Cell, Vol. 67, 853-868,

November

29, 1991, Copyright

0 1991 by Cell Press

The fat Tumor Suppressor Gene in Drosophila Encodes a Novel Member of the Cadherin Gene Superfamily Paul A. Mahoney,* Ursula Weber,’ Patricia Onofrechuk,t Harald Biessmann,t Peter J. Bryant,? and Corey S. Goodman* *Howard Hughes Medical Institute Department of Molecular and Cell Biology University of California Berkeley, California 94720 tDevelopmental Biology Center University of California Irvine, California 92717

Summary Recessive lethal mutations in the fat locus of Drosophila cause hyperplastic, tumor-like overgrowth of larval imaginal discs, defects in differentiation and morphogenesis, and death during the pupal stage. Clones of mutant cells induced by mitotic recombination demonstrate that the overgrowth phenotype is cell autonomous. Here we show that the fat locus encodes a novel member of the cadherin gene superfamily: an enormous transmembrane protein of over 5000 amino acids with a putative signal sequence, 34 tandem cadherin domains, four EGF-like repeats, a transmembrane domain, and a novel cytoplasmic domain. Two recessive lethal alleles contain alterations in the fat coding sequence, and the dominant fat allele, Gull, contains an insertion of a transposable element in the 33rd cadherin domain. Thus, this novel member of the cadherin gene superfamily functions as a tumor suppressor gene and is required for correct morpho#genesis. Introduction Cadherins are afamilyof calcium-dependent cell adhesion molecules that can mediate cell aggregation and cell sorting in vitro in a homophilic fashion (Nagafuchi et al., 1987; Edelman et al., 1987; Hatta et al., 1988; Nose et al., 1988; Mege et al., 1988; Miyatani et al., 1989). Thus far, cadherins have been found only in vertebrates. Changes in cadherin expression in vivo have frequently been correlated with morphogenetic events that appear to require changes in the adhesive properties and specificities of the cells involved (Hatta and Takeichi, 1988; Nose and Takeichi, 1986; Hatta et al., 1987). These observations have led to the hypothesis that cadherin-mediated intercellular adhesion allows the separation and/or joining of cells during development via changes in the expression of different subclasses of cadherins on the surfaces of cells (for reviews see Takeichi, 1987, 1988, 1990). Misexpression of N-cadherin in Xenopus embryos causes morphological defects in the formation of the neural tube (Detrick et al., 1990; Fujimori et al., 1990) supporting the idea that cadherins play a critical role in morphogenesis ‘by mediating selective cell-cell adhesion.

Although these “gain-of-function” experiments have provided important insights into cadherin function, a challenging problem has been to determine the normal function of cadherins during in vivo development by carrying out “loss-of-function” experiments, thus revealing the direct relationship between cadherin expression and morphogenesis. We have sought to isolate cadherins in the fruit fly Drosophila, and to identify the corresponding genetic loci, as a way of using genetic analysis to test directly the roles of cadherins in vivo. Over the past few years, the Drosophila homologs of several well-characterized vertebrate cell and substrate adhesion molecules have been cloned, suggesting that many of the adhesion molecules involved in tissue morphogenesis evolved long ago, before the spllit of the phyletic lines leading to the arthropods and chordates. Mutations have been identified in many of thesle Drosophila genes, including one of the a subunits and the j3 subunit of the PS integrins (MacKrell et al., 1988; ILeptin et al., 1989; Brower and Jaffe, 1989; Zusman et al., 1990), the a subunit of laminin (Monte11 and Goodman, 1988; C. Henchcliffe, L. Garcia-Alonso, and C. S. G., unpublished data), three immunoglobulin superfamily cell adhesion molecules (neuroglian [Bieber et al., 19891, fasciclin II [Grenningloh et al., 1990, 19911, fasciclin Ill [Snow et al., 1989; Grenningloh et al., 1990; T. Elkins, D. F’erres-Marco, and C. S. G., unpublished data]), and two novel cell adhesion molecules (chaoptin [Van Vactor et al., 19881 and fasciclin I [Elkins et al., 19901). We ‘report here on the cloning of the first Drosophila member of the cadherin gene superfamily, using the polymerase chain reaction (PCR) method (Saiki et al., 1988). Moletiular genetic analysis reveals that this oadherin-like protein is encoded by the fat gene. The fat locus is one of seven known Drosophila tumor suppressor genes, identified as,such because recessive (loss-of-function) mutations in each gene lead to excessive cell proiife’ration in the’imaginal discs or other tissues (Bryant, 1987). Mutations at two loci; lethal(2)giant larvae (/g/) (Gateff and Schneiderman, 1974; ‘Mechler et al., 1985; Klambt’et al., 1989; ‘Merz et al., 1990) and lethal(l)di largei #(+f/g)(Stewart et al., 1972; Murphy,, 1974; Woods and Bryant,‘1 989), lead to a neoplastic overgrowth phenotype,in which the single-layered epithelial structure of the imaginali’discs is lost, and in which the cells jose the ability to differenbate even after transplantation linto’wild-type hosts; both of these genes have’been cloned (Mechler et al., 1985~; Klambt et’aj., 1989; Woods’and Bryant, 1989, 1991). ~M’utatio’hs in fibe other ~eties,l/etha/(2)fat (fi) (Bryant andS$!ibigef, 19ilj,~eetha/(2)gi~~ta~~c~(/gd)(Bryant and Schubiger,, ‘1971; Br&ant #and Levinson, 198!5), ‘tumorous discs (&) (Gateff andi Mechler, ‘i989)) /et&3,)c43 (~43) (Martinet al., 1977) ‘ahd $x?s overgrown (oco)‘(Jursnich et al:,, 1990)~,,lead to hyperpiastic/‘overgrowth of the imaginal,djsr?$ these disks retain theirsingle-layered epithelial struqtur,e, and itheirabi,tity to diff&ehtiate fotlowing transplantation sinto’lwild$pe larval hosts.

