Mutations Modulating the Argos-Regulated Signaling Pathway in Drosophila Eye Development

Corresponding author: Hideyuki Okano, Division of Neuroanatomy (D12), Department of Neuroscience, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan., okano{at}nana.med.osaka-u.ac.jp (E-mail)

Communicating editor: N. TAKAHATA

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Argos is a secreted protein that contains an EGF-like domain and acts as an inhibitor of Drosophila EGF receptor activation. To identify genes that function in the Argos-regulated signaling pathway, we performed a genetic screen for enhancers and suppressors of the eye phenotype caused by the overexpression of argos. As a result, new alleles of known genes encoding components of the EGF receptor pathway, such as Star, sprouty, bulge, and clown, were isolated. To study the role of clown in development, we examined the eye and wing phenotypes of the clown mutants in detail. In the eye discs of clown mutants, the pattern of neuronal differentiation was impaired, showing a phenotype similar to those caused by a gain-of-function EGF receptor mutation and overexpression of secreted Spitz, an activating ligand for the EGF receptor. There was also an increased number of pigment cells in the clown eyes. Epistatic analysis placed clown between argos and Ras1. In addition, we found that clown negatively regulated the development of wing veins. These results suggest that the clown gene product is important for the Argos-mediated inhibition of EGF receptor activation during the development of various tissues. In addition to the known genes, we identified six mutations of novel genes. Genetic characterization of these mutants suggested that they have distinct roles in cell differentiation and/or survival regulated by the EGF receptor pathway.

THE epidermal growth factor (EGF) receptor plays important roles in cell proliferation, differentiation, and survival. Activation of the receptor and its downstream signals must be tightly regulated for cells to grow and function normally. In fruit flies, the Drosophila EGF receptor (DER) is activated by multiple ligands, including Spitz, Gurken, and Vein (PERRIMON and PERKINS 1997 ). Binding of these ligands to DER triggers the activation of the Ras1/MAPK pathway. Argos, a secreted protein with an EGF-like domain, has a structure similar to these activating ligands (FREEMAN et al. 1992 ; KRETZSCHMAR et al. 1992 ; OKANO et al. 1992 ; FREEMAN 1994 ); however, it inhibits DER activation (SCHWEITZER et al. 1995 ; SAWAMOTO et al. 1996A ). Loss-of-function argos mutants show increased numbers of cells in various tissues where cellular differentiation is triggered by DER activation (reviewed by SCHWEITZER and SHILO 1997 ). On the other hand, forced expression of argos inhibits cell differentiation.

The developing Drosophila compound eye is a very useful model system for studying the function and regulatory mechanisms of the EGF receptor signaling pathway in animal development. The compound eye is composed of ~800 units called ommatidia. Each ommatidium consists of 8 photoreceptor cells, 4 cone cells and 11 pigment cells. The differentiation and survival of these cells are dependent on signaling through the Ras1/MAPK pathway, which is triggered by the interaction of DER with secreted Spitz (FREEMAN 1996 ; DOMINGUEZ et al. 1998 ; MILLER and CAGAN 1998 ; SAWAMOTO et al. 1998 ). Regulation of DER activation by Argos is required for normal ommatidial development (reviewed by SAWAMOTO and OKANO 1996 ). Loss of Argos function results in the formation of extra ommatidial cells due to excessive differentiation and decreased cell death (FREEMAN et al. 1992 ; KRETZSCHMAR et al. 1992 ; OKANO et al. 1992 ; BRUNNER et al. 1994 ; FREEMAN 1994 ). Overexpression of Argos inhibits cellular differentiation and induces programmed cell death, resulting in a decreased number of retina cells (BRUNNER et al. 1994 ; FREEMAN 1994 ; SAWAMOTO et al. 1994 , SAWAMOTO et al. 1998 ). The DER/Ras1/MAPK pathway promotes cell survival by downregulating the expression and function of head involution defective (hid), a cell death regulator (BERGMANN et al. 1998 ; KURADA and WHITE 1998 ; SAWAMOTO et al. 1998 ). In spite of these results from genetic and biochemical experiments, the precise mechanisms by which Argos regulates the DER/Ras1/MAPK pathway, which may involve unidentified genes, remain unknown.

