Genetic Characterization of the Drosophila melanogaster Suppressor of deltex Gene: A Regulator of Notch Signaling

Corresponding author: Martin Baron, School of Biological Sciences, University of Manchester, G38 Stopford Building, Oxford Rd., Manchester M13 9PT, United Kingdom., mbaron{at}man.ac.uk (E-mail).

Communicating editor: K. ANDERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Notch receptor signaling pathway regulates cell differentiation during the development of multicellular organisms. A number of genes are known to be components of the pathway or regulators of the Notch signal. One candidate for a modifier of Notch function is the Drosophila Suppressor of deltex gene [Su(dx)]. We have isolated four new alleles of Su(dx) and mapped the gene between 22B4 and 22C2. Loss-of-function Su(dx) mutations were found to suppress phenotypes resulting from loss-of-function of Notch signaling and to enhance gain-of-function Notch mutations. Hairless, a mutation in a known negative regulator of the Notch pathway, was also enhanced by Su(dx). Phenotypes were identified for Su(dx) in wing vein development, and a role was demonstrated for the gene between 20 and 30 hr after puparium formation. This corresponds to the period when the Notch protein is involved in refining the vein competent territories. Taken together, our data indicate a role for Su(dx) as a negative regulator of Notch function.

DURING the development of multicellular organisms, cell-to-cell signaling plays an important part in specifying cell fates. One important signaling pathway is mediated by the Notch receptor, which is involved in many key cell fate decisions. Notch is a transmembrane protein that is conserved during evolution. The extracellular domain contains 36 epidermal growth factor (EGF)-like repeats and three lin12/Notch cysteine-rich repeats. The intracellular domain includes six cdc10/ankyrin repeats and a proline, glutamate, serine, threonine-rich (PEST) sequence (reviewed by ARTAVANIS-TSAKONAS et al. 1995 ).

In Drosophila melanogaster, Notch is involved in the development of the central and peripheral nervous systems, oogenesis, and eye and wing differentiation (FORTINI and ARTAVANIS-TSAKONAS 1993 ). During neurogenesis, Notch activity is required for the epidermal vs. neural cell fate decision. Cells receiving the Notch signal are inhibited from the neural fate in favor of the epidermal fate by a process termed "lateral inhibition" (ARTAVANIS-TSAKONAS and SIMPSON 1991 ; HEITZLER and SIMPSON 1991 ). Notch signaling is activated by binding of the transmembrane protein Delta to the Notch receptor on an adjacent cell (HEITZLER and SIMPSON 1991 ; REBAY et al. 1991 ). In response to Notch activation, Suppressor of Hairless, a transcription factor, stimulates the transcription of downstream genes, including those of the Enhancer of split [E(spl)] complex (SCHWEISGUTH and POSAKONY 1992 ; FORTINI and ARTAVANIS-TSAKONAS 1994 ; BAILEY and POSAKONY 1995 ; LECOURTOIS and SCHWEISGUTH 1995 ). The latter encodes seven basic helix-loop-helix nuclear proteins (DELIDAKIS and ARTAVANIS-TSAKONAS 1992 ; KNUST et al. 1992 ) that function as repressors of proneural genes (OELLERS et al. 1994 ; HEITZLER et al. 1996 ). Recent reports indicate that during the signal transduction process, a proteolytic cleavage releases the cytoplasmic domain of Notch, which is translocated to the nucleus (SCHROETER et al. 1998 ; STRUHL and ADACHI 1998 ).

Notch signaling is also involved in the development of the margin and the veins of the Drosophila wing. The wing anlage develops initially in the larval stages as an epithelial monolayer subdivided by anterior/posterior and dorsal/ventral axes, which have a major influence on the growth and patterning of the imaginal disc (DIAZ-BENJUMEA and COHEN 1993 ; BASLER and STRUHL 1994 ).

The dorsal/ventral boundary is defined during the second larval instar stage, where dorsal selector factor apterous (DIAZ-BENJUMEA and COHEN 1993 ) activates the expression of the putative secreted protein fringe (IRVINE and WIESCHAUS 1994 ). The juxtaposition of dorsal fringe-expressing and ventral non-fringe-expressing cells initiates symmetrical activation of wing margin-specific genes, e.g., wingless, vestigial, and cut along the dorsal/ventral boundary (KIM et al. 1995 , KIM et al. 1996 ; DOHERTY et al. 1996 ; MICCHELLI et al. 1997 ). Fringe inhibits Serrate-mediated Notch activation on the dorsal side of the boundary, but positively regulates Delta dependent Notch activation. In contrast, on the ventral side of the boundary, the absence of fringe allows Serrate-dependent signaling. This modulatory effect of fringe, together with positive feedback loops of Serrate and Delta expression, restrict the activation of Notch to the dorsal/ventral boundary (FLEMMING et al. 1997 ; PANIN et al. 1997 ).

The five longitudinal veins appear progressively from the distal to proximal side of the wing, as lumen between the two apposing surfaces (GARCIA-BELLIDO 1977 ). The formation of the veins requires an interplay between the Notch and Drosophila EGF receptor (DER) pathways (STURTEVANT and BIER 1995 ; DE CELIS et al. 1997 ). Differentiation of the longitudinal veins is initiated during third larval instar by the localized expression of the early vein marker veinlet (STURTEVANT et al. 1993 ) and the Notch ligand Delta (KOOH et al. 1993 ). Veinlet is thought to amplify the activity of DER, a key component in determining the fate of vein cells (STURTEVANT et al. 1993 ). Notch activity is present in the presumptive intervein cells to limit vein differentiation to a "vein competent" region (FEHON et al. 1991 ). During the pupal stage, Notch expression becomes restricted to the boundaries of the vein precursor territories. At the vein boundaries, Notch signaling activates expression of E(spl)mß, which subsequently represses veinlet expression and refines the vein-competent territories to a narrower region (DE CELIS et al. 1997 ). The role of Notch signaling in wing development is reflected by the phenotypes of Notch mutants. Notch loss-of-function mutations result in flies displaying wing margin notches and broader vein territories, while those that increase the Notch signal cause wing vein gaps (SCHELLENBARGER and MOHLER 1978 ; DE CELIS and GARCIA-BELLIDO 1994 ).

