenin beforethey begin their transcriptional activationfunction in the nucleus. However, the ‘cyto-plasmic activation model’ is difficult touphold in the face of this accumulation ofdata on the nuclear localization and func-tion of Arm/β-catenin’s binding partners.

Finally, there is an overwhelming corre-lation between Wnt pathway activity andnuclear localization of β-catenin/Arm itself.Recent studies involving chromatinimmunoprecipitation showed that underconditions mimicking Wnt signalling,β-catenin can be cross-linked to TCF tar-get-genes in vivo50,51, demonstrating theactual presence of β-catenin on these geneswhen they are actively transcribed.Furthermore, Wnt stimulation or inhibitonof GSK3 (which mimics Wnt stimulation)causes an accumulation of β-catenin inXenopus and mammalian cell nuclei15,52.Additional examples of nuclear β-cateninin cells where the Wnt pathway is activeinclude the dorsal cells of Xenopus andzebrafish (Danio rerio) embryos53,54, thecrypt cells in the mammalian intestinalepithelium39, and APC mutant colorectalcancer cells55,56 (Fig. 2c). Similarly, Arm isdetectable in the nuclei of Wg-stimulatedand GSK3-mutant cells in the Drosophilaembryo, and accumulates in epidermal cellnuclei of Axin and APC mutants22,42–44.Perhaps the most dramatic example ofnuclear β-catenin in response to Wnt path-way activity is found in sea urchin(Lytechinus variegatus) embryos57 (Fig. 2d).Evidently, activation of the Wnt pathway incells generally seems to cause the nuclearaccumulation of β-catenin/Arm. Thisnuclear translocation event may thus beimportant in moving β-catenin/Arm to its

perspective

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β-catenin

c

β-catenin

GFP–Pygo

ba

TCF-4

d

Figure 2 Nuclear location of ββ-catenin and its binding partners. a, TCF-4 (brown) is nuclear in themammalian intestinal epithelium36, even in the crypts (arrows) where Wnt signalling is active. b, Tagged Pygo, a cofactor required for transcriptional activity of Arm/ββ-catenin, is nuclear in trans-fected mammalian cells24. c, ββ-catenin accumulates in the nuclei of SW480 cells56, colorectal cancercells in which the Wnt pathway is active due to mutational inactivation of APC. d, Sea urchin embryo,showing nuclear accumulation of ββ-catenin (yellow) in vegetal cell nuclei in which Wnt pathway activi-ty is required for the specification of the vegetal cell fate57 (image provided by D. McClay).

© 2003 Nature Publishing Group

perspective

subcellular destination where it can exert itssignalling function.

What, then, might this function be?Although this undoubtedly involves thetranscriptional activation of Wnt target-genes, the details of this process remainsketchy. The unexpected discovery of twonew nuclear factors, Pygo and Lgs, dedicat-ed to transcribing Wnt target genes, hasfurther highlighted the gaps in our knowl-edge about this process. Nevertheless, thecurrently most parsimonious modelremains that Arm/β-catenin functions as anuclear scaffold to assemble various co-activator complexes and recruit these toWnt target-genes, to modify their chro-matin and render it conducive to transcrip-tion. The weight of evidence as summarisedabove — the Wnt-induced nuclear translo-cation of β-catenin/Arm, together with theconstitutive nuclear location of TCF and ofall the known co-factors of β-catenin/Arm— points to the most obvious possibilitythat β-catenin/Arm does in fact signal inthe nucleus: ‘what looks like an elephant,walks like an elephant and sounds like anelephant, probably is an elephant’.Mariann Bienz is in the Medical ResearchCouncil Laboratory of Molecular Biology,Hills Road, Cambridge CB2 2QH, UKHans Clevers is in the Netherlands Institutefor Developmental Biology, Uppsalalaan 8,3584CT Utrecht, The Netherlands e-mail: mb2@mrc-lmb.cam.ac.uk

1. Cadigan, K. M. & Nusse, R. Genes Dev. 11, 3286–3305 (1997).

2. Polakis, P. Genes Dev. 14, 1837–1851 (2000).

3. Behrens, J. et al. Nature 382, 638–642 (1996).

4. Molenaar, M. et al. Cell 86, 391–399 (1996).

5. Huber, O. et al. Mech. Dev. 59, 3–10 (1996).

6. Brunner, E., Peter, O., Schweizer, L. & Basler, K. Nature 385,

829–833 (1997).

7. Riese, J. et al. Cell 88, 777–787 (1997).

8. van de Wetering, M. et al. Cell 88, 789–799 (1997).

9. Hecht, A., Vleminckx, K., Stemmler, M. P., van Roy, F. &

Kemler, R. EMBO J 19, 1839–1850 (2000).

10. Takemaru, K. I. & Moon, R. T. J. Cell Biol. 149, 249–254 (2000).

11. Barker, N. et al. EMBO J 20, 4935–4943 (2001).

12. Chan, S. K. & Struhl, G. Cell 111, 265–280 (2002).

13. Merriam, J. M., Rubenstein, A. B. & Klymkowsky, M. W. Dev.

Biol. 185, 67–81 (1997).

