Signaling Pathway in Drosophila - NCBI

Copyright 0 1995 by the Genetics Society of America

Genetic Screens to Identify Elements of the decapentuplegic Signaling Pathway in Drosophila Laurel A. Raftery,*?+Vernon Twombly,+Kristi Whartont” and William M. Gelbartt *Cutaneous Biology Research Center, Massachusetts General Hospital and Haruard Medical School, Charlestown, Massachusetts 02129, and tDepartment of Cell and Developmental Biology, Haruard University, Cambridge, Massachusetts 02138

Manuscript received July 18, 1994 Accepted for publication September 19,1994 ABSTRACT Pathways forregulation of signaling by transforminggrowthfactor$familymembersarepoorly understood at present. The best genetically characterized member of this family is encoded by the Drosophila gene decapentuplegic (dpp),which is required for multiple events during fly development. We describe here the results of screens for genes required to maximize dpp signaling during embryonic dorsal-ventralpatterning. Screens for genetic interactions in the zygote have identified an allele oftolloid, aswellas two novel alleles of screw, a gene recently shown to encode another bone morphogenetic protein-like polypeptide. Both genes are required for patterning the dorsalmost tissues of the embryo. Screens for dpp interactions with maternally expressed geneshave identified loss of function mutations in Mothers against dpp and Medea. These mutationsare homozygous pupal lethal,engendering gut defects and severely reduced imaginal disks, reminiscent of dpp mutant phenotypes arising during other d p p dependent developmental events. Genetic interaction phenotypes are consistent with reduction of dpp activityin the earlyembryo and in theimaginaldisks. We proposethat the novel screw mutations identified here titrate out some component(s) of the dpp signaling pathway. We propose that Mad and Medea encode rate-limiting components integral to dpp pathways throughout development. EMBERS of thetransforming growthfactor p (TGF-0) superfamilyof signaling factorshave diverse developmental effects, and individual TGF-p family members can induce different responses in different cell types (reviewed by KINGSLEY 1994; MASSAGUE et al. 1994). To unravel the complex functions of a TGF-p family member in vivo, we have focused on thedecapentaplegic gene (dpp) of Drosophila rnelanogastm. dpp encodes a polypeptide that is closely related to mammalian bonemorphogeneticproteins (BMPs) 2 and 4 et al. 1988). It is (PADGETTet al. 1987, 1993; WOZNEY required for multiple events during the development of the fly, including dorsal-ventral patterning of the larval ectoderm (IRISH and GELBART1987), morphoet al. 1990; IMMERgenesis of the larval gut (PANGANIBAN GLUCK et al. 1990; HURSHet al. 1993) and proximalet distal outgrowth of the adult appendages (SPENCER al. 1982; POSAKONY et al. 1991). The molecular complexityof signaling by TGF-/3 family members manifests at multiple levels. These signaling molecules dimerize and are processed to form the small bioactive ligand molecule (DERYNKet at. 1985). Both homo- and heterodimers can form, and, inleast at one case, they have opposing biological responses

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Correspondingauthor Laurel A. Raftery, Cutaneous Biology Research Center, MGH-East, Mail code 149-3214, Bldg. 149, 13th St., Charlestown, MA 02129. Present address Division of Biology and Medicine, Brown University, Providence, RI 02912.

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Genetics 139: 241-254 (January, 1995)

(LINGet al. 1986; VALE et al. 1986). Ligand availability can be masked by binding to other polypeptides (KANZ A K ~ et al. 1990; KOGAWA et al. 1991) or mediated by et al. 1993). cell surface proteoglycans (LOPEZ-CASILLAS Different levels of ligand can induce different fates in and ANDERSON, the same cell types in vivo (FERGUSON 1992b; WHARTONet al. 1993). However, very little is known about the signal transduction events in the target cells. Two cell surface receptor ser/thr kinases, deset al. 1993; EBNERet al. ignatedas type I (ATTISANO 1993; FRANZEN et al. 1993) and type I1 receptors ( M m THEWS and VALE1991; LIN et al. 1992; ATTISANOet al. 1992),arebothrequiredfor biological response to TGF-01 (WRANAet al. 1992; CHENet al. 1993). Strong candidates for d@ type I receptors have recently been identified(BRUMMELet al. 1994;NELLEN et al. 1994; PENTONet al. 1994; XIE et al. 1994), but othersubstrates for the kinase activity have yet to be determined. We report here theresults of initial screens for dominant genetic enhancers of dpp. Similar screens for geneticinteractions have proven useful in delineating other pathways, for example the signaling pathway for induction of the R7 photoreceptor in Drosophila (SIMON et ai. 1991; DICKSONand HAFEN1993). We use the term enhancers to describe mutations in other genes that engendera more severe phenotype indpp heterozygotes thanwould be expected.We have taken advantage of the exquisitely sensitive response to levels of functional dibp gene product thatis exhibited in the dorsal-

