INTRODUCTION

Wingless/int1 (Wnt) proteins are secreted glycoproteins thathave diverse and profound roles in animal development(reviewed by Wodarz and Nusse, 1998; Dierick and Bejsovec,1999). Wnt proteins act both as short range inducers and longrange morphogens. For example, during Drosophilaembryogenesis, Wingless (Wg) secreted by cells located justanterior to the parasegment boundary within each segmentdirects adjacent cells just posterior to the boundary to maintainexpression of the selector gene engrailed (en) (DiNardo et al.,1988; Martinez Arias et al., 1988; Bejsovec and MartinezArias, 1991; Heemskerk et al., 1991; Vincent and Lawrence,1994). Later in development, Wg is secreted by defined sub-populations of cells within the primordia forming the adultappendages and acts directly on cells over a range of 20-30 celldiameters to organize their patterns of gene expression andcuticular differentiation (Struhl and Basler, 1993; Zecca et al.,1996; Neumann and Cohen, 1997; Cadigan et al., 1998).

Many conserved components of the cellular machineryresponsible for transducing Wnt proteins have been identifed,including the cytosolic proteins Dishevelled (Dsh), GlycogenSynthase Kinase 3 (GSK-3/Zeste-white3/Shaggy), Axin, APC,Slimb and Armadillo (Arm/β-catenin), as well as atranscription factor LEF (LEF/TCF/Pangolin) (reviewed byWodarz and Nusse, 1998; Dierick and Bejsovec, 1999). Theseproteins appear to define an intracellular signal transductionpathway leading from reception of Wg at the cell surface to theactivation of target genes in the nucleus.

The identity of the cell surface receptor that links Wg to thisintracellular signal transduction pathway has been less certain.In the past three years, proteins of the Frizzled (Fz) family havebecome the leading candidates for the Wnt receptors. Fzproteins contain a large extracellular N terminus containing aconserved cysteine-rich domain (CRD) followed by seventransmembrane domains and a small cytosolic C terminus(Wodarz and Nusse, 1998). In Drosophila, attention has beenfocused principally on two Fz proteins, Fz and Fz2.

The first indication that Fz proteins might serve as Wntreceptors came from the discovery that Drosphila Fz2 canconfer Wg binding activity in tissue culture cells as well asectodermal cells in vivo (Bhanot et al., 1996; Nusse et al.,1997; Cadigan et al., 1998). Fz itself was also shown to conferWg binding activity in tissue culture (Bhanot et al., 1996;Nusse et al., 1997). These binding studies are consistent witha function for Fz proteins as Wnt receptors, but do not inthemselves establish a ligand-receptor relationship.

One genetic approach, overexpression of wild-type ortruncated forms of Fz proteins, has provided additional supportfor such a relationship. Overexpression of either Fz or Fz2 inDrosophila causes phenotypes associated with ectopic Wgsignaling (Cadigan and Nusse, 1997; Tomlinson et al., 1997;Zhang and Carthew, 1998), whereas overexpression ofmembrane tethered forms of the extracellular Fz2 CRD (theputative Wg-binding domain) causes phenotypes associatedwith attenuated Wg signaling (Bhat, 1998; Cadigan et al.,1998; Zhang and Carthew, 1998). Similar results have beenobtained in vertebrates; vertebrates also express a class of Fz-

5441Development 126, 5441-5452 (1999)Printed in Great Britain © The Company of Biologists Limited 1999DEV7755

Wingless (Wg) protein is a founding member of the Wntfamily of secreted proteins which have profound organizingroles in animal development. Two members of the Frizzled(Fz) family of seven-pass transmembrane proteins,Drosophila Fz and Fz2, can bind Wg and are candidate Wgreceptors. However, null mutations of the fz gene have littleeffect on Wg signal transduction and the lack of mutationsin the fz2 gene has thus far prevented a rigorousexamination of its role in vivo. Here we describe theisolation of an amber mutation of fz2 which truncates thecoding sequence just after the amino-terminal extracellulardomain and behaves genetically as a loss-of-function allele.Using this mutation, we show that Wg signal transduction

is abolished in virtually all cells lacking both Fz and Fz2activity in embryos as well as in the wing imaginal disc. Wealso show that Fz and Fz2 are functionally redundant: thepresence of either protein is sufficient to confer Wgtransducing activity on most or all cells throughoutdevelopment. These results extend prior evidence of aligand-receptor relationship between Wnt and Frizzledproteins and suggest that Fz and Fz2 are the primaryreceptors for Wg in Drosophila.

Key words: Frizzled, Wingless, Signal transduction, Drosophilamelanogaster

SUMMARY

Wingless transduction by the Frizzled and Frizzled2 proteins of Drosophila

Chiann-mun Chen and Gary Struhl*

Howard Hughes Medical Institute, Department of Genetics and Development, Columbia University College of Physicians andSurgeons, New York, NY 10032, USA*Author for correspondence (e-mail: struhl@cuccfa.ccc.columbia.edu)

Accepted 8 September; published on WWW 9 November 1999

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related proteins composed of only the extracellular domain thatappear to function as endogenous antagonists of Wnt signaling(reviewed by Wodarz and Nusse, 1998; Dierick and Bejsovec,1999). However, these results do not show that Fz proteins arenormally required for transducing Wnt signals.

If Fz proteins function as Wnt receptors, then their activitiesshould be essential for Wnt signal transduction. However, todate, loss-of-function phenotypes have not established that thisis so. Mutations that abolish activity of Drosophila Fz, thefounding member of the Fz family, have revealed that Fz isrequired for the normal planar polarity of epithelial cells (Gubband Garcia-Bellido, 1982; Vinson and Adler, 1987; Zheng et al.,1995). However, the nature of the polarizing signal is not known,and fz mutant animals appear to transduce Wg normally.Concomitant reduction of Fz and Fz2 activity by RNA-mediatedinterference, or by the use of large genomic deletions whicheliminate zygotic, but not maternal, gene activity duringembryogenesis can cause phenotypes resembling thoseassociated with reduced Wg signalling (Bhat, 1998; Kennerdelland Carthew, 1998; Muller et al., 1999). However, the reportedphenotypes are less severe than those caused by the absence ofWg itself, leaving open the question of whether Fz proteins areessential for Wg signal transduction.

A major impediment in resolving whether Drosophila Fzproteins function as Wnt receptors has been the lack of a loss-of-function mutation in fz2, which has been proposed to encodethe primary Wg receptor (Bhanot et al., 1996; Cadigan et al.,1998). The absence of such a mutation makes it difficult to assesswhether either Fz or Fz2 can transduce Wg, and if so, whetherthey are functionally redundant or play distinct roles, e.g., intransducing Wg in different contexts, or in transducingadditional Wnts. It also hinders the assessment of otherhypotheses about Wg signal transduction, such as the suggestionthat Wg signaling might involve other receptors or coreceptors,e.g., Notch (Couso and Martinez Arias, 1994) or proteoglycans(Reichsman et al., 1996; Binari et al., 1997; Hacker et al., 1997;Haerry et al., 1997; Lin and Perrimon, 1999; Tsuda et al., 1999),or that the movement or stability of secreted Wg may depend onthe distribution of its receptors (Cadigan et al., 1998).

