RECEPTOR tyrosine kinases (RTKs) play many es- pathways that may not be essential for the functions of

Copyright 2003 by the Genetics Society of America Drosophila Gain-of-Function Mutant RTK Torso Triggers Ectopic Dpp and STAT Signaling Jinghong Li and Willis X. Li 1 Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, New York 14642 Manuscript received October 10, 2002 Accepted for publication January 29, 2003 ABSTRACT Overactivation of receptor tyrosine kinases (RTKs) has been linked to tumorigenesis. To understand how a hyperactivated RTK functions differently from wild-type RTK, we conducted a genome-wide systematic survey for genes that are required for signaling by a gain-of-function mutant Drosophila RTK Torso (Tor). We screened chromosomal deficiencies for suppression of a gain-of-function mutation tor (tor GOF ), which led to the identification of 26 genomic regions that, when in half dosage, suppressed the defects caused by tor GOF. Testing of candidate genes in these regions revealed many genes known to be involved in Tor signaling (such as those encoding the Ras-MAPK cassette, adaptor and structural molecules of RTK signaling, and downstream target genes of Tor), confirming the specificity of this genetic screen. Importantly, this screen also identified components of the TGF (Dpp) and JAK/STAT pathways as being required for Tor GOF signaling. Specifically, we found that reducing the dosage of thickveins (tkv), Mothers against dpp (Mad), or STAT92E (aka marelle), respectively, suppressed tor GOF phenotypes. Furthermore, we demonstrate that in tor GOF embryos, dpp is ectopically expressed and thus may contribute to the patterning defects. These results demonstrate an essential requirement of noncanonical signaling pathways for a persistently activated RTK to cause pathological defects in an organism. RECEPTOR tyrosine kinases (RTKs) play many es- pathways that may not be essential for the functions of sential roles in normal development as well as in the RTK under physiological conditions. According to pathogenesis. The RTK family includes a large number this scenario, signaling from a highly activated RTK of single-transmembrane cell surface receptors, includnot might cross-activate components of a pathway that is ing receptors for peptide ligands such as epidermal normally involved in or important for the RTK re- growth factor (EGF), platelet-derived growth factor sponse, or it might induce the ectopic expression of (PDGF), and fibroblast growth factor. RTK overactivacascade. a ligand that triggers such a noncanonical signaling tion, which can result from ligand overabundance or ligand-independent constitutive activating mutations, We have studied the Torso (Tor) RTK pathway in the has been linked to many cancers and other human early Drosophila embryo to address the issue of how diseases (reviewed by Robertson et al. 2000). Despite overactivation of an RTK may result in aberrant gene extensive studies, we are still far from understanding expression. The early Drosophila embryo offers a unique how regulated activation of RTKs induces precise patbecause opportunity for studying signaling pathway interactions terns of gene expression and how constitutive activation many pathways operate simultaneously during of RTKs causes aberrant gene expression patterns and development and these pathways can potentially inter- pathological conditions. Previous models suggest that a act with each other. Tor is most homologous to the constitutively activated RTK hyperactivates a canonical PDGF receptor and is responsible for determining emdownstream signal transduction pathway, the Ras/Raf/ bryonic terminal cell fates (reviewed by Duffy and Per- MEK/mitogen-activated protein kinase (MAPK) signalalong rimon 1994). The Tor protein is uniformly distributed ing cassette, and the resulting change in signaling duratial the cell membrane at the multinucleated, syncy- tion and/or intensity is sufficient to cause a qualitative blastoderm stage of the embryo. However, it is actition perturbation of gene expression patterns (Marshall vated only at the two polar regions of the early embryo 1995; Greenwood and Struhl 1997; Sewing et al. 1997; by spatially restricted ligands (Casanova and Struhl Woods et al. 1997; Ghiglione et al. 1999). However, an 1993). As a result, its downstream target genes, tailless alternative model is that the effects of high RTK signalrior (tll) and huckebein (hkb), are expressed only at the anteing may require the participation of additional signaling and posterior terminal regions of the embryo (Pig- noni et al. 1990, 1992; Weigel et al. 1990; Bronner and Jackle 1991). tll is necessary and sufficient for the 1 development of the posterior structure Filzkörper (see Corresponding author: Department of Biomedical Genetics, University of Rochester Medical Center, 601 Elmwood Ave., KMRB 2-9641, Figure 1A; Steingrimsson et al. 1991). Embryos from Rochester, NY 14642. E-mail: willis_li@urmc.rochester.edu tor mutant mothers show no posterior tll expression and Genetics 164: 247 258 (May 2003)

248 J. Li and W. X. Li are missing the Filzkörper and other posterior struc- of the classical RTK pathway, the STAT (STAT92E) and tures. In contrast, embryos from tor GOF mothers exhibit Dpp [a transforming growth factor (TGF ) homolog] expanded tll expression domains and enlarged, or occa- pathways are required by Tor GOF to exert its effects on sionally ectopic, Filzkörper material (Li et al. 2002). embryos. We have further investigated the involvement Three alleles of tor GOF have been isolated and each con- of STAT92E in Tor GOF signaling and found that STAT92E tains a point mutation in the extracellular, ligand-binding is essential for the functions of Tor GOF and is likely di- domain, presumably favoring ligand-independent rectly activated by Tor GOF (Li et al. 2002). However, sur- dimerization of the receptor (Sprenger and Nusslein- prisingly, we found that STAT92E is dispensable for Volhard 1992). Furthermore, studies of the transcrip- wild-type Tor to induce its target gene tll (Li et al. 2002). tional regulation of tll have led to the proposal that Tor Therefore, it appears that STAT92E is particularly im- controls tll expression by derepression, relieving the portant for the activity of the gain-of-function mutant repressors bound to the tll promoter via phosphorylation RTK. Dpp functions as a morphogen in the early embryo by MAPK. This allows tll activation by transcrip- to direct the establishment of the embryonic dorsoven- tional activators that have yet to be unidentified and are tral (D/V) axis. Consistent with such a role, it is ex- presumably ubiquitously present in the embryo (Liaw et pressed mainly in the dorsal central region of the embryo, al. 1995). where Tor