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ventral patterning of the embryonic ectoderm (WHARTON et al. 1993) to screen for specific dominant interactions. Our screens for dominant enhancers of dpp embryonic lethality fall into two classes. Screens for enhancers of dpp expressed in the zygote haveyielded antimorphic mutations in tolloid (tld) and smew (sew). Mutations in each of these genes have been previously described as engendering aloss of the dorsal-most structures similar to those of weak dpp alleles (FERGUSON and ANDERSON 1992a and ARORAand NUSSLEIN-VOLHARD 1992, respectively). Screens formaternal effect enhancers have yielded mutations in two additional loci, Mothers against dpp (Mad) and Medea (Med). Theselatter two genes represent aclass ofgenes not previously associated with dpp pathways, so we present preliminary characterization of their mutant phenotypes. MATERIALS AND METHODS

Fly strains and culture conditions:Except forthe mutations generated in this work, most mutations are described in FLYBASE (1994) or LINDSLEV and ZIMM(1992). We confirmed the cytology of all previously reported deficiencies. Additional scu, tld, and dpp alleles are described in ARORA and NUSSLEINVOLHARD(1992), FERCUSONand ANDEMON (1992a),and WHARTON,et al. (1993), respectively. In some publications, is referred to as tld""", as tldl(" and tld3 as tld6P4'. Df(2L)JSl7, Mad7 and Madi2 are described in J. SEKELSKY, S. NEWFELD, L. RAFTERY, E. CHARTOFF and W. GELBART(unpuhlished data). Dp(2;2)DTD48, dpp"h'Jwas used as the dpp' duplication. The Cy0 wg-lacZ enhancer trap is en11 described in PERRIMONet al. (1991). Unless otherwise stated, flies were cultured at 25" on standard cornmeal-yeast extract-dextrose medium. Most embryo collections were from eggs laid on grape juiceagar medium supplementedwith a liberal amount of yeast paste. Mud mutations for larval dissection were balanced with In(2LR)Gla, Gla Be E&; Medea mutations were balanced with TM6b, Tb, so that mutant larvae could be identified. Larvae were incubated at 18" for 3 days on well-yeasted standard medium before dissection. Embryoniclethalscreen: Males were mutagenized with ethyl methane sulfonate (EMS) as described by LEWIS and BACHEK(1968). The mutagenized d p el en bw; el' males were crossed to dpp"" D p ( l ; 2 ) ~ + ~Bl/CyO " females. Progeny males of the genotype d p el en bw/dpp""" Dp ( 1 ; 2 ) ~ + ~ B l' ;~el'/+ were individually mated to 4 net dpphd/CyO females in blocks of test tubes as described by WIESCHAUS and NUSSLEIN-VOLHARD (1986). Embryos were collected from these cultures on apple juice-agar plates, aged for 48 hr at 25"C, and then inspected under the dissecting microscope with transmitted light for broods in which >lo% of the embryos were dead. The dead embryos were examined for weak dpph'-like defects, that is, evidence of incomplete head involution and clumps of Malpighiantubulematerial nearthecenter of the embryo. Crosses that gave rise to a reasonable proportion of embryos with dpphr-like mutant phenotypes were recovered from the tubes and transferred to shell vial cultures. The resultant progeny were scored for dp, BI, and Cy phenotypes. Lines that were suspected to carry an enhancer mutation on the third chromosome were tested by crossing to dpphd/G1a ;E(z) e/ TM?, Sb. All others were retestedagainst the original dpp tester stock.