Cell 854

In this paper we report the first molecular analysis of a Drosophila gene in the hyperplastic class, the fat locus. We show that the fat gene encodes an enormous transmembrane protein of over 5000 amino acids with a signal sequence, 34 tandem cadherin. domains, four EGF-like repeats, an additional cysteine-rich domain, a putative transmembrane domain, and a novel cytoplasmic domain. Recessive lethal mutations in the fat gene lead to tumorlike growth of imaginal discs (Bryant et al., 1988), whereas recessive viable alleles lead to changes in body shape and wing vein pattern (Mohr, 1923, 1929). The results presented here show that a member of the cadherin gene superfamily functions in the control of cell proliferation by acting as a tumor suppressor gene. Results Cloning of a Cadherin-like Gene at Chromosomal Interval 24D When this study began, the sequences of three different vertebrate cadherins (E-cadherin [homologous to L-CAM and uvomorulin], P-cadherin, and N-cadherin) were available in both the same and different vertebrate species (Nagafuchi et al., 1987; Gallin et al., 1987; Nose et al., 1987; Hatta et al., 1988). The mature forms of these three vertebrate cadherins share many features in common, including four extracellular domains of about 100 amino acids, which have high sequence similarity to one another and which we call “cadherin domains,” a fifth extracellular cysteine-rich domain, a transmembrane domain, and a cytoplasmic domain. The cytoplasmic domain is the most highly conserved region of the vertebrate cadherins, containing several long stretches of amino acids that are identical in sequence among the different cadherin molecules and different species. We initially tried and failed to isolate Drosophila cadherins using oligonucleotide probes representing these highly conserved regions of the cytoplasmic domain. We then turned to the four extracellular cadherin domains, although in these domains one rarely finds more than two or three conserved amino acids in a row among the different cadherin molecules and different species. We designed degenerate PCR primers (20-mer oligonucleotides with up to 256-fold degeneracy) that would amplify one entire cadherin domain, and tested which members of these pools of primers would amplify the appropriately sized fragment from Drosophilagenomic DNA (see Experimental Procedures). One set of primers produced amplified products of the appropriate size that, when subcloned and sequenced, were shown to encode two different cadherin domains. Both cadherin domain clones contained most of the highly conserved residues that are found in the vertebrate, cadherin domains, while the variable residues showed that the two domains are clearly different from one another. We probed a Drosophila cDNA library constructed from 9-12 hr embryos (Zinn et al., 1988) with one of the two PC&lified cadherin domains and isolated a corresponding cDNA clone, which we then mapped by in situ hybridization to region 24D on the left arm of the second

chromosome. The gene encoding this cDNA is the subject of this report. A cDNA corresponding to the second PCRamplified cadherin domain was subsequently isolated and mapped to polytene chromosomal region 21D; it corresponds to a different Drosophila gene (H. Clark, P. A. M., and C. S. G., unpublished data). cDNA Sequence Encodes an Enormous Transmembrane Protein The first of several unusual features concerning this Drosophila gene product is its enormous size; the predicted protein is much larger than the three vertebrate cadherins, which typically have mRNAs of 3 to 4.5 kb encoding proteins of less than 1000 amino acids in length. The initial cDNA isolated from the 9-12 hr embryonic cDNA library was 5 kb in length. When this clone was used to probe a Northern blot containing RNA from various developmental stages, only a single large transcript approximately 15-20 kb in length was detected (data not shown), thus indicating that a single, full-length cDNA would be virtually impossible to obtain. Following the sequencing of the,initial cDNA, fragments from both the 5’ and 3’ ends were used to rescreen the 9-12 hr embryonic cDNA library as well as additional cDNA and genomic libraries (see Experimental Procedures). Through several reiterative rounds of sequencing, mapping, and rescreening, ultimately a series of overlapping clones was obtained that spanned the full lengthofthegreaterthan 15 kbopen readingframe(Figure 1). The complete 15,441 bp open reading frame, as deduced from the sequence of these overlapping clones, encodes a predicted protein of 5147 amino acids. The sequence of this deduced protein is shown in Figure 1. Another difference between the known vertebrate cadherins and the Drosophila sequence is the structure of ,the N-terminal region of the predicted ,proteins. In the Drpsophilasequence, the methionine isfollowed,byaputative signal sequence. The first cadherin domain begins immediately following the signal sequence. In contrast, the three vertebrate cadherins (E-, P-, and N-) contain, after the signal sequence, an initial stretch of about 100 amino acids (followed by dibasic residues) that are proteolytically cleaved from the N-terminus of #the mature iprotein. Two other differences, as described below, include the pr,esence of EGF-like repeats’in the Drosophila sequence and the absence of any homology between the cytoplasmic domain of the Drosophila! protein and the conserved cytoplasmic domains of the known vertebrate cadherins’(Hatta et al., 1988). The most striking difference between this Drosophila sequence and the vertebrate cadherins is the number of cadherin domains. In contrast to the four tandem domains in the vertebrate cadherins, this Drosophila gene encodes 34 contiguous cadherin domains, all of which;contain most if not all of the amino acids that are,the most highly conserved among the vertebrate cadherin domains (Figures 2A and 3B), includin,g both of ,the, proposed Ca2+:binding sites (Ringwald et al., 1987; Ozawa et al., 1990). In addition, the first Drosophilacadherin domain lacksthe several amino acids (HAV) that are uniquely found in the first cadherin domain in the vertebrate E-, P-, and N-cadherins

The fat Tumor 855

Suppressor

Gene

of Drosophila

L cad1 MERLLLLFFLLLAGRESLCcTGc~KtEttAPRGRs”~~~~~~”AAFPRRRsssssQsGEMDsRA”D~sAcFEv~EG~PRG~T”GFIPTKPKFs~RFNEPPREF~~cQviGE”KTN”“~cR 120 EGMRcHYDL”“L**aPTYPIE”R~K”~c”N~N~~~~P~Qs~A,sFsEsATsGTR~L~cAATc*c”GENGvTDaYEI”AGN”DNK~R~“TiANPsGcTs”tNtETTGN~cREsRGs”~~N~ 240 SARDGGSPPRFGYLOvNVTILD~N~~~~~FDHSDYNVSLNETAtPGTPVVTVMAS~N~tGDNSKlTYYLAET~HOFTvNPETGv~STTERVNCpOQTNVKSSASQKsCvFTvFAR~HGSP

