INTRODUCTION

Genetic and molecular analyses of embryonic development in

Drosophila have elucidated some basic mechanisms in patternformation. Based on phenotypic analyses of cuticle defects inmutant embryos, many genes required for proper segmentationof the embryo have been characterized (Nüsslein-Volhard andWieschaus, 1980). Many of these genes encode transcriptionfactors. However, at the end of the regulatory cascade, whichis at least partially determined by the segment polarity (Peiferand Bejesovec, 1992) and homeotic genes (Akam, 1989), thedevelopmental program must be carried out by realizator genesthat control cell division, migration, differentiation and othercellular functions relevant to morphogenesis (Garcia-Bellido,1977). Interestingly, few realizator genes that may carry outthis program have been identified. Although a number of geneshave been identified that are regulated by homeotic selectorgenes most of them encode regulatory proteins themselves (forreview see Botas, 1993).

Several hypotheses can be proposed to explain the apparentdifficulties in isolating realizator genes. One of the majorreasons may be that these genes have functions in manytissues and are under the control of many regulatory proteins.Hence, mutations in these genes may cause pleitropic effects

which are difficult to correlate with homeotic function.Secondly, redundant pathways may have developed, such thata single mutation in one pathway may yield no phenotype.One possible approach to isolating realizator genes is toscreen for genes that are expressed in specific patterns remi-niscent of homeotic or segment polarity genes. Enhancerdetection screens have indicated that a substantial number ofDrosophila genes are expressed in segmentally repeatedpatterns reminiscent of segmentation genes of the pair-ruleand segment polarity type (Bellen et al., 1989; Bier et al.,1989). Although some of these genes have been shown toencode known segmentation genes (Grossniklaus et al., 1992;Lee et al., 1992), most enhancer detectors map to positionsof previously uncharacterized genes and their study has notbeen pursued.

Here we describe the molecular, genetic, and functionalanalysis of a novel gene named 18 wheeler (18w), whose earlyexpression pattern is reminiscent of that of segment polaritygenes. Our observations indicate that 18W can act as a het-erotypic cell adhesion molecule and that its expression isregulated by segment polarity and homeotic genes. We proposethat 18w may be a realizator gene, carrying out part of thedevelopmental program specified by the segment polarity andhomeotic genes by acting as a cell adhesion molecule or cell-

885Development 120, 885-899 (1994)Printed in Great Britain © The Company of Biologists Limited 1994

We have isolated and characterized a novel gene, named

18wheeler (18w) for its unique segmental expression patternin Drosophila embryos and expression in cells that migrateextensively. 18 wheeler transcripts accumulate in embryosin a pattern reminiscent of segment polarity genes.Mutations in 18w cause death during larval developmentand early adulthood. Escaping mutant adults often displayleg, antenna, and wing deformities, presumably resultingfrom improper eversion of imaginal discs. Sequenceanalysis indicates that 18w encodes a transmembraneprotein with an extracellular moiety containing manyleucine rich repeats and cysteine motifs, and an intracellu-lar domain bearing homology to the cytoplasmic portion of

the interleukin-1-receptor. Expression of 18W protein innon-adhesive Schneider 2 cells promotes rapid and robustaggregation of cells. Analysis of the expression of 18w indifferent mutant backgrounds shows that it is undercontrol of segment polarity and homeotic genes. The datasuggest that the 18W protein participates in the develop-mental program specified by segmentation and homeoticgenes as a cell ahesion or receptor molecule that facilitatescell movements.

Key words: morphogenesis, segment polarity, homeotic genes,interleukin-1-receptor, leucine rich repeats, Toll

SUMMARY

The

Drosophila 18 wheeler is required for morphogenesis and has striking

similarities to Toll

Elizabeth Eldon1,2,*, Sandra Kooyer1, Diana D’Evelyn1, Molly Duman2, Patrick Lawinger3, Juan Botas3 andHugo Bellen1,3,†

1Howard Hughes Medical Institute, and 3Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030, USA2Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA

*Present address: Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, USA†Author for correspondence

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signaling protein during developmental processes that requireextensive cell movement.

MATERIALS AND METHODS

Fly stocks-Enhancer detector strains P[lac,ry+]Q26b; ry506/ry506 at 56F(Stephen Kerridge, personal communication)

-Enhancer detector strain PZ l(2)00053 cn; ry506/ry506 at 56F6-9(Karpen and Spradling, 1992; Mlodzik and Hiromi, 1992)

-Df(2R)AA21 = Df(2R)56F;57D12 associated with In(2R)56D-E;58E-F (Mackay et al., 1985)

-Df(2R)G100-L141 = Df(2R) 56D-56F a synthethic deficiencyderived from two segmental aneuploidy stocks (Lindsley and Zimm,1992)

-Tp(2;Y)jl11=60F-57F|21A-56F|YSy+;YL.YS 56F-57F|21A (Lyttle,1984)

-Tp(2;Y)jl15 = 21A-55A|57C-60F;YL.YS |55A-57C|YSy+ (Lyttle,1984)

-Tp(2;Y)R31 = a transposition of 56F-57D to the Y chromosome(Lindsley and Zimm, 1992)

-Tp(2;Y)J64 = a transposition of 56F-57A to the Y chromosome(Lindsley and Zimm, 1992)

-Df(2R)M173 = M(2)173 = a multilocus deficiency at 56F (Lindsleyand Zimm, 1992)

-Df(3R)Ubx109 = uncovers Ubx and Abd A (Lindsley and Zimm,1992)

-winglessCX4 (Lindsley and Zimm, 1992)-patchedIN (Lindsley and Zimm, 1992)-Toll 9QRE = a null dorsalizing allele of Toll caused by an inversion;

selected because 2.4% of expected Toll flies emerge (Gerttula et al.,1988)

-Df(3R) ro80b = deletes 97D1-D15, removing genes proximal anddistal to Toll, but no other dorsal group genes (Gerttula et al., 1988)

Genotypes of stocks: In(2R)AA21, apx sp/SM1, Df(2R)G100-L141/SM1, Tp(2;Y)jl11; cn bw/Df(2R)jl11, Tp(2;Y)jl15; cn bw/Df(2R)jl15, Df(2R)M173/SM5, Tp(2;Y)R31; SM5/Df(2R)R31,Tp(2;Y)J64; SM5/Df(2R)J64, Df(3R)Ubx109 e/TM6b, Ubx-lacZ,wgCX4b pr/CyO ftz-lacZ, and ptc iN cn br sp/CyO; cn 7-35/CyO;Df(3R) ro80b st e/TM2, cn l(2)00053/CyO; Tl9QRE ca/TM2.

In situ hybridizations to polytene chromosomes of strains contain-ing rearrangments using probes from the most 5

′ and 3′ sequences ofthe cloned genomic sequences showed that Df(2R)AA2 andDf(2R)M173 do not affect the 18w signal. However, transpositionsTp(2;Y)R31, Tp(2;Y)J64 and Tp(2;Y)jl15 contain 18w, whereas theircorresponding deficiencies lack 18w sequences, i.e. a hybridizationsignal is observed next to the chromocenter and half a signal can beobserved on the second chromosome. We have been unable toestablish whether Tp(2;Y)jl11 affects 18w.

