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

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

Notch and Wingless Modulate the Response of Cells to 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 of Medicine and Biomedical Science, Sheffield, United Kingdom; †Centre de Biologie du De´veloppement–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 along the 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 signalling pathways 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 Hh signalling 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 Wg signalling pathway. We propose that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. Our findings suggest a paradigm of repression of the Hh response by N and/or Wnt signalling that may be applicable to signal integration in vertebrate appendages. © 2002 Elsevier Science (USA) Key Words: wing disc; hedgehog; Notch; wingless; fused; Drosophila.

INTRODUCTION During ontogeny, cells receive a succession of signals leading to highly specific and coordinated responses, such as cell proliferation, cell migration, or terminal differentiation. Evolutionarily conserved secreted signalling molecules, including members of the Wnts, TGF␤/BMPs, FGF, EGF, and Hedgehog (Hh) families, regulate such inductive cellular interactions. These signal transduction pathways, together with those regulating Notch (N) activity, are also involved in local cell– cell interactions. Although many of the components of these pathways have been identified, how the information generated by these signals is integrated to establish precise positional information and to elicit the complex set of responses observed during development is not well understood. The wing of the adult Drosophila is a complex, three1

These authors contributed equally to this work. To whom correspondence should be addressed. Fax: ⫹44 (114) 222-2788. E-mail: [email protected]. 2

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dimensional structure characterised by the stereotypic alternation of vein (named L1–L5) and intervein tissues (Fig. 1B). The adult structure originates from the wing imaginal disc, a simple invaginated epithelial sac composed of a monolayer columnar epithelium that is continuous with the squamous epithelia of the peripodial membrane. The disc can be viewed as a series of circumferentially arranged regions. From the outermost to the central region, the disc will give rise to the mesonotum, the wing hinge, and the wing pouch region (Bryant, 1978) (Fig. 1A). During pupal stages, the discs evert into the elongated appendages with the wing pouch giving rise to the wing blade. The wing imaginal disc is derived from a small embryonic epithelial cluster and is divided into compartments (Garcia-Bellido et al., 1973). At the beginning of embryogenesis, a segregation of anterior (A) and posterior (P) cells takes place, followed by the creation of dorsal (D) and ventral (V) compartments during the second larval instar. Two homeodomain transcription factors, Apterous (Ap) and Engrailed (En), serve as selector genes for the dorsal and posterior compartments, respectively.

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FIG. 1. Modulation of Hh signalling at the intersection of the AP and DV axes. (A) Drawing of a third instar imaginal wing disc highlighting the AP boundary, the prospective margin (green) corresponding to the DV boundary, and the different tissues giving rise to the adult mesonotum (mnt), hinge region (h), and wing blade (gray). In this and all other figures, discs are shown with anterior to the left and dorsal 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) fu A 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 target responding to high dose of Hh, and Wg (green) highlighting the prospective margin. Central part of the wing pouch of wild-type, fu 1, or fu A third instar wing imaginal discs (D–O). (E) In a wild-type background, Col (red) is expressed in 7 cells immediately anterior to the AP boundary in response to Hh but is not expressed in the prospective wing margin (white arrowhead). (G) In a fu 1 background, Col expression is 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 either side of the DV boundary, here highlighted by the Wg immunostainings (green). (F, H) collier in situ hybridisation in wt (F) and fu 1 (H) backgrounds showing that the down-regulation of Col in the centre of the wing pouch in a fu 1 background is due to an absence of transcription of collier. (I) Drawing showing the expression of Col and Wg in the centre of the wing pouch in a fused background. (J) Ptc immunostaining (green) in a wild-type background. At the AP boundary, Ptc is expressed in 5 cells with an absence of activation at the DV boundary (white arrowhead). (L) In a fu 1 background, a decrease in Ptc activation is observed in the centre of the wing pouch, and Ptc expression is expanded along the AP axis to 7 cell diameters. (K, M) patched in situ hybridisation in wt (K) and fu 1 (M) backgrounds demonstrating that the down-regulation of Ptc in a fu 1 background is acting at the transcriptional level. (N) decapentaplegic (dpp) in situ hybridisation 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 fu A background, a weak but reproducible decrease of dpp expression is observed in the centre of the wing pouch and dpp expression is extended along the AP axis to around 20 cell diameters.

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FIG. 2. A region refractory to Hh signalling exists along the DV boundary. (A) hh Mrt 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 DV axis, corresponding to the prospective wing margin (arrowhead). (B) fu 1;hh Mrt disc showing the absence of Col expression in several rows of cells 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 hinge region (arrow). The clones marked by the presence of an asterisk are clones in the peripodial membrane, which do not activate Col either in the peripodial membrane itself or in the overlying disc proper. (D, D⬘) Higher magnification of a wing pouch carrying Hh-expressing clones abutting the prospective wing margin highlights that Col expression is repressed in the central row of cells marking the DV axis even when these cells are expressing Hh (arrowhead).