360

RODGRTYVTVNLLOTNOHOPIISFRFFPDGGKVATVOENAVNGTVVAAVAVKDSDSGtNGRTSVRIVSGNE~GHFRtEEAA~LHlVRVNGV~~REEIGKYN~TVVAM~OGTQARTTTAH~ _ cad5 IIOVNOVNDHEPVF~KSEYTDEDTGVNAQVHYDILSGNELKWFSMDPLTGLiVTTGQ~~REIROIVELSISARDGGPNPKFAYTOLKVl~L~~~~EAPQFs cad6 OREONVTLGEOAPpOTIVALMTATOHDPGTNGSVTFA~APSVER~~P~~~A~~A~T~~~TTRRP~~~~K~S~vE~PV~AR~~GAPTP~SATATV~LNVA~~N~~~~~F"P~~"~"S~T~~

600

OOOIKLKKEVEKERILLHVTASDKODGDNALIEYRLESGGEGLF~tDARSGA~SLRGDAPASM~WKPHYKLLVSARDAGQRRSQQDAlV~lVLKSKtEM~EC~~~~~~GvEF~MVEDHEO

840

480

720

ORNS~PNREVGIVOVKSTNGKANs~IEYOIIOGDRAQNFRIDTRSGR~STARP~DRE~QANYRLTlLASSsSSSSAAASSVSvGQC~VN~~~~~~~NAPVFALDREsEPTIS~P~NAAV

960

GO~IYiSRVRORDAGVNSRISYSLTNNPNOOFRIGPVTGVLYL~RPIRA~PGStlHVELMAT~AGSPPLSSKt~t~~~~~DVNDUTPVFDHTSYETStPETTKVNTRFFALAATDlD~G~

1080

NGRISYEIIEGNTERMFGVFPDGYLFVRAPLDREERDYYALTVSCRDAGQPSRSSVVPVVlHVl~EN~NAP~FTNSTF~F~~~~~APAQTFVGKtTAVDR~lGRNAELSFT~SsQTQDFT " " IOTRNGFlKTLRPFDREALVKVSRNAEASGEOGSLRGSMAGNYMttEATVSDNGIPRL~~KVKVKVlVTDVNDNAPEFtRAPYHVT~SEGASEGTHlMHVFT~DADEG~NGDVYYSLAKG I cad13 NEAGoFNLOSATG~LSLARRLDRESOEIHHLIVVAK~AA~K~PtsTNAS~T~VV~~EN~NAP~FT~SSSEVsV~ETSPTGT~~~RFRGS~A~~GV~s~VVFS~SAGNR~~TFH~~S~TGs 1 Cddl4 LYLHKPLDYEOITSYTLNlTASOCGTPSLSTTVLVVDDNDNPPIFPSTA~VR~lKEGIP~KTP~VTVTAD~Q~SGtNGKVsYAISK~EP~~P~GRHFGINTETGVIHT~REl~RES I cad15 IDTFRLTVV'ATDRA(IPSER[ILSTEKLVTVIVEOINDNAPVFVSMNAAILPPKFSTSKGSSTAVMOVHAKDADSSSNGtVTYEIVSGPOE~FKL~RNTGIlTFTPGP~FKQEVRYOtTLKS I cad16 TOEAV(lSERRSSEVYITIITPGSGESESSVP~FEURSKtSGSVYENEPIGTSILTVTAHtASAEIEYFVTNVTAVGSRG~V~R~FDlDAKtGlLSTAAEL~REAGPEEvEVEVYAlALGG cad17 OPRTSRTKVRVTVLOKNDSPPOFiOTPFVYNVSEOLllIGHT~ST~RAHDP~T~GSVTFttMOGH~GKFttEPSTGKLILNDT~~RETKSKiELRlRVSDGVOvTEAvATl~VSD~NDNPP I cad18 LFEDTVYSFDIPEN~ORGYOVGOIVA.ROAOLGIINA~tSvGVVSDWANDVFS~NPQTGMLT~TARtDvEEV~HY~~lVQAQ~NGOPSLSTTITVYCNVLDLNDNAPlFDPMSYSS~VFENV

PHAYDGFESSFRGSLSTLVA~~~~~ANWLSGIYRKANGAA~PSATT~G~E"~~N~GPS~~N~~GVFK~~AE~P~TNGP~~~~~~~T~VVST~R~PSSNGP~APEE~V

Figure

1. Open Readidg

Frame

Encoded

1200 1320 1440 1560 1680 1800 1920 L cad19 2040

5147

by the fat Gene

Overlapping cDNq and genomic clones were isolated and sequenced to produce a 15,441 bp open reading frame, which encodes the 5147 amino acids shown here.‘The: butative bignal sequence (aminb acids 4-35) and transmembrane domain (amino acids 4564-4609) are underlined. The first amino acid of the 54 cgdherin domains (cadl-cad34) and four EGF-like (egfl-egf4) repeats are delimited by vertical arrows. Arrowheads indicate two amino acids that ark polymorphic: amino acids 1229 and 1233 encode glycine and serine, respectively, in a cDNA clone, and serilne and glycine, respectively, in thl? cortesponding genomic clone. The complete nucleotide sequence can be found in the GenSank data base. portions of the sequence encodi$J thk following amino acids were obtained from clones isolated from the indicated libraries (see Experimental flrocedures) as fOlloWS. Eye disc cDN/$! 106-310,1199-2511. Embryonic cDNA (8-21 hr): 157-521,371 l-4300. Embryonic cDNA (g-12): 2332-39E17,4270-5146. Genomic DNA: ‘I-;9421 ~486-1278.

and are thouglt to’be importa?t in the function of these molecules (Nose e/ al., 1990). Recently, theitwo~genes encoding three vertebrate desmosomal glycdprotelns (DGI, DG2, DG3) have been cloned and shqwn ‘to encode members of the cadherin superfamily (Gdodwin et al., 1990; Collins et al., 1991; Wheeler et al., 4991; Mechanic et al., 1991; Parker et al.,

