Drosophila. The wingless signalling pathway and the patterning of the wing margin in. Juan Pablo Couso*, Sarah A. Bishop and Alfonso Martinez Arias

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1 Development 120, (1994) Printed in Great Britain The Company of Biologists Limited The wingless signalling pathway and the patterning of the wing margin in Drosophila Juan Pablo Couso*, Sarah A. Bishop and Alfonso Martinez Arias Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK *Author for correspondence SUMMARY The margin of the wing of Drosophila is defined and patterned from a stripe of cells expressing the wingless (wg) gene that is established during the third larval instar in the developing wing blade. The expression of the genes cut and achaete in a small domain in the prospective wing margin region reflects the activity of wg and probably mediate its function. Our results indicate that, in the wing margin, the wingless signal requires the activity of at least three genes: armadillo (arm), dishevelled (dsh) and shaggy (sgg) and that the functional relationship between these genes and wg is the same as that which exist during the patterning of the larval epidermis. These observations indicate that arm, dsh and sgg encode elements of a unique wingless signalling pathway that is used several times throughout development. Key words: wingless, wingless signalling pathway, wing margin, cell signalling INTRODUCTION The wingless (wg) gene of Drosophila is a member of the Wnt gene family and encodes a secreted protein homologous to the product of the Wnt-1 vertebrate proto-oncogene (Rijsewick et al., 1987). Studies of the absence of the gene (reviewed by Ingham, 1991; Hooper and Scott, 1992; Peifer and Bejsovec, 1992; Nusse and Varmus, 1992, and Martinez Arias, 1993) or of its misexpression in flies (Nordemeer et al., 1992; Struhl and Basler, 1993), mice (Tsukamoto et al., 1988) and frogs (reviewed by Moon, 1993), suggest that wg encodes a signal that is involved in cell interactions. The product of wg plays a prominent role in many of the events that pattern the larval epidermis of Drosophila, for example in the maintenance of the expresssion of the engrailed (en) gene (reviewed by Ingham and Martinez Arias, 1992; Hooper and Scott, 1992; Peifer and Bejsovec, 1992). In addition, wg is also required during the development and patterning of the nervous system (Patel et al., 1989 and unpublished obs.), Malpighian tubules (Skaer and Martinez Arias, 1992) and imaginal epidermis (Baker, 1988a; Couso et al., 1993). Experiments with a temperature sensitive allele of wg show that many of these requirements reflect spatially and temporally discrete functions for wg (Bejsovec and Martinez Arias, 1991). Thus, during the development of the imaginal discs, wg is initially required for the establishment of their primordia from the larval epidermis (Cohen, 1990) and later for their patterning during larval life (Couso et al., 1993). The multiple functions of wg, revealed by the use of temperature sensitive mutants, raise the question of whether they reflect multiple responses of the cells to a single signalling event, dependent on a single signalling pathway, or whether they are related to different signalling events mediated by different signalling systems. Studies of mutants that might be involved in the same signalling event as wg during Drosophila embryogenesis have identified a small group of genes whose activity appears to be necessary for wingless signalling (reviewed by Peifer and Bejsovec, 1992; Klingensmith and Perrimon, 1991; Martinez Arias, 1993). Mutations in two of them, armadillo (arm) and dishevelled (dsh), produce a wg mutant phenotype in a cell autonomous manner in the embryo, suggesting that their wildtype products are indeed required for proper wg function (Riggleman et al., 1990; Klingensmith and Perrimon, 1991). Mutations in another gene, shaggy/zeste white 3 (sgg), lead to a larval cuticular phenotype (Perrimon and Smouse, 1989) similar to that produced by overexpression of wg (Noordemeer et al., 1992). Molecular studies indicate that arm encodes a member of the plakoglobin gene family (Peifer and Wieschaus, 1990) and sgg a non-receptor serine threonine kinase with homology to the vertebrate GSK3 (Bourouis et al., 1990; Siegfried et al., 1990, 1992). Studies of the interrelationships between wg and sgg and wg and arm during embryogenesis have led to the suggestion that the wingless signal antagonizes the activity of the sgg gene in the maintenance of the expression of en (Siegfried et al., 1992) and that arm is required for this event (Riggleman et al., 1990; Peifer et al., 1991). Although no molecular information is yet available about dsh, genetic studies indicate that it is also involved in the implementation of the wingless signal (Klingensmith and Perrimon, 1991). Despite such specific effects, mutations in either sgg, arm or dsh are highly pleiotropic i.e. absence of any one gene results in a variety of apparently unrelated phenotypes in embryos and adults (Peifer et al., 1991; Perrimon and

2 622 J. P. Couso, S. Bishop and A. Martinez Arias Mahowald, 1987; Simpson et al., 1988; Perrimon and Smouse, 1989). Therefore, it is not clear at present if the proteins encoded in these genes are common elements of a wingless signalling pathway, or they are only functionally related to wg in the maintenance of en. Here we describe a function of wg in the patterning of the wing margin of Drosophila during the third larval instar. We show that the Wingless protein (Wg) acts as a signal for the development of the pattern elements along the wing margin and that these functions are dependent on the same elements and functional relationships that control en expression during early embryogenesis. Our results indicate that in Drosophila, the wingless signal uses a conserved group of elements that act on different transcription factors at different times during development. MATERIALS AND METHODS Genetic variations and experimental crosses The mutations and allelic combinations used in these studies are as follows. wg IL At 17 C, embryos homozygous for the temperature sensitive wg IL allele display a wild-type cuticular pattern and hatch