Secondchromosomescreen: Others of the mutagenized dp el en bw; e" males were crossed to females of one of the following genotypes: dpp"" Dp(l;2)w' 7"h Bl/CyO, dpp""" Be E&/ SM6a, or Be E&/CyO. Progeny d p el en bw/CyO; e'//+ males were individually mated to net dpph"/CyO females; the crosses were scored for the presence of Cy+ progeny. Putative enhancer lines were retested againstthe original dpp tester stock. Maternal enhancement screens: dp en bw males were mutagenized with EMS as above and mated to dppd-h'JBc EIp/SM6a females. Virgin d p cn bw/SMba, Cy females were collected and mated to dpphr4en bw/SMba, Cy; e" males. These crosses were initially screened for the presence of Cy' progeny; those crosses with few such progeny were then scored for reduction of the Cy dp phenotypic class. d p cn bw/SMba ;+/e" progeny males from each of these were crossed to Sp/CyO; G1/77M6 females to recover progeny females carrying either a mutagenized second chromosome ( d p cn bw/CyO ;eii/TM6) or a mutagenized third chromosome (Sp/SMGa ;+/TM6) for retesting. Such females were retested by crossing to the same dpf~'''~ stock. In another screen design, 400 mutagenized males were crossed to Sp/CyO; Gl/TMG females. Single virgin dp en h u / Cy0 ;+ / T M 6 females were tested as above. Fine mapping relative to dpp: Mutations that were >99.5% lethal in trans to dpphr4were tested for recombination events in the following manner: enhancer dp cn bw/net dppn""females were mated to Zn(2LR)DTD8, dpp'ir4/Cy0males. All surviving Cy' progeny from this cross should have had the held-out phenotype, unless a recombination event occurred between the enhancermutation and dpp"-"" to create an enhancer+dpp' chromosome. Any straight-winged flies were retested against the DTD tester chromosome; all that were not sterile were genotypically dpp"t"' . Mutations that gave escapers in trans to dpp"" were tested for recombination events in a similar manner, except that recombinants between the flanking markers net and dp were selected first and then tested for ho and enhancer phenotypes. Fine mapping relative to s m This experiment depended on our observation that a duplication of dpp, Dp(2;2)DTD48, dpp""', partially rescued the lethality of s d z / D f scw but did not rescue s n f 1 2 / s d 1 lethality. Thus, s d 2 Dp(2;2)DTD48, dpp"""/b pr, an EMS-induced scw null mutation, sa#' Dp(2;2)DTD48, dpp"""/Df(2L)ODIb, a deficiency removing sew, or s a d Dp(2;2)DTD48/sdiRi were generated. These females were mated to scd'/CyO males. The Cy' progeny from this cross would either be s c S 2 Dp dpp/scd' escapers, which occurred in test crosses at a frequency of 3.5% or a wild-type recombinant between the enhancer chromosome and the snu chromosome. Embryonic cuticlepreparations: Preparations of cuticles from differentiated embryos were performed as described by WIESCIIAUSand NUSSLEIN-VOLHARD (1986). Cuticles were photographed under darkfield optics using either an Olympus BHS or a Nikon Microphot FXA microscope, with either Kodak Techpan or TMAX film. Lethal interaction with dpp alleles: For zygotic interactions, dpp tester females were mated to heterozygous enhancer males; for maternal effect interactions heterozygous enhancer females were mated to tester dpp males. The flies were transferred to fresh food approximately every 3 days. For zygotic interactions, percent survivorship of the class of interest was calculated as the (number of tester chromosome/dpp mutant chromosome progeny) divided by (number dpp mutant chromosome/balancer chromosome progeny) X 100%. For maternal interactions, the denominator was (number of maternal enhancer mutant/balancer chromosome progeny). The total number of progeny scored for each of the crosses was 125-500. The dpp stocks used were: dpp"'/CyO, net dpp'" en/