1991). DGl also does not contain an N-terminal preprotein domain that is proteolytically cleaved, and moreover, does not contain the HAV sequence, but rather has the amino acids RAL in this position. Furthermore, the DG2 and DG3 proteins (alternative products of one gene) also do not contain the HAV sequence but rather have the amino acids YAT in this position. (The first cadherin domain encoded

Cell 856

VERTEBRATE

CADHERIN

DROSOPHILA

fat

EGF

Figure

DOMAINS

CADHERIN

DOMAINS

REPEATS

2. The Cadherin

Domains

and EGF

Repeats

of the Predicted

fat Gene

Product

cadherin domains. The 34 cadherin domains encoded by the fat gene (A) Homology between the predicted fat cadherin domains and vertebrate are shown aligned beneath several representative cadherin domains reproduced from the published vertebrate sequences. Nl and N2 represent the first and second cadherin domains of chick N-cadherin, respectively (Hatta et al., 1988). E2 and P2 represent the second cadherin domains found in mouse E-cadherin and mouse P-cadherin, respectively (Nagafuchi et al., 1987; Nose et al., 1987). The 34 Drosophila domains represented by Fl-F34 are contiguous, tandem cadherin domains encoded by the fat gene. To illustrate the extent of the homology among all of the fatcadherin domains, as well as between the fat and vertebrate cadherin domains, some of the most highly conserved amino acid residues are shown in reverse color. (B) Homology between the predicted fat EGF-like repeats and the EGF-like repeats found in other molecules involved in cell interactions. FATlFAT4 indicate the four contiguous EFG-like repeats that are encoded by the fat gene. Aligned below them are the first of 36 EGF-like repeats found in the Drosophila Notch predicted protein (NOTCH) (Wharton et al., 1985), the first of four EGF-like repeats found in the Drosophila Delta predicted protein (DELTA) (Knust et al., 1987), the first of 10 and of 13 EGF-like repeats found in the Caenorhabditis elegans /in-72 and g/p-l predicted proteins (LIN-12 and GLP-I, respectively) (Yochem et al., 1988; Yochem and Greenwald, 1989) and the EGF-like repeat of human endothelial leukocyte adhesion molecule 1 (ELAM-1) (Bevilacqua et al., 1989). The conserved cysteines are shown in reverse color. Numbers on the right side refer to the portions of the proteins shown here.

by the fat gene also contains the amino acids YAT but in adifferent location within thedomain, making its functional significance unclear.) Thus, the Drosophila protein described here is not the only member of the family to differ from the vertebrate E-, P-, and N-cadherins in these respects. Following the 34 cadherin domains of the Drosophila sequence, the homology to the vertebrate sequences comes to an end. There is a region of 160 amino acids that does not appear homologous to any part of the vertebrate cadherins or to anything else in the data banks. This novel sequence is followed by a region that encodes four EGFlike repeats (Figures 1,2B, and 3B), similar to those found in such cell interaction molecules as the Notch (Wharton et al., 1985), De/ta.(Knust et al., 1987) /in-72 (Yochem et al., 1988), g/p-l (Yochem and Greenwald, 1989; Austin and Kimble, 1989) and ELAM-1 (Bevilacqua et al., 1989)

gene products. Such EGF-like repeats are not found in the known vertebrate cadherins. Following the four EGF repeats is a region of about 250 amino acids, which includes 12 cysteines. Several cysteine-rich stretches do not follow the precise EGF repeat consensus, although one stretch of six cysteines (from amino acids 4325 to 4365) does have many features suggesting that it might be a more divergent EGF-like repeat. The fifth extracellular domain of the vertebrate sequences also contains four conserved cysteines. Whether any of these cysteine-rich sequences are similar in origin or func-, tion is unknown. In the Drosophila sequence, this cysteine-rich region is followed by about 200 amino acids, which contain only two cysteines. The Drosophila cDNA sequence then encodes a 26amino-acid hydrophobic putative transmembrane domain, followed by a cytoplasmic domain of 539 amino acids. The

The far Tumor

Suppressor

Gene of Drosophila

857

ss 1

putative cytoplasmic domain shows no homology to the cytoplasmic domains of the vertebrate cadherins, nor to any other sequence in the available data banks. The total length of the deduced Drosophila cDNA open reading frame is 15,441 bp, which after cleavage iof the signal sequence would encode a mature protein of 5112 amino acids, with a predicted nonglycosylated hrl, of around 560,000 daltons.

A SS

hl

1

34CADHERlN

DOMAINS

~44

4 4

B

412

c

faf cDNA CGGTCM&

1

_ _ _ 1 -412 LTR CAT ;GTAGTATGTGCCTATGCAATATTAAGAACAATTAAATAAAAi%

CATATTAAC...GATCAaCCACTCGAAGGCcAcAAAGTATAAGTGCATtGCCCACTCGAA 132 164 GGCAAAAAGTATAAGTGCATGGTC 196

5

E

faf cDNA 412 LTR 1 AGTTCATCATTACA ,&TGTGCGCACACAAAAT

10076 CAG CAA TTG GAT TAT G a 0 L D Y 3294

CTC ATA CAG GAA TAC L ID E Y

T TTG AAT H L T

Expression and Larval

Single-stranded 35S-labeled antisense RNA probes were synthesized from a 2 kb cDNA subclone and hybridized to paraffin sections of Drosophila embryos and larvae. Sense strand probes were used as controls and showed no specific hybridization. No hybridization to RNA was detected in sections of early cleavage stage or blastoderm embryos, nor in sections of embryos in the early stage6 of gastrulation. Hybridization to RNA was first detected in stage 7 embryos (staging is according to Campos-Ortega and Hartenstein [1985]), at the end of gastrulation and the be-

