notch and wingless modulate the response of cells … · notch and wingless modulate the response...

Developmental Biology 248, 93–106 (2002)doi:10.1006/dbio.2002.0720

Notch and Wingless Modulate the Response of Cellsto Hedgehog Signalling in the Drosophila Wing

Bruno Glise,* ,† ,1 D. Leanne Jones,* ,‡ ,1 and Philip W. Ingham* ,2

*MRC Intercellular Signalling Group, Centre for Developmental Genetics, University School ofMedicine and Biomedical Science, Sheffield, United Kingdom; †Centre de Biologie duDeveloppement–CNRS, 31062 Toulouse, France; and ‡Department of Developmental Biology,Beckman Center B373, Stanford University, School of Medicine, Stanford, California 94305

During Drosophila wing development, Hedgehog (Hh) signalling is required to pattern the imaginal disc epithelium alongthe anterior–posterior (AP) axis. The Notch (N) and Wingless (Wg) signalling pathways organise the dorsal–ventral (DV) axis,including patterning along the presumptive wing margin. Here, we describe a functional hierarchy of these signallingpathways that highlights the importance of competing influences of Hh, N, and Wg in establishing gene expression domains.Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc revealed that collier/knot(col/kn), patched (ptc), and decapentaplegic (dpp) are repressed at the DV boundary by N signalling. Attenuation of Hhsignalling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en)symmetrically about the DV boundary. We show that this down-regulation depends on activity of the canonical Wgsignalling pathway. We propose that modulation of the response of cells to Hh along the future proximodistal (PD) axis isnecessary for generation of the correctly patterned three-dimensional adult wing. Our findings suggest a paradigm ofrepression of the Hh response by N and/or Wnt signalling that may be applicable to signal integration in vertebrateappendages. © 2002 Elsevier Science (USA)

Key Words: wing disc; hedgehog; Notch; wingless; fused; Drosophila.

INTRODUCTION

During ontogeny, cells receive a succession of signalsleading to highly specific and coordinated responses, suchas cell proliferation, cell migration, or terminal differentia-tion. Evolutionarily conserved secreted signalling mol-ecules, including members of the Wnts, TGF�/BMPs, FGF,EGF, and Hedgehog (Hh) families, regulate such inductivecellular interactions. These signal transduction pathways,together with those regulating Notch (N) activity, are alsoinvolved in local cell–cell interactions. Although many ofthe components of these pathways have been identified,how the information generated by these signals is inte-grated to establish precise positional information and toelicit the complex set of responses observed during devel-opment is not well understood.

The wing of the adult Drosophila is a complex, three-

1 These authors contributed equally to this work.2 To whom correspondence should be addressed. Fax: �44 (114)

222-2788. E-mail: [email protected].

0012-1606/02 $35.00© 2002 Elsevier Science (USA)All rights reserved.

dimensional structure characterised by the stereotypic al-ternation of vein (named L1–L5) and intervein tissues (Fig.1B). The adult structure originates from the wing imaginaldisc, a simple invaginated epithelial sac composed of amonolayer columnar epithelium that is continuous withthe squamous epithelia of the peripodial membrane. Thedisc can be viewed as a series of circumferentially arrangedregions. From the outermost to the central region, the discwill give rise to the mesonotum, the wing hinge, and thewing pouch region (Bryant, 1978) (Fig. 1A). During pupalstages, the discs evert into the elongated appendages withthe wing pouch giving rise to the wing blade.

The wing imaginal disc is derived from a small embry-onic epithelial cluster and is divided into compartments(Garcia-Bellido et al., 1973). At the beginning of embryo-genesis, a segregation of anterior (A) and posterior (P) cellstakes place, followed by the creation of dorsal (D) andventral (V) compartments during the second larval instar.Two homeodomain transcription factors, Apterous (Ap) andEngrailed (En), serve as selector genes for the dorsal and
posterior compartments, respectively.
93

FIG. 1. Modulation of Hh signalling at the intersection of the AP and DV axes. (A) Drawing of a third instar imaginal wing dischighlighting the AP boundary, the prospective margin (green) corresponding to the DV boundary, and the different tissues giving rise to theadult mesonotum (mnt), hinge region (h), and wing blade (gray). In this and all other figures, discs are shown with anterior to the left anddorsal up. (B) Wild-type adult wing with the five longitudinal veins named L1–L5 and the anterior and posterior crossveins (a.c and p.c,respectively). (C) fuA adult wing phenotype showing a preferential fusion of veins L3 and L4 both proximally (black arrowhead) and distally(black arrow). (D) Drawing showing the centre of the wing pouch in a wild-type background with the expression of Col (red), a targetresponding to high dose of Hh, and Wg (green) highlighting the prospective margin. Central part of the wing pouch of wild-type, fu1, or fuA

third instar wing imaginal discs (D–O). (E) In a wild-type background, Col (red) is expressed in 7 cells immediately anterior to the APboundary in response to Hh but is not expressed in the prospective wing margin (white arrowhead). (G) In a fu1 background, Col expressionis extended along the AP axis to 10 cell diameters and is absent in the centre of the wing pouch in approximately 6 rows of cells on eitherside of the DV boundary, here highlighted by the Wg immunostainings (green). (F, H) collier in situ hybridisation in wt (F) and fu1 (H)backgrounds showing that the down-regulation of Col in the centre of the wing pouch in a fu1 background is due to an absence oftranscription of collier. (I) Drawing showing the expression of Col and Wg in the centre of the wing pouch in a fused background. (J) Ptcimmunostaining (green) in a wild-type background. At the AP boundary, Ptc is expressed in 5 cells with an absence of activation at the DVboundary (white arrowhead). (L) In a fu1 background, a decrease in Ptc activation is observed in the centre of the wing pouch, and Ptcexpression is expanded along the AP axis to 7 cell diameters. (K, M) patched in situ hybridisation in wt (K) and fu1 (M) backgroundsdemonstrating that the down-regulation of Ptc in a fu1 background is acting at the transcriptional level. (N) decapentaplegic (dpp) in situhybridisation in a wild-type background showing expression in around 12 cell diameters and an absence of expression at the DV boundary(black arrowhead). (O) In a fuA background, a weak but reproducible decrease of dpp expression is observed in the centre of the wing pouchand dpp expression is extended along the AP axis to around 20 cell diameters.

