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Corresponding author: Lizabeth A. Perkins, Pediatric Surgical Research Labs, Warren 10, Massachusetts General Hospital, 32 Fruit St., Boston, MA 02114., perkins{at}helix.mgh.harvard.edu (E-mail)
Communicating editor: T. SCHÜPBACH
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Previously, Csw has been demonstrated to function throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unambiguously elucidated, current evidence suggests that Csw plays more than one role during transduction of the RTK signal and, further, the molecular mechanism of Csw function differs depending upon the RTK in question. The isolation and characterization of a new, spontaneously arising, viable allele of csw, cswlf, has allowed us to undertake a genetic approach to identify loci required for Csw function. The rough eye and wing vein gap phenotypes exhibited by adult flies homo- or hemizygous for cswlf has provided a sensitized background from which we have screened a collection of second and third chromosome deficiencies to identify 33 intervals that enhance and 21 intervals that suppress these phenotypes. We have identified intervals encoding known positive mediators of RTK signaling, e.g., drk, dos, Egfr, E(Egfr)B56, pnt, Ras1, rolled/MAPK, sina, spen, Src64B, Star, Su(Raf)3C, and vein, as well as known negative mediators of RTK signaling, e.g., aos, ed, net, Src42A, sty, and su(ve). Of particular interest are the 5 lethal enhancing intervals and 14 suppressing intervals for which no candidate genes have been identified.
RECEPTOR tyrosine kinases (RTKs) control a number of diverse cellular processes including growth, differentiation, migration, and viability (reviewed in 
A combination of genetic and biochemical data has led to the following model of RTK signal transduction. Upon activation by a ligand, the RTK becomes autophosphorylated on specific tyrosyl residues. The adapter protein GRB-2 (Downstream of receptor kinase, Drk) then binds the activated receptor via its SH2 domain, leaving two SH3 domains to associate with Son of Sevenless (Sos), a ubiquitously expressed Ras guanine nucleotide exchange factor (
A further component of RTK signaling pathways in Drosophila is Corkscrew (Csw), a cytoplasmic, nonreceptor protein tyrosine phosphatase (PTP;
While a role for Csw in the RTK signaling cassette has been clearly demonstrated, the biochemical function of Csw remains to be unambiguously elucidated. Current evidence suggests that Csw plays more than one role during transduction of the RTK signal and, further, the molecular mechanism of Csw function differs depending upon the RTK in question (
Csw, therefore, plays at least two functions in the Tor pathway, as an adaptor protein for Drk and as a phosphatase to dephosphorylate the negative regulatory RasGap binding site. However, while the Csw:Tor interaction is dependent on RTK activation and tyrosine phosphorylation of the RTK, interaction between Csw and the Sev RTK is constitutive; that is, the Csw:Sev interaction is not activation dependent and does not require tyrosine phosphorylation (
For most RTKs the precise molecular mechanisms of function of Drosophila Csw and its homologues, nematode PTP-2 and vertebrate SHP-2, remain unclear; however, genetic experiments have shown that in nearly all cases these SH2-containing phosphatases serve positive functions during signal transduction (reviewed by
In this article we report the isolation and characterization of a new, spontaneously arising, viable allele of csw, which we designate cswlf. Adult homozygous cswlf flies exhibit rough eyes and a wing vein gap phenotype. Both of these phenotypes are consistent with the requirement of Csw in the Sev and EGFR pathways during eye development (
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Drosophila strains:
All flies were raised on standard Drosophila media at 25°. Chromosomes and mutations that are not described in the text, or below, can be found in FlyBase (
To date, cswlf is the 10thcsw allele to be characterized. Unlike the other 9 alleles where csw mutant animals (csw-/Y) derived from heterozygous females (csw-/+) die during early pupal stages, cswlf is not required for viability. However, all 10 alleles exhibit similar fully penetrant maternal effect phenotypes on embryonic development; that is, all embryos derived from females that lack maternal activity for any of the csw alleles fail to hatch; we refer to these as csw mutant embryos.