Northern analysisTotal RNA was isolated with acidic (pH 5.0) hot phenol (68°C)(Sambrook et al., 1989). Poly(A)+ RNA was purified with oligo-dTcellulose (Sambrook et al., 1989). About 5 µg of poly(A)+ RNA wasloaded per lane. The membrane was hybridized with a unique non-repetitive Klenow labeled 0.62 kb EcoRV-Kpn1 cDNA fragment,which does not contain the leucine rich repeats or OPA repeats.

In situ hybridization to polytene chromosomes andembryosDigoxigenin-labeled probes were prepared as described in theBoehringer-Mannheim ‘DNA labeling and detection kit: nonradioac-tive’. In situ hybridization to polytene chromosomes were performedessentially as previously described (Langer-Safer et al., 1982).Detection was adapted for digoxigenin-labeled probes. In situ hybrid-

ization to whole embryos was performed essentially as described byTautz and Pfeifle (1989).

DNA sequencingSingle stranded DNA was sequenced by dideoxy chain-terminationmethod using the automated fluorescence procedure (Applied Biosys-tems) as described by Bellen et al. (1992). The sequence of the cDNAclones was obtained with the assistance of the Institute for MolecularGenetics Sequencing Core Facility. Briefly, single strand templateswere prepared from a series of exonuclease III-mung bean nucleasegenerated deletions of the 3.2 kb and 2.6 kb cDNAs cloned in pBlue-script SK+ and KS+ (Stratagene). The sequences of 38 clonescovering both strands of these two cDNAs accounts for 5.2 kb of the5.6 kb message (Fig.7). The sequence of approximately 120 bp at the5′ end of the transcript was deduced from the sequence of a genomicclone using a primer complementary to a sequence near the 5′ end ofthe 3.2 kb cDNA. Polymerase chain reaction was used to recoverDNA representing the interval between the site of P element insertionin strain l(2)00053 and the open reading frame using primers com-plementary to the 31 bp terminal repeat of the P element and to the5′ end of the open reading frame. This product was subcloned intopBS SK+ and sequenced.

Other molecular techniquesMost standard techniques are described in Sambrook et al. (1989).Two PCR primer sets were used to determine the molecular defectsin mutants induced by P-element excision. To detect 5′ deletions aprimer derived from a sequence 750 bp upstream of PZ and a primercomplementary to the 31 bp inverted repeat of P elements were used.To detect 3′ deletions, the 31 bp P-element primer and a primer com-plementary to the 5′ end of the cDNA were used.

Production of polyclonal antibodies, immunoblots, andimmunocytochemical staining of embryos with antibodiesPolyclonal rabbit antibodies were generated against epitopes derivedfrom either the putative extracellular or the putative intracellularportion of the 18W protein. To generate polyclonal antibodies againstthe extracellular domain a bacterial fusion protein was made bycloning a 1.1 kb EcoRV cDNA fragment, corresponding to aminoacids 576-948, into the StuI site of pMal-c (New England BioLabs).A 356 bp NaeI-KpnI fragment, corresponding to amino acids 1065-1159, was filled in with Klenow and cloned into the StuI site of pMal-c. The resulting plasmids were transformed into TBI cells, the fusionproteins were overexpressed and affinity purified (New EnglandBioLabs). New Zealand white rabbits were injected with 100 µg ofaffinity purified protein and boosted at least twice before immune serawere collected and tested. Immunoblotting was essentially performedas described by Towbin et al. (1979). Extracts enriched for membraneproteins were made by homogenizing embryos in a buffer containingprotease inhibitors. After centrifugation, the pellet was resuspendedin 2× Laemmli buffer and centrifuged again. The supernatant waselectrophoresed through a 10% SDS polyacylamide gel. Proteins weretransferred onto a nitrocellulose membrane in Towbin buffer with20% methanol. Membranes were incubated in 10% goat serum andblotto in PBT. The primary antibody was used at a final dilution of1:750. Antibody binding was detected using a secondary antibodyconjugated to alkaline phosphatase at a dilution of 1:1000 and a chro-mogenic substrate.

β-galactosidase expression in embryos was detected using anaffinity purified polyclonal antibody (Cappel) and an anti-rabbitsecondary antibody conjugated to horseradish peroxidase (HRP) asdescribed by Bellen et al. (1992). Most antibodies were preincubatedwith 0- to 3-hour old embryos at a 10× higher concentration for 2-3hours at room temperature or one to several days at 4°C prior to use.

Drosophila S2 cell culture and transfectionA 5.2 kb genomic fragment was excised from λDASH#H with PvuI

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18 wheeler

and SSpI (nucleotides 273-5445 in Fig. 6), and cloned into the SmaIsite of pRmHa3 expression vector (Bunch et al., 1988). Schneider S2tissue culture cells were grown at 24°C (Snow et al., 1989) inSchneider’s Drosophila medium (GIBCO), supplemented with 15%heat inactivated fetal bovine serum (FBS; GIBCO), 100 U/ml of peni-cillin and 100 µg/ml streptomycin (PS; GIBCO). Transfections wereperformed using the calcium phosphate precipitate method (Wigler etal., 1979). Briefly, 10 ml of exponentially growing S2 cells wereplated onto 150 mm × 15 mm Petri dishes and cotransfected for 20hours with 2 ml of a calcium phosphate precipitate containing 20 µgeach of pRmHa3-18w and pPC4 to confer α-amanitin resistance.Medium containing calcium phosphate was removed from cells 16-24 hours after transfection, washed and replaced. The following day,the cells were resuspended in Schneider’s medium containing FBS,PS and 5 µg/ml α-amanitin. The medium was changed every 5th day.After 3 weeks and several passages, aggregation assays wereperformed.

Aggregation assaysS2 cells were grown to log phase in Drosophila Schneider’s mediumcontaining FBS, PS and α-amanitin. After determining cell concen-trations, the cells were collected by centrifugation and resuspended inSchneider’s medium containing FBS and PS at a density of 1×106

cells/ml. Synthesis of the 18W protein was induced by adding CuSO4to a final concentration of 0.7 mM. 2 ml of this suspension was added

to a 50 ml conical tube and gently rotated on a shaker at 100 rpm for2-4 hours at room temperature. Cell aggregation was scored using aZeiss Axiophot microscope.

Mixing experiments were performed using cells labeled with thelipophilic dyes DiI (Molecular Probes). Cells were labeled by adding10 µl of 10 mM DiI in ethanol to 1 ml of cells resuspended in BSS(50 mM NaCl, 40 mM KCl, 15 mM MgSO4, 10 mM CaCl2, 20 mMglucose, 50 mM sucrose, 2 mg/ml bovine serum albumin and 10 mMtricine, pH 6.95). The DiI-labeled S2 cells were mixed with the 18W-S2 cells and induced. Aliquots were spotted onto microscope slidesand examined by fluorescence microscopy using a Zeiss Axiophotmicroscope.