94 Glise, Jones, and Ingham

© 2002 Elsevier Science (USA). All rights reserved.

The selector gene apterous subdivides the disc into dorsaland ventral compartments, which leads to the formation ofan organising centre at the dorsoventral (DV) boundary ofthe developing wing disc (Cohen et al., 1992; Diaz-Benjumea and Cohen, 1993; Williams et al., 1993). Thissubdivision results in the activation of Notch immediatelyadjacent to both sides of the DV boundary, which in turnactivates the expression of wingless (wg) (reviewed by Irvineand Vogt, 1997). Diffusion of Wg refines expression of targetgenes along the DV axis, and both N and Wg are required fordifferentiation of wing margin structures (Couso et al.,1994; deCelis et al., 1996; Diaz-Benjumea and Cohen, 1995;Neumann and Cohen, 1997; Strigini and Cohen, 2000;Zecca et al., 1996). Several mechanisms participate inlimiting Wg expression to cells immediately adjacent to theDV boundary. Wg signalling regulates N activity via Di-shevelled, which binds to N and reduces its activity. Inaddition, Wg up-regulates the N ligands Delta and Serrate,

which act in a dominant-negative fashion, at high levels, torepress N signalling (Axelrod et al., 1996; deCelis and Bray,1997; Diaz-Benjumea and Cohen, 1995; Micchelli et al.,1997). Furthermore, the POU-domain protein Nubbin lim-its the range over which N activation induces Wg in thewing pouch by setting a threshold for N activity (Neumannand Cohen, 1998).

The engrailed (en) gene is expressed primarily in theposterior compartment, where it allows the synthesis of Hh(Tabata et al., 1992; Zecca et al., 1995; reviewed by Law-rence and Struhl, 1996). Secretion of Hh from posteriorcompartment cells induces the expression of decapentaple-gic (dpp), collier/knot (col/kn), patched (ptc), and en itself atspecific concentration thresholds in cells of the adjacentanterior compartment. Hh has at least two major roles inthe anterior–posterior (AP) patterning of the wing: an indi-rect, long-range activity mediated via its regulation of theTGF� family member, Dpp, and a short-range activity at

FIG. 2. A region refractory to Hh signalling exists along the DV boundary. (A) hhMrt third instar imaginal disc shows expression of Col (red)along the DV boundary in response to ectopic Hh synthesis. Note the absence of Col expression in the central row of cells along the DVaxis, corresponding to the prospective wing margin (arrowhead). (B) fu1;hhMrt disc showing the absence of Col expression in several rows ofcells on either side of the DV axis (arrow). (C, D) Double staining with ectopic expression of Hh, marked by the presence of the GFP staining(green), and Col (red). (C, C�) Hh induces Col expression nonautonomously in the wing pouch but is not able to activate Col in the hingeregion (arrow). The clones marked by the presence of an asterisk are clones in the peripodial membrane, which do not activate Col eitherin the peripodial membrane itself or in the overlying disc proper. (D, D�) Higher magnification of a wing pouch carrying Hh-expressingclones abutting the prospective wing margin highlights that Col expression is repressed in the central row of cells marking the DV axis evenwhen these cells are expressing Hh (arrowhead).

95Modulation of Hh Targets by N and Wg


the AP border (Blair, 1992; deCelis and Barrio, 2000; Mohleret al., 2000; Mullor et al., 1997; Strigini and Cohen, 1997;Vervoort et al., 1999; Zecca et al., 1995). The latter ismediated partly by the activation of col/kn, the Drosophilamember of the COE transcription factor family (Dubois andVincent, 2001), and is essential for correct differentiation ofintervein tissue in the medial region of the wing betweenveins L3 and L4 (Mohler et al., 2000; Nestoras et al., 1997;Vervoort et al., 1999).

Hh initiates a signalling cascade that impinges on thezinc finger protein Cubitus interruptus (Ci), a homologue ofthe vertebrate Gli transcription factors (reviewed by Ing-ham and McMahon, 2001). In the absence of Hh signalling,full-length Ci protein is cleaved into a C-terminally trun-cated form that translocates to the nucleus and inhibitstranscription of target genes such as dpp or hh itself(Aza-Blanc et al., 1997; Methot and Basler, 1999). In cellsthat receive the Hh signal, Ci cleavage is inhibited, and theprotein acts as a transcriptional activator capable of induc-ing the transcription of Hh target genes (Chen et al., 1999;Methot and Basler, 1999; Ohlmeyer and Kalderon, 1998).Regulation of Ci activity is mediated by several proteins(reviewed in Ingham and McMahon, 2001), most notably inthe context of this study, cAMP-dependent protein kinase 1(pka-C1), and the putative serine/threonine kinase Fused(Fu), which acts to maximise Hh signalling by regulating Citrafficking from the cytoplasm to the nucleus (Chen et al.,1999; Jiang and Struhl, 1998; Methot and Basler, 2000;Theodosiou et al., 1998; Wang et al., 1999, 2000; Wang andHolmgren, 1999).