Here, we describe the isolation of a likely null allele of theDrosophila fz2 gene and examine the consequences of removingeither or both the fz and fz2 gene functions during development.Our main finding is that the elimination of both Fz and Fz2activity causes an absolute loss of Wg transduction in virtuallyall of the Wg signaling processes that we have assayed, both inembryos and in the developing primordium of the adult wing. Incontrast, absence of either Fz or Fz2 activity alone, has little ifany detectable effect on Wg signaling in any tissue at any stageof development. We conclude that there is an absoluterequirement for Fz proteins in transducing Wg in most or alldevelopmental contexts, but that either Fz or Fz2 can carry thefull burden of Wg signal transduction on its own. These results,taken together with previous findings, lead us to propose that Fzand Fz2 are the primary receptors for Wg in Drosophila.

MATERIALS AND METHODS

MutationsThe following null or amorphic alleles for fz, wg, and dsh wereused: fzH51 (Jones et al., 1996), wgCX4 (Baker, 1987) and dsh75

(Manoukian et al., 1995). Other mutations are described in Flybase(http://flybase.bio.indiana.edu/).

TransgenesThe following Gal4 driver lines were used. vgQ1206-Gal4 (Gal4expressed under the control of the vestigial boundary enhancer alongthe dorsoventral compartment boundary of the wing imaginal disc,Simmonds et al., 1995; originally developed by S. Morimura and M.Hoffman). dpp-Gal4 (Morimura et al., 1996). h-Gal4 (Brand andPerrimon, 1993).

The following Flp-out transgenes were generated by standardmethods using previously described transgenes (Brand and Perrimon,1993; Basler and Struhl, 1994; Nellen et al., 1996; Zecca et al., 1996)as starting materials and the fz2, fz2C1 and GFP coding sequences(Bhanot et al., 1996; details available upon request). Tubulinα1>CD2,y+>fz2. UAS>GFP, y+>fz2. UAS>CD2, y2>fz2C1 (the fz2C1 codingsequence was introduced by replacing the wild-type coding sequencefrom the ApaI site to the termination codon with the correspondinggenomic DNA of the mutant allele, from ApaI to the newly inducedXbaI site which introduces a termination codon). In most cases, the Flp-out cassettes in these transgenes were removed prior to the experiment.

Additional transgenes employed are as follows. FRT2A (Perrimon etal., 1996). hsp70-flp UAS>CD2, y+>flu-∆Arm and UAS>CD2, y+>flu-wg (Zecca et al., 1996). hsp70-GFP and hsp70-CD2,y+ (inserted on 3L;G. S. and A. Adachi, unpublished materials, see also Jiang and Struhl,1995, 1998). UAS-flp (generated by inserting the flp coding sequencinginto the pUAST vector; Brand and Perrimon, 1993). The OvoD,w+

transgene, and its use in generating female germ line clones aredescribed in Perrimon et al. (1996).

Genetic screenMales of the genotype y; fzH51 ri FRT2A/TM2 were mutagenized withEMS and crossed to females of the genotype y; vgQ1206-Gal4 UAS-flp;hsCD2,y+ ri FRT2A/TM2.

F1 progeny of the genotype y; vgQ1206-Gal4 UAS-flp/+; fzH51 riFRT2A/ hsCD2,y+ ri FRT2A express Flp primarily in wing imaginalcells under vgQ1206-Gal4 control, especially in cells giving rise to themargin. This Flp activity mediates a high frequency of mitoticrecombination (Golic, 1991), generating clones of cells homozygousfor the mutagenized fzH51 ri FRT2A chromosome arm. The wings of theresulting F1 flies were screened for wing notches and ectopic marginbristles. Approximately 24,000 F1 flies were screened, leading to theidentification of a single candidate fz2 mutation, fz2C1(see Results). Wehave subsequently observed that animals of the same genotype as thefly in which the fz2C1 mutation was initially identified: y; vgQ1206-Gal4UAS-flp/+; fzH51fz2C1 ri FRT2A/ hsCD2,y+ ri FRT2A, are less fit thanwildtype flies, probably because of leaky expression of the UAS-flptransgene, and consequently the generation of fzH51 fz2C1 clones, inother tissues. The lack of fitness associated with this genotype mayaccount for only one fz2 mutation being obtained.

Verification of the fz2C1 mutationThe fz2C1 mutation was mapped relative to the ri and fz loci by standardgenetic methods. The UAS>fz2 and Tubα1>fz2 transgenes were eachintroduced together with the fz1H51 fz2C1ri FRT2A chromosome underthe conditions of the screen to assay for the rescue of the wing notchingphenotype associated with the double mutant clones. Genomic DNAwas obtained from fzH51 fz2C1 embryos derived from fzH51 fz2C1

germline clones, amplified by PCR, subcloned and sequenced. Thefz2C1 allele is associated with a TGG to TAG mutation in codon Trp320 which introduces an XbaI site. This restriction site polymorphismwas used subsequently to confirm the genotype of fz2C2/fz2C1 flies.

As noted in the Results, fz2C2/fz2C1 flies derived from heterozygousparents differ from their heterozygous siblings in several respects. First,they are usually developmentally delayed, typically eclosing as adults1-2 days later, and show variable survival to the adult, depending ongenetic background. Second, they tend to be smaller in size, although

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5443Wingless transduction by the Frizzled proteins

normally proportioned and patterned. Finally, they are sterile both asmales and females. None of these phenotypes resemble phenotypesknown to be associated with reductions in Wg signaling and it ispossible that they are not due to the mutation itself, but rather to secondsite mutations on the same chromsome that are unrelated to the lesionin the fz2 gene. Consistent with this possibility, these phenotypes do notappear to be fully rescued by the presence of a Tubulinα1-fz2 transgene.We also note that fz2C1 homozygotes derived from mutant fz2C1 eggshatch as morphologically normal first instar larvae (Fig. 6E), but die atvarying times during larval development.