The selector gene apterous subdivides the disc into dorsal and ventral compartments, which leads to the formation of an organising centre at the dorsoventral (DV) boundary of the developing wing disc (Cohen et al., 1992; DiazBenjumea and Cohen, 1993; Williams et al., 1993). This subdivision results in the activation of Notch immediately adjacent to both sides of the DV boundary, which in turn activates the expression of wingless (wg) (reviewed by Irvine and Vogt, 1997). Diffusion of Wg refines expression of target genes along the DV axis, and both N and Wg are required for differentiation 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 in limiting Wg expression to cells immediately adjacent to the DV boundary. Wg signalling regulates N activity via Dishevelled, which binds to N and reduces its activity. In addition, Wg up-regulates the N ligands Delta and Serrate,

which act in a dominant-negative fashion, at high levels, to repress 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 limits the range over which N activation induces Wg in the wing pouch by setting a threshold for N activity (Neumann and Cohen, 1998). The engrailed (en) gene is expressed primarily in the posterior compartment, where it allows the synthesis of Hh (Tabata et al., 1992; Zecca et al., 1995; reviewed by Lawrence and Struhl, 1996). Secretion of Hh from posterior compartment cells induces the expression of decapentaplegic (dpp), collier/knot (col/kn), patched (ptc), and en itself at specific concentration thresholds in cells of the adjacent anterior compartment. Hh has at least two major roles in the anterior–posterior (AP) patterning of the wing: an indirect, long-range activity mediated via its regulation of the TGF␤ family member, Dpp, and a short-range activity at

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the AP border (Blair, 1992; deCelis and Barrio, 2000; Mohler et al., 2000; Mullor et al., 1997; Strigini and Cohen, 1997; Vervoort et al., 1999; Zecca et al., 1995). The latter is mediated partly by the activation of col/kn, the Drosophila member of the COE transcription factor family (Dubois and Vincent, 2001), and is essential for correct differentiation of intervein tissue in the medial region of the wing between veins L3 and L4 (Mohler et al., 2000; Nestoras et al., 1997; Vervoort et al., 1999). Hh initiates a signalling cascade that impinges on the zinc finger protein Cubitus interruptus (Ci), a homologue of the vertebrate Gli transcription factors (reviewed by Ingham and McMahon, 2001). In the absence of Hh signalling, full-length Ci protein is cleaved into a C-terminally truncated form that translocates to the nucleus and inhibits transcription of target genes such as dpp or hh itself (Aza-Blanc et al., 1997; Me´ thot and Basler, 1999). In cells that receive the Hh signal, Ci cleavage is inhibited, and the protein acts as a transcriptional activator capable of inducing the transcription of Hh target genes (Chen et al., 1999; Me´ thot and Basler, 1999; Ohlmeyer and Kalderon, 1998). Regulation of Ci activity is mediated by several proteins (reviewed in Ingham and McMahon, 2001), most notably in the 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 Ci trafficking from the cytoplasm to the nucleus (Chen et al., 1999; Jiang and Struhl, 1998; Me´ thot and Basler, 2000; Theodosiou et al., 1998; Wang et al., 1999, 2000; Wang and Holmgren, 1999). The generation of a three-dimensional adult wing requires the organisation of a proximodistal (PD) axis orthogonal to the AP and DV axes. This axis runs parallel to the DV axis in the two-dimensional epithelium of the imaginal disc. Previous studies have shown that patterning along the PD axis is specified in part via a mechanism involving interactions between cells expressing wg and cells expressing dpp (Diaz-Benjumea et al., 1994). These interactions lead to the activation of the homeobox genes homothorax, aristaless, and Distal-less along the PD axis, which are involved in establishing the final shape and size of the wing (Azpiazu and Morata, 2000; Campbell and Tomlinson, 1998; Campbell et al., 1993; Casares and Mann, 2000). In this study, we have found evidence that modulation of Hh target gene expression by N and Wg signalling plays a critical role in the elaboration of pattern, especially at the distal tip of the wing. We find that Hh target gene expression along the DV boundary is repressed by N signalling. In addition, by sensitising the Hh pathway, we demonstrate that this repression also occurs by activation of the canonical Wg signalling pathway. Unlike ptc and col/kn, expression of the Hh target gene en is not repressed by N activation but is repressed by Wg activity. We propose that modulation of the Hh response along the DV axis is required to give rise to a correctly patterned threedimensional adult wing: specifically, down-regulation of

Hh targets at and around the DV boundary is necessary to allow vein tissue and bristle differentiation along the adult wing margin.