signaling is absent (Ray et al. 1991). As with RTKs in mammals, Tor signaling in Drosophila We found that mutations in components of the Dpp is relayed by the Ras-MAPK pathway. Embryos lacking Draf pathway suppressed the phenotypes associated with tor Y9, gene activity have phenotypes identical to those of tor suggesting that Dpp signaling contributes to the defects null embryos, resulting in the complete absence of the caused by Tor Y9. In addition, we found that dpp expres- posterior tll expression and posterior structures (Melnick sion is ectopically induced in tor Y9 embryos. These results et al. 1993; Li et al. 1997). Drosophila Ras1 and Draf are suggest that, in contrast to wild-type RTK that mainly structurally and functionally homologous to mamma- requires the Ras-MAPK cassette, gain-of-function RTK lian H- or K-Ras and Raf-1, respectively, as human Ras requires multiple additional signaling pathways to cause and Raf-1 can at least partially substitute these Drosoph- dramatically different biological consequences. ila proteins during development (Ambrosio et al. 1989; Lu et al. 1993; Casanova et al. 1994; W. X. Li and J. Li, unpublished data), suggesting a conservation in this MATERIALS AND METHODS portion of the RTK signaling pathway. In addition, mu- Drosophila strains and genetics: Fly stocks: Fly stocks include tations in the Ras-MAPK cassette molecules completely tor Y9 /CyO (viable and fertile due to the presence of suppres- abolish the effects of tor GOF on embryos (Ambrosio et al. sors in this particular stock; Klingler et al. 1988) and dpp H46 / CyO23. The CyO23 chromosome carries an extra copy of dpp 1989). Therefore, genetically the Ras-MAPK cassette is as a transgene in addition to the endogenous dpp, thus dupliessential for both Tor and Tor GOF molecules and appears cating the dpp locus (Wharton et al. 1993). All other stocks, to be the major, if not the sole, downstream signaling including the Bloomington deficiency kit stocks, are from the pathway that conveys Tor signals in wild-type embryos. Bloomington Drosophila Stock Center (FlyBase 1998) or as Whether additional pathways are required for Tor GOF described in Li et al. (2000, 2002). Genetic screen: To screen for suppressors of tor Y9 by autosomal remains unclear. Df stocks, virgin females of tor Y9 /CyO were crossed to males To systematically examine whether additional signal- of each individual Df stock (Df/Balancer) in vials. From the F 1 ing pathways are required for Tor GOF, we conducted a progeny of each of the above crosses, one to five virgin females genetic screen using a set of contiguous chromosomal of the tor Y9 / ; Df/ genotype (identified by the absence of deficiencies to determine what genomic regions, and Balancer and CyO) were crossed to wild-type males and tested for fertility at 18. Normally tor Y9 / females are completely subsequently what genes in these regions, may partici- sterile at 18 because they lay eggs of mostly class II phenotype pate in signaling by a Tor GOF mutant, Tor Y9. Genetic that fail to hatch (see results and Figure 1C). The presence screens for modifiers of Tor or other RTK downstream of hatched larvae in eggs laid by tor Y9 / ; Df/ females would components (such as Draf of Ras1) have previously been indicate that the sterility of tor Y9 / females is dominantly conducted and have contributed to the identification suppressed by a particular Df. Suppression is further con- firmed by retesting and examination of cuticle preparations. of a number of essential components of RTK or Ras/ To screen for X-chromosomal Df stocks, Df/Balancer females Raf signaling (Simon et al. 1991; Doyle and Bishop were used to cross with tor Y9 /CyO males. Certain CyO chromosomes 1993; Dickson et al. 1996; Karim et al. 1996; Li et al. exhibited suppression of tor Y9 and were not used as a 2000). We focused on the genomic regions that are not control. Embryos collected from tor Y9 /CyO flies crossed to wild type (Oregon R) were used as a control. known to encompass genes required for RTK signaling Effects of the zygotic dosage of dpp on tor GOF phenotypes: To test because these regions might contain genes encoding the effects of changing the dosage of dpp on tor Y9 phenotypes, novel components of RTK signaling or, more interest- we crossed tor Y9 / females to dpp H46 /CyO23 (carrying Dp[dpp ]) ingly to this study, components of other signaling pathresulting males at 18 and examined the cuticles of the embryos. The embryos from this cross should have either one or ways that are particularly important for the phenotypic three zygotic doses of dpp, since they should have a zygotic effects of the Tor Y9 mutation or of gain-of-function forms genotype of either dpp H46 / or CyO23/ (two copies of the of RTKs in general. Here we show that in addition to endogenous dpp and the duplication carried by the CyO23 the Ras-MAPK cassette and other known components chromosome) regarding the dpp locus. The embryos from

Drosophila Mutant Torso Triggers Dpp and STAT Signaling 249 Figure 1. Classification of Tor gain-of-function mutant phenotypes associated with tor Y9. Larval cuticle preparations of embryos laid by females with indicated genotypes are shown. Solid arrows point to the Filzkörper. Open arrows indicate the head skeletons and mouth hook. Triangles indicate the positions of visible ventral denticle bands. (A) A wild-type larva has eight abdominal denticle bands (A1 A8). Note the position and morphology of the Filzkörper (arrow) and head skeletons. (B) Class I cuticles are typical of embryos from tor Y9 /tor Y9 mothers; usually only a hollow shell of the vitelline membrane can be found in the cuticle preparations. Ventral denticle bands and head skeletons are usually completely absent. The Filzkörper, if present, appears diffused and mislocalized. (C) Class II cuticles are representative of embryos from tor Y9 / mothers raised at 18. These embryos have remnants of defective head skeletons and one or two partial ventral denticle bands. The Filzkörper usually appears normal in structure. (D) Class III embryos, found in the majority of tor Y9 / flies raised at 21, have normal head skeleton and Filzkörper. About four ventral denticle bands are usually present. (E) Class IV cuticles are nearly normal, with only minor defects in the central body region. Some of these embryos will hatch. such a cross are clearly divided into two phenotypic classes nant gain-of-function allele of tor that is associated with (class I and class III; see text) in an 1:1 ratio (see results a point mutation W317R in the extracellular domain of and Figure 4, A and B). This is in contrast to the control embryos (tor Y9 / females crossed to / males) that showed Tor, presumably causing ligand-independent dimerizamostly class II phenotypes (not shown; see Figure 1C). To tion of the Tor Y9 molecules (Sprenger and Nusslein- confirm this interpretation, tor Y9 / females were crossed to Volhard 1992). To make use of tor Y9 for a genetic /CyO23 males, which resulted in embryos zygotically con- screen, we characterized the temperature and dosage taining either two or three copies of dpp. Embryos from such a cross exhibited class I and II phenotypes in about equal dependency of the tor Y9 embryonic phenotypes in detail ratios, suggesting that the class I phenotype is due to the (Figure 1 and Table 1). presence of three copies of dpp. First, we found that tor Y9 is dosage sensitive. Embryos A similar strategy was used to test the effects of changing from homozygous mothers (tor Y9 /tor Y9 ) exhibit the the zygotic dosage of dpp on the tor 4021 phenotype. tor 4021 / strongest gain-of-function phenotypes, while those from females were crossed to dpp H46 /CyO23 males at 22 (intermedithe heterozygous (tor Y9 / ) or hemizygous (tor Y9 /tor ) ate temperature) and the resulting embryos were analyzed for cuticle phenotypes. females show progressively weaker phenotypes (Table Examination of embryos: Cuticle preparations were per- 1). In cuticle preparations of embryos from tor Y9 /tor Y9 formed according to a standard protocol with minor modifimothers, only a hollow shell of vitelline membrane can cations. Embryos were dechorionated with 50% Clorox, washed extensively with 0.1% Triton, mounted in Hoyer s, usually be found. No remnants of ventral denticle bands and photographed in dark-field optics. In situ hybridization or the head skeleton are visible. The pair of Filzkörper, if for dpp mrna was performed according to a standard protocol present, is diffused as two balls of light-colored material using digoxigenin-incorporated antisense RNA probes made usually located in the center of the embryo (Figure 1B). from a PCR fragment of a 500-bp dpp coding region according to the supplier s protocol. Stained embryos were photothe We designate such a phenotype as class I. In contrast, graphed using Normaski optics. majority of embryos from tor Y9 /tor show less severe cuticle phenotypes and fall into the class IV category RESULTS (see below and Table 1). Second, eggs from tor Y9 / females show a dramatic Dosage- and temperature-dependent embryonic phefemales temperature-dependent phenotypic series. At 18, tor Y9 / notypes associated with tor Y9,ator GOF allele, and the lay eggs that exhibit the most severe defects. In design of a genetic screen: tor Y9 is a cold-sensitive domi- cuticle preparations, the majority of these embryos show

250 J. Li and W. X. Li TABLE 1 Dosage and temperature dependency of tor Y9 % of embryos for each maternal genotype and temperature Phenotypic tor Y9 /tor tor Y9 / tor Y9 /tor Y9 class 18 21 25 18 21 25 18 21 25 Class I 0 0 0 13 1 0 91 84 79 Class II 11 4 0 74 23 0 9 16 20 Class III 63 57 6 12 66 16 0 0 1 Class IV 26 49 94 1 10 84 0 0 0 Total (n) 238 292 246 483 518 557 267 243 411 Total (n) represents the total number of cuticles examined for eggs collected at different temperatures and dosages of tor Y9. The percentage indicates cuticles that fall into class I IV phenotypes (see text and Figure 1 for definitions). remnants of the head skeleton, visible as brownish-col- ing a set of nearly contiguous deficiency (Df) stocks, we ored material at the anterior of the egg. Essentially all screened for genes that, when in a heterozygous state, the central structures are undifferentiated except for an could dominantly suppress tor Y9 (see materials and occasional strip of ventral denticle band with discernible methods for more detail). Among a total of 206 Df bristle patterns (Figure 1C). The Filzkörper is clearly stocks representing 70% of the Drosophila genome, identifiable and often is a little enlarged. This phenotype we identified 31 deficiencies, which define 26 genomic is less severe than the class I phenotype and we regions, that dominantly suppressed the maternal-effect designate these embryos as class II. When the temperature sterility of tor Y9 / females to various degrees (Figure 2 is raised to 21, most eggs show improved differenti- and Table 2). To determine which genes in each geno- ation such that they exhibit intact head skeleton, Filzkörper, mic region are responsible for the suppression, we se- and more structures in the central region. lected candidate genes for each deficiency region and Usually about four ventral denticle bands are identifiable used available mutant alleles to test their ability to sup- in these embryos, which we designate as class III press the sterility and embryonic defects associated with (Figure 1D). When tor Y9 / females are kept at 25, there tor Y9 at 18 in trans-heterozygotes. Such testing allowed is a significant improvement in the development of the us to determine genes responsible for the suppression embryos such that many of them hatch and become of tor Y9 in the identified genomic region. These genes nearly morphologically normal crawling larvae. The ma- should be required for the biological functions of Tor Y9. jority of these larvae show only minor defects in the Listed in Table 2 are the candidate genes we have con- central regions. The phenotypes range from missing firmed by testing the indicated mutant alleles. Cuticle one or two ventral denticle bands to indistinguishable phenotypes and classes that confirmed the suppression from wild-type larvae. These are class IV embryos (Fig- of tor Y9 for representative genes are shown in Figure 3 ure 1E). and Table 3. Most of the genomic regions identified in The temperature and dosage-dependent phenotypes our screen for suppressors of tor Y9 encompass loci that of tor Y9 indicate that the strength of this allele is modifiable, are known to function positively in Tor signaling (17/ suggesting that it might be sensitive to the dosage 26; Figure 2 and Table 2), validating the specificity of of other molecules that are required for Tor Y9 to exert this screen. its effects on embryos. If true, the tor Y9 allele would Among the genes that behaved as strong suppressors be suitable for conducting a dosage-sensitive modifier of tor Y9 are those encoding the Ras-MAK signaling cas- genetic screen. We tested the possible suppression of sette components, such as Ras1, Draf, Downstream of Raf1 tor Y9 phenotypes by reducing the dosage of Draf and (Dsor1; encoding a MEK; Tsuda et al. 1993), and rolled Ras1 genes by half. Indeed, Draf or Ras1 heterozygotes (rl; encoding a MAPK) (Brunner et al. 1994). These strongly suppressed the tor Y9 phenotype. At 18, halving genes are located at cytogenetic positions 85D21, 3A1, the maternal dosage of Ras1 or Draf in tor Y9 / females 8D3, and h41, respectively, and are disrupted by the resulted in embryos that exhibited mostly class III phenotype corresponding Dfs listed in Table 2 (FlyBase 1998). In while a significant number of the embryos exhib- addition, we also identified genes encoding components ited class IV phenotype and hatched into larvae (Figure that modulate the Ras-MAPK cassette in RTK signaling 3A; data not shown for Ras1). Therefore, tor Y9 appears as suppressors of tor Y9. These genes include corkscrew suitable for conducting a modifier screen for dosagesensitive (csw; encoding a SHP-2 protein tyrosine phosphatase; genes affecting its function. Perkins et al. 1992), downstream of receptor kinase (drk; Identification of genomic regions and genes required encoding an SH2/SH3 adaptor molecule) (Simon et al. for the effects of tor Y9 on embryonic development: Us- 1993), son of sevenless (sos; encoding the Ras guanyl-