Generation of clones for phenotypic analysis Eggs from fzH51 fz2C1 or fz2C1 germ-line cells were obtained using theFLP/FRT/ovoD method (Chou et al., 1993). Females carrying theseclones were fertilized by fzH51 fz2C1/TM3, ftz-lacZ, fzH51/TM3, ftz-lacZ,or fz2C1/TM3, ftz-lacZ males, depending on the experiment, allowingthe various mutant and wild-type progeny to be distinguished bystaining for Ftz-β-gal expression. Imaginal disc clones of fzH51 fz2C1 orfz2C1 mutant cells marked by the absence of the hsp70-GFP markergene were generated using the FLP/FRT method (Golic, 1991; Chou etal., 1993; Xu and Rubin, 1993) as follows. Larvae of the genotype yhsp70-flp; fzH51 fz2C1 (or fz2C1) ri FRT2A/hsp70-CD2,y+ hsp70-GFPFRT2A were heat shocked for 1 hour at 37°C during the first instar, orat 35°C during the early- to mid- third instar to induce the mutantclones. They were subsequently subjected to a second 37°C heat shockfor 60 minutes and allowed to recover for 1 hour at 25°C before fixationand immunostaining to induce expression of the hsp70-GFP markergene. For the experiment involving the Minute technique, the M(3)i55

mutation was introduced on the hsp70-CD2,y+ hsp70-GFP FRT2Achromosome.

Overexpression and epistasis experiments For the overexpression experiment, imaginal wing discs derived fromlarvae of the genotype UAS>fz2C1/dpp-G4 were stained for Wg and Fz2expression, and the resulting adults assayed for alterations in normalpatterning. For the epistasis experiment, females carrying h-Gal4 fz1H51

fz2C1 germline clones were crossed to males of the genotype UAS>flu-∆Arm (or UAS>flu-wg) fz1H51 fz2C1/TM3, ftz-lacZ and the resulting firstinstar larvae analyzed for defects in cuticular pattern. Under theseconditions, approximately half of the embryos inherit the TM3, ftz-lacZchromosome and develop into phenotypically wild-type larvae, whereasthe remaining half inherit the UAS>flu-∆Arm (or UAS>flu-wg) fz1H51

fz2C1 chromosome and show the phenotypes illustrated in Fig. 2D,E.

Antibody stainingStandard protocols for immunohistochemistry and immunofluorescencewere followed for both embryos and imaginal discs (Struhl and Basler,1993; Zecca et al., 1996) using mouse anti-Wg, mouse anti-En, mouseanti-Arm, rabbit anti-Vg, rabbit anti-Fz2, mouse anti-Dll, rabbit anti-Lab, rabbit anti-Eve, rabbit anti-GFP (Clontech), and rabbit anti-β-gal(Cappel) antisera (Diederich et al., 1989; Wu et al., 1995; Bhanot et al.,1996; Kim et al., 1996; Zecca et al., 1996; Neumann and Cohen, 1997;and references therein). The Armadillo mAb N27A1, En mAb 4D9 andWg mAb 4D4 antisera developed by E. Wieschaus, N. Patel and S.Cohen were obtained from the Developmental Studies Hybridomabank, Department of Biological Sciences, Iowa City, IA.

RESULTS

Isolation of a loss-of-function mutation in theDrosophila fz2 genePrevious saturation genetic screens for both maternal andzygotic factors required for normal embryonic patterning havefailed to identify any mutations in the fz2 gene, despite yieldingmultiple mutant alleles of several other genes involved in Wg

signaling, including Wg itself (Nüsslein-Volhard andWieschaus, 1980; Perrimon et al., 1989, 1996). The failure toidentify fz2 mutations in these screens might indicate that Fz2is not involved in Wg signal transduction. However, it is alsopossible that a requirement for Fz2 activity would only beapparent when both maternal and zygotic contributions areeliminated, or in the absence of other potentially redundantactivities, particularly Fz.

Recessive, loss-of-function mutations in fz cause defects inplanar polarity of epithelial cells: mutant animals are viableand generally display a normal morphology except that thebristles and hairs secreted by epidermal cells point in abnormaldirections. However, they do not appear to compromise Wgsignal transduction. To assess the possibility that Fz and Fz2have redundant roles in Wg signal transduction, we conducteda screen for new, recessive mutations that block Wg signaltransduction in fz mutant cells. Conveniently, both the fz andfz2 genes are located on the left arm of the third chromosome.This allowed us to conduct an “F1” genetic screen in whichflies carrying clones of wing cells homozygous for a fzmutation and newly induced, second site mutations on thischromosome arm were screened for phenotypes reflecting aloss in Wg signal transduction (Materials and Methods).

Wg is normally expressed in a thin stripe of cells straddlingthe dorsoventral compartment boundary of the mature wingimaginal disc, under the control of the extracellular signalsDelta and Serrate (reviewed by Irvine and Vogt, 1997). Wgemanating from these cells directs the formation of wingmargin bristles and organizes gene expression, growth andpatterning in surrounding cells of the presumptive wing blade(Zecca et al., 1996; Neumann and Cohen, 1997). Hence,mutations that block Wg signal transduction cause a loss ofwing margin bristles as well as deletions of nearby portions ofthe wing. Wg also plays a role in restricting its own expressionto cells immediately adjacent to the dorsoventral compartmentboundary, down-regulating the transcription of wg itself inneighboring cells that are close, but not next, to the D/Vboundary (Rulifson et al., 1996). When Wg signal transductionis blocked in these cells, they ectopically express Wg and as aconsequence induce nearby wild-type tissue to form ectopicmargin bristles.

Approximately 100 mutations were obtained in our screenwhich cause wing margin defects in clones of mutant cells thatare also homozygous for the fz loss of function mutation, fzH51

(see Materials and Methods). Of these, only one was associatedwith the formation of ectopic bristles in neighboring, wild-typewing tissue (Fig. 1B). This mutation, which we designate fz2C1,appears to be a loss-of-function mutation in fz2 by thefollowing criteria.

First, the mutation maps meiotically to a locationapproximately 1 centiMorgan distal to radius incompletus (ri),the expected map position given the cytological localization ofthe fz2 gene (Bhanot et al., 1996).

Second, both the wing notching and ectopic bristlephenotypes associated with fzH51 fz2C1 mutant cells arecompletely rescued when the fz2 coding sequence is expressedin these cells using either a Tubulinα1-fz2 transgene, whichshould be expressed in most or all cells, or a UAS-fz2 transgenedriven by a vg-Gal4 transgene (Fig. 1C, data not shown;Materials and Methods). All of the remaining wing notchingmutations obtained in the screen fully complement the fz2C1

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mutation in a fzH51 mutant background,indicating that they are not in the fz2 gene.

Third, the fz2C1 mutation is associatedwith a single base change in the fz2 genethat changes codon 320 from TGG to TAG(data not shown; Materials and Methods).This creates a stop codon located at thejunction between the coding sequence ofthe amino-terminal extracellular domain(which contains the CRD) and theremainder of the protein, which includesall seven transmembrane domains. It isunlikely that the resulting truncated protein, composed of justthe extracellular domain, would retain any signal transducingactivity.