Deficiency screen:
Males carrying deficiencies on the second and third chromosomes were crossed to cswlf/FM7. All progeny from this cross were genotyped and counted. Male progeny carrying both the cswlf mutation and the deficiency were scored for enhancement and suppression using three criteria: wing vein gaps, rough eye phenotype, and viability. The wing vein gaps of these flies were compared to that of males mutant for cswlf alone, and likewise for the rough eye phenotype. Viability was scored by comparing the number of males bearing cswlf and the deficiency to the number of males with cswlf and the balancer for that particular deficiency. In cases where cswlf/Y; Df/+ males were not obtained, we controlled for the possibility that the deficiency deleted a haplo-insufficient locus by determining whether the sibling FM7/Y; Df/+ males were present in expected numbers.
Phenotypic analysis of adult structures:
The eyes of live flies were examined in an Electroscan microscope under a wet vent chamber. Fixation and sectioning of the adult eyes were performed essentially as described in
Production of csw germline mosaics:
cswlf germline clones were generated using the "dominant female sterile or FLP-DF5 technique" as previously described (
In situ hybridization and immunohistochemistry:
In situ hybridization on whole-mount embryos using digoxigenin-labeled probes was performed according to
For immunohistochemistry, embryos were fixed with 4% formaldehyde and immunostained according to
All embryos were analyzed and photographed with a Nikon FXA equipped with Nomarski optics. Larval cuticles were prepared in Hoyer's mountant as described by
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Isolation of the viable allele cswlf:
Previously all known alleles of the Drosophila gene csw have been found to be lethal at the early pupal stage. We have recently isolated a recessive, viable spontaneous mutation that exhibits reproducible rough eye and wing vein gap phenotypes (Fig 1, compare A and E with B and F). The mutation displays a degree of temperature sensitivity with stronger phenotypes displayed at higher temperatures (data not shown). Linkage analysis and deficiency/duplication mapping determined that the mutation is located to the distal tip of the X chromosome in the vicinity of the previously described corkscrew (csw) locus. When placed in trans to any of the other csw alleles the resulting csw-/cswlf females were largely nonviable. However, when occasional females did emerge they exhibited eye and wing phenotypes more severe than those observed in cswlf/cswlf females (data not shown). The phenotypes of the new mutation were completely rescued by expression of the cswwild type minigene under the control of its endogenous promoter (J. A. LORENZEN, M. MELNICK and L. A. PERKINS, unpublished results). This rescue, together with the complementation analysis, allowed us to conclude that the new mutation represents a reduced activity, viable allele of csw, which we have designated cswlf.
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Sections of the adult ommatidium reveal that the rough eye phenotype results from a loss of photoreceptors, commonly R7; however, there is also occasional loss of outer photoreceptors as well (Fig 1I and Fig J). These results further support a role for Csw in both the EGF (DER; TOP) and SEV RTK pathways (
Ovary and embryonic phenotypes of cswlf:
Under favorable culture conditions 10 to 15% of females from a balanced stock are homozygous for cswlf. These homozygous cswlf females are essentially sterile and only rarely produce fertilized eggs. The eggs from homozygous cswlf mothers have shells that are partially ventralized (Fig 2). This defect, previously demonstrated to be the result of Csw function in the Torpedo EGF RTK pathway during oogenesis (
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Fertilized eggs are rarely obtained from homozygous cswlf mothers, so we chose to analyze cswlf mutant phenotypes during embryogenesis by producing cswlf germline clones using the FLP-DFS technique (
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Since the observed cuticular defects, alone, could not account for the lethality exhibited by cswlf mutant embryos, specific tissues, whose specification and/or differentiation are dependent upon RTK signaling, were examined for defects resulting from the cswlf mutation. Consistent with the mild cuticular phenotypes observed, residual cswlf activity is sufficient for normal function of the Tor RTK pathway, the first RTK pathway known to be active during embryogenesis. In this pathway, which initiates patterning of the acron (anteriormost head) and telson (posteriormost tail), we used the expression patterns of tll and hkb as indicators of a loss of anterior and posterior patterning information (
At later stages of embryogenesis, tissues whose specification and/or differentiation requires signaling by the EGFR or either of the Drosophila FGF RTKs [Breathless (Btl) and Heartless (Htl)] were assayed in cswlf mutant embryos. For specification and proper positioning of the segmentally reiterated precursor cells that give rise to both the DA1/#1 larval muscles and a subset of pericardial cells, input from both the EGF and Htl pathways is essential (
Finally, to assay the effect of the cswlf lesion on signaling by the Btl RTK, we utilized a molecular marker that highlights the tracheal lumen (
Taken together, our results suggest that the cswlf lesion is sufficient to compromise four RTK signaling pathways that are utilized throughout development. EGFR signaling is disrupted during oogenesis, embryogenesis, and imaginal stages. SEV signaling is disrupted during specification of the R7 photoreceptor, and both FGF RTKs, Btl and Htl, are disrupted during embryogenesis. Surprisingly, specification of the larval head and tail by the Torso RTK is unaffected by cswlf.