pression

SUBSTITUTION

Figure 3. Comparison of a Typical Vertebrate Cadherin with the Predicted Products of the Normal fat Gene and the fat Mutant Alleles (A) Vertebrate cadherin genes encode a signal sequence and preprotein domain (shown in solid black and horizontal lines, respectively), which are cleaved off and not found in the mature molecule. Four cadherin domains (shown’ lightly stippled) are followed by a fifth extracellular domain (showri with heavy diagonal lines), a transmembrane domain, and a cytoplasmic domain (shown with black squares). SS, signal sequence; TM, transmembrane domain. (6) Key features of the predicted gene product of the fat locus include the signal sequence (shown iv black), 34 cadherin domains (shown lightly stippled), four EGF repeats (shown with diagonal lines), and a large cytoplasmic domairl (shown tiled) that is not homologous to the vertebrate cytoplasmic’domain. ‘The position of the 412 transposon found in the coding sequence of the fat gene isolated from Gull mutant flies is shown by a triangle. The border between the fat coding sequence and the 5’ LTR of the 412 transposon is shown in detail in (C). The positions of the alterations found in the fat coding sequence of the ft”’ and W4 alleles are shown ,by open arrows, and are described in more detail in (E) and (F) below. (C) The fat open reading fraye: is interrupted at bp 10,898, which encodes amino acid 3670 in Figure 1 (the methionine at amino acid

position 1 occurs at bl, 193) in Gull hutants. The 5’ LTR of the element begins(bp 1)followingthree additional base pairs (delimited byarrows) that are not normally encoded by either the fat gene or the 412 transpobon. Stop codons that exist’in all’three reading frames of the LTR are indicated by overlines’. ‘An extda ~33 bp, present as a tandem, direct

ippeat (bp 132-164) nbt usually f&d in 412 LTRs, is shown underlined; lowercase letters in this region indicate bases that differ from the normal LTR sequence (bp ~.165-196), which immediately follows this small insertion.

Alsd missing

a& 11 bp of fat coding

sequence

(see

text). (D) The 3’end

of the GM/ bieakpoint

caused

by the insertion of the 412 the fatopen reading beginning

following

is largely

maintained,

although

the level of mRNA

increases steadily until it peaks in stage 13 (approximately 10 hr) embryos toward the end of germband retraction (Figures 4A, 4C, and 5). By this time, the mRNA is also expressed at high levels in the pharyngeal region of the foregut, as well as in the cells of the hindgut, both ectodermally derived structures; the mRNA is not detected in the embryonic central nervous system (Figure Ei). Following stage 13, the level of mRNA begins to decline until it is no longer detectable toward the end of embryogenesis. The mRNA reappears in the larva, and is restricted to the imaginal discs, although a small set of neurons in the optic lobes also express the mRNA. In third instar larvae, although no expression of mRNA is detected in the ep;idermis, all of the imaginal discs appear to express the mRNA uniformly at high levels (see Figures 4B and 4D). The Cadherin-like Protein Is Encoded by the faf Gene The region of the second chromosome to which the cDNA subclone encoding cadherin domains hybridized, 24D, contains one gene in particular of which different mutant alleles display a range of phenotypes that perturb imaginal disc development in a way that might be expected from defects in cell adhesion. Recessive lethal mutations in fat, which map to region 24D5.6-7, cause a hyperplastic, tumor-like overgrowth of the imaginal discs (Bryant et al., 1988), whereas recessive viable alleles cause minor de-

bp 10,076,

changes

element isshown byali arrow. The DNAencoding fiame resumes at bp 10,910. (E)‘The fP mutation cotisists of a 1’7 bp deletion,

Ectoderm

:ginning of germband elongation. The transcript is found exclusively in the surface ectoderm and somle other ectodermal derivatives, and is absent from the mesoderm and endoderm. As development proceeds, this pattern of ex-

f/ h’DELETION

ftsz44

of mRNA in Embryonic lmaginal Discs

which

encodes

the translational

(F) The fW bp insertion. amino acid boxed under

amino

reading

acid

3295

(shown1

boxed),

that

frame of the fal transcript.

mutation consists of a 31 bp deletion, accompanied by a 14 The deletion begins following bp 10,675, which encodes 3495, and when combined with the inserted DNA (shown the deleted region), shifts the translational rlBadinQ frame.

Cell 858

Figure

4. Expression

of fat RNA in the Drosophila

Embryo

and Larva

%-labeled single-stranded antisense RNA transcribed from a 2 kb cDNA fat subclone (corresponding to amino acids 22653013) was hybridized to sections of Drosophila embryos and third instar larvae. Following autoradiography, bright field and corresponding dark field micrographs of ([A] and [Cl) a parasaggital section of an embryo during germband shortening illustrate the expression of fat RNA in the surface ectoderm (E), in the cells of the pharyngeal region of the foregut(F), and in the hindgut (H) of the embryo. ([B] and ID]) Parasaggital section through the anterior region of a third instar larvademonstrates hybridization of the probe to the larval imaginal discs. The probe also hybridized to the genital disc (not shown). Note that the larval epidermis (E) does not express fat RNA. 8, brain.

fects in adult morphology (Mohr, 1923, 1929). We verified that the cDNA hybridizes to the cytogenetic interval containing fat by showing hybridization to the Df(2L)M-zB deficiency chromosome but not to the Df(2L)Mll deficiency chromosome (data not shown), thus localizing the gene to a small, genetically well-characterized region that contains the fat locus as well as four other known genetic complementation groups (Bryant et al., 1988). We first cloned and mapped about 60 kb of genomic DNA containing the 15.4 kb open reading frame by chromosome walking. Our preliminary studies of intronlexon boundaries indicate that there are very few introns in the genomic DNA corresponding to this cDNA; furthermore, the coding region is contained within only 17.5 kb of genomic DNA, indicating that most of the sequence is in exons (data not shown). Genomic DNA was then prepared from 22 different fat alleles and examined by probing blots of genomic DNA (using 6-nucleotide recognition restriction enzymes) with labeled fragments from the cDNA. This experiment revealed that two fat alleles, Gull (G, a spontaneous dominant allele of fat) and Gu/~ (G”, the spontaneous revertant of Gull that acts as a strong recessive fat allele), exhibit distinct restriction fragment length alterations (using four different restriction enzymes) that are different from wild type and from each other. Genomic DNA from the Gull and G” fly strains (each mutantmis maintained as a viable heterozygote over a bal-