One approach for further elucidating the mechanisms of Argos' action is to identify components that interact with argos genetically. Screens for modifiers affecting the eye phenotype caused by mutations of a gene have been used successfully to identify genes that function in a common signaling pathway (for example, HAY et al. 1995 ; DICKSON et al. 1996 ; KARIM et al. 1996 ; MA et al. 1996 ; VERHEYEN et al. 1996 ; NEUFELD et al. 1998 ). To identify genes that function in the Argos-regulated signaling pathway, we carried out a genetic screen for enhancers and suppressors of the eye phenotype induced by eye-specific overexpression of Argos. In this article, we describe the mutants isolated from this screen, which include those for both novel and known genes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Drosophila stocks:
Canton-S or w¹¹¹⁸ were used as wild-type strains. The CyO, GMR-argos and TM3, GMR-argos chromosomes were generated by transposition of the GMR-argos transgene (SAWAMOTO et al. 1998 ) onto CyO and TM3 balancer chromosomes using a transposase expressed from the P[ry⁺, 2-3] transgene (ROBERTSON et al. 1988 ). sev-Ras1^N17 (KARIM et al. 1996 ), sev-Ras1^V12 (FORTINI et al. 1992 ; KARIM et al. 1996 ), GMR-hid (GRETHER et al. 1995 ), GMR-rpr (WHITE et al. 1996 ), hs-argos (SAWAMOTO et al. 1994 ), Raf^HM7 (MELNICK et al. 1993 ), Dsor1^Su1 (TSUDA et al. 1993 ), Sos^JC2 (ROGGE et al. 1991 ), rl^Su23 (LIM et al. 1997 ), clown^4a1 (WEMMER and KLAMBT 1995 ), bulge^6d7 (WEMMER and KLAMBT 1995 ), sprouty⁵ (HACOHEN et al. 1998 ), and S²¹⁸ (KOLODKIN et al. 1994 ) were previously described. Fly stocks with multiple recessive markers and the deficiency kits for second and third chromosomes were obtained from the Bloomington Stock Center.

Plasmid construction and P-element-mediated germline transformation:
pUAS-argos was constructed by inserting the 2-kb EcoRI fragment of the argos cDNA, which includes the entire coding region (OKANO et al. 1992 ), into the EcoRI site of the pUAST vector (BRAND and PERRIMON 1993 ). The resulting pUAS-argos plasmid was injected into w¹¹¹⁸ ; Dr/TMS, Sb P[ry⁺, 2-3] embryos as previously described (SAWAMOTO et al. 1994 ). Several independent transformant lines with similar phenotypes were obtained. All data presented in this article are from a single strain, 14-1.

Genetics:
Fly cultures and crosses were performed according to standard procedures at 25°, except where otherwise noted. For heat-shock experiments, second instar larvae were collected in a vial containing medium and repeatedly heat-shocked at 36° for 1 hr with a 5-hr interval at 25°, using a temperature-programmable incubator.

Male w¹¹¹⁸ flies were subjected to mutagenesis with EMS. Two- to five-day-old males were starved for 6 hr at 25° and then fed 5–25 mM EMS in a 10% sucrose solution overnight as described previously (LEWIS and BACHER 1968 ). The males were mated to CyO, GMR-argos/Tft virgin females. Approximately 140,000 EMS-treated F₁ progenies were examined under a dissecting microscope for an enhancement or suppression of the rough-eye phenotype of CyO, GMR-argos. Putative enhancers and suppressors were backcrossed to the w¹¹¹⁸; CyO, GMR-argos/Tft stock to test for chromosomal linkage and balanced over CyO, GMR-argos or TM3, GMR-argos.

Complementation tests were performed based on lethality or by having a visible phenotype in trans-heterozygotes. As a result, three complementation groups were identified on the second and third chromosomes. Other mutations were presumed to be single hits. One of the three complementation groups was homozygous viable with apparent phenotype and the other two groups were homozygous lethal.

Mutations of enhancers and suppressors were mapped meiotically, using the markers b pr c px sp and ru h th st cu sr e ca for the second and third chromosomes, respectively. After their map positions were determined, mutant flies were crossed with deficiency stocks in the relevant regions.

Histology:
For scanning electron microscopy, flies were prepared as described by KIMMEL et al. 1990 . Semithin sections of adult heads were prepared as described by SAWAMOTO et al. 1994 . The anti-ELAV antibody was purchased from the Developmental Studies Hybridoma Bank at the University of Iowa. Immunohistochemistry of eye discs was carried out essentially as described by TOMLINSON and READY 1987 , except that discs were fixed in 4% paraformaldehyde in PBS. Cobalt sulfide staining and acridine orange staining were performed by methods described in WOLFF and READY 1991 .

