Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing

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1 evelopment 124, (1997) Printed in Great ritain The ompany of iologists Limited 1997 EV Feed-back mechanisms affecting otch activation at the dorsoventral boundary in the rosophila wing Jose F. de elis 1 and Sarah ray 2, 1 epartment of Genetics and 2 epartment of Anatomy, University of ambridge, owning Street, ambridge 2 3Y, UK Author for correspondence ( sjb32@mole.bio.cam.ac.uk) SUMMARY otch function is required at the dorsoventral boundary of the developing rosophila wing for its normal growth and patterning. We find that clones of cells expressing either otch or its ligands elta and Serrate in the wing mimic otch activation at the dorsoventral boundary producing non-autonomous effects on proliferation, and activating expression of the target genes E(spl), wingless and cut. The analysis of these clones reveals several mechanisms important for maintaining and delimiting otch function at the dorsoventral boundary. First, otch activation in the wing leads to increased production of elta and Serrate generating a positive feedback loop that maintains signalling. We propose that during normal development, wingless co-operates with otch to reinforce this positive feedback and ut, which is activated by otch at late stages, acts antagonistically to prevent elta and Serrate expression. Second, high levels of elta and Serrate have a dominant negative effect on otch, so that at late stages otch can only be activated in cells next to the ligandproducing cells. Thus the combined effects of otch and its target genes cut and wingless regulate the expression of otch ligands which restrict otch activity to the dorsoventral boundary. Key words: otch pathway, cut, wingless, cell interactions, imaginal disc ITROUTIO The otch pathway functions in diverse developmental processes ranging from the selection of neural precursors to the regulation of growth and pattern during rosophila imaginal development (Muskavitch, 1994; Artavanis-Tsakonas et al., 1995). asic components of the otch pathway have been conserved through evolution and include the elta (l) and Serrate (Ser) transmembrane proteins, that function as ligands of the otch receptor, the A-binding protein Suppressor of Hairless (Su(H)), which functions as a key transducer element, and a family of related basic helix-loop-helix proteins, which are direct targets of Su(H) and in rosophila are encoded by the Enhancer of split (E(spl)) gene complex (rev. in Artavanis- Tsakonas et al., 1995 and ye and Kopan, 1995; Lecourtois and Schweisguth, 1995). Although these fundamental components are operative in many processes, the relationship between the distribution of the ligands and receptor, and the precise cells where the pathway is activated appear complex. For example, genetic manipulations which alter the relative levels of otch and l in rosophila result in dominant phenotypes similar to those caused by reducing otch function (Vassin et al., 1985). These effects are different from those observed with other signalling pathways and indicate that normal otch signalling depends on a fine-tuned balance between the amounts of otch and its ligands. Thus it is important to detemine how the levels of ligands and receptor are regulated and how they influence the capacity of cells to send and receive the otch signal. The formation and maintenance of the dorsoventral (d/v) boundary in the rosophila wing imaginal disc is dependent on sophisticated mechanisms to guarantee restricted activation of otch over a prolonged period. otch is required here for growth of the wing and patterning of the wing-margin (Shellenbarger and Mohler, 1978), and ectopic otch activity produces ectopic margin structures and dramatic consequences on cell proliferation (Speicher et al., 1994; Kim et al., 1995; iaz-enjumea and ohen, 1995; de elis et al., 1996; oherty et al., 1996). The d/v boundary forms at the interface between two cell populations that are clonally segregated, the dorsal and ventral compartments. orsal cells express the homeotic gene apterous, which regulates expression of the secreted protein Fringe and the otch ligand Ser (ohen et al., 1992; lair et al., 1994; Irvine and Wieschaus, 1994, Kim et al., 1995). otch is specifically activated in the dorsal and ventral cells that form the d/v boundary (de elis et al., 1996) and its activation here requires two ligands, l and Ser. Ser is expressed in dorsal cells, and in misexpression studies is only effective at activating otch in ventral cells, suggesting that Ser is a dorsal to ventral signal (ouso et al., 1995; iaz-enjumea and ohen, 1995; Kim et al., 1995; de elis et al., 1996). l is necessary on both sides of the boundary, but both loss-offunction and ectopic expression experiments suggest that it is principally required in ventral cells to signal to dorsal cells (oherty et al., 1996; de elis et al., 1996). The surfacespecific requirements for l and Ser indicate that other