MATERIALS AND METHODS Drosophila Stocks Alleles used in this study were: fu 1, fu JB3, fu A, and fu RX15 (Pre´ at et al., 1993); hh Mrt (Felsenfeld and Kennison, 1995); pka-C1 H2 and pka-C1 B3 (Lane and Kalderon, 1993); wg CX4 (Baker, 1987); N l1N-ts2 (Shellenbarger and Mohler, 1975); and N 55e11 (Mohler, 1956).

Clonal Analysis Clones of mutant cells were induced by Flp-mediated mitotic recombination (Xu and Rubin, 1993). The larvae were heat-shocked for 2 h at 38°C during first and second instar and dissected during late third instar. The chromosome double mutant for pka-C1 and wg (Pan and Rubin, 1995) was kindly provided by J. Modolell. The genotypes of the flies used to make mitotic clones were as follows: pka-C1 clones: w,hsFLP1; pka-C1 H2,FRT40A/Ubi-GFP,FRT40A; dpp-lacZ; and pka-C1 wg clones: w,hsFLP1; pka-C1 B3,ck,wg CX4, FRT40A/Ubi-GFP,FRT40A.

Ectopic Expression Clones of cells which ectopically express hh or N intra were induced by the GAL4-UAS system by using a Flp-out cassette (Brand and Perrimon, 1993; Ito et al., 1997). The genotypes of the flies used were as follows: y,w,hsFLP1;Act⬎y⫹⬎Gal4,UAS-GFP/ ⫹;UAS-hh/⫹ and y,w,hsFLP1; Act⬎y⫹⬎Gal4,UAS-GFP/⫹;UASN intra/⫹. UAS-hh has been previously described (Ingham and Fietz, 1995). UAS-N intra was kindly given by D. Strutt (deCelis and Bray, 1997). The fu 1/FM7;71B-Gal4/71B-Gal4 stock was constructed to obtain larvae expressing different components of the Wg signalling pathway in an fu 1 background. The different UAS constructs used were: UAS-Wg (Hacker et al., 1997), UAS-Dsh (Neumann and Cohen, 1996), and UAS-Arm S10 (Pai et al., 1997). After crosses of the appropriate stocks, we selected and dissected third instar male larvae based on gonad morphology.

Immunohistochemistry Third instar wing imaginal discs were dissected in phosphatebuffered saline (PBS) and fixed for 20 min at room temperature in 4% paraformaldehyde in PBS. Incubation with primary antibodies was carried out overnight at 4°C by using the following dilutions: rabbit anti-Collier antibody 1:200 (Crozatier et al., 1999), mouse anti-Patched 5E10 antibody 1:500 (Strutt et al., 2001), mouse anti-Wingless 4D4 antibody 1:400 (Brook and Cohen, 1996), mouse anti-Engrailed 4D9 antibody 1:200 (Patel et al., 1989), and mouse anti-Cut 2B10 antibody (Blochlinger et al., 1990). Double stainings were done in parallel with mouse and rabbit primary antibodies. GFP labelling was observed directly without immunostaining. To count precisely the rows of cells expressing the different markers, the discs were stained, after immunostainings, with propidium iodide to mark the nuclei. Imaginal discs were mounted in PBS/glycerol and observed with a Leica SP

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confocal microscope. Capture images were manipulated with Photoshop.

RNA in Situ Hybridizations col (Crozatier et al., 1996), ptc (Ingham et al., 1991), and dpp (dppE55, a 4-kb dpp cDNA) expression were monitored by wholemount in situ hybridization using digoxigenin-labeled antisense RNA probes.

RESULTS Hh Target Genes Are Not Activated in the Prospective Wing Margin Short-range Hh signalling is mediated partly by the activation of col/kn close to the AP boundary and is essential for differentiation of intervein tissue in the medial region of the wing between veins L3 and L4 (Vervoort et al., 1999). During second larval instar, N and Wg define the DV boundary, which will eventually give rise to a specialised vein structure: the wing margin (Fig. 1A). In order to reveal possible interactions between the different signalling pathways involved in patterning the different axes in the wing, we examined the expression of the different Hh targets around the DV boundary. In wild-type third instar wing discs, we detected an absence of Col expression in the central row of cells corresponding to the prospective wing margin (Fig. 1E). In addition, Ptc up-regulation and dpp expression are not observed in these cells (Figs. 1J and 1N). The changes in Ptc, Col, and dpp expression occur at the transcriptional level as reflected by the changes in the pattern of mRNA accumulation (Figs. 1F, 1K, and 1N). These observations suggest that in wild type there is a modulation of the response to Hh signalling in the prospective wing margin (Fig. 1D). By contrast to the other Hh targets, En is activated in the cells corresponding to the prospective wing margin (see Fig. 3A).