243Enhancers Drosophila of dpp in SMGa, dpphr56cn bw/Cy023, dpphr4cn bw/Cy023, dpphr8' cn bw/ This allele is very near the minimal d p p activity threshCy023, dpphr2?en bw/Cy023, dpphrg2 cn bw/CyO23, and dpphTg3 old for viability because it demonstrates relatively little en bw/Cy023. Cy023 carries a P element constructthat rescues et al. 1993) yet exhibits subhaplo-lethality (WHARTON the haploinsufficiencyof dpp" alleles (WHARTONet al. 1993). stantial lethality in trunsheterozygous combination with Assay for number of amnioserosa cells by immunohistochemical staining for K r i i r ~ gene l product: To examine zyseveral tld alleles that had previously been shown to gotic interactions, net dpp /Cy0 wg-LacZwas used as the tester enhance a moresevere dpp allele (data not shown; FERstock. scw/Cy0 wg-lacZ stocks were used, so that dpp/sm emGuSON and ANDERSON, 1992a). bryos could be distinguished from those that carried the balBecause the dppdependent patterning of the ectoancer on the basisofwgdirectedexpressionofP-galactosiderm occurs very early in embryogenesis, we reasoned dase. dpp/+ ;tld/+ embryos were distinguished from dpp/+ ; that some of the gene products involved in dpp signal TMJ/+ embryos on the basisof the mutant phenotype. By stage 9, weakly ventralized embryos can he distinguished from transduction might be supplied during oogenesis, so wild-typeembryosbecausethegermhand is forced underthat the target cells would be poised to receive the dpp neath the amnioserosa. We used the same tester stock to exsignal. Thus, we looked for mutations in two types of amineembryoslaid by heterozygousmaternalenhancer screens. First, we screenedfor zygotic interactions, mothers. Thus, we could compare dpphr4/+ embryos with +/ where the new mutation must be expressed in the emembryosfrom the same brood. Fixedembryoswerecollected for5-12 hr and aged for 3-4hr. Embryos were stained bryo to interact with dpp. These screens demonstrated anti$ with rabbit anti-Kruppel antibody, and either rabbit that we could specifically identify genes involved in the galactosidase or mouse monoclonalanti-P galactosidase (Prodorsal-ventral patterning process. We then carried out mega, Madison, w),and detected with horseradish peroxia small-scale screen formaternal effect interactions, dase conjugated secondary antibodies (Vector, Burlingame, CA). Fixationandenzymaticdetectionwereasdescribed where the new mutation must be expressed in the (WHMrON et al. 1993) except that phosphate-buffered saline, mother. This screen proved to be quite productive. pH 7.4, was used as thebuffer.Immunohistochemically Zygotic screens: Screens for zygotic enhancers were stained embryos were examined by bright field microscopy F2 lethal screens for mutations thatdie in trans to dHhr4 on a Nikon Microphot FXA microscope, using a color chip (Figure 1). The screens were carried out in two ways. digitizingcameratodisplay the image on a video screen. Stainedamnioserosa nuclei were counted as we focused First, the test crosses were carried out in blocks of test through the embryo. At least 20 embryos of each genotype tubes, so that progeny could be examined as embryos were scored. for dpphr-likephenotypes. This type of screen had two Interactions with dppdIrkalleles: Stockswereconstructed advantages: we were directly screening for the desired mutation and the enhancer mutation to hearing the dppdLSk embryonic lethal phenotype and mutations could be he tested. Females carrying a dpphrallele were mated to these double mutant males and the progeny scored for defects in recovered on either the second or the third chromothe eye, antenna, wing and legs. The numbers of legs lacking some. However, in practice, this approach proved to be tarsal claws were scored for 213 flies of each genotype. inefficient due to the variable background of dpphr4/+ embryonic lethality. In the second type of screen, we RESULTS examined the adult progeny for loss of a phenotypic class, which biased the screen for second chromosome Enhancer screens mutations. We chose to screen for dominant mutations that enA total of 9360 mutagenized haploid genomes were hance, or exacerbate, dpp mutant phenotypes, because screened; 2260 of these were screened initially for emloss of function mutations in rate-limiting components bryonic lethality. From both screens, a total of 237 were of the dpp signaling pathway would be most likely to retested and 126 continued to show lethality in trans appear to decreasedpp activity. Such screens might also to the dpphr4 tester chromosome.These werepassed identify mutations in genes whose products increase through a series of complementation tests designed to the activity of the dpp gene product or are indirectly eliminate lethal interactions with cryptic mutations on gain of function regulated by dpp. Inaddition,rare the dpphr4tester chromosome. The meiotic recombinamutations might be obtained. In these types of screens, tion map position of each mutation was determined. such mutations might alter protein function such that In the end, we retained 13 mutations on the second an essential component is inappropriately inactivated chromosome, two alleles of scu and 11 of dpp, and one or is titrated out or a negative regulator is inappropriThe assignon the third chromosome, an allele of ately expressed. ment of allelism is described below. All of these mutaThe dorsal-ventral patterning of the ectoderm during tions engendered significant embryonic lethality in embryogenesis is sensitive to dpp gene dosage; one copy trans to dpp"", with varying degrees of ventralization of of dpp is insufficient for normal development (IRISH the cuticle (Figure 2). and GELBART 1987). Likewise, leaky missensemutations Maternal effects screens: Maternal effect enhancer in dpp show varyingdegrees of lethality in heterozygous screens were based on thefailure to recover dpphr4proganimals (WHARTON et al. 1993). We selected the receseny from motherscarrying mutagenized chromosomes, sive lethal allele dppkr4as the tester allele for our screens. regardless of the maternal second chromosome inher-