2 3242 J. F. de elis and S. ray factors which restrict the activating capabilities of Ser and l are involved in regulating otch signalling. otch activity at the d/v boundary is required for the localised expression of several genes that affect wing morphogenesis, including vestigial (vg), wingless () and cut (ct) (Kim et al., 1995; Rulifson and lair, 1995; ouso et al., 1995; Kim et al., 1996; eumann and ohen, 1996). oth vg and are expressed from very early stages at the d/v boundary and are proposed to have important roles in the subsequent development of the wing (Williams et al., 1994; iaz-enjumea and ohen, 1995; ouso et al., 1995). When clones of cells lacking otch function are restricted to one surface at the d/v boundary, they abolish and ct expression from the cells on both sides of the boundary (Rulifson and lair, 1995). This nonautonomous effect argues that otch is needed in the boundary cells on one side for them to be able to signal to the neighbouring compartment, suggesting that otch signalling also regulates the levels of available ligand. We have investigated the relationships between otch and the expression of its ligands to determine its relevance in maintaining otch activity at the d/v boundary, by manipulating different components of the otch pathway and different target genes in the developing wing. We have combined GAL4 controlled gene expression (rand and Perrimon, 1993) with FLP/FRT mediated recombination (Xu and Rubin, 1993; Zecca et al., 1995) to generate clones of marked cells where specific proteins are misexpressed, allowing us to determine how the effects relate to the site of misexpression. The phenotypes and effects on target genes show that Ser and l have cell autonomous dominant negative effects that help to restrict high levels of otch activation. In addition, otch activity regulates the transcription of its ligands, partly by means of its two target genes and cut. These mechanisms link otch signalling with the expression of its ligands, and provide a framework for understanding the dynamic expression patterns of these genes during normal development. by a series of cloning steps (details are available on request) and contains: the 3 kb oti/kpn1 abx/ubx fragment from pa2, the 17 kb MluI/ssiW1 fragment from pm8 containing the >stop-forked + > cassette, the 3 kb sshii/blunt (SpeI filled in) fragment containing GAL4, the 3 kb blunt/kspi fragment from p2 containing the IRESlacZ, the KspI/otI pwhiterabbit vector fragment. The vector contains mini-white as a selectable marker for P-element transformation. abx/ubx<frt stop-forked + FRT> GAL4-lacZ transformant flies were generated by injecting pwa>f>gil into white forked flies in the presence of transposase following standard procedures (Rubin and Spradling, 1982). Generation of GAL4-expressing clones in the wing disc lones were generated by heat-shocking larvae for 6 minutes at or hours after egg laying (AEL). The genotypes of the heat-shocked larvae were f 36a FLP1.22; abx/ubx<frt f + stop FRT> GAL4-lacZ/ UAS-X, where UAS-X is either: UAS-l, UAS-Ser, UAS- fl, UAS- ecd, UAS-i or UAS-. In the unrecombined state these flies are forked + and no GAL4 protein is expressed. Induction of FLPase catalyses site-specific recombination at the FRT sites, removing forked + and juxtaposing the disc enhancer-promoter fragment (abx/ubx) next to GAL4 (Fig. 1). This allows production of GAL4 which can drive expression of any ca cloned downstream abx/ubx FRT stop forked + FLP abx/ubx FRT Gal4-lacZ Gal4-lacZ stop forked MATERIALS A METHOS rosophila strains We used two ct alleles, ct 1 and ct n, the otch temperature sensitive allele l(1) ts, the forked allele f 36a as a cuticular marker and FLP1.22 (an X chromosome hsflp line) as a source of FLP recombinase (Xu and Rubin, 1993). The following UAS-lines were used: UAS-l (UAS- l30; oherty et al., 1996) and UAS- (a gift from A. Martinez- Arias) insertions in the second chromosome; UAS- fl, UAS- ecd (full length and otch extracellular domain insertions in the third chromosome; gifts from M. aylies), UAS- i (intracellular domain of otch, gift from M.Go and S. Artavanis-Tsakonas) and UAS-Ser (Speicher et al., 1994) insertions in the third chromosome. We also used the lacz lines ctwh1 (Jack et al., 1991), lz and vg dorsoventral enhancer (Williams et al., 1994). Generation of the FRT/GAL4-lacZ construct The starting plasmids were: pa2, containing the Ubx IRES linked to lacz (Hart and ienz, 1996); p2, containing the abx enhancer, linked to the Ubx transcription start-site and mra leader (Hart and ienz, 1996); the yeast GAL4 protein coding sequences in pks (gift from ick rown); the P-element based transformation vector pwhiterabbit (gift from. rown); and pm8 containing the FRT-hsp70 trailer sequences 16 kb forked genomic A-FRT (>stop-forked + >) cassette (Zecca et al., 1995). The plasmid pwa>f>gil was generated lone Induction Gal4/βGal lones forked lones Fig. 1. Generation of Gal4-expressing clones. (Top) Schematic representation of the construct used to generate forked cells that express Gal4 and lacz. FLP mediated recombination at the FRT sites leads to excision of the transcription terminator sequences and wildtype forked gene (amber box), fusing the abx/ubx enhancer promoter (light green box) to the dicistronic Gal4-lacZ gene (blue box). The presence of an internal ribosome entry site upstream of lacz permits translation from this gene. The abx/ubx enhancer is expressed throughout the wing disc so that all cells where this recombination event occurs will transcribe Gal4 and lacz. (ottom) Identification of Gal4-lacZ clones. The cell in which recombination is induced and its progeny express the lacz gene and can be recognised in the imaginal disc using anti-β-galactosidase (βgal) antibodies or X-gal staining (blue spots in the wing disc). lones were induced in forked mutant flies, and because they lose the wild-type copy of forked (amber box), they can be identified in the adult wing as forked cells (grey patches in the wing) in a background of forked + cells.