Previous analysis of Hh targets in fu 1 or fu A third instar imaginal discs demonstrated that Ptc expression is lowered and anterior En expression is absent when compared with wild-type wing discs (Figs. 1J–1M) (Alves et al., 1998; Sanchez-Herrero et al., 1996). Furthermore, there is an increase in the number of cells activating dpp along the AP axis (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 Ptc up-regulation in the centre of the wing pouch in approximately six rows of cells on either side of the DV boundary (Figs. 1G–1I and 1L–1M) (see also Vervoort et al., 1999). The changes in Ptc and Col expression occur at the transcriptional level as reflected by the changes in the pattern of mRNA accumulation (compare Fig. 1F with 1H and 1K with 1M). Although Ci levels are high and the protein is primarily cytoplasmic, there is no obvious variation in Ci levels 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 cells activating Col and Ptc along the AP axis in a fu background (compare Fig. 1N with 1O). This effect is probably due to the lower level of Ptc receptor, which sequesters Hh, resulting in an increased range of the Hh ligand. A weak but reproducible down-regulation of dpp expression in the centre of the wing pouch was also observed in a fu background, which is not detectable by dpp-lacZ staining (data not shown and Alves et al., 1998; Sanchez-Herrero et al., 1996). The results shown for fu 1 (Figs. 1E–1H, 1J–1O) were also observed in the fu JB3, fu A, and fu RX15 animals (data not shown). In contrast to previous studies, our data reveal that transcription of all characterised Hh targets is affected by the loss of Fu activity in the wing imaginal disc. While the levels of expression of Hh targets are lowered, there is a decrease or an absence of activation of these targets at the DV boundary and an increase in the number of cells responding along the AP axis (Fig. 1I).

fused Mutants Reveal a Modulation of Hh Signalling at the Intersection of the AP and DV Axes

A Region Refractory to Hh Signalling Exists along the Entire DV Boundary

The fused gene has previously been identified as a positive component of the Hh pathway (Ingham, 1993; Pre´ at et al., 1993) that is thought to maximise the Hh signal by regulating Ci trafficking from the cytoplasm to the nucleus (Me´ thot and Basler, 2000; Wang and Holmgren, 2000). In wings from flies carrying hypomorphic class I alleles fu 1 and fu JB3 (data not shown) and from the class II alleles fu A (Fig. 1C) and fu RX15 (data not shown) (Pre´ at et al., 1993; The´ rond et al., 1996), the L3 and L4 wing veins are fused both proximally and distally, with intervein tissue remaining in the medial portion of the wing (compare Figs. 1B and 1C). In addition, there is a posterior extension of the wing margin sensory bristles from the third to the fourth vein (Alves et al., 1998; Fausto-Sterling, 1978; Sanchez-Herrero et al., 1996).

fu mutant flies display a fusion of veins L3 and L4 at the distal tip of the wing, which corresponds to the intersection of the AP and DV signalling centres in the imaginal disc. This suggests that the developing wing is particularly sensitive to a reduction of Hh signalling at the DV boundary. To determine whether down-regulation of Hh target gene occurs only at the intersection of the AP and DV axes, we analysed Col expression in an existing gain-of-function allele of Hh called Moonrat (hh Mrt) (Felsenfeld and Kennison, 1995). In hh Mrt, in addition to its normal expression in the posterior compartment of the wing disc, hh is ectopically expressed along the DV boundary within the anterior compartment (Felsenfeld and Kennison, 1995). We examined Col expression in hh Mrt third instar wing discs and found that Col is expressed within its normal

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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-C1 H2 clones were generated by mitotic recombination and marked by the absence of the GFP staining. Arrowheads are used to indicate the DV boundary. 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-C1 H2 clones (absence of green) and Col (red) and En (blue) expression. Note the presence of En expression in the cells corresponding to the prospective wing margin in the anterior compartment. (B) Double labelling identifying the clones (absence of green staining) and Col (red). We observe an absence of Col activation 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) Double labelling identifying the clones (absence of green staining) and Ptc (red). (D) Double labelling showing amorphic ptc IIW clones (absence of green staining) and Col expression (red). Removal of Ptc function leads to activation of Col, in a cell-autonomous manner, in all cells except those contributing to the prospective wing margin (arrowhead).

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domain but also on either side of the DV axis in the anterior compartment (Fig. 2A). Again, we observed an absence of Col expression in the central row of cells along the DV axis corresponding to the prospective wing margin (Fig. 2A). Previous experiments have shown that fu mutations can suppress the hh Mrt adult phenotype and the ectopic expression of ptc-lacZ and dpp-lacZ observed in discs in this mutant background (Alves et al., 1998). In the fu 1;hh Mrt background, Col expression is absent in several rows of cells on either side of the DV axis, suggesting that Hh target gene activation is sensitive to a repressive activity all along the DV compartment boundary (Fig. 2B). To verify this result, we generated clones that express hh ectopically at random locations throughout the wing imaginal disc (Figs. 2C and 2D). In such experiments, we detected nonautonomous activation of Col in response to Hh exclusively inside the wing pouch (Figs. 2C and 2C⬘). Expression of Col was absent from the central row of cells marking the DV boundary even when Hh is expressed in these cells (arrowheads in Figs. 2D and 2D⬘). These experiments confirm that the region corresponding to the prospective wing margin cannot activate Hh target gene expression. In addition, this repression is expanded to several rows of cells on either side of the DV boundary in a fu background, even when the cells are exposed to a high dose of Hh ligand as in a hh Mrt background.