The generation of a three-dimensional adult wing re-quires the organisation of a proximodistal (PD) axis or-thogonal to the AP and DV axes. This axis runs parallel tothe DV axis in the two-dimensional epithelium of theimaginal disc. Previous studies have shown that patterningalong the PD axis is specified in part via a mechanisminvolving interactions between cells expressing wg andcells expressing dpp (Diaz-Benjumea et al., 1994). Theseinteractions lead to the activation of the homeobox geneshomothorax, aristaless, and Distal-less along the PD axis,which are involved in establishing the final shape and sizeof the wing (Azpiazu and Morata, 2000; Campbell andTomlinson, 1998; Campbell et al., 1993; Casares and Mann,2000).

In this study, we have found evidence that modulation ofHh target gene expression by N and Wg signalling plays acritical role in the elaboration of pattern, especially at thedistal tip of the wing. We find that Hh target gene expres-sion along the DV boundary is repressed by N signalling. Inaddition, by sensitising the Hh pathway, we demonstratethat this repression also occurs by activation of the canoni-cal Wg signalling pathway. Unlike ptc and col/kn, expres-sion of the Hh target gene en is not repressed by Nactivation but is repressed by Wg activity. We propose thatmodulation of the Hh response along the DV axis isrequired to give rise to a correctly patterned three-dimensional adult wing: specifically, down-regulation of

Hh targets at and around the DV boundary is necessary toallow vein tissue and bristle differentiation along the adultwing margin.

MATERIALS AND METHODS

Drosophila Stocks

Alleles used in this study were: fu1, fuJB3, fuA, and fuRX15 (Preat etal., 1993); hhMrt (Felsenfeld and Kennison, 1995); pka-C1H2 andpka-C1B3 (Lane and Kalderon, 1993); wgCX4 (Baker, 1987); Nl1N-ts2

(Shellenbarger and Mohler, 1975); and N55e11 (Mohler, 1956).

Clonal Analysis

Clones of mutant cells were induced by Flp-mediated mitoticrecombination (Xu and Rubin, 1993). The larvae were heat-shockedfor 2 h at 38°C during first and second instar and dissected duringlate third instar. The chromosome double mutant for pka-C1 andwg (Pan and Rubin, 1995) was kindly provided by J. Modolell. Thegenotypes of the flies used to make mitotic clones were as follows:pka-C1 clones: w,hsFLP1; pka-C1H2,FRT40A/Ubi-GFP,FRT40A;dpp-lacZ; and pka-C1 wg clones: w,hsFLP1; pka-C1B3,ck,wgCX4,FRT40A/Ubi-GFP,FRT40A.

Ectopic Expression

Clones of cells which ectopically express hh or Nintra wereinduced by the GAL4-UAS system by using a Flp-out cassette(Brand and Perrimon, 1993; Ito et al., 1997). The genotypes of theflies used were as follows: y,w,hsFLP1;Act�y��Gal4,UAS-GFP/�;UAS-hh/� and y,w,hsFLP1; Act�y��Gal4,UAS-GFP/�;UAS-Nintra/�.

UAS-hh has been previously described (Ingham and Fietz, 1995).UAS-Nintra was kindly given by D. Strutt (deCelis and Bray, 1997).

The fu1/FM7;71B-Gal4/71B-Gal4 stock was constructed to ob-tain larvae expressing different components of the Wg signallingpathway in an fu1 background. The different UAS constructs usedwere: UAS-Wg (Hacker et al., 1997), UAS-Dsh (Neumann andCohen, 1996), and UAS-ArmS10 (Pai et al., 1997). After crosses ofthe appropriate stocks, we selected and dissected third instar malelarvae based on gonad morphology.

Immunohistochemistry

Third instar wing imaginal discs were dissected in phosphate-buffered saline (PBS) and fixed for 20 min at room temperature in4% paraformaldehyde in PBS. Incubation with primary antibodieswas carried out overnight at 4°C by using the following dilutions:rabbit anti-Collier antibody 1:200 (Crozatier et al., 1999), mouseanti-Patched 5E10 antibody 1:500 (Strutt et al., 2001), mouseanti-Wingless 4D4 antibody 1:400 (Brook and Cohen, 1996), mouseanti-Engrailed 4D9 antibody 1:200 (Patel et al., 1989), and mouseanti-Cut 2B10 antibody (Blochlinger et al., 1990).

Double stainings were done in parallel with mouse and rabbitprimary antibodies. GFP labelling was observed directly withoutimmunostaining. To count precisely the rows of cells expressingthe different markers, the discs were stained, after immunostain-ings, with propidium iodide to mark the nuclei. Imaginal discswere mounted in PBS/glycerol and observed with a Leica SP



confocal microscope. Capture images were manipulated with Pho-toshop.

RNA in Situ Hybridizationscol (Crozatier et al., 1996), ptc (Ingham et al., 1991), and dpp

(dppE55, a 4-kb dpp cDNA) expression were monitored by whole-mount in situ hybridization using digoxigenin-labeled antisenseRNA probes.

RESULTS

Hh Target Genes Are Not Activated in theProspective Wing Margin

Short-range Hh signalling is mediated partly by the acti-vation of col/kn close to the AP boundary and is essentialfor differentiation of intervein tissue in the medial region ofthe wing between veins L3 and L4 (Vervoort et al., 1999).During second larval instar, N and Wg define the DVboundary, which will eventually give rise to a specialisedvein structure: the wing margin (Fig. 1A). In order to revealpossible interactions between the different signalling path-ways involved in patterning the different axes in the wing,we examined the expression of the different Hh targetsaround the DV boundary.

