We have investigated the role of the Notch and Wingless signaling pathways in the maintenance of wing margin identity through the study of cut, a homeobox-containing transcription factor and a late-arising margin-specific marker. By late third instar, a tripartite domain of gene expression can be identified about the dorsoventral compartment boundary, which marks the presumptive wing margin. A central domain of cut- and wingless-expressing cells are flanked on the dorsal and ventral side by domains of cells expressing elevated levels of the Notch ligands Delta and Serrate. We show first that cut acts to maintain margin wingless expression, providing a potential explanation of the cut mutant phenotype. Next, we examined the regulation of cut expression. Our results indicate that Notch, but not Wingless signaling, is autonomously required for cut expression. Rather, Wingless is required indirectly for cut expression; our results suggest this requirement is due to the regulation by wingless of Delta and Serrate expression in cells flanking the cut and wingless expression domains. Finally, we show that Delta and Serrate play a dual role in the regulation of cut and wingless expression. Normal, high levels of Delta and Serrate can trigger cut and wingless expression in adjacent cells lacking Delta and Serrate. However, high levels of Delta and Serrate also act in a dominant negative fashion, since cells expressing such levels cannot themselves express cut or wingless. We propose that the boundary of Notch ligand along the normal margin plays a similar role as part of a dynamic feedback loop that maintains the tripartite pattern of margin gene expression.

The specification of distinct cell types within the growing wing disc epithelium depends in large part on interactions between adjacent cells or cell populations. Many of these interactions subdivide the disc in a step-wise fashion. Thus, the disc is initially divided into a small number of lineage compartments, between which cells will not mix. The wing disc is first divided into anterior and posterior (A/P) compartments, and later into dorsal and ventral (D/V) compartments. Interactions between cells in adjacent compartments can then locally define specific cells at compartment boundaries; for instance, Hedgehog secreted by posterior cells signals to cells just to the anterior of the A/P boundary. Finally, boundary cells can themselves subdivide into smaller boundary-specific regions and signal to cells further from the boundary (reviewed in Blair, 1995; Lawrence and Struhl, 1996).

The Notch (N) and Wingless (Wg) signaling pathways play important roles during the development of imaginal discs (pathways reviewed in Artavanis-Tsakonas et al., 1995; Klin-gensmith and Nusse, 1994). In the wing, these pathways mediate ongoing patterning processes, which overlap in space and time. It is thought that, early in development, N and/or Wg are responsible for the reciprocal signaling between dorsal and ventral compartments, which defines cells near the D/V boundary (Couso et al., 1995; Kim et al., 1995, 1996; Rulifson and Blair, 1995; Diaz-Benjumea and Cohen, 1995; Doherty et al., 1996; de Celis et al., 1996a,b; Neumann and Cohen, 1996; Jonsson and Knust, 1996). Later in development these signals help subdivide the region near the D/V boundary into a number of distinct subregions, and maintain those subdivisions during growth and metamorphosis (Phillips and Whittle, 1993; Couso et al., 1994; Rulifson and Blair, 1995; Rulifson et al., 1996). In this paper, we investigate the distinct roles of N and Wg in establishing and maintaining margin-specific regions of gene expression, concentrating especially on the function and regulation of cut (ct).

ct encodes a homeodomain transcription factor (Blochlinger et al., 1988) with significant structural and functional similarity to several vertebrate proteins (see Ludlow et al., 1996). ct has a well-established role specifying neuronal cell fates within the embryonic peripheral nervous system (PNS), where ct is expressed in external sensory organs (Blochlinger et al., 1990). Removing ct transforms external sensory organs into chordotonal organs (Bodmer et al., 1987), while ectopic expression of ct causes chordotonal organs to differentiate as external sensory organs (Blochlinger et al., 1991). Thus, ct can function as a bimodal switch during cell fate decisions.

ct also plays a distinct role during the development of the D/V boundary in the wing imaginal disc, the site of the future margin of the wing blade. The D/V boundary is first established in mid-second instar at the junction between dorsal, apterous-expressing and ventral, non-expressing cells (reviewed in Blair, 1995). ct is one of several ‘margin-specific’ genes expressed in response to the apterous boundary (Blair, unpublished data). ct is initially expressed in a narrow row of cells, 2-5 cells wide, along the presumptive wing margin beginning at mid to late third instar (Jack et al., 1991; Blochlinger et al., 1993). This row of cells straddles the D/V boundary, and is largely coincident with the region which, slightly earlier in development, expresses wg and the vestigial (vg) intron 2 enhancer (Blair, 1993, 1994; Williams et al., 1994). These ‘edge’ cells (Couso et al., 1994) delineate a distinct subregion of margin cells located between the dorsal and ventral rows of margin bristle precursors; while the bristle precursors, like other external sensory organs, eventually express ct, they only begin doing so several hours after pupariation (Blair, 1993). Loss of ct expression from the edge cells results in the loss not only of the ct-expressing edge cells and bristles, but also of adjacent epithelial cells, which do not express ct; the cell loss is apparently due to cell death during subsequent pupal stages (Jack et al., 1991; Dorsett, 1993).

Thus, the ct transcription factor is required for a long-range signal or process that maintains cells both in and adjacent to the region of ct expression. The wing defects observed in ct mutants are similar to those caused by reductions in Wg signaling. Removing ct from both sides of the D/V boundary results in extensive notching of the margin (Jack et al., 1991; Dorsett, 1993), while reducing ct function on one side only occasionally induces notching (Santamaria and Garcia-Bellido, 1975; see Discussion). Similarly, removing Wg or the ability to receive the Wg signal from both sides of the D/V boundary results in notching of the margin; removing the ability to receive Wg from one side of the D/V results in the autonomous loss of margin bristles and proneural gene expression without notching (Baker, 1988b; Couso et al., 1994; Diaz-Benjumea and Cohen, 1995; Axelrod et al., 1996; Rulifson et al., 1996). Removing Wg function during mid to late third instar, using temperature-sensitive alleles, also results in incomplete formation and/or loss of wing margin structures (Phillips and Whittle, 1993; Couso et al., 1994; Diaz-Benjumea and Cohen, 1995). We will show below that ct is in fact required for the maintenance of margin wg expression; this loss of wg may play a role in the ct wing notching phenotype (see Discussion).