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FIGURE1.-Screens for enhancers of dpp. Full genotypes are described in MATERIALSAND METHODS. (A) F2 lethal screen for (7100) were screened for second chromosome enhancer mutations expressed in the zygote. Mutagenized second chromosomes failure to complement a dpp recessive lethal mutation, dpphr4.The table of progeny classes recovered indicates the progeny genotypes recovered from a mating with a wild-type male, and those recovered from a mating with a male carrying an enhancer mutation. +, progeny of this genotype are present; -, progeny of this genotype are missing. The presence of the dp/dp+ dpp class was initially determined by the presence of Cy+ progeny. Probable dpp alleles were eliminated by meiotic recombination mapping, as discussed in the text. (B) Maternal effect screen. Mutagenized haploid genomes (1400) were screened for maternal effect interaction with the dpp recessive lethal mutation, dpp". The table of progeny classes indicates the progeny genotypes recoveredfrom a mating with a wild-type female, and those recovered from a mating with a female carrying an enhancer mutation. We first screened for the absence of Cy' progeny to select those vials missing the dp/dp+ dpp class and then scored the progeny for the absence of the dp' dpp/SMGa, d p Cy class.

ited. We screened 1850 haploid genomes in a series of pilot screens; the major one used is depicted in Figure 1. Nine lines transmitted the enhancer phenotype on the second chromosome and eight on the third. Only those lines that gave strong enhancement, with < l o % survivors of the dpphr4progeny, were retained. The putative maternal enhancers were tested for strict maternal effect interactions, by crossing heterozygous enhancer males to dHhr4 cn bw/SM6a females. There was no enhancement when the matings were done in this direction. The location of the maternal effect enhancers was determined by meiotic recombination mapping. In the end we retained seven mutations: four alleles of Mothers againstdpp ( M u d ) onthe second chromosome and three alleles of Medea (Med) on the thirdchromosome. New dpp alleles: Eleven mutations from the zygotic screen mapped in the distal portion of the interval between net (2-0) and dp (2-13). These were expected to be mutations in dpp based both on the proximity to dpp (2-4.0) by meiotic recombination and on the zygotic lethality in trans to dpphr4.Four of these mutations were

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FIGURE7.-Extent of interaction of s c r m alleles with a series of dpp alleles. Antagonistic alleles and null alleles are grouped separately. The genotype of the cross is indicated with the dpp allele from the mother along the x-axis and the screw allele from the father indicated by color of the bar. % survivors are presented, as described in the MATERIALS AND METHODS.

the two new scw alleles,with the exception of dppCa7 (Figure 7), and sad' isalways stronger than s d 2 . In contrast, the null s m alleles, including the s d ' revertant, s a d t R ' , show no significant interaction with d@. These data demonstrate the unique character of the scw alleles recovered in this screen, namely, they are antagonistic toward dpp. We have examined maternal effect interactions with dpp for the fourMedeu alleles and a deficiency for Med, Df(3R)E40 (Figure 8 ) . Note that dpp/dpp' progeny are lost regardless of whether the Medeu+ or the Medeu mutant chromosome is inherited from the mother (as in Figure 1 ) . Unlike tld and scw, loss of Medeu function still results in an interaction with dpp. However, the interaction with dpp is stronger for Me& than for the deficiency, raising the possibility that this allele has some additional antagonistic activity against dpp. Likewise, maternal effect interactions were examined for the four Mud alleles isolated in this study, one of which is a deficiency, Df(2L)C28 (Figure 8 ) . The deficiency for Mud interacts strongly with most dpp alleles, and all alleles engender loss of both dpp/Mud and dpp/ + progeny from Mud/+ mothers. None of the alleles examined here are more severe than the deficiency. A number of other alleles have been obtained in other types of screens and all show similar interactions with dpp, although the degreeof interaction varies (J. SEKEL SKY,S. NEWFELD, L. RAF~ERY,E. CHARTOFF and W. GELB ART, unpublished data). Interaction with dpp in the imaginal disks: dpp is required multiple times during Drosophila development; in addition to dorsal-ventral patterning, it is required

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FIGURE8.-Maternal effect enhancer mutations interact with a series of dpp alleles. Each panel includes the interactions with a full series of dpp alleles for a mother heterozygous for the given mutation. Both classes of progeny heterozygous for the dpp allele were scored for survivorship, indicated by different colored bars. The dpp alleles are indicted along the x-axis. (A) M e & d + mothers. Med Df = Df(3R)E40 (B) Mad/+ mothers. Mad Df = Df(2L)C28.