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Screen for dominant modifiers of GMR-argos:
To target the expression of Argos to developing eyes, we previously generated GMR-argos transgenic flies using the pGMR vector (SAWAMOTO et al. 1998 ). The pGMR vector contains a multimer cluster of binding sites for the Zn-finger protein Glass and provides eye-specific expression by Glass-dependent promotor activity in all the cells posterior to the morphogenetic furrow in the eye imaginal discs (HAY et al. 1994 ). The adult compound eyes of the flies carrying one copy of the GMR-argos transgene showed a mild rough morphology (Fig 1B) compared with the wild-type eye (Fig 1A). Some photoreceptor cells and pigmented lattices were often lost in the GMR-argos/+ eyes (Fig 1E). The effects of argos overexpression were dose dependent: flies with two copies of the transgene had smaller eyes with a more irregular array of ommatidia containing fewer cells (Fig 1C and Fig F) than flies carrying one copy (Fig 1B and Fig E). Thus, the phenotype of GMR-argos/+ eyes is sensitive to the amount of Argos protein and also possibly to other molecules that have functions related to Argos. In addition, the phenotype of the GMR-argos flies is stable and not altered by wild-type chromosomes or balancers (data not shown). Therefore, we used the phenotype caused by one copy of the GMR-argos transgene to screen for enhancers and suppressors. As a pilot test, we crossed GMR-argos flies to a collection of flies with mapped deficiencies and scored for modification of the rough-eye phenotype. As a result, we found that the GMR-argos phenotype was modified by a number of deficiencies corresponding to regions in which known components of the DER/Ras1/MAPK pathway and genes implicated in cell death signaling are located (data not shown). These results suggest that a twofold reduction in the dose of a gene that functions in the Argos-regulated pathway should alter the signaling strength and modify the rough-eye phenotype of GMR-argos. For example, loss-of-function mutations in one copy of a gene that acts positively on the DER/Ras1/MAPK pathway should reduce signaling and enhance the GMR-argos phenotype. Conversely, loss-of-function mutations in one copy of a gene that acts negatively on the pathway should increase signaling and suppress the GMR-argos phenotype. In addition, gain-of-function mutations in such genes may also be isolated in this screen. Since homozygotes for mutations in many of the genes required for the DER/Ras1/MAPK pathway are expected to be lethal, the ability to detect mutations as heterozygotes is critical for the efficient isolation of mutations.

View larger version (153K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Phenotype of GMR-argos. (A–C) Scanning electron micrographs of the adult compound eyes of wild type (A), GMR-argos/+ (B), and GMR-argos/GMR-argos (C). (D–F) Tangential sections of the adult compound eyes of wild type (D), GMR-argos/+ (E), and GMR-argos/GMR-argos (F).

Females carrying a GMR-argos construct on the CyO balancer were mated to w¹¹¹⁸ males that had been subjected to mutagenesis with EMS. As a parental line for screening, flies with the GMR-argos transgene inserted into CyO were used to prevent recombination between the new mutations and the transgenes. F₁ progenies carrying CyO were observed under the dissecting microscope and their eye morphology, size, and color were scored for modification of the GMR-argos phenotype. To isolate dominant modifiers of GMR-argos, we screened ~140,000 F₁ progeny. Enhancer mutations were identified as those resulting in an increased roughness and smaller size of eyes (Fig 2). Suppressor mutations were identified by the occurrence of reduced ommatidial fusions and the reappearance of straight ommatidial rows (Fig 3). In this screen, we recovered three enhancers and 10 suppressors (Table 1 and Table 2). First, the chromosomal linkage of these mutations was determined. Each mutation was located on either the second or third chromosomes. The mutations were then balanced over CyO or TM3 chromosomes carrying the GMR-argos transgene. To determine allelism, complementation tests were performed among all the mutations on a given chromosome. Failure to complement was scored either by lethality or by a mutant eye phenotype in adult trans-heterozygotes. The enhancers fell into one complementation group consisting of two alleles and a mutant of a single allele (Table 1), and the suppressors fell into two complementation groups and six mutants of single alleles (Table 2). Examples of the modified phenotypes are shown in Fig 2 and Fig 3. EF2-1enhanced the GMR-argos phenotype, resulting in a smaller eye and loss of photoreceptor and pigment cells (Fig 2B and Fig E). EM3-1 acted as a weak enhancer of GMR-argos due to a reduction in retina cells and the fusion of lenses (Fig 2C and Fig F). On the other hand, SF3-2 resulted in considerable recovery of the GMR-argos phenotype (Fig 3B). The decrease in number of photoreceptor cells in GMR-argos was rescued by this mutation to a nearly wild-type appearance, although the pigment cell phenotype was not recovered (Fig 3E). In contrast with SF3-2, another single-hit suppressor, SM3-2 suppressed the GMR-argos phenotype by restoring the phenotype of pigment cells but not that of photoreceptor cells (Fig 3C and Fig F).