normally. However, the original stock wg IL cn bw sp/cyo (Nüsslein-Volhard et al., 1984) never yields homozygous wg IL pupae or adults. Various allelic combinations (unpublished observations) suggested that this pupal lethality was not associated with the wg allele but with a secondsite mutation in the original wg IL cn bw sp chromosome. For this reason, we generated a new recombinant chromosome wg IL ck pr cn that retains the wg IL allele but has lost any pupal lethals existing in the original chromosome. Although the newly obtained recombinant chromosome is homozygous lethal because of the presence of ck, adult flies that are homozygous for the wg IL allele can be obtained as heterozygous wg IL cn bw sp/wg IL ck pr cn individuals. When reared at 25 C these individuals die as embryos with a characteristic wg null phenotype but, at 17 C, wg IL homozygous adults emerge, survive and display a visible mutant phenotype (see results and Fig. 5B). Both in the temperature shift experiments and in the antibody stainings, mutant larvae or adults were identified as the Tb + Hu + progeny of a cross between flies from wg IL stocks balanced with the SM6aTM6b translocated balancer (in which the markers Tb and Hu are linked to the second chromosome). In the temperature shift experiments, the appropiate crosses were set up at 17 C and layings were collected at 5 hours intervals. The vials were transferred to 25 C at the appropriate times, either until the end of development (shifts) or for a period of time and then transferred back to 17 C (pulses). The age of the larvae at the time of shift was estimated after studying the development of the homozygous wg IL control animals continuously maintained at 17 C. Pharate adults were dissected, their wings inflated by heating in 10% NaOH, and mounted in Hoyer s for microscope examination. spd flg This mutation is homozygous viable and fertile and behaves as a weak specific regulatory allele of wg in the third instar. Homozygous spd flg flies display small wings with a severely reduced alula and defects at the wing margin (Tiong and Nash, 1990; see also results and Fig. 4). When spd flg is placed in trans with lethal alleles of wg, some defects in the wing margin can be observed and the alula is still very reduced (unpublished observations). For antibody stainings, mutant larvae were taken from a homozygous spd flg stock. A temperature sensitive condition for arm In the embryo, the phenotype of the temperature sensitive allele arm H.6 ranges from strong at 25 C to wild type at 17 C (Klingensmith et al., 1989), but the larvae raised at 17 C have very small discs and die as undifferentiated pupae. To study the adult phenotypes of loss of function of arm, we have used the synthetic temperature sensitive allelic combination arm H.6 ; arm BCD7 /+, which produces wild-type flies at 17 C and mutant phenotypes at 25 C (see results and Fig. 3). arm BCD7 is a duplication for arm that can provide partial arm function and was generated as a transformant with a transposon containing an arm minigene (Peifer et al., 1991). The temperature shifts were done as described above for wg IL. dsh v26 Mutant dsh v26 animals die as undifferentiated pupae (Perrimon and Mahowald, 1987). However, we recovered imaginal discs for antibody stainings from the mutant larvae. These were recognized by virtue of the y and w markers in a y w dsh v26 /Binsn stock. cut The ct-wing mutants are a class of viable regulatory mutants with a mutation at the ct locus that affects a specific enhancer element that drives ct expression during the third larval instar, in the stripe along the prospective wing margin (Jack et al., 1991), thus reducing or eliminating only that expression of ct, which coincides with the expression of wg (unpublished observations). In our experiments we have used the mutants FM6, ct/ct 145 and ct 6. For antibody staining of imaginal discs, mutant larvae were taken from the homozygous stock ct 6 or from a stock y w ct 145 /FM6, y w ct/y ct + y +, where mutant heterozygous females were recognized by their gonads and y mutant phenotype. Such females have no expression of Ct in the stripe along the wing margin. Control experiments with FM6 alone indicate that the results presented in this work are not due to any mutation on the balancer chromosome. Clonal analysis Flies of the appropiate genotypes were crossed and their eggs collected every 12 hours in food plates or split bottles. The vials containing larvae were irradiated with 1,000 rads using an Al filter hours after they were laid. In all cases mutant and control clones were generated, and their sizes (number of cells) were studied and compared. Because the layings were 12 hours long and the experiments were carried out in batches, the average size of the control clones was used to match comparable experiments and eliminate spurious age dispersion. To study the phenotype of arm mutant clones we have used two different mutant conditions. In the first instance we used the genotype y arm XK f 36a /M(1)O Sp; mwh jv/+. After X-rays induced mitotic recombination, mutant arm M + clones were generated and marked y f 36a, whereas mwh jv clones served as controls. We have also generated clones of the temperature sensitive allele arm H.6 (see above). To do this, animals of the genotype y arm H.6 f 36a /+; mwh jv/+ were cultured at 25 C until irradiation, and then transferred to 17 0 C. In the case of dsh, the genotypes used were y dsh v26 sn 3 /+; mwh jv/+, which produce dsh mutant clones marked y sn and control mwh jv clones. Animals of the genotype y dsh v26 sn 3 /M(1)O Sp ; mwh jv/+, were used to generate dsh M + clones. To study sgg mutant clones, twin clones were generated in animals of the genotype sgg M11-1 /y f 36a. The same recombination event produces a control y f 36a clone and an accompaning sgg mutant clone, identifiable by its phenotype (Ripoll et al., 1988; see results and Fig. 5 ). To generate clones doubly mutant for sgg and dsh, a recombinant chromosome sgg M11-1 dsh v26 was generated. The experimental genotype was sgg M11 dsh v26 /y f 36a. In this situation, because the f locus is proximal to dsh, control f clones are necessarily accompanied by a mutant sgg dsh twin clone.