formidgutmorphogenesis and proximal-distal patterning of the imaginal disks. Two types ofgenes might have been recovered in these screens, those specific to a single developmental role for dpp and those genes integral to the dpia signaling pathway. This latter class of genes might continue to show interactions with dpp at other developmental stages. To test these possibilit-

ies, we looked for zygotic interactions between the enhancer mutations and imaginal disk-specific mutations in dpp. Different crosses were used to score the effects of each enhancer gene, in part because of the zygotic lethality of many dpphralleles in transheterozygous combination with antagonistic tZd or scw alleles. For Med, en-

Enhancers Drosophila of dpp in hancement of dpp phenotypes was observed in the leg, eye, wing and antenna; Mad interactions are described elsewhere (J. SEKELSKY, S. NEWFELD, L. RAFTERY, E. CHARTOFF and W. GELBART, unpublished data). For antagonistic scw alleles, enhancement has been observed in the wing and the leg. tld"' exhibited weak enhancement of leg phenotypes in the dpp genotypes examined; however, very few adults were recovered. For each genotype,the loss oftarsal claws at thedistal tip of the legs provided a quantitative indication of an interaction. We report the average number of legs per animal that still had at least one tarsal claw, so that a low number is indicative of a genetic interaction. For the scw alleles, we scored tarsal claws in dppdi2scw/dppPx7 animals. Flies wild-type for scw had an average of 4 -C 1 legs with tarsal claws, whereas s d 2 animals had an average of 2 2 2 legs withtarsal claws, and s d . " animals had 1 t 1. In the case of tld, dppd5/dpph"6; tl&' animals had 3.5 5 2 legs with tarsal claws, whereas dppd5/dpphT56; tld' animals had 5.9 5 0.3. For Med, dppd6/dpphr4; Med4 animals had 0 5 0 legs with tarsal claws, dppd6/dpphr4; Me& had4 2 1. Theseexperiments do not permit comparisons between genes but clearly indicate that each gene can affect dpp function during development of the imaginal disks. DISCUSSION

In this report we describe a series of mutations that were identified on thebasis oftheir ability to genetically enhance embryonic lethality of a weak heterozygous dpp recessive allele. These mutations were isolated in two types of genetic screens. The first was designed to identify mutations that enhanced dpp when expressed in the zygote. This screen identified one mutation in tld and two novel mutations in scw. Both of these genes had been previously suggested to be required for the formation of dorsal structures of the embryonic ectoderm ( h o wand NUSSLEIN-VOLHARD 1992; FERGUSON and ANDERSON 1992a). The fact that the only zygotic enhancer mutations we isolated were in dorsal-ventral patterning genesvalidated the specificity ofthe screens. The second type of screen was designed to identify genes whose products were required for dpp function but which were expressed during oogenesis. We anticipated that such genes might exist because the first dpp signaling event occurs early in embryogenesis, probably just after cellularization. Thus, components of the dpp signal transduction machinery may be already present in the oocyte so that target cells are poised to respond. It was quite likely that such genes would have escaped identification in previous screens for embryonic lethal phenotypes, because the maternally expressed component would obscure the early effects ofloss of gene activity in the embryo. Likewise they mayhave been missed in screens for recessive maternal effect lethal