However, it is unclear how ct expression is established and maintained. The evidence to date suggests that ct expression is regulated either by N, wg, or both. N is required during the third instar for the formation and maintenance of adult wing margin structures, and the expression at late third instar of many margin-specific genes; reduction or loss of the N ligands encoded by Delta (Dl) or Serrate (Ser) also induces margin notching in adult wings and the loss or reduction of margin-specific gene expression (Shellenbarger and Mohler, 1978; Jack and DeLotto, 1992; Parody and Muskavitch, 1993; Speicher et al., 1994; de Celis and Garcia-Bellido, 1994a; Thomas et al., 1995; Rulifson and Blair, 1995; Kim et al., 1995; Diaz-Benjumea and Cohen; 1995; Couso et al., 1995; Doherty et al., 1996; de Celis et al., 1996a; Jonsson and Knust, 1996). ct expression is reduced in N hypomorphs and by the dominant allele of Ser (Jack and DeLotto, 1992; Thomas et al., 1995; de Celis et al., 1996a), and is lost from Suppresser of Hairless (Su(H)) clones (Neumann and Cohen, 1996). We will show below that ct is also lost from N clones. N gain-of-function mutations, or overexpression of N or N ligands, can induce ectopic ct expression (Thomas et al., 1995; Doherty et al., 1996; de Celis et al., 1996a; Neumann and Cohen, 1996; Jonsson and Knust, 1996). However, manipulations that cause gain or loss of N signaling also result in the ectopic expression or loss of wg, respectively (Thomas et al., 1995; Rulifson and Blair, 1995; Kim et al., 1995, 1996; Couso et al., 1995; Diaz-Benjumea and Cohen, 1995; Doherty et al., 1996; de Celis et al., 1996a; Neumann and Cohen, 1996; Jonsson and Knust, 1996). Since removal of Wg function using either a temperature-sensitive allele or null clones results in a loss of margin ct expression (Couso et al., 1994; Neumann and Cohen, 1996), it is possible that these N-mediated effects are indirect.

Therefore, we have used clonal analysis and temperature-sensitive mutants to directly test the role of N and wg in regulating ct expression on the presumptive wing margin. Our results indicate that ct is a direct target of N but not Wg. N function was required autonomously during mid through late third instar for ct expression on the margin. In contrast, ct expression was observed in clones that are unable to receive Wg signal. Our evidence will further suggest that wg acts indirectly to establish or maintain margin ct expression by directing expression of high levels of the N ligands Dl and Ser in cells adjacent to the ct and wg-expressing cells.

However, this presents an apparent paradox, as ct is not expressed in cells expressing high levels of Dl and Ser; Dl and Ser expression is highest in cells to either side of the edge cells and drops abruptly within the edge cells themselves. We will demonstrate that cells expressing high levels of Dl and Ser cannot express ct and wg, but are capable of triggering margin-like levels of ct and wg in adjacent cells lacking Dl and Ser. Thus, high levels of Dl and Ser appear to act in a cell autonomous dominant negative fashion. We propose that the boundary between flanking cells, which express high levels of Dl and Ser, and the edge cells, which express much lower levels, directs or maintains ct and wg expression within the edge cells.

All genetics, clone generation, gene overexpression, immunohisto-chemistry, in situ hybridization, and light and confocal microscopy were as previously described (Rulifson and Blair, 1995; Rulifson et al., 1996), with the following additions.

Primary antisera: 1/400 rabbit anti-Dsh (kindly provided by R. Nusse), 1/1000 guinea pig anti-Dl (kindly provided by M. Muskavitch), 1/1000 rabbit anti-Ser (Speicher et al., 1994; kindly provided by E. Knust), 1/2000 rabbit anti-Cut (kindly provided by K. Blochlinger), 1/1000 rat anti-Apterous (Lundgren et al., 1995; kindly provided by J. Thomas).

Mutant stocks: ctC145 is a lethal amorphic allele (Bodmer et al., 1987) that eliminates anti-Cut staining (not shown); we generated the ctC145FRT18A line. ct2s was kindly provided by P. Morcillo. Dlrev10 is a null allele (Doherty et al., 1996); the FRT82BDlrev10 line was kindly provided by D. Doherty. We used DlRF/DlB2 for Dlts experiments; DlRF is a temperature-sensitive allele (Parody and Muskavitch, 1993) and DlB2 is an amorphic allele, both kindly provided by M. Muskavitch. SerRX82 and SerRX106 are null alleles (Thomas et al., 1991; Spreicher et al., 1994). We generated the FRT82BSerRX106 line; the FRT82BSerRX82Dlrev10 was kindly provided by G. Struhl. dsh clones used svbYP17bdshv26FRT101 or y w dsh75FRT101, which gave identical results. N clones used N55e11FRT18A, Ndshclones used N1081svbYP17bdshv26FRT101, wg used FRT42DwgCX4 and sgg-zw3 used sgg-zw3D127FRT18A. Each was crossed to appropriate πM-FRT-FLP stocks.

Gene expression on the developing wing margin

The expression patterns of ct, wg and the N ligands Dl and Ser change during development of the wing disc. From mid to late second instar (60-48 hours before pupariation, BP) Ser is expressed throughout the dorsal compartment (Couso et al., 1995; Diaz-Benjumea and Cohen, 1995); Dl is expressed in both dorsal and ventral compartments (Doherty et al., 1996; de Celis et al., 1996a); wg is expressed in a ventral domain that overlaps slightly into the dorsal compartment (Couso et al., 1993, 1994; Williams et al., 1993; Phillips and Whittle, 1993; Ng et al., 1996). At early to mid third instar (48-24 hours BP), this pattern changes. Dl and Ser both are expressed at higher levels near the D/V boundary. wg is expressed throughout the prospective wing blade and, at higher levels, in a wide stripe concentrated near the margin; our results indicate that this stripe is initially stronger on the ventral side of the D/V boundary (Fig. 1A), coincident with the higher expression of Dl (Fig. 1B).

Fig. 1.

Expression patterns in wg-LacZ wing discs, stained with anti-β-gal (green). In this and subsequent figures, anterior is up and dorso-proximal left; A-C are at the same magnification; D is at lower magnification. (A) Early-mid third instar disc, stained also with anti-Ap to show dorsal compartment (red). Note wg-lacZ expression throughout the prospective wing blade and heightened expression along the margin, largely on the ventral side. (B) Mid third instar, stained also with anti-Dl (red) and anti-Ct (blue). Heightened Dl expression follows the heightened margin expression of wg-lacZ. No ct expression is detectable. (C) Mid-late third instar. The region of margin wg-lacZ expression has narrowed to the ‘edge’ cells, and most or all of the wing blade expression is lost. Dl expression has begun to fade from the edge cells, and is heightened in flanking cells and in a broad L3 prevein region. ct is now also detected in the edge cells. (D) Late third instar, stained with anti-Dl and anti-Ct. Dl is expressed in the pre-vein regions and cells flanking the margin edge cells. ct is expressed in the edge cells, as is wg-lacZ (not shown).