14. Miller, J. R. & Moon, R. T. J. Cell. Biol. 139, 229–243 (1997).

15. Hsu, S. C., Galceran, J. & Grosschedl, R. Mol. Cell Biol. 18,

4807–4818 (1998).

16. Cox, R. T. et al. Development 126, 1327–1335 (1999).

17. Cox, R. T., Pai, L. M., Kirkpatrick, C., Stein, J. & Peifer, M.

Genetics 153, 319–332 (1999).

18. Tolwinski, N. S. & Wieschaus, E. Development 128, 2107–2117

(2001).

19. Peifer, M., Orsulic, S., Sweeton, D. & Wieschaus, E.

Development 118, 1191–1207 (1993).

20. Peifer, M. & Wieschaus, E. Cell 63, 1167–1176 (1990).

21. Siegfried, E., Wilder, E. L. & Perrimon, N. Nature 367, 76–80

(1994).

22. Peifer, M., Sweeton, D., Casey, M. & Wieschaus, E. Development

120, 369–380 (1994).

23. Kramps, T. et al. Cell 109, 47–60 (2002).

24. Thompson, B., Townsley, F. M., Rosin-Arbesfeld, R., Musisi, H.

& Bienz, M. Nature Cell Biology 4, 367–373 (2002).

25. Parker, D. S., Jemison, J. & Cadigan, K. M. Development 129,

2565–2576 (2002).

26. Belenkaya, T. Y. et al. Development 129, 4089–4101 (2002).

27. Cox, R. T., Kirkpatrick, C. & Peifer, M. J. Cell. Biol. 134,

133–148 (1996).

28. Müller, H. A. & Wieschaus, E. J. Cell. Biol. 134, 149–163 (1996).

29. Montross, W. T., Ji, H. & McCrea, P. D. J. Cell. Sci .113,

1759–1770 (2000).

30. Herold, A., Truant, R., Wiegand, H. & Cullen, B. R. J. Cell. Biol.

143, 309–318 (1998).

31. Brannon, M., Gomperts, M., Sumoy, L., Moon, R. T. &

Kimelman, D. Genes Dev. 11, 2359–2370 (1997).

32. Roose, J. et al. Nature 395, 608–612 (1998).

33. Cavallo, R. A. et al. Nature 395, 604–608 (1998).

34. Levanon, D. et al. Proc. Natl Acad. Sci. USA 95, 11590–11595

(1998).

35. Bienz, M. TCF: Curr. Opin Cell Biol. 10, 366–372 (1998).

36. Korinek, V. et al. Nature Genet. 19, 379–383 (1998).

37. Galceran, J., Farinas, I., Depew, M. J., Clevers, H. & Grosschedl,

R. Genes Dev. 13, 709–717 (1999).

38. Galceran, J., Hsu, S. C. & Grosschedl, R. Proc. Natl Acad. Sci.

USA 98, 8668–8673 (2001).

39. van de Wetering, M. et al. Cell 111, 241–250 (2002).

40. Oshima, M. et al. Proc. Natl Acad. Sci. USA 92, 4482–4486

(1995).

41. Zeng, L. et al. Cell 90, 181–192 (1997).

42. Hamada, F. et al. Science 283, 1739–1742 (1999).

43. Ahmed, Y., Nouri, A. & Wieschaus, E. Development 129,

1751–1762 (2002).

44. Akong, K. et al. Dev. Biol. 250, 91–100 (2002).

45. Vleminckx, K., Kemler, R. & Hecht, A. Mech. Dev. 81, 65–74

(1999).

46. van Genderen, C. et al. Genes Dev. 8, 2691–2703 (1994).

47. Castrop, J. et al. Blood 86, 3050–3059 (1995).

48. Daniels, D. L. & Weis, W. I. Mol. Cell 10, 573–584 (2002).

49. Tutter, A. V., Fryer, C. J. & Jones, K. A. Genes Dev. 15,

3342–3354 (2001).

50. Kioussi, C. et al. Cell 111, 673–685 (2002).

51. Kirmizis, A., Bartley, S. M. & Farnham, P. J. Molec. Cancer

Therapeutics, in press (2003).

52. Yost, C. et al. Genes Dev. 10, 1443–1454 (1996).

53. Schneider, S., Steinbeisser, H., Warga, R. M. & Hausen, P. Mech.

Dev. 57, 191–198 (1996).

54. Larabell, C. A. et al. J. Cell Biol. 136, 1123–1136 (1997).

55. Munemitsu, S., Albert, I., Souza, B., Rubinfeld, B. & Polakis, P.

Proc. Natl Acad. Sci. USA 92, 3046–3050 (1995).

56. Rosin-Arbesfeld, R., Townsley, F. & Bienz, M. Nature 406,

1009–1012 (2000).

57. Logan, C. Y., Miller, J. R., Ferkowicz, M. J. & McClay, D. R.

Development 126, 345–357 (1999).

ACKNOWLEDGEMENTS

We thank M. Peifer, E. Wieschaus, R. Grosschedl, R. Moon, M. van

de Wetering and M. Freeman for discussion, and D. McClay for the

sea urchin image.

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