3 Feedback mechanisms on otch signalling 3243 of its UAS sites (rand and Perrimon, 1993). GAL4-expressing cells are detected in the adult as forked and in the imaginal disc through lacz expression which is coupled to GAL4 by an internal ribosome entry site (Hart and ienz, 1996). In the discs the clones were monitored by anti-β-galactosidase or X-gal staining unless otherwise stated. Immunocytochemistry and in situ hybridisation Immunocytochemistry was performed as described by Jennings et al. (1994). We used rabbit anti-β-galactosidase (appel), rabbit anti-ser (Speicher et al., 1994) and mouse monoclonals anti-otch (Fehon et al., 1991), anti-l (Kooh et al., 1993), anti-t (lochinger et al., 1990), anti-wg (eumann and ohen, 1996), anti-e(spl) mab323 (Jennings et al., 1994) and anti-β-galactosidase (Promega) antibodies. Secondary antibodies were from Jackson Immunological Laboratories (used at 1/250). X-gal staining and whole-mount in situ hybridisation with digoxigenin-labelled A probe was performed as described previously (ubas et al., 1991). boundary and are flanked on either side by cells containing high levels of l and Ser (Fig. 2,G,I and see Micchelli et al., 1997). Thus during third instar the ligands are first expressed at highest levels in the cells where otch is activated (based on accumulation of Wg, Vg and E(spl)) and later they become up-regulated in the cells immediately flanking this central domain. Effects of reduced otch and ct function on l and Ser expression The expression patterns of Ser and l observed during normal development are consistent with otch regulating the levels of its ligands at the d/v boundary. They further suggest that the onset of ct expression could be linked to the change in l and Ser accumulation observed late in the third larval instar. We therefore investigated whether reductions in otch or ct modify these expression patterns accordingly. Using a ts allele we RESULTS A ynamic expression of otch, elta and Serrate in the developing wing We first describe how l and Ser expression relates to otch activation at the d/v boundary during normal wing development. In early third instar discs otch appears uniform, Ser is restricted to the dorsal compartment (ouso et al., 1995) and l is present at highest levels at the d/v boundary (Fig. 2A; de elis et al, 1996; oherty et al., 1996). This domain of l coincides with that of the stripe and the vg d/v enhancer (Fig. 2E,F), which are both indicative of otch activation (ouso et al., 1995; iaz- enjumea and ohen, 1995; Kim et al., 1995; de elis et al., 1996; oherty et al., 1996; Kim et al., 1996). Throughout mid third instar both l and Ser continue to be highest at the d/v boundary although Ser expression is restricted to dorsal cells (Fig. 2H). When ct expression is first detected, the patterns of ligand expression become further modulated: both l and Ser are reduced in the cells where t is present (Fig. 3-E), which retain and vg expression (Fig. 2,,G). In late third instar t, Wg and high levels of otch (Fig. 2) are expressed in the central stripe of 2-3 cells located at the d/v l ap l+ap E F G H l+ l+ ap Ser+ap ap l+vg +Ser l+vg l Ser l+ser l+ser Fig. 2. ynamic expression patterns of l and Ser at the d/v boundary. (A) Expression of l (anti-l, green) and apterous (monitored in ap-lacz line, red) in early third instar disc. l is enriched in ventral cells but can be detected on both sides of the boundary. (-) High magnification of the d/v boundary showing l (green, ) and Ser (green, ) in relation to ap-lacz expression (red, -) in late third instar discs. oth l and Ser accumulate in dorsal and ventral cells that flank the d/v boundary, and are excluded from the central cells at the boundary (red nuclei and arrow, -). Levels of l in the dorsal and ventral compartments are similar, but levels of Ser are higher in dorsal cells. () istribution of otch (green) and Ser (red) in late third instar discs when otch is at higher levels (arrowhead) along the d/v boundary between the flanking stripes of cells containing Ser). (E-F) Expression of l (anti-l, green) in mid third instar discs coincides with the expression of two transcriptional targets of otch, (-lacz, red, E) and vg (vg boundary-enhancer driving lacz, red, F). The d/v boundary is indicated with arrows in E and brackets in F. (G) In contrast, expression of l (green) and vg (red) are complementary in late third instar discs. (H) istribution of l (green) and Ser (red) in early third instar discs showing preferential ventral accumulation of l. (I) omparison of l (green) and Ser (red) at the d/v boundary of late third instar disc shows that dorsal and ventral stripes of expression overlap (upper image), although Ser is at higher levels in the dorsal side. I