3 wingless signalling in the Drosophila wing margin 623 Immunocytochemistry Imaginal discs were fixed for minutes in cold 4% paraformaldehyde in PBS and stained, using standard procedures, with DAB. Polyclonal anti-wg antibody was provided by M. van den Heuvel and used diluted 1:250. Monoclonal anti-ac and anti- Ct antibodies were provided by S. Carroll and Y. N. Jan respectively and used diluted 1:200 and 1:500 respectively. In some experiments, gene expression was monitored with lacz reporter gene fusions, and in those cases a standard enzymatic assay coloured the β-galexpressing cells blue. For ct, the transformant ct whz, which carries the specific ct enhancer that drives expression in the edge cells only (Jack et al., 1991) was used. The transformant line neu-a101 was used to detect expression of β-galactosidase in the bristle precursors. Fig. 1. Fate map, development and differentiation of the wing margin of Drosophila. (A) The margin of the wing of Drosophila contains three domains, each with a stereotyped array of pattern elements. In both the anterior triple row, and the distal double row the sensory elements - stout bristles (open circles), slender bristles (triangles) and chemosensory bristles (solid circles) - are inervated, whereas in the posterior row the bristles (hatched circles) are not innervated. Epidermal hairs are indicated as dots. (B) Correlation of gene expression and cellular differentiation during the development of the margin. The main surface of the drawing represents a wing margin that has been opened out to allow the dorsal and the ventral surfaces to lie on the same plane. This representation permits a topological correlation between the final pattern elements on the wing and the patterns of gene expression in the third instar wing disc. A section of the latter is represented by a row of cells with the pseudostratified appearance characteristic of third larval instar disc epithelia which, after wing evagination in the pupa, adopt a squamous morphology. In the anterior margin of the adult wing (main surface), it is possible to discern several rows of pattern elements from ventral (left) to dorsal (right). Ventrally there are two rows of epidermal hairs which form the ventral side of the marginal vein (not indicated) and a row of bristles containing a sequence of one recurved chemosensory bristle every four slender mechanosensory bristles. Medially there is a row of hairs that defines the edge of the margin and a row of thickly packed stout mechanosensory bristles. Finally, on the dorsal side of the marginal vein there is another row of hairs followed by a row of chemosensory bristles each interspersed by four hairs. This pattern of bristles represents what is commonly known as the triple row and is modified as it progresses into the posterior region of the wing, first in the double and then in the posterior row (see A above and Fig. 2). This pattern of cellular differentiation is derived from the presumptive margin of the third instar imaginal discs, where it is prefigured by the patterns of expression of wingless (wg), cut (ct) and achaete (ac) (shown in fig. 3). As indicated in the figure, the expression of Wingless protein (Wg, dark stippling) and Cut protein (Ct, black nuclei) outline the edge proper, and the marginal elements are defined by the expression of Achaete protein (Ac, hatched nuclei) under the influence of Wg (apical stippling). The different location of the stippling reflects differences in the distribution of Wg, characteristic of wild-type discs. The differences between dorsal and ventral, as well as those between the different regions of the margin along the anteroposterior axis, are probably due to the activity of other genes (see text). (C) Chronology of events during the development of the margin (O Brochta and Bryant 1985; Hartenstein and Posakony 1989; Cubas et al., 1991; Blochlinger et al., 1993; Jack et al., 1991; Blair 1992). All times refer to hours after egg laying (AEL). The margin is indicated by the double line. The top line indicates the cells of the edge i.e. those that express ct and will give rise to the stripe of hairs in the middle of the margin, while the bottom line indicates the cells of the margin. The shading indicates the periods of mitotic activity in the different regions of the margin. L3e and L3l indicate third instar larval stages, early (e) and late (l). PF indicates the time of puparium formation and the symbols at the bottom, the time at which the precursors of the different sensory organs are visible. For symbols, see above, and for further details, see text.

4 624 J. P. Couso, S. Bishop and A. Martinez Arias Fig. 2. Detail of the triple row showing the edge cells. Two planes of focus through the wing margin, as seen from the ventral side of the wing, showing the row of edge cells (arrows) between the ventral row of slender (large arrow) and the dorsal row of stout (arrowheads) mechanosensory receptors. The blue color reflects β-galactosidase activity which has been expressed in the stripe of ct-expressing cells along the wing margin in the third larval instar and early pupa (Jack et al., 1991, see methods and fig. 3c) and therefore shows that the edge cells of the adult wing arise from the stripe of ct-expressing cells shown in Fig. 3A,C. RESULTS The development of the wing margin in Drosophila The wings of Drosophila develop during larval life within the dorsal imaginal discs of the second thoracic segment, the wing discs. While the establishment of the basic system of polar coordinates of positional information in these discs takes place during the first and second larval instars (Couso et al., 1993), the fine patterning of the tissue, as reflected in the determination of the sense organs and hairs that decorate the margin of the wing, is achieved during the third larval instar and the early stages of pupal development (Figs 1, 2; García-Bellido and Santamaría, 1978; Hartenstein and Posakony, 1989, 1990; Cubas et al., 1991; Rodriguez et al., 1990). Studies of wg expression in wing discs during the third larval instar have revealed a complex pattern of wg RNA (Baker, 1988b) and protein expression (Couso et al., 1993; Williams et al., 1993). A striking feature of this pattern is a stripe of wg expression along the presumptive margin Fig. 2 Fig. 3. Patterns of gene expression in the late third instar larval disc (approx. 