RESULTS

18w is expressed in a complex pattern duringembryogenesisThe 18w gene was isolated in a search for full length cDNAsof the couch potato gene (Bellen et al., 1992) because itcontains an OPA repeat sequence, which is often found inDrosophila genes involved in development (Wharton et al.,1985). Whole-mount in situ hybridization experimentsindicated that in the early phases of embryonic development

Fig. 1. Embryonic expression pattern of 18w. Whole Drosophila embryos hybridized in situ using an 18w cDNA probe. Embryos A-H areshown with the anterior end to the left and the dorsal side up; I is a view of the dorsal side. (A) Early stage 5 embryo (for staging see Campos-Ortega and Hartenstein, 1985). Note the two anterior domains of expression and the broad more posterior domain. (B) Late stage 5. Ten broadstripes are visible. Intermediate stripes between the broad stripes start to appear at the anterior end and are one to two cells wide. (C) Stage 8.Note the intense staining of the amnioproctodeal invagination and the staining around the cephalic furrow. (D) Stage 10. Twenty stripes cannow be observed; 15 stripes in the trunk, five in the head region. Not all stripes are in the plane of focus. (E) Stage 11. In addition to the stripesseen in stage 10, 18w is also expressed around the tracheal pits (rings) and a small cluster of cells in each segment located more dorsally.(F) Early stage 12. Expression in the lateral stripes fades while expression in clypeolabrum, salivary gland primordia, invaginating trachealprecursor cells, and posterior spiracles anlagen become clearly visible. (G) Stage 13. Expression is quite complex and most conspicous in thedeveloping tracheal tree. (H) Stage 15. 18w is expressed in pharynx, hindgut, salivary glands (out of focus), and the tracheal tree (out of focus).(I) Dorsal view of stage 15/16. 18w is expressed in the region of the pharynx, dorsal vessel, anal pads, and tracheal system.

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18w expression resembles that of segment polarity genes. Amore detailed analysis of its expression pattern was thereforeinitiated.

In situ hybridizations to whole-mount embryos using digox-igenin-labeled probes detect no transcripts in 0- to 2-hour oldembryos. However, just prior to cellular blastoderm formation,during nuclear elongation, expression is initiated in severaldomains (Fig. 1A). The most anterior two stripes in the pre-sumptive head region are 5-6 cells wide. As shown in Fig.1A,B, these stripes do not encircle the embryo. The third stripeis 3 cells wide and encircles the embryo. Prior to cellulariza-tion, the central region of the embryo, from 60-55% egg lengthto 30% egg length (0% is posterior), is faintly labeled. As cel-lularization proceeds, this domain rapidly subdivides in fivecircular stripes which are two to three cells wide. The posteriortwo stripes, 9 and 10, are 4-5 cells wide and are more intenselylabeled than stripes 4-8 (Fig. 1B).

At late cellular blastoderm secondary stripes begin toappear between all but the first two primary stripes. Theyappear first at the anterior end, are initially only one cell wide,and lead to the transient expression of 18 stripes during earlygastrulation (hence, 18 wheeler). The secondary stripesrapidly widen and darken (Fig. 1C), and two additional stripesappear in the head region (Fig. 1D), leading to 20 stripes thatcan then be identified during germ band elongation (Fig. 1D).During gastrulation the cephalic furrow forms immediatelyanterior to stripe 3. Stripe 10 encircles the pole cells in thepresumptive hindgut and anal region. As the ventral furrowinvaginates, 18w expression is initially retained in the pre-sumptive mesoderm.

At maximum germ band elongation the pattern becomesmore complex. Lateral expression in each stripe is lost, whileventral and dorsal expression is retained. Along the ventralmidline a small cluster of cells begins to express 18w on theposterior side of each ventral stripe. In addition, transcriptsaccumulate around each invaginating tracheal pit and at thesites of the salivary gland placodes. We also observe stainingin five regions in the presumptive head region (Fig. 1E).During germ band retraction expression becomes generallyless abundant (Fig. 1F) and more restricted to specific regionsor cell clusters. At stages 12.3 and 12.1 (for staging see Klämbtet al., 1991) the developing tracheal system, the salivary glandanlagen and the anlagen for the anal plate, the posteriorspiracles, and the clypeolabrum express 18w. In addition, a rowof cells at the leading edge of the two epidermal sheets thatconverge toward the dorsal midline are labeled. These cellsparticipate in the formation of the dorsal vessel (heart) andcontinue to express 18w into the later phases of embryonicdevelopment (Fig. 1I). In stage 15 and 16, we also observe 18wexpression in two cells in the head region, the dorsal portionof the pharynx, a portion of the stomach and the hindgut (Fig.1H). Hence, 18w is expressed in a very dynamic pattern, oftenin cells that undergo extensive migration.

To determine where the 18w transcript is expressed withrespect to known segment polarity genes like engrailed andwingless we performed double labeling experiments by firststaining embryos carrying an enhancer detector insertion in18w (PZ, see below) with β-galactosidase followed by in situhybridization with a digoxigenin engrailed -labeled probe (Fig.2 top). In a second experiment, wild-type embryos werehybridized with 18w and wingless probes (Fig. 2 middle). At

late cellular blastoderm and early gastrulation 18w is expressedin an alternating two cell / one cell wide stripe pattern. The twocell wide stripes correspond exactly to the wingless- andengrailed-expressing cells (data not shown). As shown in Fig.2, during germ band elongation 18w is expressed in a domainwhich is 3-4 cells wide in most segments. This domain spansthe parasegmental but not the segmental boundaries (Fig. 2bottom).

To determine the size, abundance and complexity of 18wmessages we performed a developmental northern analysis. Asshown in Fig. 3, a single 5.6 kb message can be detected at allstages analyzed. This message accumulates during embryoge-nesis beginning at 2-3 hours after egg laying, is abundantduring the third larval instar and pupal stages and is present inadult females.

E. Eldon and others

Fig. 2. 18w is expressed at the segmental boundaries. Top. Germband extended embryo from the l(2)00053 (=PZ) strain expressinglacZ in the 18w expressing cells (broader light blue stripes). Adigoxigenin-labeled probe detects engrailed-expressing cells (darkblue). Middle. Detail of the ventral area of a whole-mount embryohybridized with wingless and 18w probes. The dark blue, one cellwide stripe, corresponds to the wingless signal, the remaindercorresponds to the 18w signal. Bottom. At cellular blastoderm 18w isexpressed in the wingless- and engrailed-expressing cells. Duringgerm band extension 18w is expressed in stripes that are 3- to 4-cellswide that spans the parasegmental but not the segmental boundaries.

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Two P element enhancer detectors map in proximityof 18w at 56F6-9 To initiate a genetic study we localized 18w by in situ hybrid-ization to polytene chromosome bands 56F6-12. Seven defi-ciencies, some associated with transpositions, were identifiedthat potentially lack all or parts of the 56F cytologicalsubdivion (see Materials and Methods). In situ hybidizationsto chromosomes carrying these deficiencies using probesderived from the genomic walk (see below and Materials andMethods) allowed us to refine mapping of 18w to 56F6-9.

Unfortunately, the only deficiencies that fail to hybridize tothe 18w probe are associated with Y-chromosome transposi-tions. These deficiencies are of limited use for the isolation ofmutations in 18w because flies in which the deficiency segre-gates independently from the transposition are rarely viable,indicating that these deficiencies are mostly haploinsufficient.However, we identified two P-element enhancer detectors at56F: P[lacZ, ry+] Q26 (provided by Steve Kerridge) and PZl(2)00053 (provided by Allen Spradling; Karpen and

Spradling, 1992). Flies homozygous for P[lacZ, ry+]Q26(abbreviated PlacZ) are viable, whereas flies homozygous forPZ l(2)00053 (abbreviated PZ) are sublethal. Although theoriginal PZ stock was characterized as homozygous lethal,approximately 8-10% of all homozygous PZ flies surviveunder non-crowded conditions. These flies eclose 2-4 daysafter their siblings, are often small, have morphologicaldefects, are unable to fly and jump, and have very poor viabilityand low fertility.