View larger version (161K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Enhancement of the GMR-argos phenotype by the enhancer mutations. (A–C) Scanning electron micrographs of the adult compound eyes of GMR-argos/+ (A), EF2-1/GMR-argos (B), and GMR-argos/+; EM3-1/+ (C). (D–F) Tangential sections of the adult compound eyes of GMR-argos/+ (D), EF2-1/GMR-argos (E), and GMR-argos/+; EM3-1/+ (F).

View larger version (163K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Suppression of the GMR-argos phenotype by the suppressor mutations. (A–C) Scanning electron micrographs of the adult compound eyes of GMR-argos/+ (A), GMR-argos/+; SF3-2/+ (B), and GMR-argos/+; SM3-2/+ (C). (D–F) Tangential sections of the adult compound eyes of GMR-argos/+ (D), GMR-argos/+; SF3-2/+ (E), and GMR-argos/+; SM3-2/+ (F).

Classification and characterization of modifiers by genetic tests:
Modifiers isolated in screens based on eye phenotypes often contain mutations of genes that do not have functions related to the mutation used as the background for the screen. Therefore, some of the mutants isolated in our screen may not have been specifically involved in the cellular differentiation and/or survival processes regulated by Argos. Moreover, the modifiers may have included a variety of genes, since the roles of the EGF receptor pathway are pleiotropic. To classify and characterize modifiers, we carried out four genetic tests to examine the effects of modifier mutations on (1) the eye phenotype caused by argos overexpression using promoters other than GMR, (2) the eye phenotype caused by mutant Ras1 overexpression (sev-Ras1^N17 and sev-Ras1^V12), (3) the cell death induced by overexpression of hid and rpr, and (4) the wing vein phenotype caused by argos overexpression. These results are summarized in Table 1 and Table 2.

1. Interactions with sev-argos and hs-argos: Since Argos expression was induced under the control of the Glass binding sites in the GMR-argos eyes, mutations in genes including glass that regulate the expression of argos from this construct might modify the eye phenotype by increasing or decreasing the expression level of the GMR-argos transgene. For example, 11 alleles of glass mutations were isolated in a screen for modifiers of GMR-sina (NEUFELD et al. 1998 ), although any glass mutations were not isolated in our screen. To examine the effect of the modifier mutations isolated in this study on the phenotype caused by argos overexpression induced using other promoters, we crossed the modifier mutants to the flies carrying both sev-GAL4 and UAS-argos transgenes. The sev-GAL4/+ ; UAS-argos/+ flies showed a rough-eye phenotype similar to GMR-argos (data not shown). All of the mutants showed modifying effects on the sev-GAL4/+ ; UAS-argos/+ phenotype that were similar to those on GMR-argos (Table 1 and Table 2). In addition, we also crossed all the modifier mutants to hs-argos transgenic flies (SAWAMOTO et al. 1994 ), where argos expression is induced under the control of the hsp70 promoter. The eye phenotype of the hs-argos flies was also modified by all of the modifier mutations (Table 1 and Table 2). These results imply that the effects of these mutations on the GMR-argos phenotype were not dependent on the change in transcriptional activity regulated by Glass.

2. Interactions with Ras1 mutation alleles: Some of the modifiers isolated in our screen were expected to be mutants of genes encoding components of the Ras pathway. To examine whether the modifier mutations affect the phenotype caused by increased or decreased Ras1 activity, we crossed them to sev-Ras1^V12 and sev-Ras1^N17 lines. The sev-Ras1^N17 transgenic fly expresses a dominant-negative Ras1 allele under the control of the sev-enhancer/promoter and produces a rough-eye phenotype caused by the absence of the R7 cell in ~25% of the ommatidia and a lack of outer photoreceptor cells (Fig 4A and Fig D; KARIM et al. 1996 ). All the enhancer mutations showed similar enhancing effects on sev-Ras1^N17 (Table 1) and the suppressors, except SM3-5 and SM3-9, rescued the phenotype of sev-Ras1^N17 (Table 2). Examples of these effects are shown in Fig 4. The two strong enhancers of GMR-argos, EF2-1 (Fig 4B and Fig E) and EM2-4 (data not shown) enhanced the rough-eye phenotype of sev-Ras1^N17, so that the number of photoreceptor cells was decreased in >90% of the ommatidia. The two suppressors of GMR-argos, SM3-8 (Fig 4C and Fig F) and SM3-6 (data not shown), rescued the loss of R7 in almost all the ommatidia. In contrast to sev-Ras1^N17, the sev-Ras1^V12 transgenic fly expresses a constitutively activated Ras1 protein and shows a rough-eye phenotype caused by excess R7 cells and destruction of the regular ommatidial array (Fig 4G and Fig J; KARIM et al. 1996 ; KARIM and RUBIN 1998 ). It has been shown that the sev-Ras1^V12 phenotype is not affected by mutations of genes that function upsteam of Ras1 (KARIM et al. 1996 ). On the other hand, the sev-Ras1^V12 phenotype was suppressed by mutants identified as enhancers of GMR-argos, EF2-1 (Fig 4H and Fig K) and EM2-4 (data not shown), and enhanced by two suppressors of GMR-argos, SM3-6 (Fig 4I and Fig L) and SM3-8 (data not shown), suggesting that these genes may function downstream of or in parallel to Ras1.