120 hours AEL) along the presumptive wing margin. All images show details of the presumptive anterior wing margin; the orientation of the discs is always such that posterior is to the bottom and dorsal to the left. At this stage the imaginal cells form a pseudostratified epithelium and at different planes the width of the stripes of stained cells can vary, although the relationships between the patterns of gene expression shown here are constant (see fig. 1B). (A) Apical plane of focus through a disc stained with anti-ct antibody. The Ct protein is expressed in a stripe of about three cells wide along the wing margin, which will give rise to the edge cells. (B) Apical plane of focus through a disc stained with anti-ac antibody. Ac is expressed in two parallel bands on the presumptive anterior wing margin. Within the bands, the precursors of the chemoreceptors can be discerned by the bigger size of their nuclei, which accumulate high levels of Ac (arrows). Note that these precursors are surrounded by cells with lower levels of protein and that they arise close to the edge cells (see below). (C) Prospective wing margin stained to reveal simultaneously ac and ct expresssion. The blue colour shows β-galactosidase activity under the control of a ct enhancer element specific to the edge of the wing margin (Jack et al., 1991, see Methods and Fig. 2), while the red one shows Ac expression. The ct gene is expressed between the two stripes of Ac. Although different planes of focus show, for the most part, nuclei expressing one or the other gene, a degree of coexpression exists in certain domains (arrows). (D) Detail of a wg IL /+ disc grown at 25 C stained with anti-wg antibody. The staining highlights two kinds of cells, those darkly stained at the center of the stripe, which express the wg gene and therefore have accumulated the protein from the wg IL allele (see González et al., 1991), and those on either side, lightly stained, that reveal the movement of Wg. It can be seen that the stripe of wg-expressing cells is about three cells wide (unpublished observation; see also E and F). (E) Apical plane of focus through a wild-type disc stained with anti-wg antibody. High levels of stain can be observed in a stripe of three cells which are expressing the wg gene (unpublished observation), but lower levels can be detected up to two or three cells away from this stripe. Comparison of this domain with A, B and C indicates that Wg can be found over the whole field of ct- and Ac-expressing cells. (F) Medial plane of focus through a disc stained to show β- galactosidase expression in the chemoprecursors, through a reporter gene insertion in the neuralized gene (blue color; see Methods) and Wg (brown; as above). Notice that the cells with high levels of Wg are located between the rows of chemoprecursors. Therefore, the wg-expressing cells coincide with the ct-expressing cells (see B and C).

5 wingless signalling in the Drosophila wing margin 625 of the wing established around the mid-third instar larva (Fig. 4A) and which in the late third instar (ca. 120 hours after egg laying; AEL) is about three cells wide (Figs 3D, 4B). These cells, which continue to express wg after puparation (Fig. 4C), have high levels of Wingless protein (Wg) and coincide with cells that express the cut (ct) gene (Jack et al., 1991) all along the presumptive wing margin (Fig. 3 and unpublished observation). Lower levels of Wg can be detected up to three cells away on either side of the stripe of wg expression. It is likely that this stain reveals binding of Wg by cells that do not synthesize it (Fig. 3D,E). These parallel stripes overlap, in the anterior compartment, with two bands of cells that express the Fig. 3

6 626 J. P. Couso, S. Bishop and A. Martinez Arias Fig. 4. Expression of Wg in the imaginal discs during the third larval instar and pupal stages. All images at the same magnification, posterior to the bottom. (A) Mid-third instar larval wing and leg discs. The final pattern of Wg expression is established in the wing disc: a broad stripe in the notum (n), a thinner one in the prospective wing margin (arrow) and another one encircling the prospective wing blade (for a detailed fate map of the third instar wing disc showing the correspondence with adult structures see Campuzano and Modolell, 1992). (B) Late third instar wing disc (shortly before 120 hours AEL). The stripe of Wg expression in the margin (see Fig. 3) is the same as in A, but, at this stage there are two stripes over the prospective wing hinge regions (h), around the wing blade region (v, prospective ventral wing blade). (C) Dorsal view of a pupal wing 4-8 hours APF, after evagination of the wing regions shown in B to the right of the figure so that the ventral wing blade (labelled v in B) is now underneath this plane of focus. Wg is still present along the wing margin, in a stripe of about five cells wide. Note that while in the late third instar disc (B) the protein levels decrease from a central high point of expression (see also fig. 3), in the pupal wing the protein appears constrained within a bounded domain. This bounded distribution is reminiscent of that described for the parasegmental boundary during late stages of embryogenesis (González et al., 1991). (D) Late third instar disc from a spd flg mutant. Note the narrower width of the stripe of Wg in the presumptive posterior margin (bottom part of the stripe), where spd flg mutant flies display the stronger phenotype (see Fig. 5C). Achaete protein (Ac) (Fig. 3 and for a review of the achaete scute complex (AS-C) see Campuzano and Modolell, 1992). Towards the end of the third larval instar, the precursors of the sensory elements of the wing margin begin to be determined, when the chemosensory precursors can be discerned from amidst the Ac-expressing cells that are nearer to those that express wg (Fig. 3). Later, during the early pupal stages, mechanosensory precursors arise from the interface between ct- and Ac-expressing cells (Blair, 1993) and finally, the cells of the presumptive wing margin rearrange and differentiate to give rise to the array of pattern elements characteristic of the wing margin (Fig. 1; Hartenstein and Posakony, 1989). One element of this pattern not previously described is a row of epidermal cells located