4 3244 J. F. de elis and S. ray Fig. 3. Effects of otch and ct mutations on Ser and l expression. (A-) Phenotypes of two ct mutants, ct n (A) and ct 1 (). (-E) Expression of l (anti-l, green) and ct (ct-lacz, red) in early (), mid () and late (E) third instar discs. The onset of ct expression coincides with the suppression of l expression in the d/v boundary (), and in late third instar they are expressed in complementary domains (E). Low levels of overlap arises from perdurance of the ct-lacz reporter gene. (F-H) The effects of ct n on the expression of l (anti-l, green) are variable, but the gap in l expression at the d/v boundary (wild-type, F) can be missing (G) or reduced (H). (I-K) Expression of Ser (anti-ser, green) and (anti-wg, red) in wild-type (I), ct n (J) and ct 1 (K). Ser expression is reduced in both the dorsal and ventral stripes and expression is patchy. ote that in some places Ser is co-expressed with Wg (yellow and arrow). (L-) Reduction in the levels of l in l(1) ts late third instar mutant discs along the d/v boundary after 12 hours at 30.5 varies from strong (arrows in M) to moderate (L,). In contrast, levels of l are increased in proneural clusters of the thorax (L-M) and wing (asterisk in L-, compare with F). (O) Late l(1) ts disc after 12 hours at 30.5 with reduced levels of both Wg (red) and Ser (green). (P-Q) Strong (Q) and moderate (P) reduction in the levels of l along the d/v boundary (arrows) in l(1) ts early third instar discs after 12 hours at A wt ct n wt E ct 1 wt ct 1 ct 1 L M O F G H wt ct n ts I J K P Q ts ts ts wt ct n ts ts find that after 12 hours at the restrictive temperature (30.5 ) the levels of l and Ser are low along the d/v boundary (Fig. 3P-Q). The late expression flanking the d/v boundary also fails to develop (Fig. 3L-O), although expression associated with sensory organ development remains at high levels (Fig. 3L-). After 24 hours at 30.5 expression of l and Ser at the d/v boundary is further reduced, or absent (data not shown). Two different alleles were used to test whether ct influences expression of l and Ser; ct n a severe allele that produces extensive loss of margin tissue (Fig. 3A) and little or no detectable t protein at the d/v boundary (not shown) and ct 1, a hypomorphic allele that produces weak wing notching (Fig. 3). In the majority of ct n discs the late l expression is altered, in particular the normally distinct stripes of l flanking the d/v boundary are diffuse, and high levels of l persist in the cells in between (Fig. 3F-H). Similarly, in ct n mutants the boundary of Ser expression is diffuse and overlaps with expression (Fig. 3I-J). In addition Ser expression is not activated in the flanking ventral cells (Fig. 3J). The ct 1 mutation causes similar but more modest effects, the levels of t, l and Ser are reduced in the anterior/central region of the disc and some overlap of Ser and expression occurs (Fig. 3K and data not shown). In mutants with either of these ct alleles the levels of expression are also reduced (Fig. 3J-K; Micchelli et al., 1997). These results indicate that t is required for the repression of Ser and l in the central d/v cells. This function of ct may contribute to the reduced expression observed in ct mutants, because expression of l and Ser in the boundary cells interferes with otch signalling (Thomas et al., 1995; Klein et al., 1997 and see below). A i i i l l Ser l Fig. 4. Effects of i and fl -expressing clones on the expression of l and Ser. (A-) Expression of l (green) in i clones (red) monitored in mid (A) and in late () third instar discs. Open arrowhead marks the position of the normal d/v boundary. () Expression of Ser (green) in i clones (red) induced houirs AEL and monitored in late third instar discs. () Expression of l (green) in fl clones (red) monitored in late third instar discs. ottom panels are merged images, and the position of several clones in each panel has been indicated by arrows or asterisks. Fl

5 Feedback mechanisms on otch signalling 3245 onsequences of unrestricted otch activity in the wing The effects of otch mutants are consistent with otch regulating the expression of its own ligands. To test this further we examined the effects of expressing a constitutively active intracellular fragment of the otch receptor ( i ), and the full-length otch protein itself ( fl ) in clones of cells. The clones were marked in both the larva and the adult (see Fig. 1) allowing us to determine how the observed effects on gene expression and phenotypes relate to the cells where the proteins are expressed. All i -expressing clones within the wing pouch induce l and Ser expression within the clone, whether they are dorsal or ventral (Fig. 4A-) even though Ser expression in wild-type is restricted to dorsal cells. fl also induces l expression in the wing pouch (Fig. 4), but neither fl nor i upregulate l or Ser expression in thoracic clones, indicating that this A response is context dependent. To determine how the activation of Ser and l expression in i and fl clones relates to effects on known target genes, we analysed ct and expression (eg. iaz-enjumea and t ohen, 1995; Kim et al., 1995; de elis et al., 1996; oherty et al., 1996). oth were induced in i -expressing clones at all positions in the wing-pouch although not in all cells within each clone (Fig. 5A-,K-L). As expected for an activated receptor ct and expression t is mostly restricted to cells within the clone, but can also detected in the cells G immediately adjacent (Fig. 5A-) consistent with l and Ser being elevated in the clone and activating otch in adjacent cells. Similar non-autonomous effects are seen with fl -expressing cells which induce ct expression when they are close to the d/v boundary (Fig. 5- I E). Since the E(spl) genes are general targets of otch signalling (Jennings et al., 1994) we also analysed E(spl) I' expression in i and fl -expressing clones, to compare it with ct and. As expected E(spl) expression is activated in i -expressing cells at all locations in the disc (Fig. 5) confirming that the pathway downsteam of otch can be activated throughout, even though the effects on ct, and ligand expression are more