Genetics interactions of cswlf with altered forms of Ras and Raf:
Previously,
We investigated the interactions of the above transgenes in males hemizygous for cswlf (Fig 4). Further supporting a role for Csw during Ras signaling, cswlf partially suppressed the rough eye phenotypes of the activated forms of Ras and Raf, sev-RasV12 and sev-RafTorso, while it enhanced the rough eye phenotype of dominant negative Ras, sev-RasN17 (Fig 4, compare A with G, B with H, and C with I). We characterized the interactions at the cellular level by examining transverse sections through ommatidia. Consistent with the interactions observed by SEM, above, in combination with activated forms of both Ras and Raf, cswlf partially suppressed the transformation of the nonneuronal cone cells into R7 photoreceptors (Fig 4, compare D with J and F with L). Conversely, the photoreceptor loss resulting from sev-RasN17 was enhanced in combination with cswlf (Fig 4, compare E with K).
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Taken together these data suggest that in the developing eye the cswlf lesion is sufficient to alter the activities of both activated and dominant negative forms of Ras and an activated form of Raf. These phenotypes, developing within the context of signaling by both the EGFR and Sev pathways, firmly support a role for Csw as a modifier of the strengths of the inductive signals elicited by both the EGF and SEV RTKs.
Genetic interactions of cswlf with downstream transcription factors in the eye:
We have tested for genetic interactions between cswlf and two downstream effectors of Ras signaling, yan and pointed, in the developing eye. Within the context of R7 formation during signaling by the Sev RTK, Yan and Pnt antagonize each other; that is, while pnt promotes the formation of the R7 photoreceptor, yan opposes or negatively regulates R7 formation (
88, and a recessive lethal mutation, pnt07825, both dominantly enhanced the rough eye phenotype of males carrying cswlf. An enhancement of the wing vein gap phenotype of cswlf males was observed with pnt07825, but not pnt88.
Together these results suggest that the cswlf lesion is sufficient to alter the signal received by the transcription factors whose activities are essential for proper formation of the R7 photoreceptor.
Screening for modifiers of cswlf adult phenotypes:
We have utilized the cswlf mutation as a starting point to conduct a sensitized genetic screen (see MATERIALS AND METHODS) to identify chromosomal regions that are able to dominantly enhance or suppress the cswlf phenotype when their dosage is reduced by one-half. Enhancing deficiencies (Table 1) were defined as those deficiencies that result in absence, i.e., lethality, of cswlf/Y; Df/+ males or those deficiencies in which the cswlf mutant phenotype was more severe but not lethal (examples shown in Fig 1D, Fig H, and Fig L). Suppressing deficiencies (Table 2) were defined as those deficiencies that in conjunction with cswlf (genotype: cswlf/Y; Df/+) result in marked improvement of the cswlf adult phenotypes (examples shown in Fig 1C, Fig G, and Fig K). The eye and wing phenotypes of outcrossed cswlf males (genotype: cswlf/Y; +/+) were used as controls. From this screen we have identified regions on both the second and third chromosomes that encompass loci that modify the expressivity of cswlf. Frequently, the intervals containing loci that modify the cswlf phenotypes could be refined by deficiencies that do not interact with cswlf. These are included in Table 1 and Table 2 as deficiencies showing no (N) interaction with cswlf.
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Enhancers of cswlf:
Our deficiency screen identified 14 genomic regions on the second chromosome and 19 genomic regions on the third chromosome that dominantly enhanced the cswlf eye and/or wing vein phenotypes (Table 1). Consistent with a role for Csw in RTK signaling, as well as validating the screen, a significant number of the genomic regions identified encompass loci that function positively during RTK signaling or genetically interact with known components of RTK signaling. Briefly, deficiencies were identified that encode the Egfr receptor, the RTK transducers drk, dos, Ras1, rolled/MAPK, Src64B, the Egfr ligand vein, the Egfr ligand processing protein Star, and the transcription factors pnt, seven-in-absentia (sina), and seven-up (svp). We also identified enhancer interactions with deficiencies encoding candidate loci that have been previously identified as modifiers of RTK phenotypes and whose molecular identities are unknown, e.g., E(Egfr)B56 and Su(Raf)3C, or known molecules whose connection to RTK signaling is undescribed, e.g., the RNP motif containing protein split-ends (spen).