between the medial and the ventral bristles of the anterior triple row that can be traced back to the region that expresses ct and wg in the third instar disc (Fig. 2). We call these cells the edge cells and those on each side of them, the marginal cells. The spatial relationships between these pattern elements and the different patterns of gene expression superimposed on a fate map of the wing margin during the third larval instar are shown and discussed in Fig. 1B. Wingless is required for the patterning of the wing margin At 17 C, embryos homozygous for the temperature sensitive wg IL allele (for details see methods) display a wild-type cuticular pattern, hatch normally and develop into adults which survive but display some defects in the epidermis: the sternites are for the most part missing (not shown) and the margin of the wing lacks some bristles (Fig. 5B). Stronger variations of these phenotypes are observed in other combinations of wg IL with hypomorphic alleles of wg (Phillips and Whittle, 1993 and unpublished observations; see Methods). This requirement for wg in the patterning of the wing margin is emphasized by the observation that flies homozygous for the mutation spd flg, an allele of wg which specifically affects the wing margin (Tiong and Nash, 1990, Methods and Fig. 5C), show reduced levels of Wg in the corresponding presumptive region of the disc (Fig. 4D). Finally, very large clones of wg null alleles occasionally produce notches when they reach the wing margin (Baker, 1988b). Altogether, these results indicate that the pattern of wg expression during the third larval instar might be related to a function of wg in the patterning of the wing margin. To test

7 wingless signalling in the Drosophila wing margin 627 Fig. 5. Phenotypes of adult wings, oriented as Fig. 4C, distal to the right and posterior to the bottom. (A) Wild-type wing; (B) wing from a wg IL homozygous animal raised at 17 C. Bristles are lost along the entire wing margin. In the triple row, the positions of lost stout mechanoreceptors are occupied by recurved chemoreceptors. In slightly stronger mutant conditions, this results in the transformation of the the triple row into a narrow double row. (C) spd flg mutant wing. Some bristles are missing along the anterior margin and the posterior margin is completely lost. Defects in wing hinge regions that express Wg produce an abnormally shaped wing. (D) Wing from a wg IL animal shifted to 25 C shortly before 96 hours AEL. These wings have been dissected out from the pharate adults and inflated (see methods), hence the apparent defects asociated with veins. (E) Detail of the anterior wing margin of a wg IL animal shifted to 25 C at 0-4 hours APF. The wing was mounted to allow a frontal view of the streched margin. The mechanoreceptors (and posterior bristles, not shown), which should have been determined at about 12 hours APF (see Fig. 1C), have been lost and the remaining chemoreceptors are arranged in a narrow margin (compare with Figs 1B and 2). this further and to define the precise requirements for Wg in this process, we have eliminated its activity by shifting wg IL homozygous animals from 17 C to 25 C at defined times during the third larval instar (see Methods). Such shifts result in the complete inactivation of Wg (Baker, 1988a; González et al., 1991; Bejsovec and Martinez Arias, 1991) and produce different effects at the wing margin depending on their timing and length. Shifts started after mid-third instar (approx. 96 hours AEL) or at the time of pupation ( hours AEL, i.e. 0-4 hours after puparium formation, APF) produce pharate adults with a narrow margin phenotype: most bristles are missing, and the remaining ones are intermingled in two or even one row, instead of the three rows interspaced by tricomes, characteristic of the wild-type (compare Figs 5E with 1B and 2). A 24 hour-long pulse, from 96 to 120 hours AEL, produces weaker narrow margin phenotypes, very similar to those of the viable allelic combinations described above. However, shifts shortly before 96 hours AEL result in pharate adults in which the wing lacks all the features characteristic of a wild-type wing margin. In these wings, every bristle, whether non-innervated, chemo or mechanoreceptors, as well as the marginal vein, is missing; otherwise such wings bear a fair pattern of veins and sensilla (Fig. 5D). Unlike the phenotype of other mutants in Drosophila that affect the wing margin, this phenotype is not associated with notching, and the number of cells of the wing blade does not appear to be

8 628 J. P. Couso, S. Bishop and A. Martinez Arias significantly reduced. The absence of these two features, which are usually associated with extensive cell death (Fristrom, 1969; Spreij, 1971), suggests that the primary cause of the total absence of wing margin is the lack of marginal cell fates. The products of dsh and arm are required for wingless signalling during the development of the wing margin to antagonize the activity of sgg Genetic studies suggest that the products of arm, dsh and sgg are involved in wingless signalling during embryogenesis

9 wingless signalling in the Drosophila wing margin 629 Fig. 6. Phenotypes of clones of cells mutant for arm, dsh and sgg. (A) Detail of the alula and proximal posterior region of an arm mutant wing (see Methods). Notice the loss of bristles (arrows) reminiscent of the weak wg mutant phenotypes shown in Fig. 5B,C. (B,C) Small surviving arm M + clones in the anterior and posterior margin, respectively. The arm mutant cells are small and abnormal (arrowheads). Although the clones result in the loss of bristles (arrows), neighbouring wild-type cells located in the wing blade can occassionally form bristles (stars). (D,E) Clones of cells mutant for dsh in the anterior ventral and posterior dorsal wing margin, respectively. Every bristle is lost in the surface of the wing margin occupied by the mutant clone (to the right of the arrows), but wildtype neighbours can occasionaly differentiate bristles (stars). In addition, the dsh cells differentiate multiple tricomes with abnormal orientation and display abnormally highlighted contours. (F) Extra bristles in the anterior wing margin produced by a sgg mutant clone in the dorsal surface of the wing. (G) Ectopic bristles produced by a sgg mutant clone in the wing