ancer chromosome) was purified and used to construct Gull and G” genomic libraries, which were then probed with portions of the cDNA in order to obtain the corresponding genomic DNA clones. Genomic clones were obtained with both the expected wild-type and mutant patterns of restriction fragments. Portions of the Gull and G” genomic clones that exhibited the same mutant patterns of restriction fragments as was seen on the Southern blots were sequenced in order to determine the nature of the alterations in the mutant alleles. The spontaneous Gull allele (Mohr, 1923, 1929) was found to have a complete 7 kb 412 transposable element (Finnegan et al., 1978; Will et al., 1981) inserted in the 15.4 kb open reading frame of the cDNAsequence. The point of insertion of this transposon is in the 33rd cadherin domain (see Figure 3B). The 15.4 kb open reading frame is interrupted by stop codons that exist in all three reading frames of the 412 transposon long terminal repeat (LTR) at the 5’ end of insertion (see Figure 3C). Thus, translation of the mRNA encoding the predicted extracellular cadherin domains is probably terminated prematurely in the Gull mutant. Several differences exist between the structure of the 412 transposon inserted in Gull and previously described 412 insertions (Shepherd and Finnegan, 1984): First, the 5’ LTR of the 412 transposon in Gulldiffers from the normal structure in that it contains an additional 33 bp, of which

The fat Tumor 859

Figure

Suppressor

5. Expression

Gene

of Drosophila

of fat RNA in the Ectoderm

of Drosophila

Embryos

Bright field and dark field micrographs ([A] and [B], respectively) of a slightly oblique transverse section through retraction show that fat RNA is expressed in the surface ectoderm of the embryo, but not in the gut (G), somatic central nervous system (N). Section was treated as in Figure 4.

29 bp are a direct repeat of the LTR sequence. Second, there is a 3 bp insertion (CAT, Figure 3C) between the fat cDNA sequence and the S’end of the 412 LTR. Third, there is a loss of 11 bp of fat DNA at the site of insertion (see Figures 3C and 3D). More typically, 412 insertionis are characterized by the duplication of 4 bp of host DNA at the site of insertion, with the 412 transposon inserted between these duplicated bases (Will et al., 1981). The corresponding region of the wild-type gene has been sequenced to confirm that there is not an intron at the location of the insert and thus that the 412 transposon interrupts the open reading frame. A similar analysis of genomic clones derived from the G” library demonstrated that the 412 transposon described above is still present in the same location in the coding sequence. Detailed Southern analysis, using a variety of different genomic and cDNA probes that span the entire fatopen reading frame, indicates that the G”DNA contains an additional rearrangement (either an insertion or inversion) approximately 2 kb 5’to the site of the 412 transposon insertion, which would place this breakpoint in the middle of the cadherin domains. While the precise nature of this chromosomal rearrangement in the fat gene in G” is complex and not yet fully characterized, it appears likely that the 15.4 kb open reading frame jssufficiently altered in G”

an embryo following germband (S) or visceral (V) mesoderm, or

and that this allele represents a true null mutation of the fat gene. We have also prepared genomic DNA blots, using 4-nucleotide recognition restriction enzymes, from 15 of the fat mutant stocks. Using this analysis, we found allelespecific changes in the restriction fragment pattern of three recessive lethal fat alleles, f?‘, f?Z44,and VzB’, which correspond to alterations in the region encoding the extracellular portion of the predicted gene product (data not shown). Using primers that were homologous to the fat DNA sequence in the region containing the altered restriction fragments, we utilized PCR to amplify the affected region of the genomic DNA from balanced stocks carrying thefth’ and ft”‘44 mutant chromosomes. Sequencing of multiple clones obtained following the amplification procedure revealed alterations in the fat coding sequence of both the fth’ and ft”‘@ alleles. The fth’ allele contains a 1’7 bp deletion in the 30th cadherin domain (see Figure 3E), while the pZd4 allele contains a 31 bp deletion, accompanied by a 14 bp insertion, in the 32nd cadherin domain’of the predicted fat protein (see Figure 3F). Both of these alterations in the DNA encoding the fat gene product change the translational reading frame, and thus result in stop codons following shortly after the altered regionsof the fat gene. The likely consequence of these alterations is the production

Figure

6. Overgrowth

An overgrown

of the Wing

wing imaginal

lmaginal

disc from

Disc in a fat Recessive

a larva homozygous

Lethal

Mutant

for /offB13 (left) compared

of a secreted, truncated form of the fat protein, since the gene products encoded by the fth’ and ft”“‘+’ mutant alleles would retain the putative signal sequence and 29 or 31 of the extracellular cadherin domains, respectively, but terminate translation prior to the production of the EGF-like repeats and transmembrane and cytoplasmic domains (see Figure 3s). Thus, based on our analysis of the Gull and G” genomic DNA clones and the PCR-amplified regions of the fth’ and ffz44 mutants, all of which contain significant defects in the region of the open reading frame encoding cadherin domains, we conclude that the fat locus encodes the cadherin-like molecule described in this report. Mutations in the fat Gene Lead to a Ceil-Autonomous Overgrowth Phenotype The fat locus derives its name from the fat, broad thorax

with a full-grown

wild-type

wing disc (insert

in IOUver right).

and abdomen of homozygous flies carrying recessive viable mutations in this gene (Mohr, 1923). More recently, recessive lethal fat alleles have been obtained that lead to tumor-like overgrowth of the imaginal discs (Bryant et al., 1988). This overgrowth occurs as the cells of the imaginal discs continue to proliferate during an extended larval period, resulting in large discs that contain many convoluted and abnormal folds of epithelial cells (Figure 6). During metamorphosis, these discs evert and differentiate into defective adult structures that contain cuticular ingrowths and outgrowths, bristle duplications, and regions of reversed polarity. The homozygotes die during the pupal stage (Bryant et al., 1988). Clones of cells generated by mitotic recombination that are homozygous for recessive lethal mutations at the fat locus show autonomous overgrowth,, leading to large patches of mutant tissue that protrude from the body sur-

;f~;

fat Tumor

Suppressor

Gene of Drosophila

which is consistent described above.