When possible, we tested for genetic interactions between cswlf and mutations in the candidate loci. As outlined above, we observed strong genetic interactions with cswlf and known positive components of RTK signaling, Ras, Raf, and Pnt. In addition, and supporting their designated "candidate genes" status, loss-of-function mutations in several loci, dosR31, drkTZ160, Egfr1F26, rasC40b, and Star1, were observed to strongly enhance the cswlf mutant phenotypes, while the gain-of-function mutation rolledSEM strongly suppressed the cswlf phenotypes. We were satisfied to learn that, in combination with cswlf, the deficiency that contains the E(Egfr)B56 candidate, like the mutation, enhanced only the vein gap phenotype (17, also enhanced the wing phenotype of cswlf but, like E(Egfr)B56, no obvious enhancement of the cswlf rough eye phenotype was observed in combination with Src64B17.
Of particular interest are the enhancing deficiencies for which no candidate genes have been identified. Five genomic regions, three on the second chromosome and two on third chromosome, are lethal in combination with cswlf (Table 1). It is possible, however, that the lethal interactions with Df(3L)st-f13 (72C1-D1; 73A3-4) and Df(3L)Pc-MK (78A3; 79E1-2) result from the combined effects of nonlethal enhancer loci that also map to these regions.
Of the remaining nonlethal enhancing loci, most are enhancers of both the wing vein and eye cswlf phenotypes, five are enhancers of the vein gap phenotype only, and two are enhancers of the eye phenotype only. One of the latter, covered by the deficiency Df(2L)net-PMF, is an enhancer of the rough eye phenotype while also completely suppressing the wing vein phenotype, suggesting that two interacting loci are in this region. Interestingly, a candidate gene, net, maps to this region and displays ectopic veins and may thus be responsible for the suppression of the wing vein gap phenotype (see below).
Finally, there are several genomic intervals for which we expected interactions with cswlf; however, no interactions were observed. Three notable examples include the genomic intervals encoding the exchange factor Sos (34D4), the kinase Ksr (83A5), and the transcription factor Phyllopod (51A2). There are several reasons why interactions may not have been detected in these genetic intervals, the most likely being that the dosage of an interacting gene removed by the deficiency is not critical and, thus, removal of one copy would be insufficient to modify the cswlf phenotypes. Another likely possibility is that the regions deleted remove not only these positive components of RTK signaling but also nearby negative components. This latter hypothesis is thought to be the case for ksr and sos since, as expected, mutant alleles for two of these expected interacting loci, ksrS638 and sos34G, exhibit strong dominant enhancing interactions with cswlf.
Suppressors of cswlf:
Our deficiency screen identified 13 genomic regions on the second chromosome and 10 genomic regions on the third chromosome that dominantly suppressed the cswlf eye and/or wing vein phenotypes (Table 2). Among the suppressing loci 8 suppress both the wing vein and eye cswlf phenotypes, 8 are suppressors of the vein gap phenotype only, and 7 are suppressors of the eye phenotype only.
Also validating the screen, several of the genomic regions identified encompass loci that function to negatively regulate RTK signaling; that is, deficiencies were identified that encode the known negative regulators of RTK signaling argos (aos) and sprouty (sty). We also identified suppressing interactions with deficiencies encoding candidate loci that have been previously identified as modifiers of RTK phenotypes and whose molecular identities are both known, e.g., the serum response factor gene blistered (bs) and the tyrosine kinase gene Src42A, and unknown, e.g., echinoid (ed), net, and suppressor of veinlet [su(ve)], as well as a gene whose expression pattern suggests involvement in RTK signaling, e.g., the zinc finger encoding gene terminus (term).
When possible, we tested for genetic interactions between cswlf and mutations in the candidate loci. Supporting their designated candidate genes status, loss-of-function mutations in several loci, aosdelta7, net1, Src42Ak10108, and sty5, were found to suppress cswlf mutant phenotypes. Fourteen additional suppressor loci, 8 on the second chromosome and 6 on the third chromosome, contain no obvious candidate genes (Table 2).