As noted in the Introduction, vertebrates express a class ofsecreted Fz-related proteins composed of just the amino-terminal CRD-containing domain that function as endogenousantagonists of Wnt signaling. To test whether the truncatedFz2C1 mutant protein might have a similar antagonisticfunction or might alter Wg signaling, we used the Gal4/UASmethod to over-express Fz2C1 protein in a stripe of cells locatedjust anterior to the anteroposterior compartment boundary inthe wing imaginal disc (Materials and Methods). Under theseconditions, the Fz2C1 protein is expressed at levels several foldabove that of the endogenous wild-type protein; nevertheless,we could not detect any deviation from the normal distributionof Wg protein in the disc, nor from the normal pattern of theadult wing (data not shown). By contrast, over-expression ofboth the wild-type Fz2 protein as well as a GPI-linked form ofthe Fz2 extracellular domain under the same conditions causeenhanced accumulation of secreted Wg protein and cause gain-and loss-of-Wg signaling phenotypes, respectively (Cadigan etal., 1998; Zhang and Carthew, 1998; data not shown). Thus,we cannot observe any effect of the Fz2C1 mutant protein, evenwhen over-expressed, on the distribution or signaling activityof Wg, leading us to conclude that the fz2C1 mutation is anamorphic allele.

Embryos lacking both Fz and Fz2 activity cannottransduce WgTo assay the possible roles of Fz and Fz2 in Wg signaltransduction during embryogenesis, we generated embryoshomozygous for the fzH51 and fz2C1 mutations that derive fromfemale germ cells that are similarly mutant for the two genes

(Materials and Methods). Such embryos lack the maternal andzygotic contributions of both genes, and hence, should bedevoid of Fz and Fz2 activity. We refer to these embryos belowas fz− fz2− mutant embryos. To assay these embryos for Wgsignal transducing activity, we examined six well defined Wgsignaling events, two in the ectoderm, one in the visceralmesoderm, one in the endoderm, one in the central nervoussystem, and one in the somatic mesoderm. As we describebelow, these embryos appear unable to transduce Wg whenassayed for each event.

First, we examined the cuticular pattern formed by suchdouble mutant embryos. The epidermis of wild-type embryossecretes a segmented cuticle, decorated on the ventral side bystereotyped bands of patterned hairs separated by broad swathsof naked cuticle (Fig. 2A). In embryos devoid of Wg activity,or of Dsh or Arm activity, most signs of segmentation areeliminated and the ventral cuticle forms a “lawn” of hairsspanning most of the anteroposterior body axis (Nüsslein-Volhard and Wieschaus, 1980; Perrimon et al., 1989; Peifer andWieschaus, 1990; Noordermeer et al., 1994; Fig. 2B). Embryosdevoid of Fz and Fz2 activity show the same characteristic“lawn” phenotype (Fig. 2C).

Second, as noted in the Introduction, the early stripedexpression of En in the ectoderm (Fig. 3A) is labile, unlessmaintained by Wg signaling from adjacent cells across theparasegment boundary. In wg−, dsh− and arm− mutant embryos,this expression is lost within 2 hours after the onset ofgastrulation (by stage 10; DiNardo et al., 1988; Martinez Ariaset al., 1988; Bejsovec and Martinez Arias, 1991; Fig. 3B). Weobserve a similar loss of ectodermal En expression in fz− fz2−

mutant embryos (Fig. 3C).Third, Wg signaling is essential in the visceral mesoderm

for initiating a series of stereotyped constrictions that partition

C.-m. Chen and G. Struhl

Fig. 1. Loss of Wg signal transduction insomatic clones of fzH51 fz2C1cells in the wing.Clones of dsh75 (A) and fzH51 fz2C1 (B) cellscause similar wing notching phenotypesincluding the failure of mutant cells to formmargin bristles (arrowheads) and the formationof ectopic margin bristles in nearby wild-typetissue (arrows). The presence of aTubulinα1>fz2 transgene (C) is sufficient tofully rescue the wing notching phenotypesassociated with fzH51 fz2C1 mutant clones.Clones of fzH51 fz2C1cells were induced in a rimutant background, accounting for the gap inlongitudinal vein 2; the y marker used toidentify the mutant cells is not visible in thesemicrographs.

5445Wingless transduction by the Frizzled proteins

the midgut (Fig. 3E). As in embryos lacking Wg, Dsh, or Armactivity (Bienz, 1994; Fig. 3F), these gut constrictions areabsent in fz− fz2− mutant embryos (Fig. 3G).

Fourth, Wg signaling from the visceral mesoderm ofparasegment 7 up-regulates the expression of thehomeodomain gene labial in the adjacent endoderm (Fig. 3I).This up-regulation is not observed in wg-, dsh- or arm- embryos(Immergluck et al., 1990; Hoppler and Bienz, 1995; Yu et al.,1996; Fig. 3J), and similarly, it is not apparent in fz− fz2− mutantembryos (Fig. 3K).

Fifth, during development of the central nervous system, Wgsignaling is essential for specifying the neuroblasts thatgenerate the RP2 neurons in each segment (Chu-LaGraff andDoe, 1993; Bhat, 1996). These neurons can be easily visualizedbecause they express Even-skipped (Eve) protein (Fig. 3M).These Eve-expressing neurons are not present in the absenceof Wg signaling (e.g., in wg− embryos, Fig. 3N). Similarly, theyare absent in fz− fz2− mutant embryos (Fig. 3O).

Sixth and finally, during development of the somaticmusculature, Eve protein is expressed in a subset of myoblaststhat will give rise to the heart (Fig. 3Q) and the presence ofthese Eve-expressing cells is strictly dependent on Wgsignaling (Wu et al., 1995; Park et al., 1996; Figs. 3R). TheseEve-expressing cells are also absent in fz− fz2− mutant embryos(Fig. 3S).

In sum, embryos devoid of both Fz and Fz2 activity appearunable to transduce Wg in any of the several developmentalcontexts we have examined. These results indicate an absoluterequirement for these Fz proteins for Wg transduction duringembryonic development.

Distribution of Wg in fz− fz2− mutant embryosDuring normal development of the embryonic ectoderm, Wgprotein moves at least a few cell diameters from secreting cells,as assayed by the accumulation of punctate dots of Wgimmunoreactivity in neighboring, non-secreting cells (van denHeuvel et al., 1989; Gonzalez et al., 1991). We therefore

investigated whether the movement and apparent uptake ofsecreted Wg protein depends on Fz and Fz2 by examining thedistribution of Wg in fz− fz2− mutant embryos. We find thatwild-type and fz− fz2− mutant embryos show indistinguishabledistributions of punctate Wg staining during the first two hoursfollowing germ band extension (data not shown), consistentwith the view that neither Fz nor Fz2 protein are required forthe movement of secreted Wg during this phase ofdevelopment. However, the fzH51 mutation is expected togenerate a protein which is truncated after the sixthtransmembrane domain (Jones et al., 1996). Hence, if thisprotein is stable and reaches the cell membrane, it might beable to bind and regulate the movement of secreted Wg eventhough it can no longer transduce Wg signal. Wg expressiondissipates in fz− fz2− mutant embryos shortly after this earlystage, as expected given the loss of En expression inneighboring cells across the AP compartment boundary,preventing us from examining later aspects of Wg movementin these embryos.