restricted. E(spl) expression was also detected in all fl -expressing clones, not just in the few clones near the d/v boundary where ct and expression can be activated (Fig. 5F). This suggests that the high concentration of fl protein is sufficient for some degree of otch activation, and in the wing pouch correlates with the effects on l expression. The autonomous and nonautonomous effects of i on the expression of ct, and E(spl) can be correlated with some of the resulting adult phenotypes. In all regions of the wing i clones predominantly cause outgrowths. These are usually composed of both i -expressing and adjacent wild-type cells, demonstrating that otch has non-autonomous effects on the surrounding cells (Fig. 5G-H). The i -expressing clones also produce ectopic wing margin bristles which develop both throughout the clone and from wild-type adjacent cells (Fig. 5H,J). In addition, cells expressing i are smaller and have altered polarity in comparison to wild-type cells (Fig. 5I,I ), producing defects in the differentiation of bristles (not shown). The effects of expressing fl are similar but modest, outgrowths of the clone or of associated wild-type tissue are less frequent and the differentiation of E(spl) J H t E t F E(spl) Fig. 5. Effects of i clones. (A-) Expression of t (green, A-) and E(spl) proteins (green, ) occurs within i- expressing cells (red, overlap in yellow), and also in adjacent wild-type cells. (-E) Activation of ct expression (green) in fl clones (red) is limited to regions close to the d/v boundary. (F) lones of fl -expressing cells cause ectopic expression of E(spl) proteins (green) within the clone (red). Open arrowheads mark the location of the normal d/v boundary and arrows and asterisks the position of several clones in A-F. (G-J) Wing phenotypes of i clones. (G) Outgrowths of dorsal and ventral wing tissue protruding from the wing epithelium (arrows). Only part of the tissue in the outgrowth is formed by i -expressing cells. (H) Ventral outgrowth in which the distal part is formed by i -expressing cells (outline) that differentiated some bristle tormogens. The clone is surrounded by wild-type ectopic margin bristles of ventral characteristic. ote that most of the outgrowth is formed by wild-type cells. (I-I ) Ventral (I) and dorsal (I ) surfaces of the same wing. i -expressing cells (I ) have reduced cell size (compare with I) and altered cell polarity. (J) orsal outgrowth in which the i clone is surrounded by margin bristles. (K-L) Expression of in i clones. i clones (turquoise) cause patchy expression of within the clone ( mra, purple). The normal d/v boundary is labelled with an empty arrowhead. K L

6 3246 J. F. de elis and S. ray A Fig. 6. Upregulation of l and Ser in expressing clones. (A) Multiple expressing clones in the wing blade and hinge (red). The expression of l (anti-l, green) is activated at high levels within clones in the wing pouch. Only clones in the hinge (asterisk) induce massive overgrowth. () Wing pouch of the disc shown in A (2.5 higher magnification), -expressing clones (red) induce high levels of l (green) cell autonomously. Much lower levels of l are also detected in wild type cells in the periphery of the clones. () -expressing clones (labelled with anti-wg, red) induce Ser expression (green) only in the wing blade, but not in the hinge or thorax (e.g. asterisks). (-E) Ser (green) is ectopically expressed both within and in the periphery of expressing clones (labelled with anti-wg, red) in the wing pouch. lones were induced hours () and hours APF (E). E ectopic margin structures is only observed close to the normal wing margin (not shown), consistent with the activation of and ct expression in these positions. Ectopic Wingless leads to increased levels of elta and Serrate The ability of i to promote l and Ser accumulation is related to the regions of the disc where and ct expression are also induced. We therefore tested the effects of ectopic Wg, and found that both ligands are expressed at high levels within the clones of ectopic Wg when they are present in the wing pouch (Fig. 6; and see Micchelli et al., 1997). The elevated levels of Ser and l are largely restricted to the -expressing cells. However we also detect low levels of expression outside the clones, particularly with clones induced late in the third instar, indicative of Wg acting in neighbouring cells (Fig. 6E). Finally, -expressing clones do not have the same phenotypic consequences of i clones, being associated with extensive overproliferation only in the hinge region but not in the wing pouch (Fig. 6A,). The transcriptional activation of l and Ser in response to Wg raises the possibility that otch regulation of ligand expression is mediated by Wg. However, l expression is also elevated in fl -expressing clones, where Wg is not detected, suggesting that otch can affect the expression of its ligands independently of Wg. Activation of otch by different ligands: wing surface-specific and boundary effects To investigate whether ligand-producing cells have similar effects to i - or -expressing cells, we generated clones of cells expressing l and Ser. oth ligands produce wing outgrowths which contain wing margin structures at the clone boundaries. As with i, cells belonging to the clone form only a subset of the tissue in the outgrowth (Figs 7A, 8A-). There is also a clear positional specificity in the induction of outgrowths by Ser and l, as has been observed previously (ouso et al., 