The precise regulation of the Notch signal is crucial to its biological role, and a number of proteins that regulate this signal have been identified. The Hairless (H) protein negatively regulates the Notch pathway by direct inhibition of Suppressor of Hairless (BROU et al. 1994 ; BANG et al. 1995 ). Other regulatory molecules bind to the intracellular domain of Notch itself. These include dishevelled, a possible negative regulator (AXELROD et al. 1996 ), and deltex (dx), a positive regulator (MATSUNO et al. 1995 ). The deltex protein contains three domains separated by stretches of glutamine-rich sequence (BUSSEAU et al. 1994 ; MATSUNO et al. 1995 ) The N-terminal domain is responsible for binding to the intracellular domain of Notch. The middle section contains a proline-rich sequence that has been proposed to be an SH3 domain-binding site, and the C terminus contains a ring zinc-finger motif. Mutations in deltex resemble loss-of-function mutations of Notch pathway genes, displaying wing margin loss and vein thickening phenotypes (XU and ARTAVANIS-TSAKONAS 1990 ; GORMAN and GIRTON 1992 ). Coexpression of deltex and Notch in tissue culture regulates the nuclear location of Suppressor of Hairless (MATSUNO et al. 1995 ); however, the in vivo role of deltex and its mechanism of regulation are currently unknown.

While much has been learned regarding the nature of Notch signal transduction, there remain many unresolved questions as to how this signal is regulated. The analysis of genetic interactions has proven to be a valuable tool for isolating components or regulators of signaling pathways. By identifying and mapping mutations that interact with Notch mutant phenotypes, we are aiming to identify novel regulatory genes. One candidate for a regulator of the Notch pathway is Suppressor of deltex [Su(dx)]. The mutations Su(dx)¹ and Su(dx)² were originally described as second-chromosome mutants that dominantly suppressed the deltex phenotypes (MORGAN et al. 1931 ). Another Suppressor of deltex allele, Su(dx)^sp, has been identified more recently, having arisen spontaneously in an Ax^E2, dx^enu stock (BUSSEAU et al. 1994 ). We have determined the map location of the Su(dx) gene and have described four new Su(dx) alleles. Wing phenotypes for Su(dx) loss-of-function alleles and genetic interactions with Notch pathway mutants are presented. The results show that the wild-type function of Su(dx) is as a negative regulator of the Notch pathway.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Drosophila stocks and culture conditions:
The following mutations were as described previously: Su(dx)^sp (BUSSEAU et al. 1994 ), dx^enu and dx^sm (XU and ARTAVANIS-TSAKONAS 1990 ), nd (HING et al. 1994 ), and Ax^E2 (XU and ARTAVANIS-TSAKONAS 1990 ). The deficiency stock df(2L)yan^J2 was a gift from Zhi Chun Lai. The UAS-E(spl) line was provided by Jose de Celis, and the MS1096 line was as described previously (CAPDEVILA and GUERRERO 1994 ). All other mutants, balancer and compound X chromosomes, used are described in LINDSLEY and ZIMM 1992 . Flies were cultured on standard cornmeal-yeast medium in temperature-controlled incubators at 25° unless otherwise stated.

Mutagenesis screen:
Virgin female w^a nd dx^enu sn³; Su(dx)^sp and male +/Y; cn flies were collected and aged 3 days at 25°. The male flies were exposed to 3000 rads of X rays (filtered to remove the soft X-ray component). The males were then allowed to mate for 48 hr before being removed from the vials. Rescued male progeny were collected and crossed to virgin females of the genotype C1(A)y/Y; CyO/Sco. Only the female progeny from this cross were viable. CyO females C1(A)y/Y; CyO/* cn were collected and mated to +/Y; CyO/Sco males. Female C1(A)y/Y; CyO/* cn and male +/Y; CyO/* cn flies were collected and used to establish a stock balanced for the mutated second chromosome.

After the recovery of balanced stocks, males +/Y; CyO/* cn were crossed to w^a nd dx^enu sn³; Su(dx)^sp virgin females to confirm that the recovered second chromosome again failed to complement Su(dx)^sp in the lethal rescue assay. This ruled out the possibility that the original recovered male was a background escaper from lethality.

For genetic interaction analysis and complementation testing, the C1(A)y chromosome was removed from the stock, except for Su(dx)⁴, which was kept as a C1(A)y stock because of the 2:Y translocation (see RESULTS). For comparison of phenotypes, control crosses were performed by crossing mutant flies to OregonR wild-type flies.

Polytene chromosome analysis:
Male flies +/Y; CyO/* cn were crossed to virgin females of the genotype Bc/CyO. Non-CyO male progeny +/Y; * cn/Bc were crossed to wild-type females, and the culture was grown at 18°. Third-instar larvae were collected; those not displaying the dominant Bc larval marker were dissected, and the salivary glands were used to prepare spreads of polytene chromosomes. Standard methods of preparation and polytene chromosome staining were used (ASHBURNER 1989A ), and the chromosomes were inspected by phase contrast microscopy with a x100 oil immersion lens.