Fig. 1.

Expression patterns in wg-LacZ wing discs, stained with anti-β-gal (green). In this and subsequent figures, anterior is up and dorso-proximal left; A-C are at the same magnification; D is at lower magnification. (A) Early-mid third instar disc, stained also with anti-Ap to show dorsal compartment (red). Note wg-lacZ expression throughout the prospective wing blade and heightened expression along the margin, largely on the ventral side. (B) Mid third instar, stained also with anti-Dl (red) and anti-Ct (blue). Heightened Dl expression follows the heightened margin expression of wg-lacZ. No ct expression is detectable. (C) Mid-late third instar. The region of margin wg-lacZ expression has narrowed to the ‘edge’ cells, and most or all of the wing blade expression is lost. Dl expression has begun to fade from the edge cells, and is heightened in flanking cells and in a broad L3 prevein region. ct is now also detected in the edge cells. (D) Late third instar, stained with anti-Dl and anti-Ct. Dl is expressed in the pre-vein regions and cells flanking the margin edge cells. ct is expressed in the edge cells, as is wg-lacZ (not shown).

From mid to late third instar (24-12 hours BP), this pattern further refines (Fig. 1B-D). wg becomes expressed almost exclusively in a narrow stripe of cells straddling the D/V boundary termed the ‘edge’ cells (Baker, 1988a; Blair, 1993, 1994; Couso et al., 1993, 1994). Dl and Ser are expressed at higher levels immediately flanking this stripe but levels are lowered within the edge cells (Kooh et al., 1993; Speicher et al., 1994). Dl and Ser are also expressed at high levels along broad ‘prevein’ regions, and Ser retains a dorsal emphasis at late third instar. ct also becomes expressed in the edge cells (Jack et al., 1991; Blochlinger et al., 1993; Blair, 1993) at approximately the same stage as when the decrease in Dl and Ser levels within the edge cells first becomes apparent (Fig. 1B,C). Thus, by late third instar (12-0 hours BP), gene expression defines a tripartite domain of cells about the D/V boundary: the edge cell region, approximately 2-5 cells wide, and the two flanking regions (Fig. 1D). The flanking regions in the anterior also express high levels of members of the achaete-scute complex (Romani et al., 1989), and thus approximate the ‘proneural’ regions from which dorsal and ventral rows of margin sensory bristles arise. wg is also expressed in two rings surrounding the presumptive wing blade and in a stripe in the notal region of the disc. N expression at late third instar varies spatially (Fehon et al., 1991; Hing et al., 1994), but is at significant levels throughout the disc (see Fig. 4B in Rulifson and Blair, 1995).

cut is autonomously required to maintain margin wingless expression

During late third instar, ct and wg share a common domain of expression along the presumptive wing margin and many aspects of their mutant phenotypes appear consistent (see Discussion). We have therefore tested whether ct mutations alter margin wg expression.

In ct2s, a small deletion specifically disrupts the function of the ct wing margin enhancer; this enhancer is necessary and sufficient to drive ct expression along the wing margin (Jack et al., 1991; Mogila et al., 1992; Dorsett, 1993). We examined the distribution of both wg protein and RNA in ct2s wing discs at mid-late third instar and white prepupal (WPP, 0 hour BP) stages. The mid-late third instar discs displayed a nearly wild-type distribution of wg transcript and protein along the margin (Fig. 2B). In WPP discs, however, there was significant reduction of wg transcript levels, varying from thinning of the wg-expressing stripe to its complete loss (Fig. 2C). Similar results were observed using anti-Wg (not shown).

Fig. 2.

Regulation of wg expression by ct. (A-C) In situ hybridization showing levels of wg transcript along prospective wing margins. (A) Wild type, WPP stage. (B) ct2s, mid-late third instar; wg expression is nearly wild type. (C) ct2s, WPP; wg expression is almost totally eliminated. (D,E) anti-Wg staining (red) in ctclones (indicated by absence of green πM marker). (D) ctclone limited to dorsal compartment eliminates dorsal but not ventral wg expression (note thinning of normal wg-expressing stripe). (E) ctclone that crossed the D/V boundary autonomously eliminates most or all anti-Wg staining.

Fig. 2.

Regulation of wg expression by ct. (A-C) In situ hybridization showing levels of wg transcript along prospective wing margins. (A) Wild type, WPP stage. (B) ct2s, mid-late third instar; wg expression is nearly wild type. (C) ct2s, WPP; wg expression is almost totally eliminated. (D,E) anti-Wg staining (red) in ctclones (indicated by absence of green πM marker). (D) ctclone limited to dorsal compartment eliminates dorsal but not ventral wg expression (note thinning of normal wg-expressing stripe). (E) ctclone that crossed the D/V boundary autonomously eliminates most or all anti-Wg staining.

This ct requirement is cell autonomous. ctclones that inter-sected the edge cells showed cell autonomous reductions or loss of wg (Fig. 2D,E). While ctcells in some younger discs occasionally displayed traces of Wg protein, clones in older discs did not show any detectable wg expression. ct clones that crossed the compartment boundary completely lacked wg expression on both sides of the margin (Fig. 2E), while those positioned in the interior of the wing blade had no effect upon wg expression. Therefore, while the initiation of wg expression along the margin during mid third instar is not dependent on ct, maintenance of wg through late third instar is ct dependent. The expression of wg in the two rings encircling the presumptive wing and in the notum are not coincident with ct expression during third instar and were not sensitive to ct loss.

N is required for cut expression on the margin

While previous results suggest that N is involved ct expression (see Introduction), the autonomy and penetrance of this requirement has not been previously tested. Therefore, we examined ct expression in clones lacking N (Fig. 3A). Nclones confined to either the dorsal or ventral compartment caused a cell autonomous loss of ct expression. Such clones also had domineering non-autonomous effects, such that clones that abutted the D/V boundary on one side without crossing resulted in a loss of ct expression on both sides of the D/V boundary. In some cases ct expression was also lost a few cell diameters anterior or posterior to the clone; however, this may be due to the sporadic loss of ct observed in Nheterozygotes (not shown). Clones in the interior of the wing did not show any detectable phenotype. These N phenotypes are similar to those previously described in adult wings (de Celis and Garcia-Bellido, 1994a) and in activating wg and the vg second intron enhancer (Rulifson and Blair, 1995; Diaz-Benjumea and Cohen, 1995). The domineering aspects of the phenotypes are likely due to N’s earlier role in reciprocal signaling between dorsal and ventral compartments (see Introduction).