Fz and Fz2 transduce Wg via the regulation ofArmadilloMost, if not all, Wg signal transducing events involve themodification and up-regulation of Armadillo (Arm) protein(Riggleman et al., 1990; Peifer et al., 1994; Willert and Nusse,1998). We therefore performed two experiments to test whetherFz and Fz2 transduce Wg through the regulation of Arm. Theseexperiments establish that Fz and Fz2 act upstream of Arm totransduce Wg.

In the first experiment, we assayed Arm expression in fz−

fz2− mutant embryos. In wild-type embryos, Wg signaling isassociated with stabilization of Arm protein and its consequentaccumulation in a distinctive pattern of segmental stripes, eachstraddling a stripe of Wg-expressing cells (Riggleman et al.,1990; Peifer et al., 199; Fig. 4A). This up-regulation is notobserved in wg− embryos, and similarly, we find that it is absentin fz− fz2− mutant embryos (data not shown; Fig. 4B).

In the second experiment, we asked whetherexpression of a truncated, constitutively activeform of Arm, ∆Arm (Zecca et al., 1996), coulddrive the Wg signal transduction pathway in

Fig. 2. Cuticular phenotype of fzH51 fz2C1 mutantembryos. Wild-type embryos (A) form a segmentedlarval cuticle decorated with bands of thick ventralhairs separated by naked cuticle, whereas wg− (B)and fzH51 fz2C1 mutant embryos (C) formabnormally short, unsegmented larval cuticles thatbear a continuous “lawn” of ventral hairs. fzH51

fz2C1 mutant embryos in which a constitutivelyactive form of Arm, ∆Arm, is expressed inalternating segmental primordia using theGal4/UAS method, form corresponding stripes ofnaked cuticle (D), placing Fz and Fz2 upstream ofArm in the Wg signal transduction pathway. Incontrast, expression of Wg instead of ∆Arm inalternating stripes in fzH51 fz2C1 mutant embryos (E)does not rescue the lawn phenotype. Expression ofWg in alternating stripes in an otherwise wild-typeembryo (F) suppresses the normal formation ofventral hairs (compare with A) confirming that theGal4/UAS method generates ectopic Wg signaling.

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fz− fz2− mutant embryos. In this experiment, ∆Arm wasexpressed with the UAS/Gal4 method using a hairy-Gal4driver line which is active in alternating segmental primordia(Brand and Perrimon, 1993). As shown in Fig. 2D, expressionof ∆Arm in alternating segmental stripes in fz− fz2− mutantembryos causes them to form corresponding stripes of nakedcuticle. This result indicates that the constitutive activity of∆Arm bypasses the normal requirements for Fz and Fz2 inactivating the Wg transduction pathway.

As a control for the second experiment, we tested theconsequences of over-expressing Wg instead of ∆Arm in fz−

fz2− mutant embryos. As shown in Fig. 2E, expression of aUAS-wg transgene (Zecca et al., 1996) under the control of thehairy-Gal4 driver line failed to rescue the fz− fz2− mutantphenotype, even though hairy-Gal4 driven expression of the

same transgene in otherwise wild-type embryos causes ectopicWg signal transduction (Fig. 2F; Wilder and Perrimon, 1995).Thus, ventral ectodermal cells of fz− fz2− mutant embryosappear unable to transduce Wg, even when it is ectopicallyexpressed.

Presumptive wing cells lacking both Fz and Fz2activity cannot transduce WgWg signaling plays a variety of roles during development ofthe imaginal discs. Here, we focus on the roles of Fz and Fz2in the wing disc, and particularly in the wing pouch whichgives rise to the adult wing blade. As noted above, Wg isexpressed in the wing pouch of late third instar discs in a thinstripe of cells straddling the interface between the dorsal andventral compartments. Wg emanating from this stripe acts at

C.-m. Chen and G. Struhl

Fig. 3. Absence of Wg signal transduction in fzH51 fz2C1 mutant embryos. Wild-type, wg−, fzH51 fz2C1 and fz2C1 embryos are shown stained forexpression of the HOX proteins En (A-D), Labial (E-L) and Eve (M-T); the fzH51 fz2C1 and fz2C1 embryos derive from mutant female germ cellsand hence lack both the maternal and zygotic contributions from the mutant genes. En is normally expressed in stripes in the ectoderm of thethorax and abdomen (A) during stage 10 (staging as in Campos-Ortega and Hartenstein, 1985). These stripes are abolished in wg− (B) and fzH51

fz2C1 (C) embryos at the same stage, but appear normal in fz2C1 embryos (D); En is expressed in the central nervous system as well as in thegnathal segments of embryos of all four genotypes, accounting for the residual staining visible in B and C. The endoderm is normallysubdivided into discrete domains by constrictions imposed by the visceral mesoderm and Labial is expressed in one of these domains (E,I). Theconstrictions are abolished in wg− (F) and fzH51 fz2C1 (G) embryos but appear normal in fz2C1 embryos. Similarly, Labial expression is normalin fz2C1(L) embryos but diminished in wg− (J) and fzH51 fz2C1 (K) embryos. Eve is normally expressed in specific neurons in the central nervoussystem (CNS) including the RP2 neurons (arrow) and in specific subsets of cells derived from the somatic mesoderm that will form the heart(M,Q). These cells, marked by Eve expression, appear to develop normally in fz2C1 embryos (P,T), but are absent in wg− (N,R) and fzH51 fz2C1

(O,S) embryos.

5447Wingless transduction by the Frizzled proteins

short range to induce the formation of bristles that will decoratethe wing margin, and at longer range to activate the expressionof a number of genes, including Distalless (Dll) and vestigial(vg), that define the primordium of the wing blade and controlaspects of its growth and pattern (Zecca et al., 1996; Neumannand Cohen, 1997; Fig. 5A). Loss of Wg signal transduction,e.g., in clones of arm mutant cells, eliminates these outputs(Zecca et al., 1996) and the absence of vg expression inparticular is sufficient to cause presumptive wing cells to stopproliferating and to be lost from the epithelium (Kim et al.,1996).

fzH51 fz2C1 homozygotes derived from heterozygous parentsshow normal Wg responses during embryogenesis (Fig. 6C; datanot shown). However, these embryos die just before or afterhatching as first instar larvae, precluding an analysis of thedevelopment of their imaginal discs. Therefore to examine theconsequences of lack of both gene functions on Wg signaling inwing imaginal disc cells, we generated clones of geneticallymarked, double mutant cells (Materials and Methods).

The fz2C1 mutation was initially identified because clones offzH51 fz2C1 cells are associated with deletions of wing marginand wing blade tissue, as well as with ectopic margin bristlesin neighboring wild-type wing tissue (Fig. 1B). This distinctivephenotype is also observed in association with clones ofarmXM19 and dsh75 cells induced under the same conditions(Fig. 1A; data not shown), indicating that fzH51 fz2C1 wingimaginal cells are compromised for Wg signal transduction.