Figure 7. Mitotic Recombination Clones Demonstrate Overgrowth Phenotype Is Cell Autonomous

That

the fat

Starting with a heterozygous recessive mutation, mitotic recombination clones (arrow) were generated that were homozygous for /(2)ffr3 and that were also marked with the yellow mutation, thus causing the bristles within the mitotic clone to be yellow instead of brown. A translocation of y+ onto the left arm of the second chromosome (Dp(7: Z)sc’O, sc”y+; Lindsley and Grell, 1968) was used to link this marker with f(2)W. Clones were induced with 2000 rads of gamma rays at 48 * 4 hr after egg laying. The clone shown here leads to an outgrowth on the tarsus. The overgrowth is only seen in the marked cells within the mitotic recombination clone.

face (Figure 7) and/or show other aspects of the fat phenotype including increased bristle density and invagination of the integument. The surrounding heterozygous tissue is apparently completely normal and unaffected by the presence of the mutant clone. Therefore, we conclude that the recessive lethal fat mutations behave in a cell-autonomous fashion, causing defective morphogenesis and additional cell proliferation. This suggests that the fat gene product is a cell-bound rather than a diffusible protein,

with the predicted

protein

structure

The Gull Allele May Encode a Protein That interferes with Normal fat Function The fat allele called Gull is the only known dominant fat mutation, and when heterozygous it causes the wings of the adult fly to be held out at an angle to the body and frequently to curve downward like a gull-shaped wing (Mohr, 1923,1929). The holding of the wings at a divergent angle is also seen in some of the fat homozygous recessive viable mutants. Both homozygous viable fat mutants and Gull heterozygotes have wing crossveins that are spaced closer together than normal. In addition, Gull flies frequently have bristle duplications on the head and thorax (Mohr, 1923, 1929). The Gull lesion was originally thought to possibly consist of a multigene deficiency (Mohr, 1923). However, this is not the case. The phenotype of GulllDf(2L)Ml1 hemizygotes (and similarly that of Go/l/l(2Jtid transheterozygotes; data not shown) is the same as that observed in homozygous recessive lethal fat mutants, i.e., ovelrgrown discs and pupal lethality(Figure 8). These data, when combined with our molecular analysis of Gull, thus show that Gull is simply a fat allele and not a larger deficiency. The sequence of the Gull allele as described above shows that it contains a 412 transposon that interrupts the open reading frame in the 33rd cadherin domain. The effect of this transposon is likely to cause premature termination of translation, since stop codons are present in all three reading frames of the LTR of the transposable element (see Figures 38 and 3C). If this is the case, then the Gull protein could be produced as a truncated, secreted protein similar to that predicted by the sequences of the mutant fat alleles, W1 and P44, since it retains the putative signal sequence and 32 out of 34 cadherin domains, but has lost the EGF-like repeats, the transmembrane domain, and the cytoplasmic domain. Alternatively, the presence of the transposon could lead to transcription beginning within the transposon itself, producing a transcript that lacks the sequence encoding the signal sequence and most of the cadherin domains, but that encodes the EGFlike repeats and the transmembrane and cytoplasmic domains. Such a transcript would encode a truncated, membrane-bound fat protein, as opposed to a truncated, secreted form of the fat protein. Moreover, it is possible that both forms of the truncated fat protein are produced by the Gull mutation. An aberrant Gull protein could act in a neomorphic manner giving it a new function not found in the wild-type fat protein, or it could act in an antimorphic manner, iriterfering with the normal function of the fat protein. To ,distinguish between these alternatives, flies were bred to produce progeny that had one extra copy of the wildl-type fat locus in addition to the Gull mutant chromosome’and the one wild-type copy of fat present in the Gull heterolygotes. If the Gull mutant protein is behaving as a neomorbh, then an extra copy of the wild-type fat gene should have no effect on its phenotype, whereas if the Gull mutant is behaving as an antimorph, then an extra copy of the tiild-type

Cell 662

Figure

8. Overgrowth

of the Wing

Photographs of a full-grown seen in the Gull/Df(2L)M71

lmaginal

Disc in a Gull Hemizygous

Lethal

Mutant

wild-type (A) and overgrown Gu///Df(ZL)MI 7 (8) mutant wing imaginal disc demonstrate that the overgrowth mutant is similar to that seen in fat recessive lethal mutants (see Figure 6 and text for further discussion).

gene should partially suppress its phenotype, producing additional wild-type fat protein that can act to outcompete the Gull gene product. The results of this experiment show that an additional wild-type copy of the fat gene is able to markedly suppress characteristics of the Gull phenotype. Gull flies containing the extra wild-type fat gene were far less likely to have duplicated bristles than those Gull flies without an extra copy of the gene, had wing crossveins that were in a more normal position, and displayed a higher incidence of wings held in the wild-type position rather than the outstretched position (Figure 9). From these experiments, we conclude that the Gull mutant protein is acting in an antimorphic manner, such that additional wild-type copies of the fat gene can suppress the effects of the Gull mutation. This conclusion is consistent with the idea that an altered form of the fat protein may be present in Gull mutants and is somehow interfering with the normal functioning of the fat gene product. It seems likely that the fat gene rearrangements that have occurred in the GW mutant, although not completely characterized, have eliminated this hypothesized antimorphic protein and have essentially produced a null mutation. fat

Discussion Role of the fat Gene Product in Morphogenesis and Growth Control The control of cell proliferation in Drosophila imaginal discs has been shown to require contact-dependent cell interactions (Bryant, 1987). In the present report we have

phenotype

shown that the fat gene, which is required for the control of cell proliferation in the imaginal discs, encodes an enormous transmembrane protein (of over 5000 amino acids) with extensive sequence similarity to the extracellular “cadherin” domains found in the family of calcium-dependent cell adhesion molecules called cadherins (Takeichi, 1988). However, the fat sequence encodes 34 of these ~100 amino acid cadherin domains, whereas the many known vertebrate cadherins have only 4 of these domains. The predicted product of the fat gene also contains four epidermal growth factor (EGF) domains, an additional cysteine-rich domain, a transmembrane domain, and a

Genotype

Gull + 1 copy fat

I

Bristle duplications

+

+

52% (42)

I

18% (71)

I

57% (40)

I

70% (26)

I

I

29% (35)

100% (16)

Figure 9. Effect of One Dominant Phenotype

Gull + 2 copies fat

Extra

Copy

of the fat Gene

upon

I

the Gull

Flies carrying a duplication of the fat gene on the X chromosome were mated to heterozygous flies carrying the Gull mutation in order to produce progeny carrying the Gull mutation in the presence of either one or two copies of the wild-type fat gene (see Experimental Procedures). The presence of an additional wild-type copy of the fat gene markedly reduced, the severity of the Gull phenotype, as measured by the number of flies exhibiting duplicated thoracic bristles, close crossveins, and divergent wings. Crossvein distance is based on wild type being equal to 100%; sample number is in parentheses.