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genes, because such screens demand homozygous viability. Indeed, we found the pilot screens for maternal effect enhancement of dpp were quite productive; we identified seven maternal effect enhancer mutations out of 1800 haploid genomes screened.Although we obtained multiple alleles in each of only two genes, the maternal effect screens were not saturating. We did not recover alleles of other genes that can mutate to maternal effect enhancers of dpp, such as cactus (L. A. RAFTERY, unpublished data), thickvein (NELLENet al. 1994; PENTON et al. 1994) and saxophone (BRUMMEL et al. 1994; NELLENet al. 1994; XIEet al. 1994), presumably because of the small scale of the screens. Mutations obtained in these screens also interact with dpp duringdisk development, giving usconfidence that they are required for proper dpp function throughout development. We assayed for the lossof tarsal claws from the legs in flies mutant for a dppdfskregulatory mutation in trans to either a dpp missense or promoter mutation. Neither scw nor tld have previously been reported to have adult phenotypes. For scw, Medeu, and Mad, other imaginal disks, such as the wing and eye, can be affected in these or other dpp mutant combinations; tld has not been examinedas extensively. Because loss offunction in either Mad or Medea decreases apparent dpp function at two distinct developmental events, it is likely that thetwo maternal genes represent integral components of the dpp signaling system. The mutations in scw and tld are more difficult to interpret, because their antagonistic effects on dpp mutations may not represent their normal modes of action. At a minimum we can conclude that they are capable of perturbing dpp function both during embryogenesis and disk development. scw, tld, and dpp are required for fating the dorsal ectoderm and amnioserosa. In the case of dpp, there is evidence suggesting a posttranscriptional gradient of activity, in which the high point defines the dorsal-most fates (ST.JOHNSTON and GELBART 1987; FERGUSON and ANDERSON 199213;WHARTON et al. 1993).Null mutations in scw or tld remove only the dorsal-most subset of tissues that are lost in dpp null mutants; it is the dorsalmost tissues that are defined by the highest levelsof DPP. The antagonistic interaction of specific mutations in both scw and tld raises the possibility that these gene products are directly involved in raising the levelof DPP activity. However, the initial pattern of dpp transcription is unperturbed in either tld or scw mutants ( R A Y et al. 1992). We can envision specific models for the genetic interactions of tld and scw with dpp based on the polypeptides encoded by these genes (SHIMELL et al. 1991 and h o w et al. 1994, respectively). TLDis structurally similar to mammalian BMPl (SHIMMEL et al. 1991), whereas DPP is both structurally and functionally homologous to BMP2 and BMP4 (WOZNEY et al. 1988; PALXETT et al. 1993; SAMPATH et

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al. 1993). BMPl was originally isolated because it copurified with the TGF-o-like BMPs, suggesting a physical association with those molecules (WOZNEY et al. 1988). Both TLD and BMPlhave adomainthat is highly similar to the protease domain of astacin, as well as a domain with Clr/s repeats believed to be involved in protein-protein interactions (SHIMELL et al. 1991). Interestingly, most of the tZd alleles that antagonize dpp have mutations in the protease domain, suggesting that protein-protein interactions are maintained but catalyticactivityis lost (CHILDSand O'CONNOR1994; FINELLI et al. 1994). It is likely that TLD is involved in a proteolytic cascade, but whether it directly binds DPP remains to be determined. Even so, the antagonistic TLD mutations are poisoning DPP signaling, probably by nonfunctional binding to some component of the DPP signaling pathway. Molecular cloning and sequencing of scw indicates that it is another member of the TGF-P family (ARORA et al. 1994). In the early embryo it is expressed ubiquitously but only required dorsally. It is an interesting paradoxthatthe dpp expression domain is a subset of the scu expression domain yet the d p p phenotypes encompass more ventral tissue than those of scu. The antagonistic mutations in scw raise the possibility that these two gene products function in concert. The molecular similarity between DPP and SCW bolsters this notion, furthersuggesting that these two proteins could either interact with the same signal transduction pathway components or could themselves form heterodimers. Simple models for additive signaling by the two growth factors are confounded by our observation that a genetically null scw allele does not interact with dpp, even in our most sensitive assay.This observation might be discounted if the requirement for s m is not dosage sensitive. However, if the two signals were additive, the dpp scw double mutant phenotype should be more severe than either alone, and it is not (ARORA and NUss LEIN-VOLHARD 1992). We propose that the antagonistic effects of sczd' and s a d 2 are due to poisoning of DPP signaling by mutant SCW protein. In this model, the antagonistic SCW polypeptide binds nonfunctionally to DPP or some component of the DPP pathway, so that it effectively titrates out this component and reduces the pool available for functional DPP signaling. SCW-DPP heterodimer formation is possible in the cells where they are both expressed (ARORAet al. 1994).Thus, antagonistic SCW mutant protein could act directly to reduce the active pool of DPP. This antagonistic effect could mimic that of dominant negative TGF-P mutations, which prevent secretion of other TGF-P isoforms (LOPEZet al. 1992). The model for direct poisoning of DPP function by antagonistic SCW protein is supported by our observation that these are the only scu alleles rescued to viabilityby increased gene dosage of dpp'. Extra copies of