Interestingly, two deficiencies that delete the candidate locus bs (Df(2R)Px4 and Df(2R)Px2) and are strong suppressors of both the eye and vein gap phenotypes of cswlf, on their own, exhibit a dominant blistered wing and ectopic vein phenotype, which is, in turn, suppressed by interaction with cswlf (Fig 5). The dominant phenotypes of the deficiencies are due to the loss of the bs gene; however, a role for bs in eye development has not previously been reported (
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Finally, there are several genomic intervals for which we expected interactions with cswlf; however, no interactions were observed. Two notable examples include the genomic intervals encoding the GTPase-activating protein Gap1 (67D2-3) and the Ets-domain transcription factor Yan (22D1-2). As expected, however, the loss-of-function allele Gap1B2 exhibits a strong suppressing interaction with cswlf, and the gain-of-function allele yanXS-2382 (see above) exhibits a strong enhancing interaction with cswlf.
| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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cswlf is a hypomorphic allele that affects several RTK pathways:
We have isolated and characterized a novel allele of the Drosophila csw gene, cswlf, that bypasses the pupal lethality associated with all other known csw alleles and, when homozygous, results in rough eye and wing vein loss adult phenotypes (Fig 1). Complementation analysis with different alleles of csw, as well as deficiencies that remove the csw gene, always result in lethality or weakly viable adults with eye and wing phenotypes much more severe than homozygous cswlf flies. Further, the phenotypes of homozygous cswlf adults can be rescued by addition of one copy of a csw minigene. Together, these results suggest that the cswlf allele is a hypomorphic or residual activity mutation in the csw gene. This viable cswlf allele has provided a highly useful tool for both genetic and phenotypic analysis of csw function, as well as the identification of genomic regions and loci that genetically interact with csw.
Csw function had previously been shown to be essential for many developmental processes throughout the life cycle, including both the Sev and Egfr signaling pathways during imaginal development (
To investigate the effect of the cswlf lesion during embryonic RTK signaling, we mated females bearing cswlf germline clones to hemizygous cswlf males and examined the resulting cswlf/cswlf female and cswlf/Y male progeny. That cswlf is the weakest of the series of csw alleles is supported by the analysis of the expression patterns of tll and hkb, transcription factors whose expression directs the formation of larval terminal structures and is dependent on the activity of the Torso RTK (reviewed by
While the reduction in Csw function by the cswlf lesion does not show any obvious effects on Torso signaling, this mutation mildly affects signaling by other essential embryonic RTKs (Fig 3). As is the case for signaling by the Egfr during oogenesis and imaginal development, the cswlf mutation reduces the Egfr pathway signal, as evidenced by a partial fusion of denticle bands along the midline, mild twisting, and incomplete germ band shortening, all phenotypes exhibited by mutations affecting Egfr signaling (reviewed by
Collectively, our phenotypic analysis of the cswlf lesion suggests that, with the exception of the RTK Torso, this mutation affects an aspect of Csw function that is utilized within each of the RTK signaling pathways examined. This point is important in the context of our deficiency screen where this lesion was used to identify genomic regions that either enhance or suppress the cswlf adult mutant phenotypes. In this regard, we can reasonably assume that among the interacting genomic intervals that modify both the Sev and Egfr pathways required for proper eye and wing formation are candidate loci that function to modify additional RTK pathways, e.g., the Btl and Htl signaling pathways.