In wild-type third instar wing discs, we detected anabsence of Col expression in the central row of cellscorresponding to the prospective wing margin (Fig. 1E). Inaddition, Ptc up-regulation and dpp expression are notobserved in these cells (Figs. 1J and 1N). The changes in Ptc,Col, and dpp expression occur at the transcriptional level asreflected by the changes in the pattern of mRNA accumu-lation (Figs. 1F, 1K, and 1N). These observations suggestthat in wild type there is a modulation of the response toHh signalling in the prospective wing margin (Fig. 1D). Bycontrast to the other Hh targets, En is activated in the cellscorresponding to the prospective wing margin (see Fig. 3A).

fused Mutants Reveal a Modulation of HhSignalling at the Intersection of the APand DV Axes

The fused gene has previously been identified as a posi-tive component of the Hh pathway (Ingham, 1993; Preat etal., 1993) that is thought to maximise the Hh signal byregulating Ci trafficking from the cytoplasm to the nucleus(Methot and Basler, 2000; Wang and Holmgren, 2000). Inwings from flies carrying hypomorphic class I alleles fu1 andfuJB3 (data not shown) and from the class II alleles fuA (Fig.1C) and fuRX15 (data not shown) (Preat et al., 1993; Therondet al., 1996), the L3 and L4 wing veins are fused bothproximally and distally, with intervein tissue remaining inthe medial portion of the wing (compare Figs. 1B and 1C). Inaddition, there is a posterior extension of the wing marginsensory bristles from the third to the fourth vein (Alves etal., 1998; Fausto-Sterling, 1978; Sanchez-Herrero et al.,1996).

Previous analysis of Hh targets in fu1 or fuA third instarimaginal discs demonstrated that Ptc expression is loweredand anterior En expression is absent when compared withwild-type wing discs (Figs. 1J–1M) (Alves et al., 1998;Sanchez-Herrero et al., 1996). Furthermore, there is anincrease in the number of cells activating dpp along the APaxis (Fig. 1O) (Alves et al., 1998).

In addition to this phenotype, in fused mutant wing discs,we found a complete absence of Col expression and Ptcup-regulation in the centre of the wing pouch in approxi-mately six rows of cells on either side of the DV boundary(Figs. 1G–1I and 1L–1M) (see also Vervoort et al., 1999). Thechanges in Ptc and Col expression occur at the transcrip-tional level as reflected by the changes in the pattern ofmRNA accumulation (compare Fig. 1F with 1H and 1Kwith 1M). Although Ci levels are high and the protein isprimarily cytoplasmic, there is no obvious variation in Cilevels or localisation along the DV axis of fu mutant discs(Wang and Holmgren, 1999 and data not shown).

As for dpp, we observed an increase in the number of cellsactivating Col and Ptc along the AP axis in a fu background(compare Fig. 1N with 1O). This effect is probably due tothe lower level of Ptc receptor, which sequesters Hh,resulting in an increased range of the Hh ligand. A weak butreproducible down-regulation of dpp expression in the cen-tre of the wing pouch was also observed in a fu background,which is not detectable by dpp-lacZ staining (data notshown and Alves et al., 1998; Sanchez-Herrero et al., 1996).The results shown for fu1 (Figs. 1E–1H, 1J–1O) were alsoobserved in the fuJB3, fuA, and fuRX15 animals (data notshown).

In contrast to previous studies, our data reveal thattranscription of all characterised Hh targets is affected bythe loss of Fu activity in the wing imaginal disc. While thelevels of expression of Hh targets are lowered, there is adecrease or an absence of activation of these targets at theDV boundary and an increase in the number of cellsresponding along the AP axis (Fig. 1I).

A Region Refractory to Hh Signalling Exists alongthe Entire DV Boundary

fu mutant flies display a fusion of veins L3 and L4 at thedistal tip of the wing, which corresponds to the intersectionof the AP and DV signalling centres in the imaginal disc.This suggests that the developing wing is particularlysensitive to a reduction of Hh signalling at the DV bound-ary. To determine whether down-regulation of Hh targetgene occurs only at the intersection of the AP and DV axes,we analysed Col expression in an existing gain-of-functionallele of Hh called Moonrat (hhMrt) (Felsenfeld and Kenni-son, 1995). In hhMrt, in addition to its normal expression inthe posterior compartment of the wing disc, hh is ectopi-cally expressed along the DV boundary within the anteriorcompartment (Felsenfeld and Kennison, 1995).

We examined Col expression in hhMrt third instar wingdiscs and found that Col is expressed within its normal



FIG. 3. Removal of Ptc, but not Pka-C1 function leads to activation of Hh targets close to the DV boundary. (A–C) Amorphic pka-C1H2

clones were generated by mitotic recombination and marked by the absence of the GFP staining. Arrowheads are used to indicate the DVboundary. Irrespective of the AP position, the removal of Pka-C1 function, in the anterior compartment, leads to the up-regulation of Col,En, and Ptc only when the clones are generated away from the DV axis. (A) Triple labelling showing pka-C1H2 clones (absence of green) andCol (red) and En (blue) expression. Note the presence of En expression in the cells corresponding to the prospective wing margin in theanterior compartment. (B) Double labelling identifying the clones (absence of green staining) and Col (red). We observe an absence of Colactivation close to the DV boundary even when the clones are situated near the anterior limit of endogenous Col expression (arrow in B).In several clones situated at mid-distance relative to the DV boundary, we can observe a weak activation of Col (asterisk in B). (C) Doublelabelling identifying the clones (absence of green staining) and Ptc (red). (D) Double labelling showing amorphic ptcIIW clones (absence ofgreen staining) and Col expression (red). Removal of Ptc function leads to activation of Col, in a cell-autonomous manner, in all cells exceptthose contributing to the prospective wing margin (arrowhead).



domain but also on either side of the DV axis in the anteriorcompartment (Fig. 2A). Again, we observed an absence ofCol expression in the central row of cells along the DV axiscorresponding to the prospective wing margin (Fig. 2A).Previous experiments have shown that fu mutations cansuppress the hhMrt adult phenotype and the ectopic expres-sion of ptc-lacZ and dpp-lacZ observed in discs in thismutant background (Alves et al., 1998). In the fu1;hhMrt

background, Col expression is absent in several rows ofcells on either side of the DV axis, suggesting that Hh targetgene activation is sensitive to a repressive activity all alongthe DV compartment boundary (Fig. 2B).