To determine whether the enhancer detectors express thelacZ gene in a pattern that resembles the 18w gene, we stainedembryos and third instar larvae of both strains for β-galactosi-dase activity. Both strains show similar β-galactosidaseexpression patterns in embryos, and the following descriptionis derived from the PZ strain. As shown in Fig. 4B, theembryonic expression pattern faithfully mimicks theexpression pattern of the 18w message during germ bandextension. However, at subsequent stages of development, thelacZ expression pattern is much more complex than the profileof the 18w transcript because the β-galactosidase enzymeremains active whereas the transcript is only transientlypresent. In stage 14-16 embryos we observe β-galactosidaseexpression in ectodermal stripes which are 4-5 cells wide ineach segment, most of the developing tracheal tree, salivaryglands, anal pads, heart, hindgut, a small cluster of cells in eachmetamere of the central nervous system, pharynx, posteriorspiracles, and some of the ventral muscles. The similarity ofthe expression patterns and the cytological map positions of PZand PlacZ indicate that these enhancer detectors have insertednear the 18w gene.

Structure of 18w geneTo determine the structure of 18w we isolated about 30 kb ofcontinuous genomic sequence using cDNA probes. The entirecDNA (see below) hybridizes to a contiguous fragment of thewalk. Comparison of the restriction maps combined withsequencing information of genomic fragments and cDNA, and

Fig. 3. Developmental northern analysis. The 18w message is presentat all stages of development that were analyzed. At all stages except6- to 9-hour old embryos a single message of approximately 5.6 kb isdetected. All lanes contain 5 µg of poly(A)+ RNA.

(5.6 kb)

(4.6 kb)

Fig. 4. Structure of 18w. (A) The 18w cDNA hybridizes to contiguous genomic restriction fragments coveringapproximately 5.4 kb. Two P-element enhancer detectors are inserted upstream of 18w. Plac is a P[lacZ, ry+ ] enhancerdetector (O’Kane and Gehring, 1987), which is located 6 kb upstream of the putative 18w transcription start site. PZ is aP[lacZ, ry+] enhancer detector (Mlodzik and Hiromi, 1992), which is inserted 23 bp upstream of the putative

transcription start site. The small arrows indicate the direction in which the lacZ reporter gene is transcribed. The long arrow over therestriction map indicates the direction of transcription. Horizontal lines beneath the restriction map indicate genomic phage isolated with cDNAprobes. (B) Both enhancer detector strains exhibit very similar β-galactosidase expression patterns in embryos. Top inset shows the lacZexpression pattern of a PZ embryo, bottom inset shows an embryo of the same stage hybridized in situ with an 18w probe. The patterns arevirtually identical.

A B

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Northern analysis, suggests that the entire messenger RNA isencoded by a single exon of approximately 5.4 kb followed bya 200 bp poly(A)+ tail (see Fig. 4). However, we cannotexclude the presence of very small introns in the coding regionas we did not sequence the entire genomic fragment. Theputative transcription start was determined by S1 nuclease pro-tection to be 374 bases upstream of the translation initiationcodon. While we cannot exclude the presence of more 5′ exons,the deduced length of the transcript based on the sequence ofthe cDNAs and the S1 protection data (see below) is consis-tent with that determined by northern analysis (above). Inaddition, the proposed transcription initiation site matches theinitiation consensus sequence determined for Drosophila(Burtis et al., 1990).

The sites of PlacZ and PZ P-element enhancer detectorinsertion were mapped by genomic Southern analysis. PlacZ isinserted approximately 6 kb upstream of the putative tran-scription start site whereas PZ is inserted near the putative tran-scription site. The precise insertion site of PZ was determinedby sequencing a PCR amplified band generated with oneprimer from the P element and one from the cDNA. PZ maps23 bp upstream of the putative transcription start site (Fig. 4and 6). Based on its proximity to the 18w transcription unit,the PZ transposable element is the most suitable insertion toinitiate a genetic analysis and create additional mutations in18w.

18w is required for larval and adult viabilityTo show that the lethality associated with the PZ insertionchromosome is caused by the PZ transposable element, and tocreate additional mutations in 18w, the PZ enhancer detectorwas excised using the ∆2-3 transposase excision mutagenesis(Bellen et al., 1992). Approximately 100 ry− flies derived fromthe PZ ry+ strain were recovered and balanced lines wereestablished. The majority of excision chromosomes arehomozygous viable and these homozygous flies show noobvious behavioral or morphological defects. One of thesehomozygous viable revertants, ∆1-12, was characterized at themolecular level and was shown to be a precise or near-preciseexcision (see below). We conclude that the PZ insertion isresponsible for the observed lethality and the defects seen inhomozygous viable flies.

To determine whether the observed sublethal phenotype ofPZ flies corresponds to a partial or a total loss of 18w function,nine homozygous lethal or sublethal excision lines were char-acterized in more detail. Complementation tests were carried

out among homozygous lethal and sublethal lines. As shownin Table 1 all putative imprecise excision lines fail to comple-ment each other, as many fewer transheterozygous progeny arerecovered than expected, indicating that they all carry 18wmutations. However, with two exceptions (∆1-82 and ∆7-43),most newly isolated lethal or sublethal strains produce moreprogeny in transheterozygous combination with the original PZchromosome than in other transheterozygous combinations,indicating that the PZ insertion is a hypomorphic allele of 18w.

To determine the molecular nature of the excision-inducedalleles, we carried out a series of experiments summarized inTable 2. Embryos were stained immunocytochemically todetect β-galactosidase activity, genomic DNA was amplifiedby PCR and analyzed by Southern analysis to determine theextent of excisions, and embryos were hybridized in situ todetermine whether the 18w transcript was missing or altered inits expression pattern (see legend Table 2). The results indicatethat at least two mutants lack genomic fragments which containthe leader sequence. Mutant ∆7-35 flies carry a deletion ofapproximately 2.2 kb removing approximately 1.7 kb of theopen reading frame, whereas ∆1-82 mutants lack the tran-scription start site as well as most or all of the leader (seebelow). The complementation data indicate that both mutantsbehave as severe lack of function alleles when compared tohomozygous PZ flies. However, in situ hybridizations toembryos using the 5′ and 3′ end of the cDNA as a probeindicate that some truncated transcripts accumulate inhomozygous mutant embryos (M. Williams, personal commu-nication). Since a few homozygous ∆7-35 flies can berecovered (Table 2), either the truncated transcript retainsresidual function and 18w is essential for viability, or there isno absolute zygotic requirement for 18w.