Fig. 3.

Regulation of margin ct and wg by N, observed at late third instar. (A) Anti-Ct staining (red) in Nclones (indicated by absence of green πM marker, −). ct expression is eliminated both within and adjacent to clones that are in contact with margin (upper, lower clones), but not adjacent to clones not in contact with margin (middle clone). (B,C) Anti-Ct (red) and anti-Wg (green) staining in Nts. (B) Reared at permissive temperature. (C) Reared at non-permissive temperature for previous 24 hours. Reduction of margin anti-Cut staining is more extreme than reduction of anti-Wg. ct is still expressed in the precursors of the campaniform sensilla (arrow).

Fig. 3.

Regulation of margin ct and wg by N, observed at late third instar. (A) Anti-Ct staining (red) in Nclones (indicated by absence of green πM marker, −). ct expression is eliminated both within and adjacent to clones that are in contact with margin (upper, lower clones), but not adjacent to clones not in contact with margin (middle clone). (B,C) Anti-Ct (red) and anti-Wg (green) staining in Nts. (B) Reared at permissive temperature. (C) Reared at non-permissive temperature for previous 24 hours. Reduction of margin anti-Cut staining is more extreme than reduction of anti-Wg. ct is still expressed in the precursors of the campaniform sensilla (arrow).

Analysis of the temperature-sensitive N genotype, N55e11/Nts1, show that N is required for ct expression from mid to late third instar. Larvae shifted to the nonpermissive temperature from 24 to 0 hours BP showed a complete loss of ct expression along the margin (Fig. 3B,C). Larvae shifted from 12 to 0 hours BP showed thinning and incomplete loss of expression which was most pronounced at the distal tip of the margin (not shown). wg expression is also lost from such discs (Rulifson and Blair, 1995); double staining for wg and ct expression showed that ct loss was always more extreme (Fig. 3C).

Reception of the Wg signal is not required for cut expression

To test if wg is required directly for ct expression, clones were generated that were incapable of receiving the Wg signal. dsh, a ubiquitously expressed cytoplasmic protein, is required in a cell-autonomous fashion for the reception of the Wg signal (Klingensmith et al., 1994; Noordermeer et al., 1994; Theisen et al., 1994; Couso et al., 1994; Rulifson et al., 1996). We found that dsh clones that intersected the ct-expressing cells on the margin still had the capacity to express ct (Fig. 4A,B). While occasional loss was observed, especially in large clones that extended outside of the edge cell region, many clones had completely normal expression. Therefore, ct expression can be stimulated in the absence of Wg signal reception and the loss of ct in wgts wings and wg clones (Couso et al., 1994; Neumann and Cohen, 1996) is an indirect effect.

It was shown previously that dsh clones near the normal region of margin wg expression ectopically express wg at margin-like levels, indicating that Wg signaling represses wg expression in the flanking cells (Rulifson et al., 1996). Similarly, dsh clones occasionally contained ectopic ct-expressing cells along clone boundaries, in cells up to 10 cell diameters from the margin (Fig. 4B). No ectopic expression was observed outside the clones. Ectopic ct expression was less common and usually less extensive than the ectopic expression of wg (not shown).

Recent results suggest that Dsh can inhibit N activity by directly binding to the intracellular domain of N (Axelrod et al., 1996). This may account for the ectopic expression phenotype, as loss of dsh should derepress N activity, leading to increases in N-dependent ct and wg expression. To test the possibility that ct expression within clones is N dependent, we generated Ndsh double mutant clones. In contrast to dsh clones, which often expressed ct, Ndsh clones displayed the N phenotype: an autonomous loss of ct and domineering nonautonomy on the wing margin (Fig. 5A).

Fig. 4.

Regulation of ct by Wg pathway. (A,B). dshclones; ct is still expressed in these clones. Anti-Ct staining is shown in red. (A) Clones marked by absence of anti-Dsh staining (green). ct expression is slightly expanded in posterior clone. (B) Clones marked by absence of green πM marker. Ectopic ct-expressing cells are present in the anterior clone, distant from the margin (arrow). (C,C′,C″) en-GAL4 UAS-dsh disc, stained with anti-Ct (green) and anti-Wg (red). Posterior expression of dsh, driven by en-GAL4, reduces anti-Wg and eliminates anti-Ct staining. (C′) Detail of anterior. (C″) Detail of posterior. (D) Disc containing wg-expressing Ubx>f>wg+ FLP-out clones, stained with anti-Ct (green) and anti-Wg (red). Sizable FLP-out clone (*) does not express ct.

Fig. 4.

Regulation of ct by Wg pathway. (A,B). dshclones; ct is still expressed in these clones. Anti-Ct staining is shown in red. (A) Clones marked by absence of anti-Dsh staining (green). ct expression is slightly expanded in posterior clone. (B) Clones marked by absence of green πM marker. Ectopic ct-expressing cells are present in the anterior clone, distant from the margin (arrow). (C,C′,C″) en-GAL4 UAS-dsh disc, stained with anti-Ct (green) and anti-Wg (red). Posterior expression of dsh, driven by en-GAL4, reduces anti-Wg and eliminates anti-Ct staining. (C′) Detail of anterior. (C″) Detail of posterior. (D) Disc containing wg-expressing Ubx>f>wg+ FLP-out clones, stained with anti-Ct (green) and anti-Wg (red). Sizable FLP-out clone (*) does not express ct.

Fig. 5.

Regulation of margin Dl and Ser by Wg pathway. (A) Ndsh clone, marked by absence of green πM marker. Anti-Ct (red) and anti-Dl (blue) staining is eliminated in the clone. (B) dshclone, marked by absence of green πM marker. Anti-Ser (red) and anti-Dl (blue) staining is eliminated in the clone. (C) Disc containing wg-expressing Ubx>f>wg+ FLP-out clones, stained with anti-Wg (green), anti-Ser (red), and anti-Dl (blue). wg-expressing clone (*) raises Ser and Dl expression to margin-like levels. Phenotypes induced using the Ubx>f>wg+ FLP-out are largely cell autonomous, apparently due to the lower levels of wg expressed in the clones (Diaz-Benjumea and Cohen, 1995). (D) sgg-zw3clones, marked by absence of green πM marker (outlined in other panels). Anti-Ser (red) and anti-Dl (blue) staining is raised in clones to margin-like levels.

Fig. 5.