To verify this result, we generated clones that express hhectopically at random locations throughout the wing imagi-nal disc (Figs. 2C and 2D). In such experiments, we detectednonautonomous activation of Col in response to Hh exclu-sively inside the wing pouch (Figs. 2C and 2C�). Expressionof Col was absent from the central row of cells marking theDV boundary even when Hh is expressed in these cells(arrowheads in Figs. 2D and 2D�).

These experiments confirm that the region correspondingto the prospective wing margin cannot activate Hh targetgene expression. In addition, this repression is expanded toseveral rows of cells on either side of the DV boundary in afu background, even when the cells are exposed to a highdose of Hh ligand as in a hhMrt background.

Differential Response to Pka-C1 and PtcInactivation Close to the DV Boundary

To determine whether the refractory region is uncoveredspecifically in the absence of Fused activity, we analysedHh target gene activation in clones of cells mutant for othercomponents of the Hh pathway. Previous results suggestthat Pka-C1 directly regulates the proteolysis and activityof Ci (Chen et al., 1998; Wang et al., 1999). In the absenceof Pka-C1 function, Ci is not targeted for proteolytic pro-cessing, resulting in the activation of Hh targets in acell-autonomous manner. However, it has been shown thatloss of Pka-C1 is not sufficient for maximal activation of Ci(Wang and Holmgren, 1999).

We generated clones of cells lacking Pka-C1 in thirdinstar imaginal discs at different positions along the AP andDV axes. In clones in the anterior compartment, we ob-served an up-regulation of Col, Ptc, and En but only at somedistance away from the DV boundary (Figs. 3A–3C). Theseresults were obtained with two different amorphic pka-C1alleles, excluding the possibility of an allele-specific effect(Fig. 4B). Using dpp-lacZ staining, we did not observe anymodulation of its expression along the DV axis, most likelybecause of the perdurance of the �-galactosidase (data notshown). As in the fu mutant wing discs, we were unable toobserve in the pka-C1 clones any modulation of Ci levels orlocalisation along the DV axis (data not shown).

Full activation of the Hh pathway can be achieved cellautonomously by removing the activity of the Hh receptorPtc. In contrast to Pka-C1 clones, clones of cells lacking Ptc

FIG. 4. Wg antagonizes Hh target gene activation along the DVboundary. wgCX4,pka-C1B3 clones were generated by mitotic re-combination and marked by the absence of GFP expression. (A,B) Triple staining showing the wgCX4,pka-C1B3 clones (absence ofgreen staining), Col (red) and Wg (blue). (A) When Wg expressingcells are removed, we observed an up-regulation of Col expres-sion throughout the anterior clones, including in the cells closeto the DV boundary. However, we do not observe any activationof Col in the cells corresponding to the prospective wing margin(arrow). The absence of Wg synthesis is indicated by arrowhead.(B) When the clones abut on Wg-expressing cells but do notinclude them, we still observed close to the DV boundary aregion refractory to Col activation (arrow), showing that thiseffect is not allele specific. (C) Double labelling showing thewgCX4,pka-C1B3 clone (absence of green staining) and En (red). Weobserve an up-regulation of En in all the cells within the clone,including cells corresponding to the prospective wing margin.These results indicate that Wg is required for the repressionobserved close to the DV boundary when Pka-C1 function isremoved.



activate Col in all the cells within the clones, includingthose close to the DV boundary (Fig. 3D). However, withinclones spanning the DV boundary, we still observed theabsence of Col expression in cells along the prospectivewing margin (Fig. 3D, arrowhead). These results reveal thatcells in the vicinity of the DV boundary, although respon-sive to maximal Hh activity, remain refractory to thesubmaximal Hh pathway activation elicited by loss ofPka-C1 activity. Furthermore, the repression of Col, Ptc,and En seems to occur in a gradient along the DV axis insidethe clones (Figs. 3A–3C), suggesting the existence of adiffusible molecule emanating from the DV boundary thatmay mediate this repression.

Wg Antagonises Hh Target Gene Activation alongthe DV Boundary

One obvious candidate for a diffusible molecule originat-ing at the DV boundary that could affect expression of Hhtargets is Wg. To investigate this possibility, we generatedclones of cells double mutant for both pka-C1 and wgactivities. When Wg-expressing cells are removed by theclones, irrespective of their position along the AP axis, weobserved an up-regulation of Col and En expression in cellsboth at a distance from and close to the DV boundary (Figs.4A and 4C). Again, in these clones, we were unable toobserve any modulation of Ci levels or localisation alongthe DV axis (data not shown). Surprisingly, however, wenever observed Col up-regulation in cells corresponding tothe prospective wing margin (Fig. 4A). This indicates thatWg is responsible for the repression at a distance from theDV boundary but not at the wing margin itself. In contrastto Col, we observed En up-regulation in all the cellscorresponding to the clone, confirming that En is notdown-regulated at the DV boundary (Fig. 4C).