1995; Thomas et al., 1995; de elis et al., 1996; iaz- enjumea and ohen, 1995; oherty et al., 1996; Jonsson and Knust, 1996). Thus Ser-expressing clones only cause outgrowths and only induce ct and expression in the ventral wing surface while l-expressing clones do so in the dorsal wing surface. (Figs 7-,H-I, 8E-F,G-I). The dorsal/ventral specificty breaks down in two respects, however. First, ventral l clones and dorsal Ser clones that are close to the d/v boundary are able to induce ct and expression (Fig. 8H-I and data not shown). Second, ventral l-expressing cells in regions close to the hinge/pleural region also produce outgrowths although they do not induce ct and expression (Figs 7-,H-I, 8E-F,G-I). In addition to phenotypes consistent with otch activation, Ser-and l-expressing clones also produce loss-of-function phenotypes such as thickening of wing-veins and notches in the wing margin, which are most evident in positions where they do not induce outgrowths (i.e. dorsal Ser-expressing cells and ventral l-expressing cells; Fig. 8 and data not shown). Some of these phenotypes are similar to those produced by cells expressing the extracellular domain of otch, ecd which is proposed to act as a dominant negative inhibitor of otch (Rebay et al., 1993; Lieber et al., 1993; Jonsson and Knust, 1996; Klein et al., 1997). Thus ecd clones in the wing are frequently associated with nicks in the wing margin and cause the differentiation of thicker veins (Fig. 9A and data not shown). The hypothesis that high levels of l and Ser have dominant negative effects is also supported by the effects on and ct. oth gene products are only ever detected at the clone boundaries outside the region of ectopic Ser or l expression, as has

7 Feedback mechanisms on otch signalling 3247 been reported in previous ectopic expression studies (Kim et al., 1995; iaz-enjumea and ohen, 1995; oherty et al., 1996). In addition, when clones actually touch or cross the d/v boundary, both l and Ser repress the endogenous ct and expression (Figs 7-, 8E-F and data not shown). Similar effects are seen when ecd clones touch the d/v A boundary, consistent with this being a loss-of-function effect (Fig. 9-). E(spl) genes are ectopically activated by l and Ser predominantly at the clone boudaries in the wing pouch (Figs 7F-G, 8G) but endogenous E(spl) expression at the d/v boundary is inhibited. Interestingly, clones of l-expressing cells in the hinge induce high levels of E(spl) proteins both within the clone as well as immediately adjacent to it (Fig. 7E), indicating that the levels of ectopic l are not sufficient to prevent otch activation in the l-expressing cells. ISUSSIO of its ligands and are consistent with the maximal accumulation of otch ligands in the cells where otch is active in early and mid third instar discs (Fig. 10). This effect of otch could be mediated by Wg (see below), but expression of l is also y combining misexpression in clones with loss-of-function experiments we have identified several mechanisms that are used to maintain otch activity at the d/v boundary. These include otchdependent regulation of ligand expression, and ligand-mediated antagonism of otch when both proteins are present in the same cells. These mechanisms help restrict high levels of otch activation to the dorsal and ventral cells that form the d/v boundary, and could be important in other developmental systems where otch signalling generates boundaries between cell populations. E F G E(spl) E(spl) E(spl) H I J Positive and negative feedback in the otch pathway At the d/v boundary the absence of otch from one wing surface eliminates the ability of these cells to signal to the opposite surface (Rulifson and lair, 1995). Our results provide a link between otch activity and the expression of it s ligands, since we find that l and Ser are upregulated in i and fl -expressing clones, even though the levels of otch activation in the latter are not sufficient to induce expression of its target genes and ct. onversely, a reduction in otch function leads to a decrease in the expression of l and Ser at the d/v boundary. These results suggest that otch activity itself is required for maintaining the expression t t l Fig. 7. Effects of l-expressing clones. (A) orsal clone of l-expressing cells producing a large outgrowth of wing tissue in which most cells are wild type. The clone (outline) is surrounded by dorsal wing margin bristles. (-) Expression of occurs in wild-type cells abutting l-expressing clones (e.g. arrow in ). is high magnification of the margin region of the disc shown in. l-expressing clones that cross the d/v boundary cause cellautonomous repression of (asterisk in and ). (E-G) Expression of E(spl) proteins (green) within l-expressing clones (red). In the wing pouch (F-G) E(spl) proteins are detected exclusively in wild-type cells abutting dorsal clones (e.g. arrows), and this is observed both when clones are monitored in early third (G) and late third (F) instar discs. Ventral clones (asterisk) do not induce E(spl) expression. E(spl) proteins are detected within l-expressing cells in other parts of the disc, such as the hinge (E). (H-I) Expression of ct (green) is activated in wild-type cells at the boundaries of clones (red) in the dorsal surface (arrows in H,I). (J) The levels of l produced in l-expressing clones (arrows) are much higher than endogenous l levels. Open arrowhead marks the location of the normal d/v boundary in F-I.