blade. (H) Clone doubly mutant for sgg and dsh (from the arrow to the right) in the dorsal anterior wing margin, showing the epistatic sgg mutant phenotype (compare with F and D). Notice the presence of y f bristles produced by the control twin clone (arrowhead, and to the left). (I) Clone doubly mutant for sgg and dsh stradling the double row. Cells in the clone differentiate extra bristles either on the margin or on the internal blade, as do sgg mutant clones and unlike the dsh mutant clones. Some cells of the f twin can be seen to the left. Also, some cells differentiate multiple tricomes. y f bristles were never seen to be recruited into the tufts of bristles produced by sgg dsh clones, thus showing that the nonautonomous effect of dsh mutant cells on their wild-type neighbours is also supressed. (reviewed by Nusse and Varmus, 1992; Martinez Arias, 1993). To test whether the function of wg in the development of the wing margin is mediated by the same signalling pathway that functions in the early patterning events in embryos, we have analyzed the function of arm, dsh and sgg during the third larval instar (Fig. 6; Table 1). armadillo Studies with arm mutants suggest that its gene product might be required for wingless signalling not only in the embryo but also during the establishment of polar coordinates in the imaginal discs: hypomorphic mutant conditions for arm can produce pharate adults with wg-like mutant phenotypes of abnormal polar patterning in legs and thorax (Peifer et al., 1991). Unfortunately, the nature of these alleles precludes a study of arm function during the third larval instar (see Methods). To study this function, we have used the arm H.6 allele as the basis for a synthetic temperature sensitive allelic combination that produces wild-type adults at 17 C (for details see Methods). When these flies are shifted from 17 C to 25 C during the third larval instar, they produce adults with missing pattern elements along the wing margin and the alula, phenotypes which are reminiscent of weak wg hypomorphic conditions (Fig. 6A). These arm mutant adults also resemble wg mutants in that their abdomens lack sternites (not shown). To study the effects of stronger loss of arm function, we have performed a clonal analysis of the arm gene (see Methods; Table 1). Mosaic flies carrying clones of cells homozygous for the arm XK allele, which behaves as a strong but not null allele in the embryo (Peifer and Wieschaus, 1990), were generated. Our findings agree with those reported by Peifer et al. (1991) in that clones of arm mutant cells are found Table 1. Clonal analysis of the genes involved in wingless signalling in the adult fly Clones Age Wing margin studied Genotype (h. AEL) phenotype* Penetrance n y arm XK f 36a / non-viable 1.0 (38) M(1)O Sp lack of WM y arm H.6 f 36a (17 0 ) y dsh v26 sn sgg M11-1 /y f 36a ectopic WM ** 53 sgg M11-1 dsh v26 / y f 36a Age range estimation is based on the hours from egg laying to irradiation and on the mean size of the control clones (see methods). *See text and Fig. 6 for a more detailed description of phenotypes. Fraction of clones that produce a wing margin phenotype. The data for arm and dsh is based on the clones that reached the margin. For the sgg and sgg dsh experiments, the penetrance is calculated as the percentage of f clones accompanied by a twin clone of sgg bristles. In these two cases, the maximum value is lower than one because a fraction of sgg clones in the wing blade is lost (see also Ripoll et al., 1988). In all cases, however, the penetrance decreases in the latest generated clones. This can be explained by the residual presence of wild-type protein in the cells until the requirement for the gene function ends. Number of control clones. No f (arm) clone was found at this age. The arm H.6 clones were produced at 17 C. Because their weak hypomorphy (see methods) they show higher viability and lower penetrance and expressivity than the arm XK M+ clones. These dsh clones have half the average size and frequency of appearance than controls. **These sgg clones produce additional phenotypes when not at the wing margin, like ectopic vein differentiation (see also Ripoll et al, 1988). The sgg dsh clones generated at this age that do not show a sgg phenotype begin to show dsh-like phenotypes of abnormal cell differentiation (see Fig. 6). with lower frequency and smaller sizes than expected, revealing a requirement for arm in cell viability and/or cell differentiation (Table 1). In line with their results, we also find a high frequency of phenocopies of wg phenotypes among the irradiated flies. However, in addition we find that when the clones are generated late in development, surviving arm Minute + clones display visible phenotypes: abnormal cell differentiation and alterations of the patterning around the wing margin (Table 1 and Fig. 6B,C) similar to those produced by dsh mutant clones (see below). Finally, to overcome the low viability of arm mutant cells, we studied clones of the mild mutant condition arm H.6 at 17 C (see methods). In accordance with their weaker hypomorphy, these clones appear with higher frequency and larger sizes than arm XK M + clones, and produce the same wing margin phenotypes, although with lower penetrance and expressivity (Table 1). dishevelled In the absence of conditional or strong viable mutant alleles of dsh that would allow us to study the imaginal requirements for dsh in the patterning of the wing margin (Perrimon and Mahowald, 1987), we have generated clones of cells homozygous for the strongest available lethal allele, dsh v26 (Table 1). The resulting mosaic flies display different phenotypes depending on the position of the clone within the wing blade (Fig. 6E). If the dsh mutant cells lie within the wing blade they