To gain a clearer picture of the consequences of eliminatingboth fz and fz2 function, we examined Vg and Dll expression

in wing discs bearing “twin spots” formed by sibling fzH51

fz2C1/fzH51 fz2C1and +/+ clones which arise following mitoticrecombination in heterozygous fzH51 fz2C1/+ mother cells. Inthis experiment, the fzH51 fz2C1 mutant clones are marked bythe absence of expression of Green Flourescent Protein (GFP),while their sibling, wild-type clones are marked by enhancedexpression of GFP (Materials and Methods).

When mitotic recombination is induced early indevelopment, during the first larval instar, twin spots are foundin most portions of the mature wing disc (Fig. 6B). However,they are not found in the wing pouch, which is destined to formthe wing blade. Here, we find only wild-type clones (Fig. 5B),indicating that their fzH51 fz2C1 twins have been lost from theepithelium.

When mitotic recombination is induced later indevelopment, such as the beginning of the third instar, wild-type clones within the wing pouch are sometimes accompaniedby a mutant twin spot, although the mutant spot is typicallysmaller and shows abnormally low or undetectable levels of Vgand Dll expression (Fig. 5C). When mitotic recombination isinduced at later times during the third larval instar, wild-typeclones are usually accompanied by their mutant twins, whichexpress both Dll and Vg.

We also generated fzH51 fz2C1 mutant clones duringembryogenesis using the Minute technique (Morata and Ripoll,1975) to give these cells a growth advantage relative to thesurrounding wild-type tissue. Such clones can fill largeportions of either the anterior (A) or posterior (P) compartmentof the entire disc, except for the wing pouch from which theyappear to be excluded (data not shown).

We interpret these results as evidence that Wg signaltransduction is abolished in presumptive wing cells lackingboth Fz and Fz2 activity. As a consequence, cells that lack bothactivities cannot proliferate normally and are lost from theepithelium. We attribute the ability of small, late-inducedclones of fzH51 fz2C1 cells to survive and express Vg and Dllto the transient perdurance of Fz and/or Fz2 proteins in themutant cells.

We note that although fzH51 fz2C1 clones survive andcontribute to regions of the wing disc other than the wingpouch, their behavior in these regions appears abnormal. Forexample, in the prospective wing hinge region they aretypically smaller than their wild-type twins, round in shape,and have smooth borders. In addition, when such hinge clonesare positioned close to the D/V boundary, they express Vg, butnot Dll, and the same is true of large fzH51 fz2C1 clonesgenerated using the Minute technique (Fig. 5B and data notshown). In this respect the mutant cells resemble wild-typecells located more proximally along the DV boundary, whichexpress Vg in response to the activation of Notch, but do notexpress Dll. Wg signaling is required for the control of growthand pattern in portions of the wing disc other than the wingpouch (Neumann and Cohen, 1996; Ng et al., 1996), and wesuggest that the abnormal behavior of fzH51 fz2C1 clones inthese regions reflects their failure to transduce Wg.

Requirements for Fz and Fz2 activity in themesonotumThe wing imaginal discs also give rise to the fuselage of theadult second thoracic segment, the mesonotum, the anteriordorsal surface of which is decorated with a stereotyped pattern

Fig. 4. Armadillo abundance is not up-regulated in fzH51 fz2C1 mutantembryos. Arm levels are normally up-regulated in a segmentallyreiterated fashion in the ventral ectoderm in response to Wgsignaling (A), but remain uniformly low in fzH51 fz2C1 mutantembryos (B). The two embryos shown derive from eggs obtainedfrom fzH51 fz2C1 mutant germ cells fertilized by sperm from fzH51

fz2C1/TM3, ftz-lacZ males and were fixed, stained, and imaged inparallel.

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of large bristles (Fig. 7A). Wg is expressed in a longitudinalstripe in the developing half-mesonotum derived from eachwing disc and this stripe is positioned just lateral to a line offour large bristles (Phillips and Whittle, 1993). These are theanterior and posterior dorsocentral bristles and the anterior andposterior scutellar bristles. The formation of these bristles hasbeen reported to be Wg dependent (Phillips and Whittle, 1993).

We find that clones of fzH51 fz2C1 cells generated using theMinute technique during embryogenesis can fill the entiremesonotum of the adult fly although, as described above, theyfail to contribute to the wing blade. The bristles of such mutantmesonota show the characteristic frizzled polarity phenotype.However, the pattern is otherwise normal, including thepresence of both dorsocentral bristles and both scutellar bristlesat their stereotyped positions in each half (left and right) of themesonotum (Fig. 7C). To assay the Wg-dependence of thesebristles, we performed a similar experiment, this time using theMinute technique to generate large clones of wg− cells. In thiscase, clones of mutant cells that filled the mesonotum typicallyformed only a single dorsocentral bristle at a positionapproximately equidistant between the positions where the twodorsocentral bristles normally form (Fig. 7B). Similarly, theytended to form only one scutellar bristle at a site between thenormal positions of the anterior and posterior scutellar bristles.

The different bristle pattern phenotypes associated with wg−

and fzH51 fz2C1 clones could, in principle, reflectdifferent degrees of perdurance, or cell autonomy,associated with the two mutant conditions.However, an alternative possibility is that Wgmight be transduced in this context by a receptorother than Fz or Fz2.

Redundant roles of Fz and Fz2 in transducing Wgthroughout developmentAnimals homozygous for null mutations of fz survive toadulthood and show little if any evidence that they arecompromised for Wg signal transduction. To determinewhether animals homozygous for the fz2C1 mutation arecompromised for Wg signal transduction, we recombined thismutation away from the fzH51 mutation and then assayed thefate of fz2C1 homozygotes derived from heterozygous fz2C1/+parents. We find that such homozygotes can develop intonormal first instar larvae which in turn can give rise to adults,although they tend to be developmentally delayed relative totheir heterozygous siblings and give rise to abnormally small,sterile males and females (see Materials and Methods).Nevertheless, surviving fz2C1 homozygous flies are normallyproportioned and patterned, and lack any overt phenotypesassociated with reduced Wg signaling (data not shown). Thus,it appears that zygotic activity of the fz2 gene is not essentialfor Wg signal transduction, provided that a wild-type allele offz is present.