Regulation of margin Dl and Ser by Wg pathway. (A) Ndsh clone, marked by absence of green πM marker. Anti-Ct (red) and anti-Dl (blue) staining is eliminated in the clone. (B) dshclone, marked by absence of green πM marker. Anti-Ser (red) and anti-Dl (blue) staining is eliminated in the clone. (C) Disc containing wg-expressing Ubx>f>wg+ FLP-out clones, stained with anti-Wg (green), anti-Ser (red), and anti-Dl (blue). wg-expressing clone (*) raises Ser and Dl expression to margin-like levels. Phenotypes induced using the Ubx>f>wg+ FLP-out are largely cell autonomous, apparently due to the lower levels of wg expressed in the clones (Diaz-Benjumea and Cohen, 1995). (D) sgg-zw3clones, marked by absence of green πM marker (outlined in other panels). Anti-Ser (red) and anti-Dl (blue) staining is raised in clones to margin-like levels.

Wingless signaling is not sufficient to stimulate cut expression

We further tested whether Wg was able to stimulate ct expression using the Ubx>f>wg+ FLP-out construct (Diaz-Benjumea and Cohen, 1995) to generate clones of wg-expressing cells within the wing blade. The levels of wg expressed in these clones can elicit other wg-dependent events, such as the formation of margin-like bristles (Diaz-Benjumea and Cohen, 1995). However, such clones failed to express detectable levels of ct (Fig. 4D). This is consistent with the result of Neumann and Cohen (1996) using GAL4-driven UAS-wg.

The overexpression of dsh also mimics many aspects of Wg signaling (Axelrod et al., 1996), but failed to elicit ectopic ct expression. In fact, overexpressing dsh in the posterior compartment of the wing using enGal4/UASdsh resulted in a reduction of normal margin ct expression (Fig. 4C,C′,C″), even more dramatic than the reduction of wg expression observed previously (Rulifson et al., 1996). Since dsh overexpression appears to inhibit N activity (Axelrod et al., 1996), this result is consistent with our proposal that ct is a direct target of N signaling.

Wingless is necessary and sufficient for high levels of N ligands flanking the margin

If Wg signaling is not directly required for ct expression, why is ct lost in a wgts wing disc and in wg clones, and sporadically lost from dshclones? One possibility is that Wg affects N activity indirectly by regulating the levels of N ligand available near the margin. At mid to late third instar, the period during which ct is sensitive to N activity (see above), the N ligands encoded by Dl and Ser are normally expressed at higher levels immediately flanking the wg- and ct-expressing edge cells. We will show that Wg signaling is required for these high levels of Dl and Ser.

wg clones that intersected the wg-expressing edge cells often induced lowered levels of Dl and Ser (not shown). The wgphenotype was not cell autonomous, as expected since Wg from outside the clone can act over a distance of several cell diameters; Dl and Ser appeared at wild-type levels in cells two to three cell diameters away from wg-expressing cells at clone boundaries. Loss was seen both within clones that crossed the D/V boundary and those limited to either compartment.

dsh clones of any size within the flanking cells displayed cell autonomous reduction of Dl and Ser (Fig. 5B). Identical loss was observed in Ndsh clones (Fig. 5A), indicating that the loss was not due to derepression of N signaling. Dl and Ser expression along the presumptive wing veins was not reliably lost from dshclones.

Heightened Wg signaling was also sufficient to activate high levels of Dl and Ser expression. wg overexpressing clones, generated using the Ubx>f>wg+ FLP-out construct (Diaz-Benjumea and Cohen, 1995), expressed high levels of both Dl and Ser (Fig. 5C). Cells lacking the Shaggy-zeste white 3 (Sggzw3) serine-threonine kinase mimic reception of the Wg signal (Siegfried et al., 1992; Blair, 1994) and, in the adult wing, form margin-like bristles (Simpson et al., 1988). sgg-zw3 clones throughout the wing pouch contained elevated levels of Dl and Ser (Fig. 5D). Although the anti-Dl and anti-Ser staining appeared concentrated in the center of some sgg-zw3 clones, we feel that this apparent non-autonomy is an artifact, caused by the apical concentration of Dl and Ser and the abnormal arrangement of cells within these clones (see Blair, 1994).

N ligands are required for normal ct and wg expression on the wing margin

As with N, Dl helps direct normal ct and wg expression during late third instar. DlRF is temperature sensitive (Parody and Muskavitch, 1993), although DlRF/Dl discs showed partial reduction of ct and wg expression even when reared at the permissive temperature (Fig. 6A). This reduction became more extreme, however, when larvae were shifted to the non-permissive temperature for the previous 18 hours, and frequent gaps were observed (Fig. 6B).

Fig. 6.

Regulation of margin ct and wg by Dl and Ser. (A,B) DlRF/Dl discs, stained with anti-Ct (green) and anti-Wg (red). (A) Disc reared at permissive temperature, showing slight thinning and occasional breaks in ct and wg expression, especially near the prospective distal tip. (B) Disc reared at non-permissive temperature for the previous 18 hours. Thinning of ct and wg expression is more extreme, and breaks in expression are more frequent and widespread. (C,D) Large Dl(C) and Ser(D) clones, marked by absence of green πM marker, which crossed the D/V boundary. Nearly normal anti-Ct staining (red) is retained in the clones. (E) Large DlSerdouble mutant clone, marked by absence of green πM marker, which crossed the D/V boundary. Anti-Ct (red) and anti-Wg (blue) staining is disrupted in the clone, except along the clone boundaries.

Fig. 6.

Regulation of margin ct and wg by Dl and Ser. (A,B) DlRF/Dl discs, stained with anti-Ct (green) and anti-Wg (red). (A) Disc reared at permissive temperature, showing slight thinning and occasional breaks in ct and wg expression, especially near the prospective distal tip. (B) Disc reared at non-permissive temperature for the previous 18 hours. Thinning of ct and wg expression is more extreme, and breaks in expression are more frequent and widespread. (C,D) Large Dl(C) and Ser(D) clones, marked by absence of green πM marker, which crossed the D/V boundary. Nearly normal anti-Ct staining (red) is retained in the clones. (E) Large DlSerdouble mutant clone, marked by absence of green πM marker, which crossed the D/V boundary. Anti-Ct (red) and anti-Wg (blue) staining is disrupted in the clone, except along the clone boundaries.