8 3248 J. F. de elis and S. ray Fig. 8. Effects Ser-expressing clones. (A-) In the ventral wing surface Ser-expressing clones (outline) induced early (48-72 hours AEL) are associated with extensive outgrowths in which a A E large fraction of cells are wild type. All ventral clones induce ectopic wing margin elements at their boundaries, which mainly develop from adjacent wild-type F cells. () Ser-expressing clones in the dorsal wing surface (outline) cause the differentiation of thicker veins. (E,F) Expression of ( mra, purple) in wild-type cells abutting Ser-expressing clones (blue) primarily occurs in the ventral surface of the disc, but also when dorsal clones are located close to the d/v boundary (arrows in F). In addition endogenous expression is suppressed when clones span the d/v boundary (asterisk, E). (G) E(spl) proteins (green) are detected in wild-type cells surrounding Ser-expressing clones (red) in the ventral surface. In posterior clones E(spl) proteins are restricted to G E(spl) H t I t cells adjacent to the clone, whereas in anterior clones broader expression is detected associated with developing margin bristles. H-I) ct expression (anti-t, green) is activated in wild-type cells next to ventral clones, and next to dorsal cells located close to the d/v boundary (arrow, I is a 2.5X magnification of H). The position of the normal d/v boundary is labelled in E-I with empty arrowheads. induced in situations where Wg is not present (such as fl clones), indicating that Wg-independent mechanisms can direct expression of l and Ser in response to otch activity. A second phase of ligand expression at the d/v boundary is established by late third instar when high levels of l and Ser are present in cells flanking the boundary and not in the boundary itself (Fig. 2 and Micchelli et al., 1997). Although otch is not activated in the flanking cells, this late ligand expression is disrupted when otch function is reduced, indicating that it is still otch dependent. We propose that the combined actions of Wg and t, which are expressed in response to otch, are required to mediate this effect. The elimination of l and Ser expression from the boundary cells correlates with the appearance of t, and in ct mutants l and Ser expression is not properly down-regulated in these cells. Since ut encodes a transcription factor that belongs to a family of repressors (Jack and elotto, 1995), this could be a direct effect on l and Ser transcription (Fig. 10). In the wing pouch Wg promotes l and Ser expression mostly cell autonomously, but low levels of ligand expression are also detected outside the clones. This implies that only high levels of Wg are effective at promoting Ser and l expression, and suggests that the accumulation of l and Ser in domains flanking the boundary cells could be directed by Wg acting non-autonomously. This is supported by the observation that dishevelled, a downstream component of the pathway, is required in the flanking cells for the late pattern of Ser and l expression (Micchelli et al., 1997). Thus, the co-ordinated activities of Wg and t, with t repressing ligand expression in the central cells where otch is activated and Wg activating ligand expression in the flanking cells, can explain how the late patterns of l and Ser expression are generated in response to otch (Fig. 10). The linking of otch activity to ligand expression ensures that otch signalling is stably maintained at the d/v boundary. Vertebrate elta-like ligands are required for the formation/maintenance of somite borders, and accumulate at the presumptive boundary in a otch-dependent manner, indicating that positive regulation of ligand expression could be generally important when otch is involved in maintaining stable boundaries (ettenhausen et al., 1995; Hrabe de Angelis et al., 1997; Jen et al., 1997). A positive relationship between otch activity and ligand expression contrasts with the model proposed for neurogenesis, where it has been suggested that repression of ligand expression in cells where otch is activated could be important for amplifying differences in ligand and receptor levels between neighbouring cells (Heitzler et al., 1996). Activation of otch at other times in development can inhibit l expression (Hinz et al., 1994; Parks et al., 1997) consistent with such a negative feedback loop, although no modulation in l expression during neural precursor selection has yet been observed (Parks et al., 1997). The contrasting effects of otch activity on l expression are also evident when otch function is reduced; because l levels are

9 Feedback mechanisms on otch signalling 3249 A ap Ser Su(H) Su(H) l l t Fig. 9. Ectopic expression of a dominant negative form of otch, ecd. (A) lones of ecd -expressing cells are associated with strong wing margin loss. (-) They also eliminate () and ct () expression cell autonomously when they extend into the d/v boundary (arrows). lones were detected with X-gal () and anti-β galactosidase antibody (red in ). decreased at the d/v boundary but elevated in proneural clusters. Similarly, l and Ser expression is induced in wing but not in thoracic i clones. The effects of otch signalling on ligand expression are thus context dependent, and specific otch targets participate in determining whether otch signalling activates or represses ligand expression. Mechanisms affecting otch-ligand interactions When otch ligands are expressed ectopically, ct and expression is activated in the adjacent cells, never within the ligand-expressing cells themselves (Figs 7, 8; Kim et al., 1995; iaz-enjumea and ohen, 1995; oherty et al., 1996). In addition, when l- or Ser-expressing cells intrude on the normal d/v boundary, the endogenous ct and expression is no longer detected. These observations suggest that otch ligands interfere with activation of the receptor when they are present at high levels in the same cells. This is a mechanism that could operate normally to regulate otch signalling at the d/v boundary in late