Electron microscopy:
The flies were dehydrated in 100% ethanol and subsequently gold coated. Electron microscopy was performed on a SEM 360 Cambridge instrument.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Mapping of Su(dx):
A recessive viable mutation in Notch, notchoid (nd), has a strong genetic interaction with deltex, and flies homozygous for both nd and dx are not viable (XU and ARTAVANIS-TSAKONAS 1990 ). We have found that this lethal interaction can be rescued dominantly by Su(dx)^sp at 18°, but at 25° rescue required two copies of Su(dx)^sp (data not shown). Rescue was also achieved by a heteroallelic combination of Su(dx)^sp and Su(dx)² (data not shown). Thus, it was expected that a deficiency of Su(dx) over Su(dx)^sp would also rescue lethality. This was the basis of a deficiency screen to map the Su(dx) gene and generate new alleles of Su(dx). Full details of the crosses used to perform the screen and rescue the mutant alleles are described in MATERIALS AND METHODS. After isolation and balancing of the mutated second chromosome, each line was checked for rescue of the nd-dx interaction. Four mutations were obtained that failed to complement Su(dx)^sp in this rescue assay and that had visible cytological chromosome aberrations at the tip of 2L (listed in Table 1). Su(dx)⁷ and Su(dx)⁵² were independently derived deficiencies that were identical at the cytological level (n.b., the Su(dx)⁵² stock has subsequently been lost). Su(dx)⁵⁶ was an inversion with a breakpoint within the range of the two deficiencies. In the case of Su(dx)⁴, a modification of the crosses used to establish a stable line was necessary. The cross of C1(A)y/Y; CyO/Su(dx)⁴ cn with +/Y; CyO/Sco resulted in no viable female C1(A)y/Y; CyO/Su(dx)⁴ cn progeny. However, viable male and female progeny were recovered when both male and female parents carried the second and Y chromosomes from the original mutated male. Subsequent polytene chromosome analysis revealed that Su(dx)⁴ was a reciprocal 2:Y translocation. The translocation breakpoint lay within the range of the Su(dx)⁷ and Su(dx)⁵² deficiencies. Higher-resolution mapping of Su(dx) was achieved by testing deficiency stocks available in the region. The results, summarized in Table 1, place the Su(dx) locus between 22B4 and 22C2.

Characterization of Su(dx) alleles:
No phenotypes for Su(dx) alleles have been reported previously, other than their interactions with other mutations such as deltex (BUSSEAU et al. 1994 ). Here we describe phenotypes for Su(dx) in the wing veins and identify the developmental stage at which Su(dx) activity is required. We also describe genetic interactions with Notch pathway mutants.

Wing vein phenotypes: Homozygous Su(dx)^sp flies have a wild-type vein pattern at 25°. However, when they were kept at 29°, a recessive wing vein gap phenotype appeared (Figure 1B). The phenotype was manifested most often in veins L.IV and L.V, distal to the posterior cross-vein. Gaps were found frequently in L.II as well, but never in L.III. Su(dx)⁴, Su(dx)⁷, and Su(dx)⁵⁶ displayed wing vein gaps at 29° when placed over Su(dx)^sp (Figure 1C). At 25°, the same combinations of alleles had intact longitudinal veins, but forked or incomplete cross-veins (Figure 1D and Figure E).

View larger version (52K): In this window In a new window Download PPT slide   Figure 1. Phenotypes of Su(dx). (A) Wild-type wing; the longitudinal veins are numbered II, III, IV, and V. (B) Flies homozygous for Su(dx)sp cn at 29° display wing vein gaps in L.II, L.IV, and L.V. (C) At 29°, Su(dx)7 cn/Su(dx)56 cn flies display wing vein gaps like the other heteroallelic combinations of Su(dx). (D) At 25°, Su(dx)7 cn/Su(dx)56 cn flies have normal longitudinal veins, but they have a forked posterior cross-vein (arrow). (E) At 25°, Su(dx)7 cn/Su(dx)sp cn flies show the same forked cross-vein (arrow).

Developmental staging of Su(dx) function in the wing veins: The temperature-sensitive wing vein gap phenotype of the Su(dx)^sp mutant allowed us to determine the developmental stage in which Su(dx) functions during wing vein formation. Temperature shift analysis of Su(dx)^sp was performed using white prepupae that were collected from stocks kept at 25°, aged, and shifted to 29° at specific time points. The results have revealed a critical stage between 24 and 28 hr after puparium formation (APF) at 25° (Table 2A). Pupae that were shifted to the restrictive temperature before this stage resulted in most adults displaying a wing vein gap phenotype. A temperature upshift after 28 hr APF allowed normal development of the veins.

Shift-down experiments were used to define the start of the temperature-sensitive period of Su(dx)^sp. White prepupae were collected from stocks bred at 29° and transferred to 25°. Veins developed normally in most flies when the temperature downshift occurred before 14–16 hr APF (Table 2B). Increasing penetrance and severity of phenotype were evident when pupae were kept for longer times APF at 29°. A period of 22–24 hr APF was the minimum time at 29° required for most pupae to develop a phenotype indistinguishable from flies cultured permanently at 29°.

Thus, temperature-shift analysis has revealed a temperature-sensitive period between 16 and 22 hr APF at 29° and 24–28 hr APF at 25°, during which Su(dx)^sp affects wing vein formation. Because pupal development is ~80% slower at 25° than at 29° (ASHBURNER 1989B ), the temperature-sensitive period of Su(dx)^sp can be extrapolated to ~20–28 hr APF at 25°.

Interaction with deltex: deltex mutations result in thickened veins and wing margin loss phenotypes that are expected from a reduction in Notch signaling. The new alleles Su(dx)⁴, Su(dx)⁷, and Su(dx)⁵⁶ were compared with Su(dx)^sp for their ability to suppress dx^enu and dx^sm phenotypes. The new alleles of Su(dx) suppressed the phenotypes of both dx^enu (data not shown) and dx^sm (see Figure 2); however, the Su(dx)^sp allele was the strongest suppressor even though Su(dx)⁷ is a deficiency.