Drosophila Mutant Torso Triggers Dpp and STAT Signaling 251 Figure 2. Genomic regions and candidate genes required for the functions of Tor Y9. Chromosomal arms are represented by solid bars with cytological divisions indicated by numbers. Chromosomal deficiencies screened are represented by boxes that span the genomic region deleted in the deficiency. Open boxes represent the screened deficiencies that did not suppress tor Y9. Solid boxes indicate those that suppressed the maternal effect of tor Y9 heterozygotes. Enhancers are not indicated. Candidate genes in each genomic region are indicated. nucleotide exchange factor; Simon et al. 1991), leonardo contain novel RTK signaling components or novel molecules (leo; encoding 14-3-3 ; Kockel et al. 1997; Li et al. 1997), especially required for Tor Y9 signaling. Identifica- 14-3-3ε (Chang and Rubin 1997), hsp83 (encoding an tion of such genes is ongoing. HSP90 homolog; van der Straten et al. 1997), and tor Involvement of Dpp and STAT92E in signaling by itself. Finally, many of the known downstream target Tor GOF : Testing of candidate genes allowed us to identify genes of Tor or transcription factors involved in Tor three signaling molecules not previously known to be signaling were identified in our genetic screen. These required for the function of Tor GOF. Mothers against dpp genes include the immediate Tor target genes tailless (Mad; a Smad homolog; Newfeld et al. 1996), located (tll) and huckebein (hkb), and additional transcription at cytogenetic position 23D3, is among the genes deleted factors implicated downstream of Tor function, such as by both Df(2L)23C;23E3-6 and Df(2L)S2590, which tailup (tup; Strecker et al. 1991), lilliputian (lilli; Tang suppressed tor Y9 (Table 2). We tested Mad k00237,ap-element et al. 2001; Wittwer et al. 2001), and brachyenteron (byn; allele, and found that it behaved similarly to the Singer et al. 1996). deficiencies and suppressed tor Y9 in trans-heterozygotes The most important results of this screen relate to (Figure 3H; Table 3). thickveins (tkv; encoding a TGF the genes not previously known to be involved in RTK type I receptor homolog; Brummel et al. 1994; Nellen signaling. Testing of the mutant alleles led to the surprising et al. 1994; Penton et al. 1994), located at 25D1-2, is discovery that two components of the Dpp pathway disrupted by the two overlapping deficiencies, Df(2L) and one component of the JAK/STAT pathway are required sc19-4 and Df(2L)cl-h3, both of which modestly sup- for Tor Y9 functions (see below). Since these genes pressed tor Y9 (Table 2). We tested tkv 7, a strong allele were not identified in the numerous previous genetic of tkv, and found that embryos derived from tor Y9 /tkv screens, it is thus possible that they might be particularly females exhibit significantly less severe phenotypes than important for gain-of-function mutant RTK (such as do those derived from tor Y9 / females, suggesting that Tor Y9 ). tkv 7 dominantly suppressed tor Y9 (Figure 3G; Table 3). We were unable to identify a gene(s) responsible for The identification of two genes in the Dpp pathway as some of the Df regions that suppressed tor Y9 (question dominant suppressors of tor Y9 indicates that the Dpp marks in Figure 2 and Table 2). These regions may pathway might be required for tor Y9 signaling. In addi-

252 J. Li and W. X. Li TABLE 2 Deficiencies and mutant alleles that suppressed tor Y9 Suppression Mutant Suppression Deficiency Breakpoints of tor Y9 Candidate gene Vertebrate homolog allele tested of tor Y9 Df(1)sc-J4 1B2 14; 3A3 Draf or polehole (phl), Raf, SHP-2/protein tyrosine Draf 11 29, corkscrew phosphatase csw KN27 Df(1)64c18 2E1 2; 3C2 Draf Raf Draf 11 29 Df(1)JC19 SF6; 3C5 Df(1)lz-90b24 8B5 6; 8D8 9 Downstream of Raf1 MEK1/2 Dsor1 r1 Df(2L)C246 11D E; 12A1 2? Df(2L)net-PMF 21A1; 21B7 8? Df(2L)C144 23A1 2; 23C3 5 lilliputian Transcription factor lilli 3E8 Df(2L)23C; 23E3-6 23C; 23E3 6 Df(2L)S2590 23D2; 23E3 Mother against dpp Smad Mad k00237 Df(2L)sc19-8 24C2 8; 25C8 9 bowel Zinc-finger transcription factor bowl L26 Df(2L)sc19-4 24A5; 25E5 Df(2L)cl-h3 25D2 4; 26B2 5 thickveins TGF receptor type I tkv 7 Df(2L)b87e25 34B12 C1; 35B10 C1 son of sevenless Ras guanylnucleotide exchange factor sos e4g Df(2L)cat-255rv64 35F 36A; 36D? Df(2L)TW137 36C2 4; 37B9 C1 tailup Homeodomain transcription factor tup 1 ln(2r)bw 41A B; 42A2 3 rolled ERK/MAPK rl EMS698 Df(2R)cn9 42E; 44C torso RTK tor XR1 DF(2R)X1 46C; 47A1 Df(2R)stan1 46D7 9; 47F15 16 leonardo 14-3-3 leo P1188 Df(2R)CX1 49C1 4; 50C23 D2 downstream of receptor SH3/SH2 adaptor protein drk TZ160 kinase Df(2R)BSC11 50E6 F1; 51E2 4? Df(3L)M21 62F; 63D Hsp83 HPS90/chaperonin ATPase Hsp83 e6d Df(3L)AC1 67A2; 67D13? Df(3L)vin5 68A2; 69A1 Df(3L)vin7 68C8 11; 59B4 5 brachyenteron Brachyury family transcription factor byn 5 Df(3R)ME15 81F3 6; 82F5 7 huckebein Zinc-finger transcription factor hkb A321R1 Df(3R)p712 84D4 6; 85B6? Df(3R)by10 85D8 12; 85E7 F1 Ras1 Ras Ras1 C40B Df(3R)DG2 89E1 F4; 91B1 2 14-3-3ε 14-3-3ε 14-3-3 e 24 Df(3R)H-B79 92B3; 92F13 marelle or STAT92E STAT mrl 06346 Df(3R)B81 99C8; 100F5 tailless Nuclear hormone receptor tll L10 transcription factor Deficiencies (Dfs) that dominantly suppressed tor Y9 are listed. The chromosomal region uncovered by each Df is indicated as cytological breakpoints as reported (FlyBase 1998). The degree of suppression by each Df is indicated by the number of s, which reflects the hatching rate of eggs laid by females of a particular tor Y9 / ; Df/ genotype, as follows:, 1%;, 1 5%;, 5 10%;, 10%. More than 200 eggs were counted for each genotype. Candidate genes and their mammalian homologs for each Df are listed. Only those candidate genes that were positively confirmed by using the listed mutant alleles are shown. The degree of suppression by each allele tested is also indicated by the number of s, which was determined by the severity of cuticle phenotypes.