10 630 J. P. Couso, S. Bishop and A. Martinez Arias show several abnormalities in their differentiation related to a function of dsh in tissue polarity (Adler, 1992 and J. P. C. unpublished observations). However, when the clone reaches the margin of the wing, dsh mutant cells display an additional autonomous phenotype: they do not differentiate as pattern elements of the margin and instead behave as if they were in the middle of the wing blade i.e. they develop tricomes and non-pigmented cuticle. Interestingly, in wings bearing such marginal clones of dsh mutant cells, wild-type cells located a few cell diameters away from the wing margin but which lie adjacent to dsh mutant cells are induced to produce ectopic bristles characteristic of the margin (Fig. 6D,E). Such phenotypes are also produced by clones of other alleles of dsh (N. Perrimon, personal communication) and, depending on their size, by arm mutant clones (Fig. 6B,C). The loss of pattern elements of the wing margin in clones of cells mutant for arm and dsh are identical to those observed when wg function is inactivated from the middle of the third larval instar, suggesting that, just as in the patterning of the larval epidermis, dsh and arm are necessary for proper wg function. This suggestion is reinforced when the mutant phenotypes are studied at the level of gene expression (see below). Furthermore, the autonomy of the arm and dsh phenotypes of loss of marginal pattern elements indicate that their wild-type functions are required for the implementation of the wingless signal in the marginal cells. shaggy Clones of sgg mutant cells in the wing blade autonomously differentiate tufts of bristles characteristic of the wing margin (Ripoll et al., 1988; Simpson et al., 1988, Fig. 6F,G and Table 1), suggesting that the function of sgg is antagonistic to that of wg, arm and dsh. The marginal phenotype of sgg mutant cells is associated with the ectopic expression (Blair, 1992) and function (Simpson and Carteret, 1989) of products of the AS- C, e.g. ac. However, in our experiments, staining of imaginal discs bearing clones of sgg failed to show ectopic expression of wg (not shown; Blair, 1994). Because sgg mutant clones produce the ectopic appearance of wing margin without recourse to wg expression, it can be concluded that sgg does not act upstream of wg. To understand the relationship between the wingless signal and the activity of sgg, we have generated clones of cells simultaneously mutant for both sgg and dshs, which display viable and readily visible cell autonomous phenotypes (see Methods). These clones do not produce any dsh mutant phenotype at the wing margin, either autonomously or nonautonomously. Moreover, cells mutant for both sgg and dsh display the same phenotype as cells mutant for sgg alone: autonomous ectopic appearance of wing margin anywhere in the wing (Fig. 6 and Table 1). These results show that sgg is epistatic over dsh and therefore allow us to conclude that all the function of dsh in the patterning of the wing margin is mediated through sgg, and more precisely that, in the wild type, the wingless signal represses the function of sgg in the target cells. The products of ct and ac implement wg function at the wing margin The most conspicuous phenotype in the wing margin, resulting from the absence of wg function, is the loss of sensory elements. It is known that the expression and function of the products of the AS-C are required in clusters of cells from which the precursors of the peripheral nervous system of the fly, including both the chemo- and mechanoreceptors of the wing margin, develop (Campuzano and Modollel, 1992; Dominguez and Campuzano, 1993). In the hypothesis that wg is directing the patterning of the wing margin, it would be expected that one aspect of wg function would be to control the expression of the AS-C products, e.g. Ac, along the presumptive margin in the wing discs. When we examine the distribution of Ac in wg mutant conditions, we find a strong correlation between the mutant phenotype and the distribution of Ac in the marginal cells (Figs 5, 7). Conditions that lead to the complete absence of the margin completely abolish Ac expression (Fig. 7B), whereas conditions that produce a narrow margin lead to a sparse distribution of Ac in narrower stripes (Fig. 7C). A similar observation has been made recently with another hypomorphic allelic combination of wg (Phillips and Whittle, 1993). In addition, in mutant dsh v26 discs (see Methods), the expression of Ac is eliminated in the regions where Wg is normally expressed (Fig. 7). This corroborates the conclusions of the study of mutant phenotypes (see above), that the dsh product is involved in the transduction of the wingless signal. The integrity of the wing margin is also affected by mutations at the ct locus that eliminate or reduce only the expression of Cut protein (Ct) in the stripe that outlines the edge cells along the prospective wing margin (Jack, 1985; Jack et al., 1991; Blochlinger et al., 1993). The correlations between the phenotypes and patterns of expression of ct and wg might suggest that the activity of Ct in the edge cells of the wing margin is involved in the control of wg expression. However, antibody stainings of ct mutant discs show a wild-type distribution and abundance of Wg, despite the total absence of Ct expression in the edge cells. Moreover, the pattern of expression of wg monitored by a β-galactosidase reporter insertion at the wg locus also remains unaltered in a ct mutant background (see Methods). These observations have led us to test whether ct expression in the edge cells is dependent on the activity of wg. To do this we have monitored the expression of Ct in wing discs with various allelic combinations of wg. In all cases, the expression of Ct is reduced in correlation with the strength or spatial specificity of the wg mutant condition used. Thus, when wg IL animals are shifted to 25 C before mid-third instar, they show complete absence of Ct in the edge cells (Fig. 8). Also, in spd flg mutant discs, which display reduced levels of Wg (Fig. 4D), there are correspondingly low levels of Ct in those cells (not shown). Our observations suggest that low levels of Wg may activate Ac expression at the margin and this raises the question of why it is that the higher levels of Wg present in the edge cells do not direct Ac expression in this region. One possibility is that genes that are expressed between the two main bands of Ac prevent this expression (see Cubas and Modollel, 1992). To test this we have studied the expression of Ac in ct mutant discs. In these discs, there is Ac expression in many of those cells of the edge that in the wild type never express it (compare Figs 3B and 7D). This result indicates that Wg can direct the expression of Ac at the margin but that this expression is prevented, at least partially, by the activity of Ct.