Clonal loss of either Dl or Ser alone also occasionally induced loss of margin ct or wg expression. However, such loss was not reliable. Even in large clones that crossed the D/V boundary, loss or reductions in ct or wg expression were sporadic and, in many clones, ct or wg expression was almost normal (Fig. 6C,D). In adults, ventral Dlor dorsal Serclones that contact the D/V boundary can induce loss of margin and adjacent tissues (Couso et al., 1995; Diaz-Benjumea and Cohen, 1995; Doherty et al., 1996; de Celis et al., 1996a); although little or no loss is observed in some smaller adult clones, clones that cross the D/V boundary reliably induce extensive notching (de Celis et al., 1996a). Our results in imaginal discs suggest that some of the notching observed in adults may be due to cell loss during early pupal stages, as occurs in N/+ and SerD/+ flies (Jack and DeLotto, 1992; Thomas et al., 1995).

Comparison of single mutant clones with double mutant DlSer clones show that Dl and Ser functions are partially redundant. While clonal loss of Dl or Ser alone had only variable effects, all DlSer double mutant clones that crossed the D/V boundary completely lacked normal margin ct and wg expression, except 1-2 cell diameters inside the clone boundaries, (Fig. 6E). The rescue at clone boundaries was expected, as cell-bound Dl and Ser should be able to signal to cells immediately inside the clone, and there are also indications that normal Ser can be secreted (Couso et al., 1995). Clones observed in pupal wings (24-36 hours AP) showed wing notching phenotypes, associated with clones apparently limited to either the dorsal or ventral compartments (not shown). As expected from the phenotypes of Dl and Ser single mutant clones (Kim et al., 1995; Couso et al., 1995; Diaz-Benjumea and Cohen, 1995; Doherty et al., 1996; de Celis et al., 1996a), adult wings containing unmarked DlSer clones had regions with lost margin bristles, notched margins and thickened veins.

High levels of Dl and Ser have dominant negative effects on ct and wg expression

If Dl and Ser drive ct and wg expression along the margin, why is there no expression in the regions of highest Dl and Ser expression, that is, in the cells flanking the edge cells? The temperature-sensitive experiments indicate that N and Dl function are required at the same stage when high levels of Dl and Ser are expressed in the flanking cells; the flanking cells must be relatively insensitive to Dl- and Ser-mediated N signaling. The phenotypes of our DlSerclones show that it is in fact the high levels of Dl and Ser that render these cells insensitive.

In order to rule out effects due to earlier functions of Dl and Ser, small clones were generated during third instar. DlSer double mutant clones that lay within or adjacent to the normal margin region of high Dl and Ser expression (approximately 5 cell diameters to either side of the D/V boundary) ectopically expressed ct and wg (Fig. 7A,B). This was true even in extremely small clones of approximately four cells in either the dorsal or ventral flanking regions. Such expression was limited to DlSercells at the edges of the clones. Thus, cells expressing high levels of Dl and Ser can signal to adjacent cells and stimulate ct and wg expression, but cells expressing high levels of Dl and Ser are themselves incapable of receiving the signal. These phenotypes differ from those caused by interfering with earlier apterous-driven signaling between dorsal and ventral compartments, as dorsal clones lacking apterous induce ct and wg expression both within and outside the clone (Blair, unpublished data).

Fig. 7.

Regulation of margin ct and wg by Dl and Ser. Clones marked by absence of green πM marker, stained with anti-Cut (red) or, in A, F, with anti-Wg (blue), or, in H, with anti-HRP. (A,B) Small DlSer double mutant clones, generated during third instar. Ectopic ct and wg expression is observed in clones near the margin, and over slightly wider region in middle of wing in region of vein Dl and Ser expression (arrow in B, Dl/Ser expression not shown). (C) Small Dl clones. No ectopic ct expression is observed. (D) Small Serclones. Occasional ectopic ct expression is observed; this expression is rarer than in DlSerdouble mutant clones, and primarily in dorsal clones. (E). DlSerdouble mutant clone, stained with anti-Ct (red) and anti-Dl (blue). Note expression extends further from the margin on the edge facing the Dl+Ser+/Dl+Ser+ twin spot. Dl expression also appears heightened next to the ct-expressing cells (arrow). (F) Larger DlSerdouble mutant clone, generated during second instar. Ectopic expression extends further from the margin than in A. Also, note that expression occurs only along clone boundary, and is biased towards the margin. (G) Ectopic anti-Scute staining (red) within and adjacent to DlSerdouble mutant clones. (H) Detail of pupal wing (24-36 hours AP), just posterior to anterior margin, containing DlSer clones. Clones contain scattered neurons, visualized with anti-HRP (red).

Fig. 7.

Regulation of margin ct and wg by Dl and Ser. Clones marked by absence of green πM marker, stained with anti-Cut (red) or, in A, F, with anti-Wg (blue), or, in H, with anti-HRP. (A,B) Small DlSer double mutant clones, generated during third instar. Ectopic ct and wg expression is observed in clones near the margin, and over slightly wider region in middle of wing in region of vein Dl and Ser expression (arrow in B, Dl/Ser expression not shown). (C) Small Dl clones. No ectopic ct expression is observed. (D) Small Serclones. Occasional ectopic ct expression is observed; this expression is rarer than in DlSerdouble mutant clones, and primarily in dorsal clones. (E). DlSerdouble mutant clone, stained with anti-Ct (red) and anti-Dl (blue). Note expression extends further from the margin on the edge facing the Dl+Ser+/Dl+Ser+ twin spot. Dl expression also appears heightened next to the ct-expressing cells (arrow). (F) Larger DlSerdouble mutant clone, generated during second instar. Ectopic expression extends further from the margin than in A. Also, note that expression occurs only along clone boundary, and is biased towards the margin. (G) Ectopic anti-Scute staining (red) within and adjacent to DlSerdouble mutant clones. (H) Detail of pupal wing (24-36 hours AP), just posterior to anterior margin, containing DlSer clones. Clones contain scattered neurons, visualized with anti-HRP (red).

Little ectopic expression was observed in either dorsal or ventral Dl clones (Fig. 7C), underscoring the partial redundancy of Dl and Ser in both the dorsal and ventral compartments. Ectopic expression was observed in some Ser clones, but this was not as common as in DlSerclones, and was seen primarily in clones on the dorsal side where Ser expression is highest (Fig. 7D). Thus, the simultaneous absence of Ser uncovers a requirement for Dl in both the dorsal and ventral wing; the simultaneous absence of Dl strengthens the Ser phenotype in the dorsal wing, and uncovers a requirement for Ser in the ventral wing.