Differential Response to Pka-C1 and Ptc Inactivation Close to the DV Boundary To determine whether the refractory region is uncovered specifically in the absence of Fused activity, we analysed Hh target gene activation in clones of cells mutant for other components of the Hh pathway. Previous results suggest that Pka-C1 directly regulates the proteolysis and activity of Ci (Chen et al., 1998; Wang et al., 1999). In the absence of Pka-C1 function, Ci is not targeted for proteolytic processing, resulting in the activation of Hh targets in a cell-autonomous manner. However, it has been shown that loss of Pka-C1 is not sufficient for maximal activation of Ci (Wang and Holmgren, 1999). We generated clones of cells lacking Pka-C1 in third instar imaginal discs at different positions along the AP and DV axes. In clones in the anterior compartment, we observed an up-regulation of Col, Ptc, and En but only at some distance away from the DV boundary (Figs. 3A–3C). These results were obtained with two different amorphic pka-C1 alleles, excluding the possibility of an allele-specific effect (Fig. 4B). Using dpp-lacZ staining, we did not observe any modulation of its expression along the DV axis, most likely because of the perdurance of the ␤-galactosidase (data not shown). As in the fu mutant wing discs, we were unable to observe in the pka-C1 clones any modulation of Ci levels or localisation along the DV axis (data not shown). Full activation of the Hh pathway can be achieved cell autonomously by removing the activity of the Hh receptor Ptc. In contrast to Pka-C1 clones, clones of cells lacking Ptc

FIG. 4. Wg antagonizes Hh target gene activation along the DV boundary. wg CX4 ,pka-C1 B3 clones were generated by mitotic recombination and marked by the absence of GFP expression. (A, B) Triple staining showing the wg CX4 ,pka-C1 B3 clones (absence of green staining), Col (red) and Wg (blue). (A) When Wg expressing cells are removed, we observed an up-regulation of Col expression throughout the anterior clones, including in the cells close to the DV boundary. However, we do not observe any activation of 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 not include them, we still observed close to the DV boundary a region refractory to Col activation (arrow), showing that this effect is not allele specific. (C) Double labelling showing the wg CX4 ,pka-C1 B3 clone (absence of green staining) and En (red). We observe 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 repression observed close to the DV boundary when Pka-C1 function is removed.

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activate Col in all the cells within the clones, including those close to the DV boundary (Fig. 3D). However, within clones spanning the DV boundary, we still observed the absence of Col expression in cells along the prospective wing margin (Fig. 3D, arrowhead). These results reveal that cells in the vicinity of the DV boundary, although responsive to maximal Hh activity, remain refractory to the submaximal Hh pathway activation elicited by loss of Pka-C1 activity. Furthermore, the repression of Col, Ptc, and En seems to occur in a gradient along the DV axis inside the clones (Figs. 3A–3C), suggesting the existence of a diffusible molecule emanating from the DV boundary that may mediate this repression.

Wg Antagonises Hh Target Gene Activation along the DV Boundary One obvious candidate for a diffusible molecule originating at the DV boundary that could affect expression of Hh targets is Wg. To investigate this possibility, we generated clones of cells double mutant for both pka-C1 and wg activities. When Wg-expressing cells are removed by the clones, irrespective of their position along the AP axis, we observed an up-regulation of Col and En expression in cells both at a distance from and close to the DV boundary (Figs. 4A and 4C). Again, in these clones, we were unable to observe any modulation of Ci levels or localisation along the DV axis (data not shown). Surprisingly, however, we never observed Col up-regulation in cells corresponding to the prospective wing margin (Fig. 4A). This indicates that Wg is responsible for the repression at a distance from the DV boundary but not at the wing margin itself. In contrast to Col, we observed En up-regulation in all the cells corresponding to the clone, confirming that En is not down-regulated at the DV boundary (Fig. 4C). To verify this result, we ectopically expressed Wg in wild-type and fu 1 backgrounds using the Gal4/UAS system (Brand and Perrimon, 1993). Ectopic expression of Wg driven in the wing pouch results in a decrease in Col expression. This effect is most evident around the DV boundary close to the endogenous Wg expression domain and in the anterior-most row of Col-expressing cells, where Hh signalling input is lowest due to the distance of the source of Hh (arrowhead and arrow in Fig. 5A⬘; compare with Fig. 5B). Ectopic expression of Wg in a fu 1 background results in the complete absence of Col expression in the wing-pouch (Figs. 5C and 5C⬘), confirming the ability of Wg to down-regulate Col in the absence of Fused activity. To investigate whether the canonical Wg signalling pathway is involved in the repression of Hh targets along the DV boundary, we ectopically expressed several components of the Wg pathway in wild-type and fu 1 backgrounds. Ectopic expression of Dishevelled (Dsh), which participates in both the planar cell polarity and canonical Wg signalling pathways (Axelrod et al., 1998), causes an almost complete suppression 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 of antagonising the expression of col, ptc, and en over a short distance on either side of the DV boundary. However, Wg is not responsible for the cell-autonomous down-regulation of col and ptc within the prospective wing margin.