To verify this result, we ectopically expressed Wg inwild-type and fu1 backgrounds using the Gal4/UAS system(Brand and Perrimon, 1993). Ectopic expression of Wgdriven in the wing pouch results in a decrease in Colexpression. This effect is most evident around the DVboundary close to the endogenous Wg expression domainand in the anterior-most row of Col-expressing cells, whereHh signalling input is lowest due to the distance of thesource of Hh (arrowhead and arrow in Fig. 5A�; comparewith Fig. 5B). Ectopic expression of Wg in a fu1 backgroundresults in the complete absence of Col expression in thewing-pouch (Figs. 5C and 5C�), confirming the ability of Wgto down-regulate Col in the absence of Fused activity.

To investigate whether the canonical Wg signalling path-way is involved in the repression of Hh targets along the DVboundary, we ectopically expressed several components ofthe Wg pathway in wild-type and fu1 backgrounds. Ectopicexpression of Dishevelled (Dsh), which participates in boththe planar cell polarity and canonical Wg signalling path-ways (Axelrod et al., 1998), causes an almost completesuppression of Col expression in a fu background (Fig. 5D).Similar results were obtained by using an activated form of

Armadillo (Arm), the Drosophila �-catenin homologue (Fig.5E) (Cavallo et al., 1997).

These results indicate that Wg signalling is capable ofantagonising the expression of col, ptc, and en over a shortdistance on either side of the DV boundary. However, Wg isnot responsible for the cell-autonomous down-regulation ofcol and ptc within the prospective wing margin.

Notch Signalling Is Required for Repression of HhTargets at the Presumptive Wing Margin

We have demonstrated that activation of Col and Ptcexpression does not occur in a wild-type wing along the DVboundary and that this repression is not dependent on Wg(Fig. 4). A candidate molecule that could mediate cell-autonomous repression along the wing margin is Notch.

To investigate the capacity of N to repress Hh target geneexpression, we raised animals homozygous for a conditionalthermosensitive allele of N (Nts2) at the permissive tempera-ture and then shifted the mutant larvae to the nonpermis-sive temperature (29°C) at different stages during larvaldevelopment. Shifts at the beginning of the L2 stage, beforethe creation of the DV compartment boundary, resulted inabnormal wing discs that lacked detectable Col expressionin the anterior compartment, indicating the need to specifywing pouch identity in order to achieve activation of thesetargets. When Nts2 larvae were shifted to the nonpermissivetemperature at the beginning of the L3 stage, we obtainedsmall wing pouch structures. Col and Ptc expression inthese discs was not repressed in cells corresponding to theprospective wing margin (Figs. 6A and 6B). Moreover, sur-viving adults exhibit notches at the distal tip of the wings(Fig. 6J), reminiscent of the most sensitive phenotype ob-served in the wings of adult flies heterozygous for N55e11, anull allele of N (Fig. 6I). When a dominant active form of N(Nintra) was ectopically expressed within the wing pouch, weobserved a cell-autonomous down-regulation of both Coland Ptc (Figs. 6C and 6D). By contrast, expression of En wasnot affected by Nintra expression in either the posteriorcompartment or the anterior compartment in response toHh (Fig. 6E), consistent with the fact that en is normallyactivated in the prospective wing margin.

To determine whether there are competing influences ofN and Hh signalling in the prospective wing margin, weoverexpressed a stabilised form of Ci (CiU) (Methot andBasler, 1999) in Hh responding cells using the ptc-Gal4driver. This resulted in activation of Col in the cellscorresponding to the prospective wing margin (compareFigs. 6F and 6G). Moreover, the differentiated wings of suchflies exhibit variable defects at their distal tips, fromnotches (Fig. 6K and arrowhead in Fig. 6L) to absence ofbristles with remaining vein tissue (Figs. 6L and 6M). Thislatter phenotype is reminiscent of the effects of weakhypomorphic wingless alleles that affect bristle formationwithout notching (Couso et al., 1994). These results indi-cate that, in the prospective wing margin, N, Wg, and Hhsignalling exist in a delicate balance. In the wild type wing



disc, N repression is dominant over Hh activation, leadingto the differentiation of vein tissue at the margin, while Wgsignalling counteracts Hh signalling to allow proper veintissue and bristle formation.

DISCUSSION

Short-range Hh signalling, partly through activation ofCol function, is essential for correct AP patterning anddifferentiation of L3–L4 intervein tissue (Vervoort et al.,1999). N and Wg first define the DV boundary and latersubdivide the region near this boundary into a number ofdistinct subregions that will eventually differentiate intowing margin bristles and vein tissue (Couso et al., 1994).These signals overlap spatially and temporally and lead toopposite fates. We propose that in and close to the DVboundary, N, Wg, and Hh signalling exist in a delicatebalance to allow vein tissue, bristle, and sensory organdifferentiation along the adult wing margin.

The Hh target genes col/kn and ptc, in contrast to en, arerepressed in a wild type wing in cells corresponding to thepresumptive wing margin. We have demonstrated, usingboth gain- and loss-of-function experiments, that this re-pression is mediated by N signalling and that its inhibitionresults in aberrant morphogenesis of the wing. Hh signal-ling, achieved either by overexpression of Hh or loss of Ptcactivity, is not sufficient to give maximum activation of Hhtargets in cells of the prospective wing margin, suggestingthat a finely tuned balance of activation and repression isrequired to achieve the appropriate biological output. How-ever, overexpression of a stabilised form of Ci under theptc-Gal4 driver resulted in the activation of Col in theprospective wing margin and defects in wing margin differ-entiation, indicating that N repression can be overcome byhyperactivity of the Hh signalling pathway. N signallingmay lead to the repression of col, ptc, and dpp directly or itmay act indirectly by affecting the ability of Ci to act as atranscriptional activator. Since expression of en, whichrequires the highest level of Hh signalling and Ci activity,appears immune to N repression, we favour the formerpossibility.