third instar because elimination of l and Ser in clones close to the d/v boundary results in ectopic ct expression within the clones (Micchelli et al., 1997). The molecular basis of these dominant negative effects is poorly understood but presumably involves direct protein-protein interactions between otch and its ligands occurring in the same cell. This function could be disrupted by Abruptex, gain of function alleles of otch (de elis and Garcia-ellido, 1994a), which result in ectopic expression of ct and close Ser l ct l Ser l Ser Fig. 10. Model for otch activation at the dorsal ventral boundary. The positive and negative interactions between otch and its ligands and the observed regulation of ligand expression can be integrated with the temporal evolution of the expression of Ser, l and otch to provide a model for otch signalling at the d/v boundary. The surface specific effects of the ligands suggest that, in early third instar discs (top), dorsal Ser (purple) activates otch (green) in the adjacent ventral cells and conversely ventral l (black) activates otch (green) in adjacent dorsal cells. This leads to the accumulation of Wg, and we postulate that the combined activities of Wg and otch maintain Ser and l expression at the d/v boundary. At this stage the levels of l and Ser would not be sufficient to inhibit otch signalling. At later stages (bottom), the expression of ligands is eliminated from d/v boundary cells. This correlates with the appearance of t at the d/v boundary and the effects of ct mutants on l and Ser expression suggest that t participates in their repression in these cells. The presence of Wg in boundary cells will direct expression of l and Ser in adjacent flanking cells, where the proteins inhibit otch activation (red otch). otch accumulates preferentially in cells at the d/v boundary, which could be a consequence of otch signalling, and would re-enforce the asymmetry between signalling and receiving cells. Although the basis for the precise timing of the observed transitions in expression patterns are far from clear, the use of sequential mechanisms would ensure that the polarity and intensity of signalling at the d/v boundary is maintained during a long period of time. to the d/v boundary (de elis et al., 1996). These effects confirm that the otch protein is involved in restricting its own activation during normal development, as the Abruptex mutations are aminoacid substitutions in specific EGF repeats of the otch extracellular domain (Kelley et al., 1987). The combination of l and Ser mediated repression of otch within the same cell, and activation of otch in the adjacent cell explains how otch signalling can be maintained in a stripe of cells. As with previous experiments (ouso et al., 1995; Thomas et al., 1995; iaz-enjumea and ohen, 1995; Kim et al., 1995; de elis et al., 1996; oherty et al., 1996; Jonsson and Knust, 1996), we detect strong surface-specific bias in the response to l and Ser in the wing blade (with l mainly operative in dorsal and Ser in ventral cells) implying the existence of other factors that modify the ability of Ser and l to activate otch. The activity of these factors would be restricted to dorsal or ventral ct l Ser

10 3250 J. F. de elis and S. ray cells and possible candidates include the secreted molecules Fringe and Wg. is initially expressed preferentially in ventral cells and appears to enhance the efficiency with which Ser activates the expression of the otch-target gene vg (ouso et al., 1995). onversely, fringe is expressed in dorsal cells (Irvine and Wieschaus, 1994) and recent results support a role for Fringe in attenuating the ability of Ser to activate otch (Panin et al., 1997). Although the surface specific effects of the ligands may be sufficient to explain the restriction of otch activity early, when Ser is restricted to dorsal cells and l is highest ventrally, the presence of the ligands in the flanking cells of both compartments at later stages necessitates the involvement of other mechanisms, such as the dominant negative effects discussed above. Autonomous and non-autonomous responses to otch activation The extensive proliferation of i -expressing cells is compatible with a cell autonomous requirement for otch in cell proliferation (de elis and Garcia-ellido, 1994b). However, a large component of the outgrowths produced by i -expressing clones in the wing blade arises from neighbouring wild-type cells. Furthermore, ectopic expression of either l or Ser also causes overgrowth, despite the fact that high levels of otch activation are only evident in the cells adjacent to the Ser- or l-expressing clones. These observations indicate that limited activation of otch can have non-autonomous consequences on proliferation and wing margin patterning. It has been proposed that both effects are mediated by the secreted Wg protein acting as a long-range morphogen (iaz-enjumea and ohen, 1995). However, two observations indicate that Wg cannot be the only factor involved in mediating cell proliferation. First, we find that clones expressing fl and l in some regions outside the wing pouch result in overgrowths, even though no activation of expression is observed. Second, clones of expressing cells do not have the same effects on proliferation in the pouch as i -, l- or Ser-expressing clones, although they cause dramatic overgrowth in the hinge. The different behaviour of -, fl -, l- and Ser-expressing clones is paradoxical, because - and fl -expressing clones within the wing pouch produce l and Ser yet their effects on the behaviour of adjacent wild-type cells are not the same as l- or Ser-expressing clones. Thus, either the absolute amounts of ligand are critical in determining whether or not a response can be elicited and/or the signalling capacity of the ligands is susceptible to the presence of other molecules, such as Wg and otch. We thank M. Ashburner in whose laboratory part of this work has been done. 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