To characterize the defects in 18w mutant embryos westained homozygous ∆1-82 mutant embryos for β-galactosi-dase activity. Since these embryos retain the lacZ gene fromthe enhancer detector under the control of the 18w regulatoryelements (see Table 2) all tissues that are potentially affectedby the lack or partial lack of 18W protein can be identified. Noobvious defects were seen in internal tissues of homozygous∆1-82 embryos. However, more than 90% of larvae homozy-gous or transheterozygous for ∆1-82 or ∆7-35 die as second orthird larval instars, and only 1-3% of the expected tran-sheterozygous mutant adults eclose (e.g. 106 ∆1-82/∆7-35versus 8,848 ∆1-82/CyO or ∆7-35/CyO). These flies eclose 2-4 days later than their siblings, and most adult flies die withina few days. Approximately 60% of the adult flies (∆1-82/∆7-

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Table 1. Complementation data of lethal and sublethal 18w mutations1-11 1-14 1-15 1-24 1-82 2-62 2-63 6-81 7-35 7-43 PZ

1-11 0/1291-14 0/117 1/1621-15 7/131 17/93 2/2261-24 5/160 18/185 19/181 0/1471-82 7/137 3/115 2/124 6/143 0/1712-62 5/78 5/151 22/214 9/98 1/151 0/1192-63 8/105 5/185 0/132 7/194 3/123 1/197 0/1726-81 5/135 0/147 3/130 2/227 0/193 18/181 0/129 0/1347-35 6/100 4/151 6/104 0/134 3/132 1/62 2/150 3/104 0/2227-43 1/118 0/99 3/114 7/150 6/169 4/150 4/159 11/103 1/131 3/176PZ 9/114 7/125 23/78 26/178 1/136 13/94 11/143 12/55 22/115 1/134 6/131

Number of homozygous or transheterozygous mutant flies that survive/total number of balanced flies scored.

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35) show morphological defects in at least one appendage: leg,antenna, wing, or haltere. Similar defects were also observedin several other transheterozygote adult 18w flies. The mostfrequent defects consist of bent and swollen leg segments (e.g.kinked femurs), fused leg segments, and shortened tarsi (seeFig. 5B,C). Antennae are often abnormally positioned, appearlighter and are significantly larger than normal, wings arefolded or bent, and halteres are abnormally shaped andsometimes duplicated. These defects are indicative of problemsassociated with imaginal disc eversions which requiresextensive cell movement. It should be noted that both enhancerdetector strains (PZ and P[lacZ, ry]) express the β-galactosi-dase gene abundantly in the antennal, leg (Fig. 5A) and wingimaginal discs.

18 wheeler encodes a protein with extensivehomology to Toll and the Interleukin-1-receptorTo obtain a full length cDNA for the 18w transcript, the orig-inally recovered cDNA was used to screen a 9- to 12-hourembryonic library (Zinn et al., 1988) and five other cDNAswere isolated. A 3.2 kb and a 2.6 kb cDNA were sequencedand they account for approximately 5.2 kb of the transcript.Sequence data show that the combined cDNAs terminate witha short poly(A) tail preceeded by a polyadenlyation additionsite and contain a 4.2 kb open reading frame (ORF) whichinitiates at the most 5′ end of the 2.6 kb cDNA but lacks aninitiator methionine. However, 7 bases upstream from the endof the cDNA within the genomic sequence is an AUG codon(Fig. 6). Several lines of evidence suggest that translationinitiates at this methionine. First, all three reading framescontain stop codons upstream of this methionine; second, theresidues immediately upstream of the first methionineconform with the Drosophila translational initiationconsensus (Cavener, 1987); third, the first 27 amino acidsencoded by the ORF are hydrophobic and have the charac-teristic features of a cleaved signal peptide (von Heijne,1986).

The putative protein encoded by the ORF is 1389 amino acid

long. This corresponds to an apparent relative molecular massof 155×103 in the absence of signal peptide cleavage and otherpost-translational modifications. A Kyte and Doolittlehydropathy analysis shows that the putative 18W proteincontains two hydrophobic regions that are underlined in Fig.7. The first corresponds to the putative signal peptide and thesecond to a transmembrane domain. We therefore propose thatthe 18W protein contains an extracellular domain, a membranespanning domain, and an intracellular domain. The putativeextracellular region is unusually rich in leucine residues(165/971, 17%), and contains 16 putative N-glycosylation sitesand a single glycosaminoglycan site (aa 50-54).

A search of the GenBank protein sequence database usingFAST-A and BLAST-A revealed several sequence motifs.Following the signal sequence is a large extracytoplasmicdomain with 22 consecutive 24-amino acid leucine-rich repeats(LRRs). As shown in Fig. 7B, the first repeat starts at aa 89and the 22 following LRR repeats are aligned with it. LRRshave been identified in a number of proteins from a widevariety of organisms. In Drosophila, genes like Toll(Hashimoto et al., 1988), slit (Rothberg et al., 1988), Chaoptin(Reinke et al., 1988), tartan (Chang et al., 1993) and connectin(Nose et al., 1992) all contain these motifs. They are also foundin a yeast adenylate cyclase (Yamawaki-Kataoka et al., 1989),and a number of proteins identified from vertebrate sources,such as leucine-rich-α2glycoprotein (Jalkanen et al., 1988),cartilage proteoglycans (Krusius and Ruoslahti, 1986), andglycoprotein 1b (GP1b). The 22 LRR repeats are flanked by acarboxy-terminal domain that contains cysteines (see Fig.7A,C). This domain is also found in a number of LRR-con-taining proteins: e.g. Slit, Toll, GPIb and Glycoprotein 1X(GPIX) (Rothberg et al., 1990; Schneider et al., 1991). Thecarboxy-terminal domain is followed by a second cysteine richdomain named the amino-flanking domain as it precedes thenext run of LRRs (Rothberg et al., 1990). This domain is alsofound in Slit, GPIb, GPIX, Decorin, Biglycan, Fibromodulin(FM, a collagen-binding protein) and oligodendrocyte-myelinglycoprotein (OMgp) (Rothberg et al., 1990). Finally, the

Table 2PCR

Strain Lethal/Viable 5′ 3′ lacZ mRNA Southern Conclusion

PZ SL(9%) + + + ∆ PZ Insertion∆1-11 L + + − NT PZ Internal excision∆1-12 V − − NT + wt Revertant∆1-14 SL(0.5%) − − − NT ∆ Excision +?∆1-15 SL(3%) + + NT NT NT Internal excision∆1-24 L + + + NT NT Internal excision∆1-82 SL(4%) + − + −/+ ∆ Partial excision

Deletion of 5′ end∆2-62 L + + + NT NT Internal excision∆2-63 L + + + NT NT Internal excision∆6-81 L + + − NT ∆ Internal excision∆7-35 SL(0.5%) − − − − ∆ Excision, deletion

Rearrangement∆7-43 SL(2%) + + + +/− PZ Internal excision

Lethal/Viable: L, lethal; SL, sublethal; V, viable. Lethality was determined on 400 progeny. Hence, strains that are marked as L, may not be L but SL if moreprogeny were tested. Strain ∆1-12 was included because it is a wild-type revertant. PCR: a primer complementary to 31 bp inverted repeat of both ends of P-elements and a primer 5′ or 3′ of the P-element were used. lacZ: X-gal staining; NT, not tested. mRNA: whole-mount in situ hybridization. +, wild type; +/−,reduced; −/+, severely reduced; ∆, altered pattern; −, little or no message. Southern: Probe is 1.4 kb Cla1 fragment (see Figure 5). PZ; Cla1-HindIII = 0.7 and 0.9kb; Cla1-EcoR1 = 1.3 and 3.0 kb; Cla1 = 1.9 and 2.7 kb. Wild type, wt = 1.4 kb for restriction enzymes shown above. ∆ = not PZ or wt like. e.g. ∆ for ∆1-82;Cla1-HindIII = 0.7 and 1.2 kb; Cla1-EcoR1 = 1.3 and 1.2 kb; Cla1 = 1.9 and 1.2 kb. ∆ for ∆7-35; Cla1-HindIII = 2.9 kb; Cla1-EcoR1 = 3.9 kb; Cla1 = 4.8 kb.