Role of Csw in the RTK signaling cassette:
We tested for interactions between components of the pathway and cswlf. Uniformly, we observed that loss-of-function or dominant negative mutations in positive transducers of the RTK signal enhanced the cswlf phenotypes, while gain-of-function or activated forms of positive transducers suppressed the cswlf phenotypes. Similarly, loss-of-function mutations in negative transducers of the RTK signal suppressed the cswlf phenotypes and a gain-of-function mutation in a negative transducer, yan, enhanced the cswlf phenotypes. These results firmly support previous work demonstrating a role for Csw as a positive mediator of RTK pathway signaling, and our results are consistent with previous reports that have suggested that Csw appears to function either upstream or downstream of Ras1 and/or D-Raf depending upon the RTK under investigation (
Use of cswlf in a sensitive genetic screen:
cswlf is a viable mutation with easily scored adult phenotypes, thus making it an ideal sensitized genetic background with which to perform an F1 screen for modifier loci. The efficacy of the screen is indicated by the detection of several loci containing known components of the Ras pathway, e.g., Aos, Dos, Drk, Egfr, Pnt, Ras1, Rl/MAPK, Star, and Sty. The screen detected both modifiers of the adult phenotypes and also lethal interactions. This was to be expected, as our phenotypic analysis shows that cswlf reduces the efficiency of various RTK signals throughout development, not just in the eyes and wing veins. Some of the modifier loci included the map positions of mutations previously detected in genetic screens for modifiers of the Ras pathway; e.g., E(Egfr)B56 and Spen/E(raf)3A were detected in screens for modifiers of Egfr and Raf phenotypes (
Interestingly, we also observed enhancing and suppressing genetic interactions with genomic intervals containing the nonreceptor tyrosine kinases Src64B and Src42A, respectively. With regard to Src64B, we tested a small deletion that encompasses the Src64B gene, Df(3L)10H as well as the adult viable hypomorphic mutation Src64B17, both of which enhance the wing phenotype of cswlf; however, unlike the deficiency Df(3L)10H, we observed no obvious enhancement of the cswlf rough eye phenotype in combination with Src64B17. While in Drosophila the role of Src64B in RTK signaling has not been broadly explored, ectopic expression studies have suggested that Src64B plays a positive role during photoreceptor differentiation (
Other enhancer loci did not appear to have any obvious candidate interacting genes, including four of the lethal interactions on the second chromosome and two lethal interactions on the third chromosome. For some of these loci we were able to confirm the interaction with overlapping deficiencies (see Table 1), demonstrating that the modifiers most likely reside in the deficiency location and not elsewhere on the chromosome. Similarly, a number of the nonlethal modifier loci were also located in overlapping deficiencies.
Among the suppressor loci we identified are net, a mutation with a vein promoting phenotype; ed, which has previously been shown to be a suppressor of reduced Egfr signaling; and sty, a known negative regulator of multiple RTK pathways throughout development. In addition, two strong suppressors were contained within overlapping deficiencies that removed the bs gene. The latter deficiencies result in dominant wing blistering and ectopic vein phenotypes, which in turn are suppressed by cswlf, suggesting that the interacting allele is indeed bs, a finding we have confirmed by testing interactions with a number of bs alleles. Bs has previously been shown to act autonomously in the intervein cells of the pupal wing in order to limit the width of the wing veins and is a Drosophila homologue of the mammalian serum response factor, a MADS-box containing transcriptional regulator (
Perspective:
The fortuitous isolation of a novel, viable allele of csw, a known positive transducer of multiple RTK initiated signals, has provided a powerful tool to identify autosomal genomic intervals that genetically enhance or suppress the adult csw mutant phenotypes. It is anticipated that several of the autosomal intervals identified will yield hitherto unknown transducers and/or regulators of RTK signaling, each of which may provide clues toward the identification of new or novel ways to regulate the activities of these universally conserved, essential signaling pathways.
| FOOTNOTES |
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1 These authors contributed equally to this work. 
| ACKNOWLEDGMENTS |
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For antibodies we thank M. Krasnow, T. Volk, Dave Kosman, and John Reinitz. For fly stocks we thank the Bloomington Stock Center, Y. Hiromi, N. Perrimon, J. Price, G. Rubin, F. Takahashi, and C.-t Wu. We thank C. Gilpin for assistance with electron microscopy and W. Li for sharing data prior to publication. L.F. is supported by the Biotechnology and Biological Sciences Research Council. M.B. is supported by the Medical Research Council and the Wellcome Trust. J.A.L. is supported by Postdoctoral Fellowships from the National Institutes of Health, F32GM17901, and the American Cancer Society, Massachusetts Division. L.A.P is supported by a National Science Foundation grant IBN-9904606 and by the Department of Surgery at the Massachusetts General Hospital.
Manuscript received March 2, 2000; Accepted for publication June 21, 2000.
| LITERATURE CITED |
|---|
| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
ALLARD, J. D., H. C. CHANG, R. HERBST, H. MCNEILL, and M. A. SIMON, 1996 The SH2-containing tyrosine phosphatase Corkscrew is required during signaling by Sevenless, Ras1 and Raf. Development 122:1137-1146[Abstract].
ALLARD, J. D., R. HERBST, P. M. CARROLL, and M. SIMON, 1998 Mutational analysis of the SRC homology 2 domain protein-tyrosine phosphatase Corkscrew. J. Biol. Chem. 273:13129-13135
ASHBURNER, M., 1989 Drosophila: A Laboratory Handbook and Manual, Vols. 1 and 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
BAKER, N. E. and G. M. RUBIN, 1989 Effect on eye development of dominant mutations in Drosophila homologue of the EGF receptor. Nature 340:150-153[Medline].