To examine further the functional redundancy between fzand fz2, we performed the following three experiments. First,we fertilized eggs derived from homozygous fzH51 fz2C1 germcells with sperm from heterozygous fz2C1/+ or fzH51/+ males.The resulting fz2C1/fzH51 fz2C1 or fzH51/fzH51 fz2C1 progeny lack

C.-m. Chen and G. Struhl

Fig. 5. Wing imaginal cells devoid of both Fz and Fz2activity cannot transduce Wg. Vestigial (Vg, blue) andDistalless (Dll, red) are expressed by presumptive wingcells in response to Wg which is secreted by a thinstripe of cell straddling the dorsoventral compartmentboundary in the wing pouch (A). Clones of fzH51 fz2C1

cells marked by the loss of expression of GreenFlourescent Protein (black in a background of greenGFP expressing cells), and by the presence of a wild-type twin “spot” of homozygous hsp70-GFP cells(bright green in a background of less intensely stainedhsp70-GFP/+ cells), fail to contribute to the wing bladewhen they are induced early, during the first larvalinstar (B). However, they do give rise to other portionsof the wing disc. Double mutant clones in thepresumptive wing hinge region, which surrounds thewing pouch, show abnormal behavior. They tend to beround in shape, to have smoother borders than theirwild-type twin spots, and when located close the DVcompartment boundary, to express Vg, but not Dll(arrows; see text). Clones of fzH51 fz2C1 cells inducedlater, during the early third instar, can survive in thepresumptive wing blade, but they tend to be smallerthan their wild-type twin spots and show low or noexpression of Vg or Dll (C). Clones of single mutantfz2C1 cells induced during the first intar larval stage canproduce large clones in all areas of the wing imaginaldisc, including the presumptive wing blade, and shownormal expression of Vg and Dll (D).

5449Wingless transduction by the Frizzled proteins

any functional Fz or Fz2 protein derived from maternal geneexpression, but do express functional Fz or Fz2 protein,depending on which wild-type gene is introduced paternally.In both cases, the embryos appear to develop normally (e.g.Fig. 6D,F) and form phenotypically wild-type larvae (data notshown), indicating that either protein can transduce most or allWg signaling events during embryogenesis.

The second experiment we performed was to generatehomozygous fz2C1 embryos from eggs derived fromhomozgyous fz2C1 germ cells, and then test these embryos fortheir ability to transduce Wg using the six assays describedabove for analyzing Wg signaling in fzH51 fz2C1 mutantembryos. Although these fz2C1 mutant embryos should bedevoid of functional Fz2 protein, we find that they areindistinguishable from wild-type embryos in their ability totransduce Wg by all six assays (Fig. 3D,H,L,P,T; data notshown). In addition, they show the normal increase in Armabundance in response to Wg signaling (data not shown).

Third, we examined the behavior of clones of fz2C1 cells inthe wing imaginal disc. In experiments similar to thoseperformed for the fzH51 fz2C1 genotype, we find that the fz2C1

clones and their wild-type sibling clones are found throughoutthe wing disc, including the wing pouch, and are of similar sizeand appearance irrespective of the time they were induced (Fig.5D). When the Minute technique was used, the resulting fz2C1

clones filled large portions of either the A or P compartment,including the entire A or P portion of the wing pouch (data notshown). In both cases, the mutant clones show normalexpression of Dll and Vg, and contributed to phenotypicallynormal adult wings. Indeed, by inducing a high frequency offz2C1 clones early in development using the Minute technique,we were able to generate flies in which virtually all cells in thethorax and head are mutant (the Minute technique is noteffective in the abdomen, which remains mosaic). These fliesappeared phenotypically normal in all respects (data notshown).

Thus, we conclude that Fz and Fz2 proteins are functionallyredundant, with either protein being able to bear the full burdenof Wg signal transduction in most, if not all, contextsthroughout development.

DISCUSSION

A large body of circumstantial evidence has implicated Fzproteins as Wnt receptors during animal development (seeIntroduction). However, the consequences of eliminating the

Fig. 6. Redundant roles of Fz and Fz2 duringembryogenesis. En is expressed in stripes ofectodermal cells in the thoracic and abdominalsegments during most of embryonicdevelopment (A). fzH51 fz2C1 mutant embryosderived from the fertilization of fzH51 fz2C1 eggsby fzH51 fz2C1 sperm are devoid of functional Fzand Fz2 protein and fail to maintain stripedexpression of En in the thoracic and abdominalectoderm (B; see also Fig. 3C). This phenotypeis “paternally rescued” by the presence of eithera wild-type fz or fz2 allele introduced whendouble mutant eggs are fertilized by fz2C1 orfzH51 mutant sperm (D,F; see Materials andMethods). It is also rescued in mutant fz2C1

embryos derived from the fertilization of fz2C1

eggs fertilized by fz2C1 sperm (E), and inhomozygous fzH51 fz2C1 embryos derived fromheterozygous females (C).

Fig. 7. Bristle patterning inthe mesonotum in theabsence of Fz and Fz2activity. The mesonotum ofwild-type flies (A) bears astereotyped pattern ofmechanosensory bristles,including two dorsocentralbristles (arrowheads) and twoscutellar bristles (asterisks)that arise just lateral to anarrow longitudinal stripe ofWg expressing cells (notshown). B and C showmesonota in which most orall cells belong to large wg−

or fzH51 fz2C1 clonesgenerated using the Minutetechnique. The pattern ofthese bristles depends on Wgsignaling, as only a singlebristle of each pair tends toform in mesonota composedof wg− cells (B). However,the pattern of all four bristlesappears normal in mesonotacomposed of fzH51 fz2C1 cells(C).

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endogenous activities of these proteins have been studied inrelatively few cases, and the results so far have failed toestablish an absolute requirement for Fz proteins in Wnt signaltransduction. In Drosophila, most Wnt signaling events involvea single Wnt, Wg (D-Wnt1), and two Fz proteins, Fz and Fz2,which have been proposed as candidate receptors. However,null mutations of the fz gene do not cause phenotypes that arerelated to reductions or loss of Wg signaling activity.Moreover, loss-of-function mutations have not been reportedfor the fz2 gene, making it difficult to assess the requirment forFz2 in Wg signal transduction, either alone or in conjunctionwith Fz. As a consequence, considerable uncertainty remainsabout the normal roles of these proteins in Wg reception.

Here, we report the isolation of a likely null allele ofDrosophila fz2. Using this mutation together with an amorphicmutation in fz, we find an absolute requirement for Fz proteinsin mediating most responses to Wg signaling duringdevelopment. We also show that Fz and Fz2 are functionallyredundant with respect to Wg signaling: the presence of eitherprotein appears sufficient to transduce most if not all Wgsignals throughout development. Finally, we find that Fz andFz2 function upstream of Arm, a protein which acts withincells to transduce Wg. Taken together with previous evidencethat Fz and Fz2 have Wg binding activities (Bhanot et al., 1996;Nusse et al., 1997; Cadigan et al., 1998), our results argue thatthe two proteins constitute the primary Wg receptors inDrosophila. These results have several implications which weconsider below.