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extracellular moiety contains an additional five LRR followedby a carboxy flanking domain.

The intracellular moiety of 18W is about 380 aa long, andconsists of two domains. The first domain shows significanthomology to the cytoplasmic domain of the interleukin-1-receptor (IL-1R). The IL-1R has been sequenced in mouse,human and chicken (Guida et al., 1992) and was shown to be

homologous to the intracellular portion of the Toll protein(Schneider et al., 1991). As shown in Fig. 7D, a comparisonof the IL-1R, Toll and 18W indicates that there are six stretchesof amino acids that are more conserved than the remainingamino acids. These regions of higher conservation probablycorrespond to functional domains since point mutations insome of these conserved regions lead to non-functionalproteins (Heguy et al., 1992; Schneider et al., 1991). Thesecond domain consists of a glutamine-rich OPA repeat(Wharton et al., 1985). OPA repeats are found in manyDrosophila genes and their significance is not known.

The 18W protein is a 170×103 Mr membraneassociated proteinTwo polyclonal antibodies (A and B) were raised againstmaltose binding fusion proteins, containing either 374 aminoacids of the putative extracellular moiety (=A) or 118 aminoacids of the putative intracellular portion of 18W (=B). Bothsera recognize a single 170×103 Mr band in immunoblots ofmembrane preparations. A band of the same Mr was alsoobserved after labeling embryonic extracts with biotin andimmunoprecipitating proteins using the A antibody (data notshown). The estimated Mr based on the primary sequence isabout 155×103, but since 18W may be modified at as many as16 asn-glycosylation sites, a putative glycosaminoglycan siteand many other cytoplasmic phosphorylation sites, these Mrdifferences can easily be reconciled. Both antibodies recognizea single band with the same electrophoretic mobility inmembrane fractions. Hence, we conclude that the antiseraspecifically recognize the 18W protein. Unfortunately,immunocytochemical staining of embryos using these antiseraindicates that they fail to recognize the native 18W protein inwhole-mount embryos under the conditions used.

18W is a heterophilic cell adhesion moleculeTwo proteins containing LRR-repeats, Chaoptin andConnectin, have been shown to be homophilic cell adhesionmolecules (Krantz and Zipursky, 1990; Nose et al., 1992),whereas Toll was shown to be a heterophilic cell adhesionmolecule (Keith and Gay, 1990). In order to determine whether18W is a cell adhesion molecule we transfected non-adherentS2 cells with the entire 18w open reading frame under thecontrol of the inducible metallothionein promoter (Bunch et al.,1988) and co-transfected with a selectable marker (Jokerst etal., 1989). Immunoblots with the antiserum raised against anextracellular moiety show that S2 cells do not express 18W,and that induced 18W-S2 cells express high levels of 18Wprotein whereas uninduced 18W-S2 cells only produce lowlevels of 18W protein (not shown). As shown in Fig. 8, induced18W-S2 cells form many aggregates which often contain 15-30 cells, and sometimes 100-200 cells. These aggregates formwithin a 2- to 4-hour period after onset of induction. Induceduntransfected S2 cells and S2 cells transfected with the α-amanitin resistance gene and a control pRmHa3 not contain-ing the 18w ORF (but the neuromusculin gene in the oppositeorientation (Kania et al., 1993)) do not aggregate. Uninduced18W-S2 cells form few small aggregates but express low levelsof 18W when assayed by western analysis. We thereforeconclude that 18W acts as a cell adhesion molecule.

To establish whether 18W act as a homophilic or heterophiliccell adhesion molecule, S2 cells were labeled with a fluorescent

E. Eldon and others

Fig. 5. LacZ expression in imaginal discs and morphological defectsof legs of surviving flies mutant for 18w. (A) LacZ expressionpattern in leg imaginal disc. LacZ is expressed in rings of tissue thatare darker in the center of the leg imaginal disc, and lighter in theperiphery. (B) First thoracic leg of a surviving ∆1-82/∆7-35 male.Arrow indicates fused tarsal segments. (C) Leg of surviving∆182/∆7-35 fly. Arrow indicates distorted and bloated leg segments.

89318 wheeler

Fig. 6. Sequence of 18w. As no complete cDNA was obtained, thepresented sequence is a composite of genomic and cDNA sequences(see Materials and Methods). The 18w gene encodes a 5.6 kb transcriptwith a 5′ leader of 375 nucleotides (nt), an ORF of 4,167 nt, a trailer of898 nt, and a poly(A) tail. The enhancer detector PZ(P[lacZ,ry+]l(2)00053) is inserted 23 nucleotides upstream of theputative transcription start site, which is inferred from RNAse protectionassays. Note that the putative AGTTT transcription start site fullyconforms to the Drosophila consensus (Burtis et al., 1990). Our dataindicate that the 18w gene is encoded by a single exon but we cannotexclude the presence of another 5′ small exon, nor can we exclude thepresence of one or two very small introns in the ORF. Based on thehydropathy and sequence analysis we propose that 18w encodes aprotein with an extracellular, a transmembrane, and a cytoplasmicdomain. The signal peptide and the transmembrane domain areunderlined as is the polyadenylation signal. For structural motifs seeFig. 7. The Genbank submission number for 18w is L23171.

894 E. Eldon and others

89518 wheeler

lipophilic dye (DiI) and mixed with unlabeled 18W-S2 cells(Fig. 8C,D). The majority of cell aggregates were shown tocontain mixed populations of DiI-labeled and unlabeled cells,indicating that 18W is a heterophilic cell adhesion molecule. Todetermine whether the number of labeled S2 cells in theseaggregates is proportional to the number of labeled cells thatare mixed with unlabeled 18W-S2 cells, the ratio oflabeled/unlabeled cells was determined in aggregated and non-

aggregated cells in two separate experiments. In the first exper-iment 20% of all cells were labeled but only 10% of the cellsin aggregates were labeled. In the second experiment 27% ofall cells were DiI-labeled S2 cells and 27% of all cells in theclusters were labeled. When similar experiments are performedwith homophilic cell adhesion molecules like Fasciclin I, II orIII, fewer than 5% of cell aggregates contain DiI-labeled S2cells (data not shown). These observations indicate that 18Wcan act as a heterophilic cell adhesion molecule.