Drosophila Mutant Torso Triggers Dpp and STAT Signaling 253 Figure 3. Representative cuticle phenotypes of tor Y9 and a suppressor mutant combination. Representative cuticles from eggs laid by the indicated trans-heterozygotes at 18 are shown. These cuticles fall into class III or IV categories (see text and Table 3 for more detail). (A) Draf 11-29 / ; tor Y9 /. (B) tor Y9 / ; Ras1 C40B /. (C) csw KN27 / ; tor Y9 /. (D) tor Y9 /leo P1188. (E) tor Y9 / ; tll L10 /. (F) tor Y9 / ; hkb A321R1 /. (G) tor Y9 /tkv 7.(H)tor Y9 /Mad k00237. (I) tor Y9 / ; mrl06346/. (J) tor Y9 /tor XR1. Interestingly, tor XR1, a null allele, did not completely suppress tor Y9. Most of the tor Y9 / tor XR1 larvae exhibited collapsed head skeletons similar to those of tor XR1 /tor XR1 (not shown). tion to the two components of the Dpp pathway, we STAT92E is essential for the functions of Tor GOF and is found that STAT92E [also known as marelle (mrl), it likely activated directly by Tor GOF (Li et al. 2002). encodes the Drosophila STAT and is located at 92E1 2; Tor GOF induces ectopic dpp expression: To further Hou et al. 1996; Yan et al. 1996] is responsible for the understand the involvement of the Dpp pathway in Tor Y9 suppression of tor Y9 by Df(3R)H-B79. (Figure 3I; Tables signaling, we examined whether the dpp gene itself is 2 and 3). We have further investigated the involvement required by Tor Y9 to cause embryonic defects. Since of STAT92E in Tor GOF signaling and demonstrated that the dpp locus is haplo-insufficient, one cannot test the TABLE 3 Percentage of embryos in each phenotypic class at 18 % of embryos in each phenotypic class Maternal genotype Class I Class II Class III Class IV Total (n) Draf 11 29 / ; tor Y9 / 1.1 18.4 59.6 20.9 359 tor Y9 / ; Ras1 C40B / 3.2 19.7 50.3 26.8 157 csw KN27 / ; tor Y9 / 5.6 31.0 47.9 15.5 71 tor Y9 /leo P1188 1.6 5.2 25.4 67.8 193 tor Y9 / ; tll L10 / 10.8 36.6 39.8 12.9 93 tor Y9 / ; hkb A321R1 / 16.7 44.9 26.9 11.5 78 tor Y9 /tkv 7 8.2 52.8 27.7 11.3 195 tor Y9 /Mad k00237 4.6 48.5 34.0 12.9 217 tor Y9 / ; mrl 06346 / 8.0 56.4 28.0 7.6 225 tor Y9 /tor XR1 0.0 2.1 63.0 34.9 238 Total (n) represents the total number of cuticles examined for eggs collected at 18 from the females of indicated genotypes. The percentage indicates cuticles that fall into class I IV phenotypes (see text and Figure 1 for definitions).

254 J. Li and W. X. Li Figure 4. Involvement of dpp in the effects of tor GOF mutations on embryos. Lowering and increasing the copy number of dpp suppressed (A and C) and enhanced (B and D) tor Y9 and tor 4021 phenotypes, respectively. (E) Wild-type patterns of dpp expression. (F) dpp expression in tor Y9 embryos. Note that dpp mrna is nearly uniformly expressed in tor Y9 embryos. (G) Elimination of zygotic tll [by Df(3R)tll-e and Df(3R)tll-g trans-heterozygotes] did not completely suppress tor Y9. Note the defective head skeletons (arrow) and a reduced number of the ventral denticle bands. (H) Additional reduction in the maternal Mad gene product (using Mad k00237 ) resulted in a nearly complete suppression of tor Y9. Note the appearance of head skeletons (arrow) and a complete set of the ventral denticle bands. This phenotype is nearly identical to that of tll zygotic null embryos. maternal effects of dpp by using a Df or a null allele in ods) and found that, at an intermediate temperature a heterozygous female. However, the requirement for (22 ), increasing and decreasing the copy number of dpp in the early embryos is purely zygotic. Therefore, dpp enhanced and suppressed tor 4021 phenotypes, respectively the effects of varying the copy number of the paternal (Figure 4, C and D). At 22, embryos from dpp gene on tor Y9 embryonic phenotype could be tested. tor 4021 / females differentiated only rudimentary cuticular At 18, embryos that are zygotically dpp H46 / (containing structures and exhibited mostly class I phenotype one copy of dpp ) and from tor Y9 / females (not shown). Reducing dpp dosage by half in tor 4021 / exhibited class III phenotype (suppressed tor Y9 ; Figure embryos resulted in mostly class II type cuticles (Figure 4A), while those containing three copies of dpp showed 4C). Conversely, a 50% increase in dpp dosage enhanced mostly class I phenotype (enhanced tor Y9 ; Figure 4B; see the phenotypes of tor 4021 / embryos so that most materials and methods). This is in contrast to the of them did not differentiate any discernible cuticles embryos with two copies of dpp (wild type regarding (Figure 4D). Therefore, changes of dpp dosage modify the dpp locus), which showed mostly class II phenotypes the phenotypes of at least two tor GOF alleles, suggesting at 18 (see Figure 1C). Therefore, increasing and decreasing that the Dpp pathway is generally involved in Tor GOF the copy number of dpp enhances and sup- signaling. presses tor Y9 phenotype, respectively. Since dpp affects tor GOF phenotypes in a dosage-depen- To investigate whether the dpp pathway is generally dent manner, we reasoned that dpp might be ectopically involved in Tor GOF signaling or is specific for tor Y9,we induced in tor GOF embryos, triggering aberrant activation tested genetic interactions between dpp and another of the Dpp pathway, which in turn contributes to the tor GOF allele, tor 4021 (Sprenger and Nusslein-Volhard patterning defects associated with tor GOF. This reasoning 1992), which is the strongest extant tor GOF allele and is led us to examine the dpp mrna expression patterns associated with a Tyr-to-Cys amino acid change in the in tor Y9 embryos. Consistent with the above speculation, extracellular domain of Tor, presumably causing ligand- embryos derived from tor Y9 / females indeed showed independent dimerization (Sprenger and Nusslein- levels higher than those of wild type as well as ectopic Volhard 1992). Similar to tor Y9, tor 4021 is cold sensitive dpp mrna expression (Figure 4, E and F). and its phenotypes are most severe at low temperatures Both ectopic tll and dpp may account for the full (e.g.,18 ; Sprenger and Nusslein-Volhard 1992). We biological effects of Tor Y9 : It has been proposed that examined embryos from tor 4021 females with different the induction of ectopic expression of the Tor target paternal dpp copy numbers (see materials and meth- gene tll in the central region of the embryo by Tor GOF