View larger version (156K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Effects of enhancer and suppressor mutations on the phenotype induced by constitutively active and dominant-negative Ras1 mutations. (A–C) Scanning electron micrographs of the adult compound eyes of sev-Ras1N17/+ (A), sev-Ras1N17/EF2-1 (B), and sev-Ras1N17/+ ; SM3-8/+ (C). (D–F) Tangential sections of the adult compound eyes of sev-Ras1N17/+ (D), sev-Ras1N17/EF2-1 (E), and sev-Ras1N17/+ ; SM3-8/+ (F). (G–I) Scanning electron micrographs of the adult compound eyes of sev-Ras1V12/+ (G), sev-Ras1V12/EF2-1 (H), and Ras1V12/+; SM3-6/+ (I). (J–L) Tangential sections of the adult compound eyes of sev-Ras1V12/+ (J), sev-Ras1V12/EF2-1 (K), and Ras1V12/+ ; SM3-6/+ (L).

3. Interactions with GMR-hid and GMR-rpr: Each of the three apoptotic activators, rpr (WHITE et al. 1994 ), head involution defective (hid; GRETHER et al. 1995 ), and grim (CHEN et al. 1996 ), can induce programmed cell death when overexpressed. It is likely that these factors induce cell death through the activation of Dapaf-1/DARK, a recently identified protein homologous to CED-4 and Apaf-1 (KANUKA et al. 1999 ; RODRIGUEZ et al. 1999 ), and multiple caspases (FRASTER and EVAN 1997 ; INOHARA et al. 1997 ; SONG et al. 1997 ; CHEN et al. 1998 ; DORSTYN et al. 1999 ). The DER/Ras1/MAPK pathway promotes cell survival by regulating the expression and function of hid (BERGMANN et al. 1998 ; KURADA and WHITE 1998 ; SAWAMOTO et al. 1998 ). Since the decreased number of cells in the GMR-argos eyes is caused by excessive cell death (SAWAMOTO et al. 1998 ), we expected to isolate mutations of genes involved in the regulation of cell-death signaling in this screen. To examine the effects of the modifier mutations on cell death, we crossed them to GMR-rpr and GMR-hid (GRETHER et al. 1995 ; CHEN et al. 1996 ; WHITE et al. 1996 ). The eye discs of both GMR-hid and GMR-rpr, which had a small-eye phenotype (Fig 5A and Fig G), showed increased numbers of dying cells posterior to the morphogenetic furrow (Fig 5D and Fig J). SF3-2 considerably suppressed the small-eye phenotype and cell death in the eye discs induced by both GMR-hid and GMR-rpr (Fig 5B and Fig E and Fig H and Fig K). The hid-induced cell death was markedly suppressed by three of the suppressors, including SM3-9 (Fig 5C and Fig F; Table 2). The rpr-induced cell death was enhanced by EF2-1 and EM2-4 (data not shown) and suppressed by three of the suppressors, including SM3-2 (Fig 5I and Fig L; Table 2). These observations suggest that these modifiers, which affect the programmed cell death induced by hid and/or rpr, are mutations of genes involved in cell death.

View larger version (122K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Effects of the modifier mutations on the excessive cell death caused by overexpression of hid and rpr. (A–C) Scanning electron micrographs of the adult compound eyes of GMR-hid/+ (A), GMR-hid/SF3-2 (B), and GMR-hid/SM3-9 (C). (D–F) Acridine orange staining of eye imaginal discs from third instar larvae of GMR-hid/+ (D), GMR-hid/SF3-2 (E), and GMR-hid/SM3-9 (F). (G–I) Scanning electron micrographs of the adult compound eyes of GMR-rpr/+ (G), GMR-hid/SF3-2 (H), and GMR-hid/SM3-2 (I). (J–L) Acridine orange staining of eye imaginal discs from third instar larvae of GMR-rpr/+ (J), GMR-hid/SF3-2 (K), and GMR-hid/SM3-2 (L). Anterior at left; dorsal at top.