11 wingless signalling in the Drosophila wing margin 631 Fig. 7. Ac expression in imaginal discs hours AEL. (A) Wildtype wing disc. A complex pattern of Ac expression can be appreciated over a low background in the anterior compartment. Note the two parallel bands along the anterior prospective wing margin (arrowhead), the clusters in the prospective notum (star), and the cluster of the vein sensilla (arrow). (B) Disc from a wg IL homozygous animal shifted to 25 C before 96 hours AEL. Ac expression is abolished in the wing margin and in some clusters in the notum (star). Overall expression is reduced. The arrow points to the remnants of the cluster of the vein sensilla. (C) Disc from a dsh v26 mutant. Symbols as in A and B. Ac expression is lost in those regions that require Wg, like the wing margin and some notal clusters (compare with B). (D) Ac expresion in a wg IL mutant exposed to a pulse of restrictive temperature that gives rise to narrow margin phenotypes as in Fig. 5E. Notice that the bands of Ac expression are very reduced (compare with Fig. 2B) but that some cells (probably the precursors of the chemoreceptors) still express high levels of Ac. (E) Ac expression in a ct 145 /FM6, ct 6 mutant disc. The bands of Ac are disorganized, and there are more cells expressing Ac in the region between the original stripes than there are in wild-type discs (compare with Fig. 3B). DISCUSSION The wingless signalling pathway in the wing margin In the absence of wg function during the third larval instar in Drosophila, wings develop normally but the cells on the margin do not make the bristles and veins characteristic of the wild type. Clones of wg mutant cells generated at this time do not show a mutant phenotype probably because their small size allows rescue by diffusion of the wild-type protein from neighbouring wild-type cells (Baker, 1988a). However, at the wing margin, clones of cells mutant for arm or dsh display phenotypes similar to those caused by the loss of wg during the third larval instar (Figs 5 and 6). Because these mutant phenotypes reflect the absence of wg function in a cell autonomous manner, it can be concluded that arm and dsh functions are required in the cells that receive and interpret the wingless signal. The additional non-autonomous domineering influence that mutant arm or dsh cells exert on their wild-type neighbours suggest that, in the wild type, once cells within the marginal field have become determined as wing margin elements by the wingless signal, a process of lateral inhibition prevents their neighbours from doing so (Couso and Martínez Arias, unpublished data). Thus, the loss of bristles in such clones seems to

12 632 J. P. Couso, S. Bishop and A. Martinez Arias be due to a failure in a process of determination rather than in one of differentiation. In contrast with the behaviour of cells mutant for arm or dsh, clones of sgg mutant cells in the wing autonomously differentiate ectopic marginal pattern elements (Ripoll et al., 1988; Simpson et al., 1988 and Fig. 6). This phenotype suggests that in the wild type, where the function of wg, arm and dsh is to pattern the wing margin, that of sgg is to antagonize this process. A functional relationship between both activities is demonstrated by clones of cells simultaneously mutant for sgg and dsh. These clones display an epistatic sgg phenotype in all regions of the wing, including the margin, indicating that the wild-type function of dsh in the wing margin is to repress the activity of sgg. These results lead to a model in which the Wgdependent patterning of the wing margin is instructed by a signalling pathway that receives and transduces the wingless signal through the dsh gene product, in order to repress the activity of sgg. This model is consistent with two observations, our finding that the expression of Ac is eliminated in wg and dsh mutants, and the ectopic expression of Ac in the absence of sgg function (Blair, 1992). Our results also suggest that the arm gene product is necessary for this process. Although it has been shown that the Armadillo protein is regulated by Wg both in the embryo (Riggleman et al., 1990) and the wing margin (Peifer et al., 1991), at present it is not clear whether arm acts upstream or downstream of sgg. The functional relationships that we have described between wg, arm, dsh and sgg at the wing margin resemble those that have been found during early embryogenesis, when the wingless signal is used to establish and maintain the expression of en in the neighbouring cells of the anterior region of each parasegment. In the embryo, mutations in arm and dsh lead to a loss of en expression (Perrimon and Mahowald, 1987; van den Heuvel et al., 1993; Peifer et al., 1991), as do mutations in wg (Bejovec and Martinez Arias, 1991), whereas absence of sgg function results in the ectopic expression of en in a wgindependent manner (Perrimon and Smouse, 1989; Siegfried et al., 1992). Thus, in the wild type, it seems that the localized expression of en in the embryo and the patterning of the wing margin during the third larval instar rely on the wg-dependent inactivation of sgg activity within a small domain around the source of Wg protein. Fig. 8. Ct protein expression in wing imaginal discs. (A) Wild-type late third instar disc. Ct is found in the adult myoblasts (not shown), in developing sensory organs (arrow) and in the stripe along the wing margin (Bloechinger et al., 1993). (B) Wing disc from a homozygous wg IL animal shifted to 25 C before 96 hours AEL. Ct expression is absent from the wing margin but remains in the myoblasts (arrowhead) and in developing sensory organs, both of which are not affected by wg mutant conditions (arrow). (C) Wing from a wg IL homozygous animal, 4-8 hours APF, treated as in B. Ct expression cannot be detected on the edge cells, nor at either side of it, correlating with the absence of sensory organ development over the wing margin. However, Ct expression can still be detected in the developing sensilla of the third vein (arrow), which are present in the differentiated mutant wing (see Fig. 4E). The patterning of the wing margin Our results together with those of others (García-Bellido and Santamaría, 1978; Hartenstein and Posakony, 1989; Rodriguez et al., 1990; Cubas et al., 1991; Blochlinger et al., 1993; Jack et al., 1991; Blair, 1992) demonstrate that the patterning of the wing margin is a progressive event. It is initiated during the third larval instar with the deployment of the wg and ac gene products and continues until the differentiation of hairs and sense organs in the pupa. Using temperature sensitive wg mutants we have shown that Wg is required continuously for the patterning of the wing margin: initially to establish a marginal field within which patterning of cells will take place, and later for the progressive and spatially restricted commitment of cells to the particular fates that make up the final pattern. The establishment of a marginal field requires the expression of Wg in a stripe across the prospective wing blade half way through the third larval instar. Later, cells that express high