the dpp gene are reported to partially ameliorate the cuticular phenotype of weaks m mutants without rescuing lethality (FERGUSON and ANDERSON 1992a), andinjection of dpp RNA rescues a small amount of amnioserosa in scw nulls (ARORA et al. 1994). However, four copies ofwild-type dpp do not shift the lethal phase for sau null mutants. This suggests that DPP cannot substitute completely for SCW function. The two additional genes that were identified in the maternal effect enhancer screen had not been implicated previously in embryonic dorsal-ventral patterning. One gene,Mothers against dpp (Mad) was not previously identified by other means. The other, Medea, was previously represented by a single allele, 1(3)SG70, which had been isolated in a screen for larval/pupal lethals on the third chromosome (SHEARN and GAREN 1974). The mutations that we characterized in either gene are zygotic lethal during prepupal phase; presumably the maternal contribution of gene product is sufficient for embryonic survival. The phenotypes of larvae mutant for either gene are strikingly similar. The larvae have severely reduced imaginal disks, variable reductions in the size of the larval brain lobes, shorter gastric caecae and midgut abnormalities. These phenotypes parallel those of different types of dpp mutations: dpp'ho mutations engender altered midgutmorphogenesis (IMMERCLUCK et al. 1990; PANGANIBAN et al. 1990) and dpp'l''h mutations engender small imaginal disks (SPENCER et al. 1982) and reduced mitotic activity in the larval brain (KAPHINGST and KUNES1994). Indeed,in both Mad and Medea, mutant embryos fail to form the second midgut constriction (Mad:J. SEKELSKY, S. NEWFELD,L. RAFTERY, E. CHARTOFF and W. GELBART, unpublisheddata; Medea: R. WISOTZKEY and L. A. RAFTERY, unpublished observations), as has been observed for dpp'h7'mutants. This observation strongly supports our hypothesis that Mad and Medea are integral components of a minimal d p p pathway that is reused throughout development. The functions of Mad and Medea gene products are presently unknown. Mad has been cloned but has no informative sequence motifs. However, there are three transcription units in the nematode, Ceanorhabditis ekgans, with a high degree of similarityto Mad (J.SEKELSKY, S. NEWFELD,L. ~ F I - E R Y ,E. CHARTOFF and W. GELBART, unpublished data). The cloning of the Medea gene is underway. We are presently exploring whether these genes function upstream or downstream of dpp and whether we can detect interactions with other DPP/ BMP signaling components in Drosophila. Our data suggest that Mad and Medea encode functions that are used in dpp signaling at several developmental events and that scu and tld functions may also be used repeatedly. However, the developmental outcomes for different dpp target cell populations can be quite different. For example, the dorsal-most cells of the early embryo, the amnioserosa, stabilize the expres+

Enhancers of dpp in Drosophila

sion of the homeodomain gene zen and become squamous upon receiving the DPP signal (RUSHLOWand ARORA 1990; ARORA and NUSSLEIN-VOLHARD 1992). In contrast, the visceral endoderm expresses the homeodomain gene labial and initiates an invagination (1" MERGLUCK 1990; PANGANIBAN et al. 1990). Thus, there must be tissue-specific molecules interacting with the DPP signal transduction pathway, whichwe did not uncover in these experiments. This may be due to the variability of dpp phenotypes, so that interactions between more distant components of the pathway do not clearly manifest under the stringent requirements of such a screen. It remains to be seen how such tissuespecific outcomes are engendered by the same signal. We are deeply indebted to CAROLE LABONNEand DAVIDWELCH for assisting us with the screens, to LINDATANNER for assisting with the counts of amnioserosa cells, to RUSSELLNICHOLS for assisting with fine mapping of maternal enhancers and to ROBERT WISOTZKEY for cytology. We thank CHRISRUSHLOW for anti-Kr antibody and BOB HOLMGREN for rabbit anti-0 galactosidase. We thank LORRAINEand DIANELUKASfor the continuoussupply of fly food. We thank KAVITA ARORA, CHIP FERGUSON, NEIL KRUEGER, MIKEO'CONNOR andRICK PADGETI for discussion of results before publication. We thank the Drosophila Stock Center at theUniversity of Indiana, ALLANSHEARN, JANICE FISHER-VYLE and KEN TARTOFfor providing us with fly stocks. During most of this work, L.A.R. was supported by a Charles A. King Trust postdoctoral fellowship, K.A.W. by a National Research Service Award (NRSA) postdoctoral fellowship, and V.T. by an NRSA predoctoral training fellowship. This work was supported by a NationalInstitutes of Health grant toW.M.G. and a grant from theShiseido Co. of Japan, Ltd. to the Massachusetts General Hospital/Harvard Medical School Cutaneous Biology Research Center (L.A.R.).

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