Notch Signalling Is Required for Repression of Hh Targets at the Presumptive Wing Margin We have demonstrated that activation of Col and Ptc expression does not occur in a wild-type wing along the DV boundary and that this repression is not dependent on Wg (Fig. 4). A candidate molecule that could mediate cellautonomous repression along the wing margin is Notch. To investigate the capacity of N to repress Hh target gene expression, we raised animals homozygous for a conditional thermosensitive allele of N (N ts2) at the permissive temperature and then shifted the mutant larvae to the nonpermissive temperature (29°C) at different stages during larval development. Shifts at the beginning of the L2 stage, before the creation of the DV compartment boundary, resulted in abnormal wing discs that lacked detectable Col expression in the anterior compartment, indicating the need to specify wing pouch identity in order to achieve activation of these targets. When N ts2 larvae were shifted to the nonpermissive temperature at the beginning of the L3 stage, we obtained small wing pouch structures. Col and Ptc expression in these discs was not repressed in cells corresponding to the prospective wing margin (Figs. 6A and 6B). Moreover, surviving adults exhibit notches at the distal tip of the wings (Fig. 6J), reminiscent of the most sensitive phenotype observed in the wings of adult flies heterozygous for N 55e11, a null allele of N (Fig. 6I). When a dominant active form of N (N intra) was ectopically expressed within the wing pouch, we observed a cell-autonomous down-regulation of both Col and Ptc (Figs. 6C and 6D). By contrast, expression of En was not affected by N intra expression in either the posterior compartment or the anterior compartment in response to Hh (Fig. 6E), consistent with the fact that en is normally activated in the prospective wing margin. To determine whether there are competing influences of N and Hh signalling in the prospective wing margin, we overexpressed a stabilised form of Ci (Ci U) (Me´ thot and Basler, 1999) in Hh responding cells using the ptc-Gal4 driver. This resulted in activation of Col in the cells corresponding to the prospective wing margin (compare Figs. 6F and 6G). Moreover, the differentiated wings of such flies exhibit variable defects at their distal tips, from notches (Fig. 6K and arrowhead in Fig. 6L) to absence of bristles with remaining vein tissue (Figs. 6L and 6M). This latter phenotype is reminiscent of the effects of weak hypomorphic wingless alleles that affect bristle formation without notching (Couso et al., 1994). These results indicate that, in the prospective wing margin, N, Wg, and Hh signalling exist in a delicate balance. In the wild type wing

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

DISCUSSION Short-range Hh signalling, partly through activation of Col function, is essential for correct AP patterning and differentiation of L3–L4 intervein tissue (Vervoort et al., 1999). N and Wg first define the DV boundary and later subdivide the region near this boundary into a number of distinct subregions that will eventually differentiate into wing margin bristles and vein tissue (Couso et al., 1994). These signals overlap spatially and temporally and lead to opposite fates. We propose that in and close to the DV boundary, N, Wg, and Hh signalling exist in a delicate balance to allow vein tissue, bristle, and sensory organ differentiation along the adult wing margin. The Hh target genes col/kn and ptc, in contrast to en, are repressed in a wild type wing in cells corresponding to the presumptive wing margin. We have demonstrated, using both gain- and loss-of-function experiments, that this repression is mediated by N signalling and that its inhibition results in aberrant morphogenesis of the wing. Hh signalling, achieved either by overexpression of Hh or loss of Ptc activity, is not sufficient to give maximum activation of Hh targets in cells of the prospective wing margin, suggesting that a finely tuned balance of activation and repression is required to achieve the appropriate biological output. However, overexpression of a stabilised form of Ci under the ptc-Gal4 driver resulted in the activation of Col in the prospective wing margin and defects in wing margin differentiation, indicating that N repression can be overcome by hyperactivity of the Hh signalling pathway. N signalling may lead to the repression of col, ptc, and dpp directly or it may act indirectly by affecting the ability of Ci to act as a transcriptional activator. Since expression of en, which requires the highest level of Hh signalling and Ci activity, appears immune to N repression, we favour the former possibility. Analysis of Hh target gene expression close to the DV boundary in fu mutants revealed that activation of col and dpp and up-regulation of ptc transcription is lost from the centre of the wing pouch when Hh signalling is attenuated. Inactivation of fu results in a complete loss of en expression from the anterior compartment; however, we found that the ectopic activation of en induced by submaximal Hh pathway activation (achieved by removing Pka-C1 activity) is similarly sensitive to inhibitory influences emanating from the DV boundary. In each case, the repression of Hh targets extends up to six rows of cells away from the DV boundary in all the anterior compartment. We have demonstrated that this down-regulation is due to activation of the canonical Wg signalling pathway. At what level is this global inhibition of Hh targets by Wg signalling achieved? One