Evidence against subsets of Wg receptors whichmediate distinct Wg responsesPrevious studies of wg mutations have raised the possibilitythat different receptors mediate distinct Wg outputs, perhapsthrough direct interactions with different functional domains inWg protein, or in different developmental contexts. Forexample, some partial loss-of-function mutations in wg havebeen reported to block the specification of “naked” ventralcuticle between each band of ventral hairs secreted by theventral ectoderm of the embryo, whereas others alter only thepattern of distinct hair types within each band (reviewed byDierick and Bejsovec, 1999). These different responses haveled to the proposal that each output reflects transduction of Wgby a different receptor (Hays et al., 1997). Similarly, Fz itselfhas been proposed to play a role in Wg signal transductionduring embryogenesis, but not during the development of theimaginal discs where Fz2 has been proposed to be the primaryWg receptor (Kennerdell and Carthew, 1998; Zhang andCarthew, 1998). Our evidence contradicts these proposals byshowing that Fz and Fz2 are each capable of mediatingvirtually all Wg responses during normal development.

Evidence against other Wg receptorsCircumstantial evidence has also been presented in support ofcandidate Wg receptors other than Fz and Fz2. In particular,loss-of-function mutations in the transmembrane receptorNotch have been shown to mimic many of the phenotypesassociated with reduction or loss of Wg signaling, consistentwith a receptor-ligand relationship (Couso and Martinez Arias,1994). In addition, at least two other Fz-like proteins have beenidentified in Drosophila (M. Boutros and M. Mlodzik; A. Satoand K. Saigo, as cited by Bhat, 1998), raising the possibility

that these other proteins can transduce Wg in at least somecontexts. However, the complete absence of detectable Wgsignal transduction in cells devoid of Fz and Fz2 activity invirtually all of the contexts we have assayed suggests that theseother proteins are normally not capable of transducing Wgwhen both Fz and Fz2 are absent. Hence, we suggest that ifthese, or other, proteins have a role in Wg reception, it wouldbe through the modulation of Fz and Fz2 activities. Forexample, they could facilitate or antagonize interactionsbetween Wg and Fz proteins on the outside of the cell, or theycould modulate interactions between Fz proteins and theirdown-stream effectors on the inside of cell.

We note that our results do not rule out the possibility thatadditional Wg receptors will exist and play significant roles inat least some developmental contexts. One possible case issuggested by our finding that clones of fzH51 fz2C1 cells whichpopulate the mesonotum form the normal pattern ofdorsocentral and scutellar bristles, a response which appears tobe at least partially dependent on Wg signaling (Phillips andWhittle, 1993). Hence, Wg-dependent patterning of thesebristles may depend on additional Wg receptors other than Fzand Fz2. However, our results suggest that such examples willbe relatively rare exceptions to the general role of Fz and Fz2as Wg receptors. Our results also leave open the possibility thatFz and Fz2 function in the context of a larger receptor complexthat includes other components which are similarly essentialfor binding and transducing Wg.

Potential roles of Fz proteins in generating andinterpreting gradients of secreted WgThe distribution of Fz2 protein is generally complementary tothat of Wg itself, peaking in cells far from the source ofsecreted Wg, but expressed at low levels in cells close to thesource (Cadigan et al., 1998). This, and related observations onthe effects of over-expression of Fz2, have led to the proposalthat the distribution of Fz2 plays a significant role inmodulating the spread and accumulation of Wg, once secreted(Cadigan et al., 1998). However, our finding that clones offz2C1 cells in the wing disc, and indeed, entirely fz2C1 mutantwing discs, can give rise to phenotypically normal wingschallenges this proposal. It is possible that the Fz2C1 mutantprotein, which is composed of just the N-terminal extracellulardomain, might retain the ability to modulate the movement andaccumulation of Wg protein, even though it lacks transducingactivity. However, we think this unlikely because we fail todetect any effect of over-expression of the mutant protein oneither Wg signaling or the distribution of Wg protein duringwing development. Hence, the ability of fz2C1 mutant wingdiscs to develop normally can be interpreted as evidence thatFz2 is not required to modulate the spread or accumulation ofextracellular Wg.

Wg appears to function as a gradient morphogen duringimaginal disc development, inducing discrete outputs in termsof gene expression and pattern as a function of its concentration(Zecca et al., 1996; Neumann and Cohen, 1997; Cadigan et al.,1998). Hence, the ability of animals lacking either zygotic Fzor Fz2 activity to develop into normally patterned flies suggeststhat both proteins can transduce the same concentration of Wgwith similar effectiveness, yielding the same outputs for a giveninput concentration. It is possible that Fz or Fz2 have similar,innate capacities for binding and transducing Wg. Alternatively,

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5451Wingless transduction by the Frizzled proteins

their abilities to bind and transduce Wg may depend on otherproteins that facilitate these interactions, allowing them tointerpret a given Wg gradient in the same way.

Planar polarity and the mode of action of FzMost epidermal cells of the adult fly differentiate structuressuch as hairs or bristles, which derive from polarizedextensions of their cytoskeletons. In general, these structureshave a common orientation, all pointing in the same direction,a phenomenon referred to as planar polarity (Nubler-Jung,1987). Planar polarity is altered in cells lacking Fz activity,presumably because the mutant cells are unable to respondproperly to an as yet unknown polarizing signal.

Our evidence that Fz is one of two primary Wg receptorshas implications for its role in planar cell polarity. The moststraightforward of these is the possibility that the polarizingsignal is a Wnt which can also bind to the Wg binding site ofFz, but once bound, induces an intracellular response that isdistinct from that elicited by Wg. Curiously, previous studieshave implicated the same cytosolic protein, Dsh, in transducingboth the polarity signal and the conventional Wg signal(Theisen et al., 1994; Neumann and Cohen, 1997; Tomlinsonet al., 1997; Axelrod et al., 1998). Our present findings focusfurther attention on the relationship between Dsh andtransduction of both Wg and the putative polarizing signalbecause they indicate that Fz alone is capable of transducingboth outputs through the agency of Dsh, each in response to adifferent ligand.

The dual roles of Fz and Dsh in transducing both Wg andthe polarizing signal pose the question of how distinct ligandscan activate different transduction pathways through theiractions on the same receptor. It is possible that Fz activity ismodulated by accessory proteins which influence which ligandit will bind, and what transduction pathway it will activate inresponse to ligand. For example, the structurally related,serpentine calcitonin-receptor-like receptor (CRLR) hasrecently been shown to bind either of two ligands dependingon the presence of specific receptor-activating-modifyingproteins (RAMPs) (McLatchie et al., 1998). A similarmechanism might account for how Fz can transduce both Wgand the polarizing signal through the activation of distinctintracellular pathways even in the same cell.

We thank A. Adachi for technical assistance, and S.-K. Chan, Y.Chen, H.-M. Chung, I. Greenwald, R. Mann, A. Tomlinson, and M.Zecca for advice and discussion, and S. Carroll, S. Cohen, M. Frasch,T. C. Kaufman, and R. Nusse for antibodies. C-M. C. is supported bythe College of Physicians and Surgeons, Columbia University and bythe Columbia University Skin Diseases Research Core Center (NIHP30 AR44535). G. S. is an HHMI Investigator.

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