18w expression is controlled by segmentation andhomeotic genesSince 18w is expressed in a pattern which resembles that ofsegment polarity genes, its expression may be controlled bysegment polarity genes. To test this hypothesis we studied theexpression pattern of 18w in two segment polarity mutants:wingless and patched. Initial accumulation of 18w transcriptappears normal in wg mutants. However, by full germ bandextension (stage 10), the ventrolateral 18w stripes that arenormally three to four cells wide are reduced to stripes that areonly one to two cell wide (data not shown). These changesappear well before cell death is seen in wingless mutants(Perrimon and Mahowald, 1987). In addition, in embryosmutant for the segment polarity gene patched, the domain of18w expression is expanded (Fig. 9A,B). In patched embryos,the domain of wingless expands anteriorly and is flanked by anectopic domain of engrailed expression (Ingham et al., 1991).Ectopic expression of 18w in patched mutants corresponds tothe domains of ectopic expression of wingless and engrailed.This expansion is consistent with the observation that in wildtype embryos 18w expression is coincident with that ofwingless and engrailed. The reduced 18w expression inwingless mutants and the ectopic expression in patchedmutants suggests that wingless and engrailed positively

Fig. 7. Deduced structure of 18W and comparison of Toll and 18Wdomains. (A) Comparison of 18W and Toll protein. Schematicrepresentation of the 18W and Toll proteins. Since manyextracellular matrix proteins (ECM) have a rod-like conformation(Engel, 1989), we have depicted them as such. The 18W proteincontains a signal peptide followed by a domain that contains aglycosaminoglycan site. The extracellular domain consists of 22leucine-rich repeats (LRRs) followed by a carboxy-flanking domain,an amino-flanking domain, 5 LRR, and a carboxy-flanking domain.Single LRRs have been shown to form β-sheets and two or moreLRRs may form antiparallel sheets (Krantz et al., 1991). (B) LRRrepeats in 18W. The 24 amino-acid LRR of the 18W protein arecompared. The consensus was taken from Rothberg et al. (1990).(C) Amino-flanking domain. This domain preceeds the second run ofLRRs in Toll and 18W as well as in some other proteins like Slit,Decorin, Biglycan (see Rothberg et al., (1990) for the consensus).(D) Carboxy-flanking domain. This domain was previously identifiedin Slit, Toll and some glycoproteins (see Rothberg et al., 1990, forconsensus). (E) Interleukin-1-receptor domain. Both Toll and 18Whave significant homology to the cytoplasmic portion of the IL-1-R.The domains were aligned visually starting with the alignment ofToll and the chicken IL-1R presented by Guida et al. (1992). Byaltering the alignments slightly we find six areas that show strikingsequence conservation rather the four as initially presented by Guidaet al. (1992). These similarities are indicated in bold type. Similaramino acids are shown as +, and the similarities were according toMiyata et al. (1979).

Fig. 8. Schneider 2 cells thatexpress 18W showheterophilic cell adhesionproperties. (A) UntransfectedS2 cells treated with 0.7 mMCuSO4. (B) Cells transfectedwith pRmHa3-18w (18W-S2)and treated with 0.7 mMCuSO4. Note the large clustersof cells. Uninduced 18W-S2cells also tend to aggregatebut to a much lesser extent(not shown). (C) Anenlargement of clusters ofinduced 18W-S2 cells.(D) Same clusters as in C.Untransfected S2 cells werelabeled with lipophilic dye DiI(red and fluorescent). Notethat clusters contain asignificant number of labeledcells indicating that 18W isheterophilic cell adhesionmolecule.

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regulate the zygotic 18w expression within the ventrolateralstripes. Other genes must regulate most of the other domainsof 18w expression as these domains do not seem to be affectedin wingless and patched mutants.

The cytological bands (56F6-9) to which 18w maps corre-spond to one of the most prominent binding sites of the Ubxprotein on polytene chromosomes (Juan Botas, personal com-munication). To test whether the 18w expression pattern isaffected in Ubx mutant embryos we analyzed the expressionpattern of 18w by in situ hybridization to whole-mountembryos in homozygous Df(3R)Ubx109 mutant embryos. Thisdeficiency uncovers the Ubx and abdominal A genes, but notthe Abdominal B gene. As shown in Fig. 9C, at the end of germband retraction (stage 15), three patches of 18w expression canbe seen in the developing tracheal system. The most anteriorpatch located in the posterior region of the first thoracicsegment corresponds to the primordia of the anterior spiracles.The second and third thoracic patches are fainter and are alsoassociated with the developing tracheal tree. Expression of 18win abdominal segments of wild-type embryos is much lessprominent but the outline of the developing tracheal tree canstill be seen. Fig. 9D shows that in Df(3R)Ubx109 mutantembryos nine patches of similar intensity can be observed inthe three thoracic and first six abdominal segments Theseobservations indicate that 18w expression is in part controlledby the bithorax complex genes.

DISCUSSION

18w encodes a transmembrane protein with celladhesion propertiesSequence analysis indicates that 18W has a hydrophobic signalpeptide, an extracellular domain containing many leucine richrepeats (LRRs) and their associated cysteine motifs, a trans-membrane domain, and a cytoplasmic domain with homologyto the interleukin-1-receptor. Western analysis indicates that18w indeed encodes a membrane associated protein. Inaddition to many sites of potential N-linked glycosylation, theextracellular domain contains a glycosaminoglycan site andhence may be a proteoglycan. Interestingly, proteoglycanshave been proposed to facilitate cell migration during tissuemorphogenesis (Laurent and Frazer, 1986). We conclude thatthe structural properties of the 18W protein are compatiblewith a role in cell migration and morphogenesis.

Expression of 18W in non-aggregating S2 cells leads torapid aggregation of these cells. Low levels of expression of18W may be sufficient to cause cell aggregation sinceuninduced cells transfected with an 18w construct express lowlevels of 18W and tend to aggregate. Our data also indicatethat 18W is a heterophilic cell adhesion molecule. Severalother proteins with extracellular LRRs have been shown tobehave as cell adhesion molecules in this specific cell adhesionassay (Hashimoto et al., 1988; Nose et al., 1992; Reinke et al.,

E. Eldon and others

Fig. 9. Expression of 18w in segmentation and homeotic mutants. (A) Wild-type expression pattern of 18w in a stage 10 embryo.(B) Expression of 18w in stage 10 patched IN mutant embryo. The three to four cell wide domains of expression seen in stage 10 wild typeembryos are expanded by two cells to 5- to 6-cells wide bands in patched mutants. (C) Wild-type expression pattern of 18w in stage 15embryos. Focus is on the tracheal tree. Note the three patches in thoracic segments 1-3. (D) Expression pattern of 18w in an embryohomozygous for a deficiency that uncovers the Ubx and abdominal A genes. Note the nine dark patches similar in intensity to the thoracicpatches in wild-type embryos.

89718 wheeler

1988) but only the Toll protein has heterophilic cell adhesionproperties (Keith and Gay, 1990). It is interesting to note thatboth proteins may be structurally quite similar (see below) andthat Toll has previously been proposed to play a role in cellsthat undergo extensive migrations during early development(Gerttula et al., 1988).

Temporal and spatial requirements for 18w functionThe 18w gene is first expressed in stripes along the anterior-posterior axis. These stripes appear in three distinct phases ina manner similar to those of segment polarity genes likewingless (Baker, 1988). The first stripes are those found at thetermini, stripes 1-3 and stripes 9 and 10. This is followed byexpression in six two- to three-cell wide stripes, then in theeight one-cell wide stripes. This pattern arises slightly later for18w than for wg, whose full complement of parasegmentalstripes is present prior to the onset of germ band extension. Ourdata suggest that wingless and engrailed are positive regula-tors of 18w expression as expansion of their domains ofexpression leads to a concomitant expansion of 18wexpression.

During later phases of embryonic development theexpression pattern is quite dynamic, and cells that express 18woften undergo extensive cell movement. For example, 18w isstrongly expressed in the anlagen of the tracheal system, thetracheal placodes. These cells divide, invaginate and undergoextensive migrations as no mitosis seems to occur in the devel-oping tracheal tree (Campos-Ortega and Hartenstein, 1985;Klämbt et al., 1992). 18w mRNA decays during the develop-ment of the tracheal tree, but β-galactosidase protein can bedetected easily during the development and differentiation ofmost of the tracheal tree in PZ enhancer detector embryos.