Analysis of Hh target gene expression close to the DVboundary in fu mutants revealed that activation of col anddpp and up-regulation of ptc transcription is lost from thecentre of the wing pouch when Hh signalling is attenuated.Inactivation of fu results in a complete loss of en expressionfrom the anterior compartment; however, we found that theectopic activation of en induced by submaximal Hh path-way activation (achieved by removing Pka-C1 activity) issimilarly sensitive to inhibitory influences emanating fromthe DV boundary. In each case, the repression of Hh targetsextends up to six rows of cells away from the DV boundaryin all the anterior compartment. We have demonstratedthat this down-regulation is due to activation of the canoni-cal Wg signalling pathway. At what level is this globalinhibition of Hh targets by Wg signalling achieved? One

possibility is that Hh target genes are regulated by theopposing effects of activator and repressor complexes actingdirectly via cis-acting sequences. Such a situation haspreviously been described for the stripe gene, which isdirectly activated by Hh signalling and repressed by Wgsignalling in the Drosophila embryo (Piepenburg et al.,2000). This mechanism would imply the existence of cis-acting sites for both the Ci and dTCF/pangolin proteinswithin the regulatory regions of each of the Hh targetgenes—col, ptc, dpp, and en—that we have analysed, apossibility that cannot currently be verified due to theabsence of sequence information for all of these regions. Analternative scenario, however, is that Wg signalling modu-lates the activity of the Hh signalling pathway itself,perhaps at the level of the Su(fu)/Fu/Ci protein complex toaffect localisation and/or activity of Ci. Consistent withthis possibility, recent studies have provided evidence for adirect protein–protein interaction between the vertebrateSu(fu) and �-Catenin/Armadillo proteins (Meng et al.,2001). Such an interaction might mediate the attenuation ofHh signalling activity, for instance, by promoting the cyto-plasmic retention of Ci. It should be noted, however, thatwe have not detected any modulation of Ci distributionalong the DV axis using the available anti-Ci antibodies inthe fu mutant backgrounds, the pka-C1, or the pka-C1;wgclones.

Signal Integration and Appendage Outgrowth inOther Systems

The paradigm of modulation of Hh target gene expressionby N and/or Wg may be applicable to signal integration inother systems. Hh signalling plays a remarkably similarrole in patterning the Drosophila wing and vertebratelimbs. Specifically, Hh is involved in both short- andlong-range signalling, elaborating upon preexisting informa-tional cues to regulate anterior–posterior patterning, distaloutgrowth, and proliferation. One of the vertebrate homo-logues of hh, sonic hedgehog (shh), is expressed in the zoneof polarizing activity (ZPA), which is found at the posterioredge of the limb bud (Riddle et al., 1993). Here, Shh isthought to control the expression of molecules, such asfibroblast growth factor-4 (fgf-4), which is required for limboutgrowth (Capdevila and Johnson, 2000). Interestingly,Notch-1 expression and activity in the vertebrate limblocalises to the apical ectodermal ridge (AER), a thickeningof the ectoderm along the distal tip of the limb bud, andmolecular and misexpression studies have suggested that Nsignalling may regulate the number of AER progenitor cells.Homologues of one of the primary downstream effectors ofN signalling in the wing, cut, have also been characterisedin the chick as participating in limiting the AER (Cux-1)and regulating polarising activity derived from the ZPA(Cux-2) (Tavares et al., 2000). Furthermore, several verte-brate Wnts, homologues of Drosophila Wg, have also beenimplicated in the formation of the AER. It is possible that asimilar signalling hierarchy exists in the vertebrate limb to



allow for proximal–distal patterning and limb outgrowth.According to our model, modulation of the expression ofShh targets near the intersection of the ZPA and AER in thedeveloping vertebrate limb may be required for the differ-entiation of distal features.

During mammalian tooth development, Shh and Wnt-7bare expressed in reciprocal patterns and a balance betweenthe two activities is required for the formation of bound-aries between oral and dental ectoderm. Overexpression ofWnt-7B in domains of endogenous Shh expression has beenshown to repress ptch1 transcription and prevent tooth budformation, while overexpression of Shh suppresses thiseffect (Sarkar et al., 2000).

The positioning and outgrowth of vertebrate epithelialappendages, such as feathers, whiskers, and hair, is anotherexample where proper integration of positional informationis essential for proximal–distal patterning. Overexpressionof Wnt-7a in an in vitro feather reconstitution model leadsto the inhibition of elongation along the PD axis and loss of

AP asymmetry in the presumptive feather bud. Thesechanges were correlated with diffuse Notch-1 and Shhexpression, which are normally localised to the middle andposterior of the bud, respectively (Widelitz et al., 1999). Theloss of localized Shh expression and loss of AP asymmetryas a consequence of ectopic expression of Wnt are reminis-cent of the effect on Shh and hair follicle morphogenesis inmice expressing a stabilized form of �-catenin in the skin(Gat et al., 1998).