4. Effects on wing vein development: In addition to eye development, the DER/Ras1/MAPK pathway also plays important roles in several other developmental processes (for review, SCHWEITZER and SHILO 1997 ). The development of the wing vein is another well-studied model system for investigating the function of the EGF receptor pathway in cellular differentiation. Overexpression of Argos under control of the hsp70 promotor results in loss of the wing veins (Fig 6B; SAWAMOTO et al. 1994 ) due to inhibition of the Ras signaling pathway (SAWAMOTO et al. 1996A ). We examined whether the dominant enhancers and suppressors of GMR-argos could modify the wing vein loss induced by argos overexpression. An enhancer mutation, EF2-1, dominantly enhanced the loss of the L2 and L3 veins in the hs-argos wings (Fig 6C). Moreover, heterozygotes for four of the suppressor mutations, including SF3-1 (Fig 6D; Table 2), could completely restore this vein phenotype. These results suggest that these modifiers, which altered the wing vein phenotype of hs-argos, are mutations of genes regulating cell differentiation, not only in the eye, but also in other tissues, including the wings.

View larger version (85K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Effects of the modifier mutations on the wing vein phenotype caused by overexpression of argos. Photomicrographs of adult wings from wildtype (A), hs-argos/+; hs-argos/+ (B), hs-argos/EF2-1; hs-argos/+ (C), and hs-argos/+; hs-argos/SF3-1(D).

Mutations of known genes:
Each mutation was mapped by meiotic recombination using several recessive markers and deficiency stocks (see MATERIALS AND METHODS). After the map positions were determined, each mutant was crossed to a number of mutant flies with defects in known genes located near the mutations to determine the allelism. All three complementation groups were found to be mutations of known genes that had been previously implicated in the DER/Ras1/MAPK pathway.

EF2-1 and EM2-4 were allelic to Star(S). We have reported previously that S enhanced the argos overexpression phenotype and suppressed the loss-of-function argos phenotype in various tissues (SAWAMOTO et al. 1996A , SAWAMOTO et al. 1996B ), suggesting that argos and S function in a common pathway. In fact, S interacts with components of the EGF receptor signaling pathway (KOLODOKIN et al. 1994). Moreover, S is involved in the processing of Spitz, an activating ligand for DER (PICKUP and BANERJEE 1999 ). SM3-5 and SM3-9 failed to complement sprouty (spry) mutations. spry is a regulator for EGF receptor signaling in Drosophila tracheal development (HACOHEN et al. 1998 ). Recently, spry mutants were also isolated as regulators of EGF receptor signaling in a genetic screen similar to ours (CASCI et al. 1999 ). Spry binds to Drk and Gap1 and acts as an intracellular inhibitor of Ras signaling (CASCI et al. 1999 ; KRAMER et al. 1999 ). SF3-2 and SF3-3 were found to be new alleles of the gene clown (WEMMER and KLAMBT 1995 ). One allele of the clown gene, clown^4a1 has been identified as a suppressor of a gain-of-function mutation of the bulge gene (WEMMER and KLAMBT 1995 ). Among the remaining single-hit mutations, SF3-1 failed to complement the loss-of-function allele of bulge (WEMMER and KLAMBT 1995 ). Loss-of-function mutants of both bulge and clown suppress the eye phenotype caused by argos overexpression (WEMMER and KLAMBT 1995 ), suggesting that they have an antagonistic effect on EGF receptor signaling similar to that of argos. Thus, all the mutations of known genes isolated in this work are likely to function in DER/Ras1/MAPK pathway, indicating that our screen was highly specific for isolating genes in this pathway.

KARIM et al. 1996 performed a nearly saturated screen for modifiers of sev-Ras1^V12 to identify genes that function downstream of Ras1 and isolated numerous modifier mutations. Interestingly, mutants of the four known genes (S, spry, clown, and bulge) identified in our screen have not been isolated as modifiers of sev-Ras1^V12. Since we used a phenotype induced by overexpression of Argos, a diffusible protein that functions upstream of Ras1, our screen could have detected mutations of the genes both downstream and upstream of Ras1. For example, spry, identified in this screen, is known to act upstream of Ras1 (CASCI et al. 1999 ).