View larger version (35K): In this window In a new window Download PPT slide   Figure 2. Genetic interactions between Su(dx) and deltex. (A) deltex males, dxsm t2 v/Y; cn/+, display deltas at the tip of the longitudinal veins and small notches in the distal wing margin. (B) dxsm t2 v/Y; Su(dx)7 cn/+ flies. Su(dx)7, like the other Su(dx) alleles, dominantly suppresses the deltex phenotype. (C) dxsm t2 v/Y; Su(dx)sp,cn flies. Only the Su(dx)sp allele shows complete suppression of deltex.

Interaction with Notch: Null mutations of Notch, such as Notch^54l9, show a dominant phenotype of loss of wing margin at the distal tip of the wing (LINDSLEY and ZIMM 1992 ). Su(dx)^sp, Su(dx)⁷, and Su(dx)⁵⁶ mutants dominantly suppressed the strength and penetrance of the wing margin phenotype of Notch. The strength of the interactions varied among the different alleles, with Su(dx)^sp showing the strongest suppression (Figure 3).

View larger version (87K): In this window In a new window Download PPT slide   Figure 3. Genetic interactions between Su(dx) and N54l9. (A) y wa N54l9/+; cn/+ flies display notches at the distal wing margin. (B) The dominant interaction with Su(dx)sp,cn suppresses the Notch wing margin phenotype. (C) y wa N54l9/+; Su(dx)7 cn/+ flies also show suppression of the Notch phenotype, as y wa N54l9/+; Su(dx)56 cn/+ flies (D) do.

Interaction with nd: Su(dx) mutations were tested for interaction with nd, a recessive Notch allele with a wing margin loss phenotype (Figure 4A) that is similar to the loss of one copy of Notch (HING et al. 1994 ). Su(dx)⁴, Su(dx)⁷, and Su(dx)⁵⁶ alleles dominantly suppressed the wing margin phenotype of nd (Figure 4B). Su(dx)^sp, however, in addition to suppressing the margin phenotype, interacted dominantly to produce wing vein gaps (Figure 4C). Additional phenotypes were observed in flies that were homozygous for both nd and Su(dx)^sp. This combination was near lethal, but escapers were further enhanced for the vein gap phenotype and displayed horizontally held out and downward arching wings (Figure 4D).

View larger version (43K): In this window In a new window Download PPT slide   Figure 4. Genetic interactions between Su(dx) and nd. (A) wa nd/Y flies display distal margin notches. (B) wa nd/Y; Su(dx)7 cn/+ flies. Su(dx)7 dominantly suppresses the nd phenotype. (C) wa nd/Y; Su(dx)sp/+ flies show suppression of the nd wing margin phenotype, but this interaction also results in a wing vein gap phenotype. (D) wa nd/Y; Su(dx)sp /Su(dx)sp flies show a strong enhancement of the wing vein gap phenotype and also have horizontally held out and downwardly arching wings. This phenotype is also observed in AxE2/Y; Su(dx)sp/Su(dx)sp flies (data not shown). (E) Female wa nd/+ flies appear wild type. (F) wa nd/+ ; Su(dx)sp cn/Su(dx)sp cn flies display vein gaps. (G) The same phenotype is apparent in wa nd/+; Su(dx)sp cn/Su(dx)56 cn flies.

Homozygous Su(dx)^sp also interacts with heterozygous nd to produce wing vein gaps (Figure 4F). Su(dx)⁷ and Su(dx)⁵⁶ failed to complement Su(dx)^sp for the wing vein gap phenotype in combination with nd/+ (Figure 4G). Su(dx)⁴ could not be tested for complementation in this assay because of the 2:Y reciprocal translocation. We have also tested the original Su(dx)¹ and Su(dx)² alleles in this assay and have shown that they fail to complement Su(dx)^sp. It is interesting to note that the interaction of Su(dx) with nd is not a straightforward suppression of the wing margin phenotype. The new wing vein gap phenotype introduced implies a hyperactivation of Notch signaling in this combination of mutants.

Interaction with a gain-of-function Notch allele (Ax^E2): The Abruptex class of mutations in Notch is defined by a wing vein gap phenotype (LINDSLEY and ZIMM 1992 ) thought to result from an increase in Notch signaling (HEITZLER and SIMPSON 1993 ; DE CELIS et al. 1996 ). Flies homozygous for Ax^E2 display a loss of vein at the distal tip of L.V (Figure 5A). This phenotype was weakly enhanced by heterozygous Su(dx)^sp, Su(dx)⁴, Su(dx)⁷, and Su(dx)⁵⁶ mutations (data not shown), and it was strongly enhanced by homozygous Su(dx)^sp at 25°. This double-homozygous combination of Ax^E2; Su(dx)^sp was found to be poorly viable, but escapers that had strong wing vein gaps were observed (Figure 5B). These flies also had held-out and downward arching wings, a phenotype that was indistinguishable from the homozygous nd; Su(dx) combination. Heteroallelic combinations of Su(dx)⁴, Su(dx)⁷, and Su(dx)⁵⁶ with Su(dx)^sp showed a similarly strong enhancement of Ax^E2. Females heterozygous for Ax^E2 did not have wing vein gaps in a wild-type genetic background (Figure 5C). However, in combination with homozygous Su(dx)^sp or in heteroallelic combinations of Su(dx)^sp, Su(dx)¹, Su(dx)², Su(dx)⁷, and Su(dx)⁵⁶, Ax^E2/+ females displayed a strong loss-of-vein phenotype (Figure 5D).

View larger version (93K): In this window In a new window Download PPT slide   Figure 5. Genetic interactions between Su(dx) and AxE2. (A) Male y AxE2/Y flies display a L.V distal wing vein gap. (B) A severe enhancement of the wing vein gap at 25° is seen in the near-lethal combination y AxE2/Y; Su(dx)sp cn/Su(dx)sp cn. (C) Female y AxE2/+ flies appear wild type. (D) Female y AxE2/FM7c; Su(dx)sp cn/Su(dx)sp cn flies have wing vein gaps at 25°.