BALDARELLI, R. M., P. A. MAHONEY, F. SALAS, E. GUSTAVSON, and P. D. BOYER et al., 1988 Transcripts of the Drosophila blastoderm-specific locus, terminus, are concentrated posteriorly and encode a potential DNA-binding finger. Dev. Biol. 125:85-95[Medline].
BEGEMANN, G., A. M. MICHON, L. VAN DER VOORN, R. WEPF, and M. MLODZIK, 1995 The Drosophila orphan nuclear receptor Seven-up requires the Ras pathway for its function in photoreceptor determination. Development 121:225-235[Abstract].
BEIMAN, M., B. Z. SHILO, and T. VOLK, 1996 Heartless, a Drosophila FGF receptor homolog, is essential for cell migration and establishment of several mesodermal lineages. Genes Dev. 10:2993-3002
BIGGS, W. H. and S. L. ZIPURSKY, 1992 Primary structure, expression, and signal-dependent tyrosine phosphorylation of a Drosophila homolog of extracellular signal-regulated kinase. Proc. Natl. Acad. Sci. USA 89:6295-6299
BRONNER, G. and H. JÄCKLE, 1991 Control and function of terminal gap gene activity in the posterior pole region of the Drosophila embryo. Mech. Dev. 35:205-211[Medline].
BRONNER, G., Q. CHU-LAGRAFF, C. Q. DOE, B. COHEN, and D. WEIGEL et al., 1994 Sp 1/egr-like zinc-finger protein required for endoderm specification and germ layer formation in Drosophila. Nature 369:664-668[Medline].
BRUNNER, D., K. DUCKER, N. OELLERS, E. HAFEN, and H. SCHOLZ et al., 1994 The ETS domain protein Pointed-P2 is a target of MAP kinase sevenless signal transduction pathway. Nature 370:386-389[Medline].
BUFF, E., A. CARMENA, S. GISSELBRECHT, F. JIMENEZ, and A. M. MICHELSON, 1998 Signalling by the Drosophila epidermal growth factor receptor is required for the specification and diversification of embryonic muscle progenitors. Development 125:2075-2086[Abstract].
CARMENA, A., S. GISSELBRECHT, J. HARRISON, F. JIMENEZ, and A. M. MICHELSON, 1998 Combinatorial signaling codes for the progressive determination of cell fates in the Drosophila embryonic mesoderm. Genes Dev. 12:3910-3922
CARTHEW, R. W. and G. M. RUBIN, 1990 seven in absentia, a gene required for specification of R7 cell fate in the Drosophila eye. Cell 63:561-577[Medline].
CASANOVA, J., M. FURRIOLS, L. A. MCCORMICK, and G. STRUHL, 1995 Similarities between trunk and spatzle, putative extracellular ligands specifying body pattern in Drosophila. Genes Dev. 9:2539-2544
CASCI, T., J. VINOS, and M. FREEMAN, 1999 Sprouty, an intracellular inhibitor of Ras signaling. Cell 96:655-665[Medline].
CHOU, T.-Z. and N. PERRIMON, 1996 The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster.. Genetics 144:1673-1679[Abstract].
CLEGHON, V., U. GAYKO, T. D. COPELAND, L. A. PERKINS, and N. PERRIMON et al., 1996 Specific phosphotyrosine residues on the Drosophila Torso RTK mediate both positive and negative signaling events that are compensatory. Genes Dev. 10:566-577
CLEGHON, V., P. FELDMAN, C. GHIGLIONE, T. D. COPELAND, and N. PERRIMON et al., 1998 Opposing actions of CSW and RasGAP modulate the strength of Torso RTK signaling in the Drosophila terminal pathway. Mol. Cell 2:719-727[Medline].
CLIFFORD, R. J. and T. SCHÜPBACH, 1989 Coordinately and differentially mutable activities of torpedo, the Drosophila melanogaster homolog of the vertebrate EGF receptor gene. Genetics 123:771-787
COOPER, J. A., M. A. SIMON, and S. J. KUSSICK, 1996 Signaling by ectopically expressed Drosophila Src64 requires the protein tyrosine phosphatase Corkscrew and the adapter Downstream of Receptor Kinases. Cell Growth Differ. 7:1435-1441[Abstract].