The Role of fused in Hh Signalling

Expression of Hh target genes requires activation of Ci bya multistep process. First, Ci proteolysis must be blocked.The cleavage of Ci into its truncated repressor form ispromoted by Pka-C1-mediated phosphorylation. Removalof Pka-C1 leads to stabilisation of Ci; however, ectopictarget gene expression accomplished in such a manner doesnot reflect maximal Hh signalling. This is indicated by the

FIG. 5. Activation of the canonical Wg signalling pathway affects expression of Hh target genes along the DV boundary. Using the wingpouch specific 71B-Gal4 driver, we ectopically expressed several components of the Wg pathway in wild-type (A, A�) and fu1 backgrounds(C–E). (A, A�) Ectopic expression of Wg (green) in a wild-type background results in a decrease of Col staining (red), evident in the mostanterior row of cells (arrowhead) and around the DV boundary (arrow), compared with wild-type Col staining (B). In a fu1 background (C,C�), there is a complete absence of Col expression in the wing-pouch. (D) Ectopic expression of wild-type Dsh leads to a strongdown-regulation of Col expression in a fu background. The effect of Dsh expression is weaker than that of Wg expression, most likely dueto the patchy expression of the 71B-Gal4 driver as revealed in (A) and (C). (E) Similar results were obtained by ectopic expression of adominant-active form of Arm (ArmS10). These results suggest that Wg, signalling via the canonical Wg signalling pathway, is involved inthe modulation of Hh target genes along the DV boundary.



absence of Col and Ptc expression close to the DV boundaryin clones where Pka-C1 activity has been removed. Inaddition to preventing Ci cleavage, there is another Hh-dependent activation step that requires Fused kinase activ-ity (Methot and Basler, 2000; Wang and Holmgren, 2000).As fu is dispensable in animals where Suppressor of fused[Su(fu)] is also inactive and as Su(fu) has been shown to playa role in blocking Ci nuclear accumulation, it is hypoth-esised that Fu kinase activity is required in cells to facilitatenuclear localisation of activated Ci (Methot and Basler,2000; Price and Kalderon, 1999; Wang and Holmgren, 1999).As fu has been implicated in localisation of Ci, the down-regulation of Hh targets away from the margin describedhere may reflect the inability of activated Ci to enter thenucleus efficiently. A decrease in Ci activity is reflected in

the decreased transcription of col, ptc, and dpp and anabsence of en activation in the anterior compartment in a fumutant background.

Recent results indicated that Hh signalling in the embryo,leading to ptc transcription and dorsal epidermal morphogen-esis, occurs independently of Fu activity (Therond et al., 1999).However, Fu activity is indispensable in the embryo in Hhtarget cells expressing Wg, leading to the hypothesis thatactivation of Hh targets is repressed by an unknown localisedfactor in wg-expressing cells (Therond et al., 1999). We specu-late that Fu activity could be required in Wg-expressing cellsin the embryo to overcome a repressive effect of Wg on Hhtarget gene activation. Further studies will be needed todetermine whether this model can be extended to all thedevelopmental processes involving Hh signalling.

FIG. 6. Notch signalling is involved in repression of Hh targets at the presumptive wing margin. (A, B) Shifting of Nts2 larvae tononpermissive temperature (29°C) at the beginning of L3 stage results in the activation of Col (A) and Ptc (B) expression in the cellscorresponding to the prospective wing margin. This shift is also associated with defects in cell proliferation leading to a smaller wing pouch.(C, D) Ectopic expression of a dominant active form of N (Nintra), labelled by the presence of the GFP expression (green) results in thecell-autonomous down-regulation of Col (C, C�) or Ptc (D, D�) but not of En (E, E�). (D, D�) The arrowheads mark the absence of Ptc stainingat the DV boundary. (E, E�) The asterisks indicate a Nintra clone at the margin in the posterior compartment, and the arrows indicate a Nintra

clone in the anterior compartment in cells activating En in response to Hh signalling. There is no down-regulation of En in either clone.(F, G) Expression of Col (red) and Cut (green), an N-dependent/Wg-independent target used to mark the cells which receive N signalling atthe DV boundary. (F) Wild-type wing disc. (G) The expression of a stabilised form of Ci (CiU) under the control of the ptc-Gal4 driver inwild-type leads to an overactivation of Hh signalling at the AP-DV intersection. Col is now detectable in the cells corresponding to theprospective wing margin, as shown by the colocalisation of Col and Cut (yellow staining, white arrow). (H) Distal tip of a wild-type wingshowing the double row margin with vein tissue and innervated sensory bristles present between longitudinal veins L3 and L4. (I) Wing ofa N55e11/� fly, showing that one of the most sensitive N phenotypes is an absence of vein tissue and sensory bristles tissue in the most distalpart of the wing. (J) Wing from a Nts2 fly. Larvae were shifted to the nonpermissive temperature (29°C) at the beginning of L3 stage. Theseflies present preferential notches at the tip of the wings. (K–M) Wing phenotypes of flies expressing a stabilised form of Ci (CiU) under thecontrol of the ptc-Gal4 driver at 29°C. (L) and (M) are focused on the most distal tip of the wings. The strength of the phenotype is variableranging from a complete notch (K), to an absence of sensory bristles between veins L3 and L4 (L), a partial absence of vein structure (L, blackarrowhead), or to an absence of sensory bristles only in the posterior part of the L3–L4 margin (M). These results suggest a delicate balancebetween the N, Wg, and Hh pathways to allow vein tissue and sensory organ differentiation along the adult wing margin.



ACKNOWLEDGMENTS

We thank Drs. D. Strutt and J. Modolell and the BloomingtonStock Centre for providing fly strains; Dr. A. Vincent for theanti-Col antibody. FlyBase database for genes and alleles descrip-tion. We are grateful to Drs. P. Blader, M. Crozatier, and A. Vincentfor critical reading of the manuscript. We thank B. Savelli for hishelp in the preparation of the figures. This work was funded by aWellcome Programme grant (to P.W.I.). B.G. was supported by anEMBO fellowship (ALTF 270-1997) and by a fellowship from the“Fondation pour la Recherche Medicale.” D.L.J. was supported bya Human Frontiers Research Program postdoctoral fellowship andis now a Lilly Fellow of the Life Sciences Research Foundation.

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Received for publication April 22, 2002Accepted May 7, 2002

Published online June 25, 2002



notch and wingless modulate the response of cells … · notch and wingless modulate the response...

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