Interaction with other Notch pathway genes: We have examined the genetic interactions of Su(dx) with H, Dl, Ser, and E(spl)mß. H/+ displayed a small LV vein gap phenotype at a low penetrance in a wild-type background. The penetrance of the vein gap phenotype was increased by a dominant interaction of Su(dx)^sp with H/+ (data not shown), and this phenotype was strongly enhanced when homozygous for Su(dx)^sp (Figure 6B). The bristle loss phenotype of Hairless was also examined. Counting of microchaetae and macrochaetae revealed an enhanced loss-of-microchaetae phenotype in a homozygous Su(dx) background (see Table 3); however, a significant reduction of the average number of macrochaetae present was not observed.

View larger version (44K): In this window In a new window Download PPT slide   Figure 6. Genetic interactions between Su(dx) and H1. (A) H1/+ flies display a narrower or missing distal tip of vein L.V. (B) The vein gap phenotype is strongly enhanced in a Su(dx)sp/Su(dx)sp background.

The phenotype of the Dl¹⁷¹ allele in a wild-type background is illustrated in Figure 7A. Thickening of the veins can be observed, particularly at the distal ends and around the anterior and posterior cross-veins. Genetic interaction with heterozygous Su(dx)^sp resulted in suppression of the vein-thickening phenotypes, particularly at the distal tips of the veins (Figure 7B). We did not find a significant suppression of the wing margin phenotype of Ser¹, a dominant mutation in the second Drosophila Notch ligand.

View larger version (48K): In this window In a new window Download PPT slide   Figure 7. Genetic interaction of Su(dx)sp with Delta. (A) Dl171/+ flies display vein thickening at the distal tips of the longitudinal veins and around the anterior and posterior cross-veins. (B) Wings from Dl171/+; Su(dx)sp/+ flies showing suppression of the vein-thickening phenotypes.

To test for genetic interactions with E(spl)mß, we have used a transgenic line that ectopically expresses the E(spl)mß protein. During the refinement of the wing vein territories, Notch signaling results in accumulation of E(spl)mß protein in the vein boundary cells, which represses vein differentiation. Thus, Notch activation can be mimicked by ectopic expression of E(spl)mß, which causes a vein gap phenotype when expressed in the wing (DE CELIS et al. 1997 ). We have used the Gal4-expressing line MS1096 to drive the expression of E(spl)mß in the developing wing disc. These flies have wing vein gap and bristle loss phenotypes. MS1096; UAS-E(spl)mß/CyO flies were crossed to either wild-type or Su(dx)^sp males. Su(dx)^sp was found to strongly enhance the wing vein gap phenotype of the E(spl)mß-expressing progeny (Figure 8B). We also observed a strong enhancement of the loss-of-macro- and -microchaetae phenotype (Figure 8B). In a wild-type background, ectopic expression of E(spl)mß resulted in an average loss of 2.3 macrochaetae compared with an average loss of 13.4 macrochaetae in a Su(dx)^sp/+ background (P < 0.001). The strength of the enhanced wing vein and bristle loss phenotypes was comparable to the expression of two copies of the UAS-E(spl)mß transgene in a wild-type background (Figure 8C).

View larger version (104K): In this window In a new window Download PPT slide   Figure 8. Genetic interactions between Su(dx) and ectopic expression of E(spl)mß. MS1096; UAS-E(spl)mß/CyO flies were crossed to either wild-type or Su(dx)sp males, and the non-CyO progeny were examined for wing vein gaps and bristle loss phenotypes. (A) MS1096/Y; UAS-E(spl)mß/+ flies have wing vein and microchaetae and macrochaetae loss phenotypes. (B) MS1096/Y; UAS-E(spl)mß/Su(dx)sp flies have a strongly enhanced wing vein phenotype, as well as microchaetae and macrochaetae loss phenotypes. (C) Phenotypes of MS1096; UAS-E(spl)mß/UAS-E(spl)mß flies. Increasing the dosage of E(spl)mß gives strong phenotypes comparable to those resulting from enhancement by Su(dx)sp.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Four new Su(dx) mutations with visible cytological abnormalities have been obtained, and the Su(dx) gene has been mapped between 22B4 and 22C2 on the second chromosome. We have demonstrated that Su(dx) mutants have phenotypes that are independent of mutations in other genes. A forked cross-vein phenotype is observed at 25°, and a longitudinal vein gap phenotype is apparent at 29°.

The new alleles of Su(dx) described in this paper dominantly suppressed the deltex phenotype and failed to complement Su(dx)^sp in genetic interactions with different Notch alleles and with Hairless. By comparison of Su(dx) alleles with the deficiency Su(dx)⁷, we conclude that the phenotypes observed result from a loss-of-function of the Su(dx) gene product. In complementation tests over the Su(dx)^sp allele, the phenotype of the deficiency Su(dx)⁷ is not significantly different from those of Su(dx)⁴ and Su(dx)⁵⁶. This suggests that the latter alleles may be near to null mutations. Su(dx)⁴ is lethal over Su(dx)⁷; however, it is premature to speculate on the null phenotype because it is possible that additional genes are removed by the combination of these mutations.

Su(dx)^sp is an antimorphic allele:
Su(dx)^sp consistently displayed a stronger phenotype in its genetic interactions compared to the other loss-of-function alleles of Su(dx), including the deficiency Su(dx)⁷. In a dominant interaction, Su(dx)^sp more completely suppressed the Notch and deltex phenotypes. At 18°, one copy of Su(dx)^sp can rescue the nd-dx lethal interaction, whereas the deficiency Su(dx)⁷ is unable to do this on its own (data not shown). Furthermore, Su(dx)^sp dominantly interacts with nd to produce wing vein gaps, while for the other Su(dx) mutants, two alleles are required to produce this phenotype. These results indicate that Su(dx)^sp is an antimorphic allele.