Rarely, DlSer clones outside the margin region also expressed ectopic ct and wg. Interestingly, the regions in which such expression appeared correlated with regions of high Dl and Ser expression: they were more common in the endogenous domain of Dl and Ser flanking the veins, most notably in the domain between L3 and L4 (Fig. 7B), and along the edge of clones that abutted Dl+Ser+/Dl+Ser+ twin spots (Fig. 7E). In some cases, the ectopic ct and wg expression was flanked by margin-like levels of Dl and Ser expression, apparently ectopic, in the cells outside the clone (Fig. 7E). Our results above suggest that the Wg secreted by the clone is inducing these high levels of Dl and Ser expression in adjacent cells.

There was, in most cases, a bias towards ectopic expression in clones and at clone boundaries nearest the margin (Fig. 7B,E,F). Interestingly, when clones were generated earlier in development, ct- and wg-expressing cells were formed further from the margin (Fig. 7F). It is possible that the distribution of N ligands controls this region of competence, since both Dl and Ser are more generally expressed in the wing blade at earlier stages (see above). However, other factors, such as Scalloped, may be play a role in the distal bias (see Discussion).

Previous work has shown that ectopic Wg signaling can induce the formation of margin-like regions of proneural gene expression in the anterior of the wing and bristle formation in both anterior and posterior (Simpson et al., 1988; Blair, 1992; Diaz-Benjumea and Cohen, 1995; Axelrod et al., 1996). We have observed that DlSer clones can also induce ectopic anterior proneural gene expression (scute) outside the normal proneural regions (Fig. 7G), presumably induced by the ectopic wg expressed in such clones. Ectopic proneural gene expression was not limited to the DlSer cells, as would be expected from the long-range action of Wg secreted by DlSer cells. Anti-HRP-labeled neurons are formed in dorsal and ventral DlSerclones near the anterior margin of pupal wings (Fig. 7H), but only within the clones, so the levels of proneural gene expression outside the clone are apparently insufficient to induce neuronal development. Ectopic margin-like bristles were found near the anterior and posterior margins of adult wings containing unmarked clones (not shown).

A novel role for Cut on the wing margin

Our study indicates that ct acts during late larval and pupal stages to maintain wg transcription along the presumptive wing margin, and that this role is cell autonomous. Interestingly, this suggests a basis for the long-range effects of ct mutations. During pupal stages, ct mutants lose not only ct-expressing cells but also adjacent epithelial cells, resulting in a notched wing phenotype; these effects may be explained by the loss of the secreted Wg morphogen. Many aspects of ct and wg phenotypes appear consistent. wg is required during mid to late third instar to properly pattern and maintain the presumptive wing margin (Phillips and Whittle, 1993; Couso et al., 1994; Diaz-Benjumea and Cohen, 1995). Reducing ct function during third instar induces similar margin defects (Dorsett, 1993).

However, ct loss only affects the maintenance, not the initiation, of wg expression, and it is unclear whether such a late loss of Wg expression can induce the same amount of cell loss observed along the margins of pupal ct mutant wings. It has also been reported that hypomorphic ct6 clones that do not cross the D/V boundary can in 21% of the cases induce notching and bristle loss both within the clone and in the adjacent wild-type compartment (Santamaria and Garcia-Bellido, 1975). Our results show that such clones should affect wg expression only within the clone. However, when wg clones are limited to one compartment, defects have not been observed (Baker, 1988b; Diaz-Benjumea and Cohen, 1995), suggesting that Wg secreted in the other compartment is sufficient to maintain the margin. Thus, while the loss of Wg is undoubtedly a component of the ct phenotype, it is possible that ct is required for processes on the margin in addition to wg maintenance.

Regulation of cut on the wing margin

ct expression on the margin depends on both N and Wg activity. However, our evidence indicates that only the N requirement is direct. We demonstrated that N is required in a cell autonomous fashion to maintain ct expression on the margin. Thus, ct appears to be like other margin-specific genes: N or Su(H) is required autonomously for the margin expression of wg, the vg second intron enhancer, and certain members of the E(spl) complex (Rulifson and Blair, 1995; Diaz-Benjumea and Cohen, 1995; Doherty et al., 1996; Kim et al., 1996; de Celis et al., 1996a; Neumann and Cohen, 1996).

In contrast, the requirement for wg is indirect. Clones lacking dsh, which are unable to receive the Wg signal (Klin-gensmith et al., 1994; Noordermeer et al., 1994; Theisen et al., 1994; Couso et al., 1994; Rulifson et al., 1996), can continue to express ct. Moreover, ectopic expression of wg or dsh was not sufficient to induce margin-like ct expression (see also Neumann and Cohen, 1996). This is consistent with the previous finding that ct is not expressed sgg-zw3 clones; in other respects, sgg-zw3 cells in the wing blade mimic reception of Wg signals (Blair, 1994). Although Wg signaling is neither necessary nor sufficient for ct expression, it is possible that direct Wg signaling contributes to ct expression. However, the complete loss observed in temperature-sensitive Wg mutations (Couso et al., 1994; Neumann and Cohen, 1996) must be in a large part due to indirect effects.

Regulation of N ligands by Wg provides a plausible mechanism for this indirect effect. We showed that Dl and Ser were required to activate or maintain ct and wg expression and, in addition, that reception of the wg signal can directly modulate the levels of the N ligands Dl and Ser expressed in the cells adjacent to the margin. Dl and Ser expression was lowered or lost in dshclones and was heightened after wg over-expression or the loss of sgg-zw3. It is likely that universal removal of Wg function using a temperature-sensitive allele would also reduce the levels of N ligands expressed adjacent to the margin, and thus the levels of N-dependent ct expression on the margin. It is interesting to note that the loss of N ligands observed in dshclones does not occur because of the heightened N activity thought to be induced by the loss of dsh (Axelrod et al., 1996; Rulifson et al., 1996). Heightened N activity is thought by some to downregulate the levels of N ligands, but Dl and Ser levels were lowered in both dshand Ndshclones.

Although margin-specific genes are all directly sensitive to N activity, they are not expressed in identical patterns. wg, the vg second intron enhancer, and various E(spl) complex members are all expressed at higher levels near the margin beginning in late second or early third instar, while ct is not expressed until mid to late third instar. Each is also expressed in different sized domains that change during development. Some of these differences might be due to different sensitivities to N signaling. For example, ct expression might require higher levels or more sustained N activity. This is consistent with our finding that ct was more sensitive to Nts than wg.