possibility is that Hh target genes are regulated by the opposing effects of activator and repressor complexes acting directly via cis-acting sequences. Such a situation has previously been described for the stripe gene, which is directly activated by Hh signalling and repressed by Wg signalling in the Drosophila embryo (Piepenburg et al., 2000). This mechanism would imply the existence of cisacting sites for both the Ci and dTCF/pangolin proteins within the regulatory regions of each of the Hh target genes—col, ptc, dpp, and en—that we have analysed, a possibility that cannot currently be verified due to the absence of sequence information for all of these regions. An alternative scenario, however, is that Wg signalling modulates the activity of the Hh signalling pathway itself, perhaps at the level of the Su(fu)/Fu/Ci protein complex to affect localisation and/or activity of Ci. Consistent with this possibility, recent studies have provided evidence for a direct protein–protein interaction between the vertebrate Su(fu) and ␤-Catenin/Armadillo proteins (Meng et al., 2001). Such an interaction might mediate the attenuation of Hh signalling activity, for instance, by promoting the cytoplasmic retention of Ci. It should be noted, however, that we have not detected any modulation of Ci distribution along the DV axis using the available anti-Ci antibodies in the fu mutant backgrounds, the pka-C1, or the pka-C1;wg clones.

Signal Integration and Appendage Outgrowth in Other Systems The paradigm of modulation of Hh target gene expression by N and/or Wg may be applicable to signal integration in other systems. Hh signalling plays a remarkably similar role in patterning the Drosophila wing and vertebrate limbs. Specifically, Hh is involved in both short- and long-range signalling, elaborating upon preexisting informational cues to regulate anterior–posterior patterning, distal outgrowth, and proliferation. One of the vertebrate homologues of hh, sonic hedgehog (shh), is expressed in the zone of polarizing activity (ZPA), which is found at the posterior edge of the limb bud (Riddle et al., 1993). Here, Shh is thought to control the expression of molecules, such as fibroblast growth factor-4 (fgf-4), which is required for limb outgrowth (Capdevila and Johnson, 2000). Interestingly, Notch-1 expression and activity in the vertebrate limb localises to the apical ectodermal ridge (AER), a thickening of the ectoderm along the distal tip of the limb bud, and molecular and misexpression studies have suggested that N signalling may regulate the number of AER progenitor cells. Homologues of one of the primary downstream effectors of N signalling in the wing, cut, have also been characterised in 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 vertebrate Wnts, homologues of Drosophila Wg, have also been implicated in the formation of the AER. It is possible that a similar signalling hierarchy exists in the vertebrate limb to

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FIG. 5. Activation of the canonical Wg signalling pathway affects expression of Hh target genes along the DV boundary. Using the wing pouch specific 71B-Gal4 driver, we ectopically expressed several components of the Wg pathway in wild-type (A, A⬘) and fu 1 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 most anterior row of cells (arrowhead) and around the DV boundary (arrow), compared with wild-type Col staining (B). In a fu 1 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 strong down-regulation of Col expression in a fu background. The effect of Dsh expression is weaker than that of Wg expression, most likely due to the patchy expression of the 71B-Gal4 driver as revealed in (A) and (C). (E) Similar results were obtained by ectopic expression of a dominant-active form of Arm (Arm S10). These results suggest that Wg, signalling via the canonical Wg signalling pathway, is involved in the modulation of Hh target genes along the DV boundary.

allow for proximal– distal patterning and limb outgrowth. According to our model, modulation of the expression of Shh targets near the intersection of the ZPA and AER in the developing vertebrate limb may be required for the differentiation of distal features. During mammalian tooth development, Shh and Wnt-7b are expressed in reciprocal patterns and a balance between the two activities is required for the formation of boundaries between oral and dental ectoderm. Overexpression of Wnt-7B in domains of endogenous Shh expression has been shown to repress ptch1 transcription and prevent tooth bud formation, while overexpression of Shh suppresses this effect (Sarkar et al., 2000). The positioning and outgrowth of vertebrate epithelial appendages, such as feathers, whiskers, and hair, is another example where proper integration of positional information is essential for proximal– distal patterning. Overexpression of Wnt-7a in an in vitro feather reconstitution model leads to the inhibition of elongation along the PD axis and loss of