It is intriguing that an antimorphic allele that displays strong genetic interactions with Notch pathway mutants has a relatively weak phenotype on its own. This raises the possibility that there is functional overlap with other genes. This is not without precedent in Notch signaling. For example, the E(spl) family of DNA-binding genes has been shown to have a partially redundant function in mediating the consequences of Notch activation (DELIDAKIS and ARTAVANIS-TSAKONAS 1992 ).

Developmental stages of Su(dx) function:
Analysis of the phenotypes of Su(dx) and its interactions with Notch pathway mutants has shown that Su(dx) functions at different stages of development: the developing wing margin, wing vein differentiation, and macro- and microchaetae development. In addition, the held-out wing phenotype seen in interactions with Ax^E2 and nd mutations is suggestive of a role for Su(dx) in muscle formation. Notch signaling has previously been implicated in muscle development (BAKER and SCHUBIGER 1996 ; ANANT et al. 1998 ).

The temperature-sensitive wing vein gap phenotype of Su(dx)^sp was used to determine the developmental stage in which Su(dx) functions in vein formation. Temperature shift analysis has revealed a temperature-sensitive period between 20 and 28 hr APF at 25°. This pupal stage coincides with the apposition of dorsal and ventral wing surfaces, during which the veins are established (FRISTROM et al. 1993 ). An important role for Notch signaling in the process of vein formation is evident. Notch activity is first required in the specification of wing vein precursors in the imaginal disc (FEHON et al. 1991 ). Second, during pupal development, Notch signaling is essential in determining the correct thickness of the veins and maintaining the vein territories (SCHELLENBARGER and MOHLER 1978 ; DE CELIS et al. 1997 ; HUPPERT et al. 1997 ). The latter role is reflected by the gradual shift in Notch expression from the intervein regions to a sharp border of cells along the veins by 24 hr APF. The expression of Notch and one of its target genes, E(spl)mß, is maintained in these cells 35 hr APF (DE CELIS et al. 1997 ). In addition, the Notch ligand Delta was shown to be required for vein cell specification between 20 and 30 hr APF at 25° (HUPPERT et al. 1997 ). Thus, the developmental stage requiring Su(dx) activity is consistent with a role in regulating the Notch signal during wing vein formation.

Su(dx) is a negative regulator of the Notch pathway:
A number of observations indicate that the wild-type function of Su(dx) is as a negative regulator of the Notch pathway. The temperature-sensitive wing vein gap phenotype described in this paper is similar to that observed for gain-of-function Abruptex alleles of Notch. Complementation tests over the deficiency have shown that the Su(dx) mutants described result in a loss of function of Su(dx). This is an important prerequisite for interpreting the wild-type function of Su(dx).

The haplo-insufficient phenotype of Notch is suppressed by Su(dx) mutations, as is the mutation of Delta, the Notch ligand. In contrast, the gain-of-function Ax^E2 mutation of Notch is enhanced by Su(dx). This is similar to the known genetic interactions of Hairless with these Notch mutants (BANG et al. 1995 ). Hairless is a negative regulator of the Notch pathway, and it functions by binding to and inhibiting Suppressor of Hairless, a Notch-responsive transcription factor (BROU et al. 1994 ). The fact that Su(dx) enhanced the Hairless phenotype indicates that the two genes are regulating the Notch signal in the same direction. Similarly, the observed suppression of deltex is as expected. Because deltex is a positive regulator of Notch function, its mutation should be compensated by a mutant that leads to a hyperactivation of the Notch signal.

Activation of the Notch pathway can be mimicked by ectopic E(spl)mß expression in the wing, which results in gaps in the veins. The strength of this phenotype is dependent on the dosage of the expressed E(spl)mß, and the phenotype is enhanced in a Su(dx) mutant background. We hypothesize that the Su(dx) mutation leads to an elevation of Notch signaling and increased expression of endogenous E(spl)mß, which augments the ectopically expressed protein levels. However, we cannot rule out the alternative possibility that the enhanced phenotype may be caused by an upregulation of the downstream response to the activity of expressed E(spl)mß.

Support for a negative regulatory function for Su(dx) also comes from comparison of Su(dx) phenotypes with those resulting from ectopic expression of activated Notch and wild-type deltex proteins. It is possible to make a constitutively activated Notch receptor by expressing a truncated form that lacks the extracellular domain (REBAY et al. 1993 ). The Notch pathway can also be upregulated by overexpression of wild-type deltex (MATSUNO et al. 1995 ). When activated Notch or wild-type deltex are expressed under control of a heat shock promoter 0–24 hr APF, a wing vein gap phenotype appears. In both cases, this phenotype is strongly enhanced in a heterozygous nd background, similar to the interaction between Su(dx) mutants and nd. Thus, the Su(dx) mutation mimics an elevation of the Notch signal.

Taken together, our data indicate a role for Su(dx) as a negative regulator of the Notch pathway. The existence of feedback regulatory loops in the control of Notch signaling makes the position of Su(dx) protein in the Notch pathway difficult to define through genetic analysis. Su(dx) mutants were first identified through their interaction with deltex. It cannot be concluded that the corresponding proteins interact directly, however, especially as we have shown significant genetic interactions of Su(dx) with a number of Notch pathway genes. The precise function of Su(dx) will only be resolved through cloning of the gene and analysis of its function at the molecular level, which is in progress. In a recent mutagenesis screen, we have detected a number of enhancers of Su(dx) that may be alleles of functionally related genes. It is likely, therefore, that the further characterization of Su(dx) and its interacting mutations will be fruitful for the understanding of Notch pathway regulation.