Expression of 18w in cells undergoing extensive morpho-genetic movements is also observed later in development. β-galactosidase is readily detected in the imaginal discs of thirdinstar larvae from both the PlacZ and the PZ enhancer detectorstrains, suggesting that 18w functions in the development ofthe imago. Interestingly, β-galactosidase is expressed in aseries of concentric rings in leg imaginal discs as well as inmost other imaginal anlagen. These rings of tissue evaginateduring pupation to give rise to the segments of the legs. Theouter-most rings, which give rise to the proximal coxa andtrochanter segments, stain the faintest, and staining intensityincreases progressively towards the center of the discs, thendecreases sharply in the very middle (Fig. 5A). Similar stainingpatterns can also be observed in the antennal imaginal discs.We propose that the distortions and segment fusions weobserve in the appendages of surviving mutant adults arecaused by defective cell movements during disc evagination.Since most homozygous mutants die as larvae, these defectsmay only represent mild external defects that are paralleled bysimilar but difficult to detect internal defects. Given the rangeof tissues in which 18w is expressed, we propose that the larvaedie due to an accumulation of relatively subtle defects duringmorphogenetic movements.

Comparison of 18w and TollAlthough Toll is best known for its maternal role in dorsal-ventral pattern formation, it has a significant zygotic role aswell (Gerttula et al., 1988; Ip et al., 1993). The 18w and Tollgenes share striking features with respect to zygotic expression

patterns, message size, overall protein structure, cell adhesionproperties and mutant phenotypes. A comparison of theexpression patterns of 18w and Toll (Gerttula et al., 1988;Hashimoto et al., 1991) indicates that the genes are coex-pressed in many tissues: the tracheal placodes, some midlinecells, the developing salivary glands, the epidermis at the inter-segmental furrows, the pharynx, the cells that will form thedorsal vessel, the Malpighian tubules, and the hindgut.Although the expression patterns show differences, the genesare frequently coexpressed in regions undergoing extensivecell movement (Gerttula et al., 1988).

At the molecular level, both genes encode a single messageof approximately the same size: 5.6 kb (18w) versus 5.4 kb(Toll) (Hashimoto et al., 1988). Both proteins share similarmotifs throughout most of the protein (Figs 6 and 7), includingthe IL-1R homology in the cytoplasmic domain (Schneider etal., 1991). Interestingly, both proteins behave as heterophiliccell adhesion molecules when expressed in cultured Schneider2 cells (Keith and Gay, 1990). This implies that they must beable to interact with one or more proteins that are constitutivelyexpressed by Schneider 2 cells.

Mutations in both genes cause similar zygotic defects. Mosthomozygous Toll progeny of a Toll /+ × Toll /+ cross (Toll =amorphic or hypomorphic allele) die as first, second or thirdinstar larvae with no obvious phenotype except occasionaltracheal tree defects (Gerttula et al., 1988). Since Toll and 18wshare so many features and homozygous Toll mutant femalesare sterile, we tested the fertility of homozygous 18w mutantfemales that carry severe loss of function or null alleles (∆7-35 and ∆1-82). Although most homozygous mutant femalesdie, a few females produced offspring indicating that, unlikeToll, maternally derived 18w is not essential for the develop-ment of the embryo. Similar conclusions were derived byproducing germ line clones in females using the dominantfemale sterile technique (Perrimon et al., 1984). Few irradiatedmosaic 18w/Fs(2)D females lay eggs that do not contain mater-nally produced 18w transcript or protein. Most of their progenydie when homozygous for 18w, but some that carry a pater-nally derived wild-type copy of 18w survive (data not shown).We conclude that 18w is probably not required in the femalegerm line but that the zygotic transcript is required for properdevelopment. However, it is possible that the 18w mutants arenot complete loss of function mutations as some reduced levelsof severely truncated transcripts are still produced in thesemutants.

The similarities between Toll and 18w prompted us to inves-tigate whether 18w is a dominant enhancer of Toll/Toll orwhether Toll is a dominant enhancer of 18w/18w. Doublemutant strains were constructed, and their embryos werestained with antibodies that mark specific tissues in which 18wis expressed. We were unable to pinpoint consistent defects indouble mutant embryos. There were no obvious genetic inter-actions between various mutant alleles of 18w and Toll asassayed by an increase or decrease in the number of 18w/18wor Toll/Toll flies suriving in the presence of a single mutantcopy of the other gene. However, flies that lack zygotic Tolland have only one copy of 18w (PZ/+; Tl9QRE/Df) had a highincidence of melanotic tumors which were not observed inother flies derived from the same cross (data not shown). Thisis particularly interesting since melanotic tumors are associatedwith cellular immune responses, and the IL-1R domain of 18w

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may play a role in this response. Indeed, some recent obser-vations indicate that upon infection with bacteria or othercompounds, a protein related to Dorsal and NF-kB, namedDIF, is translocated from the cytolplasm to the nuclei of fatbody cells (Engström et al., 1993; Ip et al., 1993; for reviewsee Hultmark, 1993). Once in the nucleus, DIF may induce orpromote transcription of Drosophila immune response genes,the cecropins. It will be interesting to determine whether 18Wplays a role in this response.

The similarities in structure, expression pattern, and zygoticphenotype between Toll and 18w are remarkable since bothproteins show less than 30% identity. It is tempting to speculatethat the similarities are not restricted to these structural andfunctional features only, but that both proteins are alsoactivated by a ligand which will trigger intracellular responsesusing a cascade of protein interactions similar to the one thatis downstream of Toll in dorsoventral pattern formation (forreview see Chasan and Anderson, 1993) or in the immunesystem (Ip et al., 1993).

In this work we describe the isolation and characterizationof a novel gene named 18 wheeler (18w). 18w encodes a trans-membrane protein with an extracellular domain and an intra-cellular moiety. Cell aggregation assays using Schneider 2cells show that 18W can act as a heterophilic cell adhesionmolecule. Genetic and phenotypic analyses show that 18W isrequired for larval viability, adult appendage formation andadult viability. The 18w gene is often expressed in tissues thatparticipate in extensive cell movements, and the morphologi-cal defects correlate with some of the domains of 18wexpression. We propose that 18W is required for proper cellmotility and that it carries out part of the developmentalprogram specified by segmentation and homeotic genes as acell adhesion or a membrane receptor for cell signaling.

We thank Allan Spradling, Steve Kerridge, and Cahir O’Kane forsending us the P-element enhancer detector strains, Nathalie De Santisfor sequencing portions of the 18w cDNAs, Michael Edwards forproducing antibodies, the core sequencing facility of the Institute forMolecular Genetics for sequencing, and the Indiana Stock Center andKathy Matthews for sending us so many fly stocks promptly. Wethank Randy Smith for sequence analysis and Michael Williams forfurther molecular analysis of excision mutants. We are grateful to AdiSalzberg, Karen Schulze, and Troy Littleton for comments, and JudiColeman for secretarial support. P. L. and J. B. were supported by agrant from the American Cancer Society. E. E. was supported by theHoward Hughes Medical Institute. H. B. is an assistant investigatorof the Howard Hughes Medical Institute.

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(Accepted 3 January 1994)