Drosophila Mutant Torso Triggers Dpp and STAT Signaling 255 Figure 5. Test of genetic interaction with a gain-of-function allele of Egfr. Wings of flies with indicated genotypes are shown. (A) Wildtype wing. Wings from Egfr Elp homozygous (B) or heterozygous (C) flies exhibit extra vein tissues (arrow). This phenotype is dominantly suppressed by stat92e (D) and Ras1 (G), moderately suppressed by Draf (H), but not suppressed by Mad (E) and tkv (F). accounts for most, if not all, of the phenotypic defects dpp and tll are essential for the defects associated with associated with tor GOF (Klingler et al. 1988; Strecker tor GOF. et al. 1989). This is based on the suppression of the Involvement of Dpp and STAT92E in gain-of-function segmentation defects associated with tor GOF alleles by mutant EGF receptor signaling: To investigate whether zygotic tll mutations. It has been shown that tor GOF ; tll/ the Dpp and STAT92E pathways are also involved in tll embryos exhibited phenotypes that were similar to tll/ gain-of-function mutant EGF receptor (EGFR) signal- tll embryos, including restoration of the ventral denticle ing, we tested mutations in components of these pathways bands in the central region that would have been disrupted for their ability to suppress the phenotypes of a by tor GOF (Klingler et al. 1988). We retested the gain-of-function mutation in Egfr, Ellipse (Elp or Egfr Elp ). suppression of tor GOF by tll zygotic null embryos and Egfr Elp encodes a hyperactive EGFR molecule, causing found that tor Y9 ; tll/tll embryos (n 34) exhibited se- a rough eye phenotype and the appearance of extra verely defective head skeletons and a reduced number wing veins (Baker and Rubin 1989; Brunner et al. of denticle bands (Figure 4G). Such phenotypes were 1994). By comparing the phenotypes of Egfr Elp / flies more similar to tor Y9 embryos than to tll/tll embryos, with those of trans-heterozygotes for Egfr Elp and mutant because tll/tll embryos retain head skeletons and ante- alleles of the Dpp and STAT92E pathways, we found rior ventral denticle bands (not shown; also see Klingler that the extra wing vein and rough eye phenotypes were et al. 1988). This suggests that tll zygotic null muta- dominantly suppressed by stat92e, but not by Mad or tions cannot completely suppress tor Y9 phenotype. To tkv (Figure 5; not shown). This indicated that STAT92E assess the contribution of the Dpp pathway to tor GOF is involved in EGFR Elp signaling, although we cannot phenotypes, we tested the effects of reducing the maternal rule out the involvement of the Dpp pathway simply by Mad gene dosage on tor GOF ; tll/tll embryos. We found the lack of genetic interactions. Therefore, it appears that the phenotypes of tor Y9 /Mad; tll/tll embryos (n that STAT92E not only is involved in Tor GOF signaling 27) were nearly indistinguishable from those of tll/tll but also may be involved in signaling by at least another embryos (Figure 4H), indicating that the tor Y9 phenotype gain-of-function mutant RTK, EGFR Elp. was more completely suppressed by eliminating zygotic tll expression and simultaneously reducing Dpp signaling. Therefore, the ectopic tll expression induced DISCUSSION by Tor GOF cannot account for all the embryonic defects To understand the signaling downstream of a constitutively caused by Tor GOF. The finding that the Dpp pathway is activated RTK, we genetically screened for genes required for the tor GOF phenotypes suggests that both required for the functions of Tor Y9, a gain-of-function

256 J. Li and W. X. Li Figure 6. Model for the involvement of STAT and the Dpp pathway in mediating Tor GOF signaling. In wild-type embryos, Tor is active only in the two poles due to spatially restricted ligand supply and induces tll expression by inactivating the repressors (R) or derepression (see text for more details). In addition, Tor is able to induce dpp at least in the posterior region by similar derepression mechanisms. The expression of dpp in the dorsal central region of the embryo is independent of Tor but is dependent on the lack of active nuclear repressor Dorsal that represses dpp (see text). In tor GOF embryos, Tor GOF, due to its ligand-independent activity, is active in all regions of the embryo and is able to antagonize repressors (R) of both tll and dpp via either MAPK or other unidentified mechanisms (not shown in the model). In addition, Tor GOF is capable of activating STAT, which is able to directly bind to the promoter regions of tll and therefore enhances its transcription. mutant RTK. Results from this screen demonstrated dependent on Tor to antagonize the repression by Dorsal that, in addition to the evolutionarily conserved Ras- (Ray et al. 1991). Consistent with this model, dpp MAPK signaling cassette, gain-of-function RTK requires mrna is expressed in the whole posterior region (including multiple noncanonical pathways to exert its full biological ventral) of the early embryo (Figure 4E; also see effects. In particular, we found that Tor GOF possibly Ray et al. 1991). This terminal dpp expression domain is directly causes STAT92E activation (Li et al. 2002) and absent in tor mutant embryos (Ray et al. 1991), sug- triggers the Dpp pathway as a result of a global derepres- gesting that the ability to activate dpp expression is intrinsic sion of dpp expression via unrestricted activity of Tor Y9. to Tor. However, the bulk of dpp expression does Taken together, these results demonstrate that a gain- not appear to require input from Tor signaling. Consistent of-function mutant RTK is capable of utilizing multiple with this, tor mutant embryos exhibit normal D/V signaling pathways to augment its potency. patterning in the central region of the embryo. On the We have analyzed the possible role of STAT92E in basis of these observations, we infer that Tor is not mediating wild-type Tor functions and found that muta- required for the