Phenotypic analysis of the clown mutants:
We examined phenotype of the clown mutants in more detail, since two alleles isolated in our screen are adult viable. clown has been implicated in argos functioning (WEMMER and KLAMBT 1995 ); however, its precise roles in eye development have remained largely unknown. To better understand clown function, we analyzed the phenotypes of the two new alleles of clown, clown^SF3-2 and clown^SF3-3, isolated as suppressors of GMR-argos in our present screen. Homozygotes for the clown^SF3-2 and clown^SF3-3 mutations are viable and have extremely rough eyes (Fig 7A) showing a characteristic "white and red" appearance due to loss of pigments (data not shown) similar to that of clown^4a1 (WEMMER and KLAMBT 1995 ). The phenotype of clown^SF3-2 was more severe than those of clown^4a1 and clown^SF3-3. Flies carrying clown^SF3-2 in trans to a deficiency lacking the 68C-D region, where clown has been mapped, showed a phenotype indistinguishable from the clown^SF3-2 homozygotes (data not shown). Therefore, it is likely that clown^SF3-2 is an amorphic allele, and clown^4a1 and clown^SF3-3 are hypomorphic alleles.

View larger version (105K): [in this window] [in a new window] [Download PPT slide]   Figure 7. Analysis of the eye phenotype of the clown homozygous mutant. (A and B) Scanning electron micrograph (A) and tangential section (B) of the adult compound eye of a clownSF3-2 homozygous mutant. (C and D) Cobalt sulfide staining of developing retinas from wild-type (C) and clownSF3-2/clownSF3-2 (D) pupae at 40 hr after puparium formation (APF). Arrowhead shows increased numbers of pigment cells and arrow shows an ommatidium with extra cone cells. (E–H) Anti-ELAV staining of eye discs from the wild-type (E and F) and clownSF3-2/clownSF3-2 (G and H) third instar larvae. F and H are higher magnification views. (I and J) Anti-ELAV staining of developing retinas from wild-type (I) and clownSF3-2/clownSF3-2 (J) pupae at 40 hr APF.

To study the function of clown in eye development, we analyzed the phenotype of the null allele clown^SF3-2 in detail. Sections through the adult compound eyes revealed that the normal structure of the ommatidia was disrupted almost completely (Fig 7B). To analyze the development of cone and pigment cells, we stained pupal retinas with cobalt sulfide. The wild-type ommatidium is composed of four cone cells, two primary pigment cells, six secondary pigment cells, and three tertiary pigment cells (Fig 7C). In the clown^SF3-2 mutant, the number of secondary and tertiary pigment cells was increased (Fig 7D). The increase in pigment cells may be caused by the inappropriate differentiation of excess cells and/or by impaired cell death. Cone cells in the clown^SF3-2 mutant were deformed and their number and arrangement in the ommatidia were irregular (Fig 7D). These defects are similar to the phenotype of clown^4a1 (WEMMER and KLAMBT 1995 ).

We then analyzed the differentiation of photoreceptor cells in the clown^SF3-2 mutant. Eye imaginal discs from third instar larvae were stained with an antibody against a neuronal marker ELAV. In the wild-type eye discs, a regular succession of neuronal differentiation was observed (Fig 7E and Fig F). Posterior to the morphogenetic furrow, relatively normal five-cell preclusters developed in the clown^SF3-2 mutant (Fig 7G and Fig H). However, the adjacent clusters lacked ELAV-positive cells, resulting in the formation of a region with no photoreceptor cells. At the posterior region of the eye discs, clusters with variable numbers of ELAV-positive cells were observed. Anti-ELAV staining of pupal retinas showed that photoreceptor cells degenerated in the clown^SF3-2 eyes during pupal development (Fig 7I and Fig J). The precise mechanisms by which the clown mutation causes such a complex phenotype are unclear. Interestingly, however, this phenotype is similar to that of Ellipse, a gain-of-function DER mutation (BAKER and RUBIN 1989 , BAKER and RUBIN 1992 ), and the phenotype caused by the overexpression of secreted Spitz (FREEMAN 1996 ). Therefore, it is possible that the loss of clown function resulted in a hyperactivation of the EGF receptor signaling in the photoreceptor cell precursors, causing this phenotype. This hypothesis is supported by our observation that clown mutations suppressed the GMR-argos photoreceptor cell phenotype (Fig 3E).

Adult flies homozygous for the putative null allele clown^SF3-2 are viable and fertile. Except for the phenotype affecting the compound eyes, they do not show any visible morphological defects on the body surface. Therefore, clown is unlikely to be required for the development of other tissues in which cellular differentiation is triggered by EGF receptor signaling. To examine whether clown is involved in the development of other organs, we examined its genetic interaction with components of the DER/Ras1/MAPK pathway in wing vein development. Inhibition of DER activation by overexpression of argos under the control of the hsp70 promoter results in partial loss of wing veins (SAWAMOTO et al. 1994 ; Fig 8A). Flies homozygous for clown^SF3-2 have normal wing vein patterns (data not shown). However, the wing vein phenotype induced by argos overexpression was significantly suppressed by halving the dose of the clown gene (Fig 8B). We then examined the effect of the clown^SF3-2 mutation on the phenotypes caused by hyperactivity of the DER/Ras1/MAPK signaling. The flies carrying gain-of-function mutations for both Sos and Dsor1 had wings with extra veins (Fig 8C). In addition, the wings of the rl^Su23 mutants showed a similar extra vein phenotype (Fig 8E). These phenotypes were considerably enhanced by halving the dose of the clown gene (Fig 8D and Fig F). These results indicate that the clown gene product has a redundant function in the regulation of wing vein development possibly through antagonizing the DER/Ras1/MAPK pathway.