	ACKNOWLEDGMENTS

We thank Iain Dawson for advice and help with polytene chromosome analysis, and Bob Diederich, Mark Fortini, Tian Xu, Mike Cornell, Luke Alphey, Jenny Gleason, and Roger Wood for valuable discussion. Samantha Newby gave us valuable assistance with the electron microscopy. We thank Zhi Chun Lai and Gerry Rubin for supplying the yan^J2 deficiency stock, Jose de Celis for supplying the UAS-E(spl)mß line, and Matthew Freeman for the MS1096 Gal4 line. M.B. is supported by a Zeneca Senior Fellowship, M.F. by the Biotechnology and Biological Sciences Research Council Cell Commitment and Determination (BBSRC CAD) initiative, and D.E. by a Medical Research Council Realising Our Potential Award (MRC ROPA). S.A.T. is supported by Howard Hughes Medical Institute. We also acknowledge the support of the Royal Society.

Manuscript received December 8, 1997; Accepted for publication August 24, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ANANT, S., R. SUDIPTO, and K. V. RAGHAVAN, 1998  Twist and Notch negatively regulate adult muscle differentiation in Drosophila. Development 125:1361-1369[Abstract].

ARTAVANIS-TSAKONAS, S. and P. SIMPSON, 1991  Choosing a cell fate: a view from the Notch locus. Trends Genet. 7:403-408[Medline].

ARTAVANIS-TSAKONAS, S., K. MATSUNO, and M. E. FORTINI, 1995  Notch signaling. Science 268:225-232[Abstract/Free Full Text].

ASHBURNER, M., 1989a Drosophila: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

ASHBURNER, M., 1989b Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

AXELROD, J. D., K. MATSUNO, S. ARTAVANIS-TSAKONAS, and N. PERRIMON, 1996  Interaction between Wingless and Notch signaling pathways mediated by dishevelled. Science 271:1826-1832[Abstract].

BAILEY, A. M. and J. W. POSAKONY, 1995  Suppressor of Hairless directly activates transcription of Enhancer of split complex genes in response to Notch receptor activity. Genes Dev. 9:2609-2622[Abstract/Free Full Text].

BAKER, R. and G. SCHUBIGER, 1996  Autonomous and nonautonomous Notch functions for embryonic muscle and epidermis development in Drosophila. Development 122:617-626[Abstract].

BANG, A. G., A. M. BAILEY, and J. W. POSAKONY, 1995  Hairless promotes stable commitment to the sensory organ precursor cell fate by negatively regulating the activity of the Notch signaling pathway. Dev. Biol. 172:479-494[Medline].

BASLER, K. and G. STRUHL, 1994  Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368:208-214[Medline].

BROU, C., F. LOGEAT, M. LECOURTOIS, J. VANDEKERCKHOVE, and P. KOURILSKY et al., 1994  Inhibition of the DNA-binding activity of Drosophila Suppressor of Hairless and of its human homolog, KBF2/RBP-J kappa, by direct protein-protein interaction with Drosophila Hairless. Genes Dev. 8:2491-2503[Abstract/Free Full Text].

BUSSEAU, I., R. J. DIEDERICH, T. XU, and S. ARTAVANIS-TSAKONAS, 1994  A member of the Notch group of interacting loci, deltex encodes a cytoplasmic basic protein. Genetics 136:585-596[Abstract].

CAPDEVILA, J. and I. GUERRERO, 1994  Targetted expression of the signaling molecule Decapentaplegic induces pattern duplications and growth alterations in Drosophila wings. EMBO J. 13:4459-4468[Medline].

DE CELIS, J. F. and A. GARCIA-BELLIDO, 1994  Roles of the Notch gene in Drosophila wing morphogenesis. Mech. Dev. 46:109-122[Medline].

DE CELIS, J. F., A. GARCIA-BELLIDO, and S. J. BRAY, 1996  Activation and function of Notch at the dorsal-ventral boundary of the wing imaginal disc. Development 122:359-369[Abstract].

DE CELIS, J. F., S. J. BRAY, and A. GARCIA-BELLIDO, 1997  Notch signaling regulates veinlet expression and establishes boundaries between veins and interveins in the Drosophila wing. Development 124:1919-1928[Abstract].

DELIDAKIS, C. and S. ARTAVANIS-TSAKONAS, 1992  The Enhancer of split [E(spl)] locus of Drosophila encodes seven independent helix-loop-helix proteins. Proc. Natl. Acad. Sci. USA 89:8731-8735[Abstract/Free Full Text].

DIAZ-BENJUMEA, F. and S. M. COHEN, 1993  Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila. Cell 75:741-752[Medline].

DOHERTY, D., G. FEGER, S. YOUNGER-SHEPHERD, L. Y. JAN, and Y. N. JAN, 1996  Delta is a ventral to dorsal signal complementary to Serrate, another Notch ligand, in Drosophila wing formation. Genes Dev. 10:421-434[Abstract/Free Full Text].

FEHON, R. G., K. JOHANSEN, I. REBAY, and S. ARTAVANIS-TSAKONAS, 1991  Complex cellular and subcellular regulation of Notch expression during embryonic and imaginal development of Drosophila: implications for Notch function. J. Cell. Biol. 113:657-669[Abstract/Free Full Text].

FLEMMING, R. J., Y. GU, and N. A. HUKRIEDE, 1997  Serrate-mediated activation of Notch is specifically blocked by the product of the gene fringe in the dorsal compartment of the Drosophila wing imaginal disc. Development 124:2973-2981[Abstract].

FORTINI, M. E. and S. ARTAVANIS-TSAKONAS, 1993  Notch: neurogenesis is only part of the picture. Cell 75:1245-1247[Medline].

FORTINI, M. E. and S. ARTAVANIS-TSAKONAS, 1994  The Suppressor of Hairless protein participates in Notch receptor signaling. Cell 79:273-282[Medline].

FRISTROM, D., M. WILCOX, and J. FRISTROM, 1993  The distribution of PS integrins, laminin A and F-actin during key stages in Drosophila wing development. Development 117:509-523[Abstract].

GARCIA-BELLIDO, A., 1977  Inductive mechanism in the process of wing vein formation in Drosophila. Wilhelm Roux's Arch. Dev. Biol. 182:93-106.