AP asymmetry in the presumptive feather bud. These changes were correlated with diffuse Notch-1 and Shh expression, which are normally localised to the middle and posterior of the bud, respectively (Widelitz et al., 1999). The loss of localized Shh expression and loss of AP asymmetry as a consequence of ectopic expression of Wnt are reminiscent of the effect on Shh and hair follicle morphogenesis in mice 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 by a multistep process. First, Ci proteolysis must be blocked. The cleavage of Ci into its truncated repressor form is promoted by Pka-C1-mediated phosphorylation. Removal of Pka-C1 leads to stabilisation of Ci; however, ectopic target gene expression accomplished in such a manner does not reflect maximal Hh signalling. This is indicated by the

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FIG. 6. Notch signalling is involved in repression of Hh targets at the presumptive wing margin. (A, B) Shifting of N ts2 larvae to nonpermissive temperature (29°C) at the beginning of L3 stage results in the activation of Col (A) and Ptc (B) expression in the cells corresponding 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 (N intra), labelled by the presence of the GFP expression (green) results in the cell-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 staining at the DV boundary. (E, E⬘) The asterisks indicate a N intra clone at the margin in the posterior compartment, and the arrows indicate a N intra 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 at the DV boundary. (F) Wild-type wing disc. (G) The expression of a stabilised form of Ci (Ci U) under the control of the ptc-Gal4 driver in wild-type leads to an overactivation of Hh signalling at the AP-DV intersection. Col is now detectable in the cells corresponding to the prospective wing margin, as shown by the colocalisation of Col and Cut (yellow staining, white arrow). (H) Distal tip of a wild-type wing showing the double row margin with vein tissue and innervated sensory bristles present between longitudinal veins L3 and L4. (I) Wing of a N 55e11/⫹ fly, showing that one of the most sensitive N phenotypes is an absence of vein tissue and sensory bristles tissue in the most distal part of the wing. (J) Wing from a N ts2 fly. Larvae were shifted to the nonpermissive temperature (29°C) at the beginning of L3 stage. These flies present preferential notches at the tip of the wings. (K–M) Wing phenotypes of flies expressing a stabilised form of Ci (Ci U) under the control 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 variable ranging from a complete notch (K), to an absence of sensory bristles between veins L3 and L4 (L), a partial absence of vein structure (L, black arrowhead), or to an absence of sensory bristles only in the posterior part of the L3–L4 margin (M). These results suggest a delicate balance between the N, Wg, and Hh pathways to allow vein tissue and sensory organ differentiation along the adult wing margin.

absence of Col and Ptc expression close to the DV boundary in clones where Pka-C1 activity has been removed. In addition to preventing Ci cleavage, there is another Hhdependent activation step that requires Fused kinase activity (Me´ thot 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 play a role in blocking Ci nuclear accumulation, it is hypothesised that Fu kinase activity is required in cells to facilitate nuclear localisation of activated Ci (Me´ thot and Basler, 2000; Price and Kalderon, 1999; Wang and Holmgren, 1999). As fu has been implicated in localisation of Ci, the downregulation of Hh targets away from the margin described here may reflect the inability of activated Ci to enter the nucleus efficiently. A decrease in Ci activity is reflected in

the decreased transcription of col, ptc, and dpp and an absence of en activation in the anterior compartment in a fu mutant background. Recent results indicated that Hh signalling in the embryo, leading to ptc transcription and dorsal epidermal morphogenesis, occurs independently of Fu activity (The´rond et al., 1999). However, Fu activity is indispensable in the embryo in Hh target cells expressing Wg, leading to the hypothesis that activation of Hh targets is repressed by an unknown localised factor in wg-expressing cells (The´rond et al., 1999). We speculate that Fu activity could be required in Wg-expressing cells in the embryo to overcome a repressive effect of Wg on Hh target gene activation. Further studies will be needed to determine whether this model can be extended to all the developmental processes involving Hh signalling.

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ACKNOWLEDGMENTS We thank Drs. D. Strutt and J. Modolell and the Bloomington Stock Centre for providing fly strains; Dr. A. Vincent for the anti-Col antibody. FlyBase database for genes and alleles description. We are grateful to Drs. P. Blader, M. Crozatier, and A. Vincent for critical reading of the manuscript. We thank B. Savelli for his help in the preparation of the figures. This work was funded by a Wellcome Programme grant (to P.W.I.). B.G. was supported by an EMBO fellowship (ALTF 270-1997) and by a fellowship from the “Fondation pour la Recherche Me´ dicale.” D.L.J. was supported by a Human Frontiers Research Program postdoctoral fellowship and is now a Lilly Fellow of the Life Sciences Research Foundation.

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Received for publication April 22, 2002 Accepted May 7, 2002 Published online June 25, 2002