regulation of dpp expression in the central tions in mrl have minimal, if any, effect on the expression region of the wild-type embryo. In contrast, Tor GOF pospattern of the Tor target gene tll (Li et al. 2002). These sesses ligand-independent activity and is not confined results suggest that STAT92E is particularly important to the terminal regions. It has been shown that, Tor, for Tor GOF yet may not be required for the functions of via the Ras-MAPK signaling pathway, antagonizes the wild-type Tor or that signaling from wild-type Tor to function of Dorsal as a transcriptional repressor (Rusch STAT92E branches off and does not feed into tll. Dpp and Levine 1994; Hader et al. 2000), and Dorsal norsignaling, on the other hand, is essential for the pat- mally represses dpp expression in the ventral region. terning of the D/V axis of the early embryo. dpp is Activation of Tor throughout the whole embryo thus expressed in the dorsal 40% of the egg along the D/V would allow derepression of dpp globally (Figure 6). axis and is repressed in the ventral and lateral regions This is analogous to the derepression of tll throughout by a nuclear Dorsal gradient that has the highest concen- all regions of the embryo. However, we have shown that tration in the ventralmost region (Roth et al. 1989; STAT92E is required in the induction of the ectopically Rushlow et al. 1989; Ray et al. 1991). In contrast, Tor induced tll (Li et al. 2002). It remains to be investigated is activated in 15% of the egg length in the posterior whether the induction of ectopic dpp requires STAT92E region along the anterior/posterior axis. There is a min- activation. imal overlap between the expression domains of dpp We thank Christina Ficicchia, Healani Calhoun, and Russell Laand Tor activation in the posterior domain of wild-type France for assistance; the Bloomington Drosophila Stock Center embryos. The posterior portion of dpp expression is (Bloomington, IN) for providing the deficiency kit stocks and various

Drosophila Mutant Torso Triggers Dpp and STAT Signaling 257 strains; and Drs. Dirk Bohmann, Hucky Land, and Vladic Mogila for Li, W., E. M. Skoulakis, R. L. Davis and N. Perrimon, 1997 The comments on the manuscript. J.L. is a recipient of the Wilmot Cancer Drosophila 14-3-3 protein Leonardo enhances Torso signaling Research Fellowship from the James P. Wilmot Foundation. This study through D-Raf in a Ras 1-dependent manner. Development 124: was supported by a Howard Hughes Medical Institute Research Re- 4163 4171. Li, W., E. Noll and N. Perrimon, 2000 Identification of autosomal sources Program (grant 53000237) and a grant from the National regions involved in Drosophila Raf function. Genetics 156: 763 Institutes of Health (R01 GM65774) to W.X.L. 774. Li, W. X., H. Agaisse, B. Mathey-Prevot and N. Perrimon, 2002 Differential requirement for STAT by gain-of-function and wildtype receptor tyrosine kinase Torso in Drosophila. Development LITERATURE CITED 129: 4241 4248. Liaw, G. J., K. M. Rudolph, J. D. Huang, T. Dubnicoff, A. J. Courey Ambrosio, L., A. P. Mahowald and N. Perrimon, 1989 Requireet al., 1995 The torso response element binds GAGA and NTFment of the Drosophila raf homologue for torso function. Nature 1/Elf-1, and regulates tailless by relief of repression. Genes Dev. 342: 288 291. 9: 3163 3176. Baker, N. E., and G. M. Rubin, 1989 Effect on eye development Lu, X., T. B. Chou, N. G. Williams, T. Roberts and N. Perrimon, of dominant mutations in Drosophila homologue of the EGF 1993 Control of cell fate determination by p21ras/ras1, an receptor. Nature 340: 150 153. essential component of torso signaling in Drosophila. Genes Dev. Bronner, G., and H. Jackle, 1991 Control and function of terminal 7: 621 632. gap gene activity in the posterior pole region of the Drosophila Marshall, C. J., 1995 Specificity of receptor tyrosine kinase signalembryo. Mech. Dev. 35: 205 211. ing: transient versus sustained extracellular signal-regulated ki- Brummel, T. J., V. Twombly, G. Marques, J. L. Wrana, S. J. Newfeld nase activation. Cell 80: 179 185. et al., 1994 Characterization and relationship of Dpp receptors Melnick, M. B., L. A. Perkins, M. Lee, L. Ambrosio and N. Perrimon, encoded by the saxophone and thick veins genes in Drosophila. 1993 Developmental and molecular characterization of muta- Cell 78: 251 261. tions in the Drosophila-raf serine/threonine protein kinase. De- Brunner, D., N. Oellers, J. Szabad, W. H. Biggs, III, S. L. Zipursky et al., 1994 A gain-of-function mutation in Drosophila MAP kinase velopment 118: 127 138. activates multiple receptor tyrosine kinase signaling pathways. Nellen, D., M. Affolter and K. Basler, 1994 Receptor serine/ Cell 76: 875 888. threonine kinases implicated in the control of Drosophila body Casanova, J., and G. Struhl, 1993 The torso receptor localizes as pattern by decapentaplegic. Cell 78: 225 237. well as transduces the spatial signal specifying terminal body Newfeld, S. J., E. H. Chartoff, J. M. Graff, D. A. Melton and pattern in Drosophila. Nature 362: 152 155. W. M. Gelbart, 1996 Mothers against dpp encodes a conserved Casanova, J., M. Llimargas, S. Greenwood and G. Struhl, 1994 cytoplasmic protein required in DPP/TGF-beta responsive cells. An oncogenic form of human raf can specify terminal body pat- Development 122: 2099 2108. tern in Drosophila. Mech. Dev. 48: 59 64. Penton, A., Y. Chen, K. Staehling-Hampton, J. L. Wrana, L. Atti- Chang, H. C., and G. M. Rubin, 1997 14-3-3 epsilon positively reguprotein type I receptors in Drosophila and evidence that Brk25D sano et al., 1994 Identification of two bone morphogenetic lates Ras-mediated signaling in Drosophila. Genes Dev. 11: 1132 1139. is a decapentaplegic receptor. Cell 78: 239 250. Dickson, B. J., A. van der Straten, M. Dominguez and E. Hafen, Perkins, L. A., I. Larsen and N. Perrimon, 1992 corkscrew encodes 1996 Mutations modulating Raf signaling in Drosophila eye a putative protein tyrosine phosphatase that functions to transdevelopment. Genetics 142: 163 171. duce the terminal signal from the receptor tyrosine kinase torso. Doyle, H. J., and J. M. Bishop, 1993 