GORMAN, M. J. and J. R. GIRTON, 1992  A genetic analysis of deltex and its interaction with the Notch locus in Drosophila melanogaster.. Genetics 131:99-112[Abstract].

HEITZLER, P. and P. SIMPSON, 1991  The choice of cell fate in the epidermis of Drosophila. Cell 64:1083-1092[Medline].

HEITZLER, P. and P. SIMPSON, 1993  Altered EGF-like sequences provide evidence for a role of Notch as a receptor in cell fate decisions. Development 117:1113-1123[Abstract].

HEITZLER, P., M. BOUROUIS, L. RUEL, C. CARTERET, and P. SIMPSON, 1996  Genes of the Enhancer of split and achaete-scute complexes are required for a regulatory loop between Notch and Delta during lateral signaling in Drosophila. Development 122:161-171[Abstract].

HING, H. K., X. SUN, and S. ARTAVANIS-TSAKONAS, 1994  Modulation of wingless signaling by Notch in Drosophila. Mech. Dev. 47:261-268[Medline].

HUPPERT, S. S., L. J. JACOBSEN, and M. A. T. MUSKAVITCH, 1997  Feedback regulation is central to Delta-Notch signaling required for Drosophila wing vein morphogenesis. Development 124:3283-3291[Abstract].

IRVINE, K. D. and E. WIESCHAUS, 1994  Fringe, a boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell 79:595-606[Medline].

KIM, J., K. D. IRVINE, and S. B. CARROLL, 1995  Cell recognition, signal induction and symmetrical gene activation at the dorso ventral boundary of the developing Drosophila wing. Cell 82:795-802[Medline].

KIM, J., A. SEBRING, J. J. ESCH, M. E. KRAUS, and K. VORWERK et al., 1996  Integration of positional signals and regulation of wing formation and identity by Drosophila vestigial gene. Nature 382:133-138[Medline].

KNUST, E., H. SCHRONS, F. GRAWE, and J. A. CAMPOS-ORTEGA, 1992  Seven genes of the Enhancer of split complex of Drosophila melanogaster encode helix loop helix proteins. Genetics 132:505-518[Abstract].

KOOH, P. J., R. G. FEHON, and M. A. MUSKAVITCH, 1993  Implications of dynamic patterns of Delta and Notch expression for cellular interactions during Drosophila development. Development 117:493-507[Abstract].

LECOURTOIS, M. and F. SCHWEISGUTH, 1995  The neurogenic Suppressor of Hairless DNA-binding protein mediates the transcriptional activation of the Enhancer of split complex genes triggered by Notch signaling. Genes Dev. 9:2598-2608[Abstract/Free Full Text].

LINDSLEY, D. L., and G. G. ZIMM, 1992 The Genome of Drosophila melanogaster. Academic Press, New York.

MATSUNO, K., R. J. DIEDERICH, M. J. GO, C. M. BLAUMUELLER, and S. ARTAVANIS-TSAKONAS, 1995  Deltex acts as a positive regulator of Notch signaling through interactions with the Notch ankyrin repeats. Development 121:2633-2644[Abstract].

MICCHELLI, C. A., E. J. RULIFSON, and S. S. BLAIR, 1997  The function and regulation of cut expression on the wing margin of Drosophila: Notch, wingless and a dominant negative role for Delta and Serrate. Development 124:1485-1495[Abstract].

MORGAN, T. H., A. H. BRIDGES, and J. SCHULTZ, J. SCHULTZ, 1931  The constitution of the germinal material in relation to heredity. Year Book Carnegie Inst. 30:408-415.

OELLERS, N., M. DEHIO, and E. KNUST, 1994  bHLH proteins encoded by the Enhancer of split complex of Drosophila negatively interfere with transcriptional activation mediated by proneural genes. Mol. Gen. Genet. 244:465-473[Medline].

PANIN, V. M., V. PAPAYANNOPOULOS, R. WILSON, and K. D. IRVINE, 1997  Fringe modulates Notch-ligand interactions. Nature 387:908-912[Medline].

REBAY, I., R. J. FLEMING, R. G. FEHON, L. CHERBAS, and P. CHERBAS et al., 1991  Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell 67:687-699[Medline].

REBAY, I., R. G. FEHON, and S. ARTAVANIS-TSAKONAS, 1993  Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor. Cell 74:319-329[Medline].

SCHELLENBARGER, D. L. and J. D. MOHLER, 1978  Temperature-sensitive periods and autonomy of pleiotropic effects of l1 N^ts, a conditional Notch lethal in Drosophila. Dev. Biol. 62:432-446[Medline].

SCHROETER, E. H., J. A. KISSLINGER, and R. KOPAN, 1998  Notch-1 signaling requires ligand induced proteolytic release of intracellular domain. Nature 393:382-386[Medline].

SCHWEISGUTH, F. and J. W. POSAKONY, 1992  Suppressor of Hairless, the Drosophila homolog of the mouse recombination signal-binding protein gene, controls sensory organ cell fates. Cell 69:1199-1212[Medline].

STRUHL, G. and A. ADACHI, 1998  Nuclear access and action of notch in vivo. Cell 93:649-660[Medline].

STURTEVANT, M. A. and E. BIER, 1995  Analysis of the genetic hierarchy guiding wing vein development in Drosophila. Development 121:785-801[Abstract].

STURTEVANT, M. A., M. ROARK, and E. BIER, 1993  The Drosophila rhomboid gene mediates the localized formation of wing veins and interacts genetically with components of the EGF-R signaling pathway. Genes Dev. 7:961-973[Abstract/Free Full Text].

XU, T. and S. ARTAVANIS-TSAKONAS, 1990  deltex, a locus interacting with the neurogenic genes, Notch, Delta and mastermind in Drosophila melanogaster. Genetics 126:665-677[Abstract].