Our dsh and dsh-overexpression phenotypes could also be interpreted in this manner. Dsh can inhibit N activity, perhaps by direct N-Dsh binding (Axelrod et al., 1996). Thus, the N targets wg and ct should react to changes in Dsh levels. As expected, clones lacking dsh expand the domain of wg expression, while dsh overexpression reduces normal margin wg expression (Rulifson et al., 1996). ct reacted similarly to such manipulations, but appeared to require higher levels of N activity than wg. In dsh clones, ectopic ct expression was observed less frequently and was restricted to a smaller domain within the clone than was expression of wg. ct expression on the wing margin was also more sensitive to suppression by overexpression of dsh than wg.

However, it is likely that additional factors regulate margin-specific gene expression. Recent reports suggest that ct is directly regulated by Scalloped, a transcription factor that is expressed throughout the wing blade but at higher levels near the margin (Campbell et al., 1992; Williams et al., 1993), as Scalloped binds to the wing margin enhancer of ct (Morcillo et al., 1996). Although ct-expressing cells autonomously require N and Su(H) activity (Neumann and Cohen, 1996; this study), little or no Su(H) binding is found to the ct wing margin enhancer (Morcillo et al., 1996). It is thought that most or all N signaling is mediated by the Su(H) transcription factor (reviewed in Artavanis-Tsakonas et al., 1995). Thus, ct may be regulated by some other Su(H) target.

Activation and repression: dual roles for Dl and Ser

At mid to late third instar, the N ligands Dl and Ser are expressed at high levels in cells flanking the ct- and wg-expressing edge cells. Temperature-sensitive alleles were used to show that N and Dl activities were required during this period for ct and wg. Nonetheless, ct and wg are not expressed in the flanking cells.

Previous analyses have suggested that Dl and Ser can act both as activators and repressors of N. Overexpression of Dl or Ser induces ectopic margin-like gene expression, but only in cells adjacent to the overexpressing cells, and normal margin gene expression is also inhibited in the overexpressing cells (Speicher et al., 1994; Thomas et al., 1995; Kim et al., 1995; Couso et al., 1995; Diaz-Benjumea and Cohen, 1995; Doherty et al., 1996; de Celis et al., 1996a,b; Jonsson and Knust, 1996). Overexpression of Dl or Ser can also induce other N loss-of-function phenotypes (Sun and Artavanis-Tsakonas, 1996; T. R. Parody and M. A. T. Muskavitch, personal communication), and raising the dosage of Dl+ can increase the severity of N defects and decrease hypersensitive NAbruptex defects (de la Concha et al., 1988; de Celis and Garcia-Bellido, 1994b). Our experiments show that even wild-type levels of Dl and Ser can both activate and repress N targets during normal development. When SerDl double mutant clones were generated in the flanking region, where Ser and Dl levels were highest, ectopic wg and ct expression was induced within the clone in cells adjacent to wild-type cells. The phenotype was not due to some earlier function of Ser and Dl in wing development, as it held even in very small clones generated during the third instar. Rather, cells expressing high levels of Dl and Ser can signal to adjacent cells and induce ct and wg expression, but are themselves incapable of receiving the signal. Signaling is therefore highest at sharp boundaries of ligand expression.

We suggest that a similar event occurs along the normal margin at the boundary between the edge and flanking cells (Fig. 8). We suppose that signaling between dorsal and ventral compartments biases cells at the D/V boundary to take on the edge cell fate. This signaling could be mediated by high levels of N activity, or some unknown signal. Cells immediately flanking the D/V boundary would express their edge cell bias by expressing ct, higher levels of wg and lower levels of N ligand, and would themselves become relatively insensitive to Wg signal. Hypothetically, ct expression itself may be responsible for making these cells insensitive to Wg. Once this bias between the edge cells and the flanking cells is established, the signaling between the two regions would sharpen and maintain the boundary between them. Wg secreted by the edge cells would induce high levels of N ligand in the flanking cells, as shown in this study, and also repress wg expression in those cells, as shown previously (Rulifson et al., 1996). The insensitivity of the edge cells to Wg would, however, prevent these events from occurring in the edge cells. N activity would be repressed in the flanking cells by the high levels of N ligand, but would be high in the edge cells through the non-autonomous activity of Dl and Ser in the flanking cells, rein-forcing the expression of wg and ct.

Fig. 8.

Model of reciprocal signaling between edge and flanking cells on the wing margin. See text.

Fig. 8.

Model of reciprocal signaling between edge and flanking cells on the wing margin. See text.

One implication of our results is that high levels of ligand expression alone cannot be taken to indicate high levels of N activity, even in the wild-type wing. Rather, boundaries of ligand expression may be more critical. Apparently, such boundaries must also be fairly sharp. wg-FLPout and sgg-zw3 clones induce higher N ligand levels, but the boundaries between such clones and the lower but still substantial levels of ligands in surrounding cells do not elicit ct or wg expression. Moreover, ligand misexpression driven using ptc-GAL4 is more effective at the sharp posterior boundary of ptc expression than at the fuzzy anterior one (Kim et al., 1995; Couso et al., 1995; Diaz-Benjumea and Cohen, 1995; Doherty et al., 1996; Jonsson and Knust, 1996)

But how could Dl and Ser autonomously repress N activity? A number of studies have sought to illuminate the nature of the interactions that exist between N and its ligands Dl and Ser, as dimerization or multimerization of receptors or ligands constitutes a potentially rich site for regulating the N pathway. In vitro studies using an aggregation assay in S2 cells show that Dl-expressing cells can bind in a homotypic fashion, but N-expressing cells do not (Fehon et al., 1990). Thus, it is possible that situations that favor homotypic Dl-Dl binding could sequester ligand from the N receptor. Dominant negative phenotypes can also be accentuated by expression of extracellular fragments of N ligand, so the activity of ligands may be modified outside the cell (Sun and Artavanis-Tsakonas, 1996; T. R. Parody and M. A. T. Muskavitch, personal communication). However, our data and previous studies suggest that cells with high levels of ligand expression can signal to adjacent cells; all ligand cannot be sequestered in such cells, nor can such ligand be in a permanently inactivated form. Nor does the evidence as yet support the idea that cells receiving high levels of N activity become desensitized, as hypersensitive NAbruptex mutations, or overexpression of wild-type or activated forms of N, does not disrupt development of the normal margin (Doherty et al., 1996; de Celis et al., 1996a,b; Kim et al., 1996). The molecular basis of cell autonomous repression thus remains an interesting puzzle.

We thank Dan Rohwer-Nutter for technical assistance, the Keck Neural Imaging Center for use of its confocal microscope, and Drs H.-M. Chung, J. Kim, and S. Carroll for discussions. This work was supported by grants from the NIH (R01-NS28202) and NSF (IBN-9305209).

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