DE CELIS, J. F., 1998 Positioning and differentiation of veins in the Drosophila wing. Int. J. Dev. Biol. 42:335-343[Medline].
DICKSON, B., F. SPRENGER, D. MORRISON, and E. HAFEN, 1992 Raf functions downstream of Ras1 in the sevenless signal transduction pathway. Nature 360:600-603[Medline].
DICKSON, B. J., A. VAN DER STRATEN, M. DOMINGUEZ, and E. HAFEN, 1996 Mutations modulating Raf signaling in Drosophila eye development. Genetics 142:163-171[Abstract].
FLYBASE,, 1999 The FlyBase database of the Drosophila Genome Projects and community literature. Nucleic Acids Res. 27:85-88. Available from http://flybase.bio.indiana.edu/
FREEMAN, M., C. KLAMBT, C. S. GOODMAN, and G. M. RUBIN, 1992 The argos gene encodes a diffusible factor that regulates cell fate decisions in the Drosophila eye. Cell 69:963-975[Medline].
FRISTROM, D., P. GOTWALS, S. EATON, T. B. KORNBERG, and M. STURTEVANT et al., 1994 blistered: a gene required for vein/intervein formation in wings of Drosophila. Development 120:2661-2671
FUKUDA, M., Y. GOTOH, and E. NISHIDA, 1997 Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase. EMBO J. 16:1901-1908[Medline].
FURRIOLS, M., A. CASALI, and J. CASANOVA, 1998 Dissecting the mechanism of Torso receptor activation. Mech. Dev. 70:111-118[Medline].
GARCIA-BELLIDO, A. and J. DE CELIS, 1992 Developmental genetics of veination pattern. Annu. Rev. Genet. 26:277-304[Medline].
GAUL, U., G. MARDON, and G. M. RUBIN, 1992 A putative Ras GTPase activating protein acts as a negative regulator of signaling by the sevenless receptor tyrosine kinase. Cell 68:1007-1019[Medline].
GHIGLIONE, C., N. PERRIMON, and L. A. PERKINS, 1999 Quantitative variations in the level of MAPK activity control patterning of the embryonic termini in Drosophila. Dev. Biol. 205:181-193[Medline].
GISSELBRECHT, S., J. B. SKEATH, C. Q. DOE, and A. M. MICHELSON, 1996 heartless encodes a fibroblast growth factor receptor (DFR1/DFGF-R2) involved in the directional migration of early mesodermal cells in the Drosophila embryo. Genes Dev. 10:3003-3017
HERBST, R., P. M. CARROL, J. D. ALLARD, J. SCHILLING, and T. RAABE et al., 1996 Daughter of Sevenless is a substrate of the phosphotyrosine phosphatase Corkscrew and functions during Sevenless signaling. Cell 85:899-909[Medline].
HERTOG, J. D., 1999 Protein-tyrosine phosphatases in development. Mech. Dev. 85:3-14[Medline].
HSU, J. C., C.-T. WU, N. PERRIMON, and J. M. BAI, 1998 Echinoid, a negative regulator of the Drosophila EGF receptor signaling pathway. A. Dros. Res. Conf. 39:193A.
KARIM, F. D., H. C. CHANG, M. THERRIEN, D. A. WASSERMAN, and T. LAVERTY et al., 1996 A screen for genes that function downstream of Ras1 during Drosophila eye development. Genetics 143:315-329[Abstract].
KHOKHLATCHEV, A. V., B. CANAGARAJAH, J. WILSBACHER, M. ROBINSON, and M. ATKINSON et al., 1998 Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell 93:605-615[Medline].
KOLODKIN, A. L., A. T. PICKUP, D. M. LIN, C. S. GOODMAN, and V. BANJEREE, 1994 Characterization of Star and its interactions with Sevenless and Egf receptor during photoreceptor development in Drosophila. Development 120:1731-1745[Abstract].
KRAMER, S., M. OKABE, N. HACOHEN, M. A. KRASNOW, and Y. HIROMI, 1999 Sprouty: a common antagonist of FGF and EGF signaling pathways in Drosophila. Development 126:2515-2525[Abstract].
LAI, Z.-C. and G. M. RUBIN, 1992 Negative control of photoreceptor development in Drosophila by the product of the yan gene, an ETS domain protein. Cell 70:609-620[Medline].