Torso, a receptor tyrosine Cell 70: 225 236. kinase required for embryonic pattern formation, shares sub- Pignoni, F., R. M. Baldarelli, E. Steingrimsson, R. J. Diaz, A. strates with the sevenless and EGF-R pathways in Drosophila. Patapoutian et al., 1990 The Drosophila gene tailless is ex- Genes Dev. 7: 633 646. pressed at the embryonic termini and is a member of the steroid Duffy, J. B., and N. Perrimon, 1994 The torso pathway in Drosoph- receptor superfamily. Cell 62: 151 163. ila: lessons on receptor tyrosine kinase signaling and pattern Pignoni, F., E. Steingrimsson and J. A. Lengyel, 1992 bicoid and formation. Dev. Biol. 166: 380 395. the terminal system activate tailless expression in the early Dro- FlyBase, 1998 FlyBase: a Drosophila database. Flybase Consortium. sophila embryo. Development 115: 239 251. Nucleic Acids Res. 26: 85 88. Ray, R. P., K. Arora, C. Nusslein-Volhard and W. M. Gelbart, Ghiglione, C., N. Perrimon and L. A. Perkins, 1999 Quantitative 1991 The control of cell fate along the dorsal-ventral axis of variations in the level of MAPK activity control patterning of the the Drosophila embryo. Development 113: 35 54. embryonic termini in Drosophila. Dev. Biol. 205: 181 193. Robertson, S. C., J. A. Tynan and D. J. Donoghue, 2000 RTK Greenwood, S., and G. Struhl, 1997 Different levels of Ras activity mutations and human syndromes when good receptors turn bad. can specify distinct transcriptional and morphological conse- Trends Genet 16: 265 271 [Erratum. Trends Genet. 16 (8): 368]. quences in early Drosophila embryos. Development 124: 4879 Roth, S., D. Stein and C. Nusslein-Volhard, 1989 A gradient of 4886. nuclear localization of the dorsal protein determines dorsoventral Hader, T., D. Wainwright, T. Shandala, R. Saint, H. Taubert et pattern in the Drosophila embryo. Cell 59: 1189 1202. al., 2000 Receptor tyrosine kinase signaling regulates different Rusch, J., and M. Levine, 1994 Regulation of the dorsal morphogen modes of Groucho-dependent control of Dorsal. Curr. Biol. 10: by the Toll and torso signaling pathways: a receptor tyrosine 51 54. kinase selectively masks transcriptional repression. Genes Dev. 8: Hou, X. S., M. B. Melnick and N. Perrimon, 1996 Marelle acts 1247 1257. downstream of the Drosophila HOP/JAK kinase and encodes Rushlow, C. A., K. Han, J. L. Manley and M. Levine, 1989 The a protein similar to the mammalian STATs. Cell 84: 411 419 graded distribution of the dorsal morphogen is initiated by selec- [Erratum. Cell 85 (2)]. tive nuclear transport in Drosophila. Cell 59: 1165 1177. Karim, F. D., H. C. Chang, M. Therrien, D. A. Wassarman, T. Sewing, A., B. Wiseman, A. C. Lloyd and H. Land, 1997 High- Laverty et al., 1996 A screen for genes that function down- intensity Raf signal causes cell cycle arrest mediated by p21cip1. stream of Ras1 during Drosophila eye development. Genetics Mol. Cell. Biol. 17: 5588 5597. 143: 315 329. Simon, M. A., D. D. Bowtell, G. S. Dodson, T. R. Laverty and Klingler, M., M. Erdelyi, J. Szabad and C. Nusslein-Volhard, G. M. Rubin, 1991 Ras1 and a putative guanine nucleotide 1988 Function of torso in determining the terminal anlagen of exchange factor perform crucial steps in signaling by the sevenless the Drosophila embryo. Nature 335: 275 277. protein tyrosine kinase. Cell 67: 701 716. Kockel, L., G. Vorbruggen, H. Jackle, M. Mlodzik and D. Boh- Simon, M. A., G. S. Dodson and G. M. Rubin, 1993 An SH3-SH2- mann, 1997 Requirement for Drosophila 14-3-3 zeta in Rafsevenless SH3 protein is required for p21ras1 activation and binds to dependent photoreceptor development. Genes Dev. 11: 1140 and Sos proteins in vitro. Cell 73: 169 177. 1147. Singer, J. B., R. Harbecke, T. Kusch, R. Reuter and J. A. Lengyel,

258 J. Li and W. X. Li 1996 Drosophila brachyenteron regulates gene activity and van der Straten, A., C. Rommel, B. Dickson and E. Hafen, 1997 morphogenesis in the gut. Development 122: 3707 3718. The heat shock protein 83 (Hsp83) is required for Raf-mediated Sprenger, F., and C. Nusslein-Volhard, 1992 Torso receptor activ- signalling in Drosophila. EMBO J. 16: 1961 1969. ity is regulated by a diffusible ligand produced at the extracellular Weigel, D., G. Jurgens, M. Klingler and H. Jackle, 1990 Two terminal regions of the Drosophila egg. Cell 71: 987 1001. gap genes mediate maternal terminal pattern information in Steingrimsson, E., F. Pignoni, G. J. Liaw and J. A. Lengyel, 1991 Drosophila. Science 248: 495 498. Dual role of the Drosophila pattern gene tailless in embryonic Wharton, K. A., R. P. Ray and W. M. Gelbart, 1993 An activity termini. Science 254: 418 421. gradient of decapentaplegic is necessary for the specification of Strecker, T. R., S. R. Halsell, W. W. Fisher and H. D. Lipshitz, dorsal pattern elements in the Drosophila embryo. Development 1989 Reciprocal effects of hyper- and hypoactivity mutations in 117: 807 822. the Drosophila pattern gene torso. Science 243: 1062 1066. Wittwer, F., A. van der Straten, K. Keleman, B. J. Dickson and Strecker, T. R., M. L. Yip and H. D. Lipshitz, 1991 Zygotic genes E. Hafen, 2001 Lilliputian: an AF4/FMR2-related protein that that mediate torso receptor tyrosine kinase functions in the Dro- controls cell identity and cell growth. Development 128: 791 800. sophila melanogaster embryo. Proc. Natl. Acad. Sci. USA 88: Woods, D., D. Parry, H. Cherwinski, E. Bosch, E. Lees et al., 1997 5824 5828. Raf-induced proliferation or cell cycle arrest is determined by Tang, A. H., T. P. Neufeld, G. M. Rubin and H. A. Muller, 2001 the level of Raf activity with arrest mediated by p21cip1. Mol. Transcriptional regulation of cytoskeletal functions and segmen- Cell. Biol. 17: 5598 5611. tation by a novel maternal pair-rule gene, lilliputian. Develop- Yan, R., S. Small, C. Desplan, C. R. Dearolf and J. E. Darnell, Jr., ment 128: 801 813. 1996 Identification of a Stat gene that functions in Drosophila Tsuda, L., Y. H. Inoue, M. A. Yoo, M. Mizuno, M. Hata et al., 1993 development. Cell 84: 421 430. A protein kinase similar to MAP kinase activator acts downstream of the raf kinase in Drosophila. Cell 72: 407 414. Communicating editor: T. Schüpbach