View larger version (120K): [in this window] [in a new window] [Download PPT slide]   Figure 8. Genetic interaction of clown with argos, Dsor1, Sos, and rolled in wing vein development. Photomicrographs of adult wings are shown. (A) hs-argos/+; hs-argos/+. (B) hs-argos/+; hs-argos/clownSF3-2. (C) Dsor1Su1/Y; SosJC2/+. (D) Dsor1Su1/Y; SosJC2/+; clownSF3-2/+. (E) rlSu23/+. (F) rlSu23/+; clownSF3-2/+. Anterior at top. clownSF3-2 enhances formation of extra veins in Dsor1Su1/Y; SosJC2/+ and rlSu23/+. The additional ectopic veins between L2 and L3 are marked by arrows.

To determine the epistasis between argos and clown, the effect of argos overexpression was examined in flies homozygous for the clown^SF3-2 mutation. The eyes of GMR-argos/+ ; clown^SF3-2/clown^SF3-2 flies showed a phenotype indistinguishable from that of clown^SF3-2/clown^SF3-2 (data not shown). This observation indicates that clown functions downstream of or in parallel to argos. Since clown mutations suppressed the dominant-negative Ras1 allele but did not affect the phenotype caused by a constitutively activated Ras1 (Table 2), the clown gene product may play an important role in the Argos-mediated inhibition of the EGF receptor activation upstream of Ras1.

Mutations of novel genes:
Results from complementation tests revealed that the other six modifiers were mutations of novel genes. Since the results from characterizing the mutants of known genes showed a high specificity of this screen for identifying genes involved in a common pathway with argos, we expect that these novel genes play similar important roles. SM3-6 and SM3-8 enhanced Ras1^V12 and suppressed Ras1^N17, suggesting that they are mutations of novel genes that function downstream of Ras1 as negative regulators. SM3-2 may be involved in the cell-death signaling pathway regulated by rpr, since it suppressed cell death induced by rpr but not by hid. As shown in Fig 3F, suppression of the GMR-argos phenotype by SM3-2 was observed only in pigment cell death. Therefore, SM3-2 may function in pigment cell precursors that are known to undergo programmed cell death in normal eye development. EM3-1, SM2-1, and SM3-3 altered the Ras1^N17-induced defects due to photoreceptor differentiation but did not affect cell death induced by rpr and hid, or the wing vein phenotype caused by argos overexpression. It is possible that these three genes are involved in cellular differentiation during ommatidial development. Further characterization of these mutants and molecular cloning of the genes would unequivocally clarify their functions in cellular differentiation and/or survival regulated by the DER/Ras1/MAPK pathway.

	ACKNOWLEDGMENTS

We are grateful to Hiroshi Akimaru, Yasushi Hiromi, Kenji Matsuno, Shigeru Morimura, Yasuyoshi Nishida, Masataka Okabe, and Leo Tsuda for discussion and/or technical instructions; John M. Abrams, Andreas Bergman, Bruce A. Hay, Shigeo Hayashi, Jui-Chou Hsu, Yasushi Hiromi, Felix D. Karim, Christian Klämbt, Phani Kurada, Todd Laverty, Armen S. Manoukian, Yasuyoshi Nishida, Ryusuke Niwa, Masataka Okabe, Gerald M. Rubin, Herman Steller, Tadashi Uemura, Kristin White, the Umeå Stock Center, and the Bloomington Stock Center for the fly stocks used in this work; the Developmental Studies Hybridoma Bank for the anti-ELAV antibody; Sachiyo Miyao for scanning electron microscopy and preparation of fly head sections; and Ritsuko Shimamura for making the fly medium. This work was supported by grants from the Japanese Ministry of Science, Education, Sports and Culture; Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation; Strategic Promotion System for Brain Science, Science and Technology Agency of Japan; and Human Frontier Science Program. A.T. was supported by the Japan Society for the Promotion of Science.

Manuscript received October 13, 1999; Accepted for publication December 7, 1999.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

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