The fat Tumor Suppressor Gene of Drosophila 663

cytoplasmic domain (Figures 1,2, and 3B). If, indeed, this protein exists as a single uncleaved polypeptide chain, it would be an enormous transmembrane protein-one of the largest in the fruit fly. There is a high degree of homology between the predicted cadherin domains of the Drosophila molecule and the four extracellular cadherin domains of the known vertebrate cadherins, especially in the amino acids that are conserved among the vertebrate domains, including the putative calcium-binding sites (Ringwald et al., 1987; Ozawa et al., 1990). In contrast, the cytoplasmic domain of the Drosophila molecule shows no homology to the cytoplasmic domains of the vertebrate cadherins. Furthermore, the vertebrate cadherins do not contain EGF-like repeats, and they are considerably smaller than the predicted fat protein. Thus, this enormous transmembrane protein encoded by the fat gene is probably not the Drosbphila homolog of one of the already known vertebrate cadherins, but rather defines a new type of cadherin-like molecule of a much larger size. Our results indicate that the cadherins, previously thought to comprise a gene family of closely related cell adhesion molecules in vertebrates, are actually members of a larger gene superfamily that is defined by the presence of cadherin domains; thus, the Drosophila fat gene is a member of this superfamily. Whether a true vertebrate homolog of the Drosophila fat gene exists is presently unknown. This is the first gene isolated in Drosophila, and for that matter outside of the chordates, which encodes true cadherin domains. It was previously reported that one of the neoplastic tumor suppressor genes in Drosophila, lethal(2)giant larvae (Igl), has similarities in sequence to the extracellular region of vertebrate cadherins (KIBmbt et al., 1989). However, this reported similarity is only a few amino acids in length over a few short stretches whose relative order does not conform to the cadherin domain structure, thirs making the potential relationship between /g/ and cddherins weak at best. We do n’ot know if this cadherin-like molecule actually functions as a calcium-dependent adhesion molecule. All of ‘the molecules containing cadherin domains described thus far function as homophilic adhesion molecules, and the cadherin domains themselves have been shown to mediate the adhesion. However, we will have to express the fat protein in tissue culture cells to directly test the hypothesis that this cadherin-like molecule mediates &ilcium-dependent homophilic adhesion. Moreover, it is not yet known whether the entire 15.4 kb open reading frame is t&slated into a single polybeptide, and to what ddgree this large protein is further processed. However, the cell-autonomous behavior of mtitant fat clones prodticed by rhitotic recombination in lawal imaginal idiscs is consistent iith the proposed in vivo fu’nction of thefatgene prpduct asan integral membrane proiein. Thus, besed on th& cell-autbnomous behavior of mutalnt fat clones ihduced bi’,mitotic recombination, the disc od’ergrowth phenotype sf,~.!(2)fi homozygotes, and the structbre of the predicted fat; protein, we conclude that the f$ gene encbdes a cabherin-like transmembrane proteiri that is required for

contact-dependent interactions controlling cell proliferation and morphogenesis in the imaginal discs. The Drosophila fat gene is the first member of the cadherin gene superfamily that has been shown ‘to function in the control of cell proliferation in vivo. Do thle vertebrate cadherins have a similar function? While trxperimental studies on cadherins in vertebrates have not yet revealed a clear role for these molecules in the control of cell proliferation, such a function has not often been specifically tested. The clearest case comes from studies ‘of the effects of peptides homologous to the N-terminal region of E-cadherin. Peptides as small as 17-mers, when added to the apical side of kidney epithelial cell monolayers, cause overgrowth and formation of ectopic clusters of cells (Liaw et al., 1990). The fat gene is not the first tumor suppressor gene that encodesaprotein withsequencesimilaritytocelladhesion molecules. One of the tumor suppressor genes deleted in human colorectal carcinomas (called the DCC gene) encodes a deduced protein with multiple immunoglobulin domains and fibronectin type Ill repeats, similar to the group of cell adhesion molecules that includes NCAM, fasciclin II, Ll, and others (Fearon et al., 1990). In the case of the DCC gene, just as with the fat gene, direct evidence that the gene encodes a cell adhesion molecule has not yet been obtained, but in both cases the sequencftsimilarityto the domains of well-characterized cell adhesion molecules is striking. The tumor suppressor quality of the DCC and fat genes may mean simply that proper cell adhesion is a necessary prerequisite for the exchange of growth-control signals between cells, or alternatively, and perhaps more interestingly, it could signify that some cell adhesion molecules have an important cell signaling role that extends beyond their adhesive function. In regard to this potential cell signaling function, there are two interesting differences betwee? this predicted fat protein and the known vertebrate cadherins. First, the fat protein contains four EGF repeats, while tl-ie vertebrate cadherins do not. It will be interesting in the future to see if the EGF repeats of the fat product are required in transformant constructs to rescue the cell proliferation phenotjrpe of fat recessive lethal alleles. Second, the cytoljlasmic domain of the Drosophila protein shares no homology with the highly conserved cytoplasmic domains of the known vertebrate cadherins (Hatta et all., 1988; Takeichi, 1988), suggesting that it might have effector functions in the cell that are different from the well-known vertebrate cadherins. The fat locus is one of seven known Drosophila tumor su’ppressor genes that function in imaginal discs (Bryant, 19’87) and is the best-characterized of the five genes in which mutations produce hyperplastic overgrowth. Twentytwb alleles of the fat locus are known, including recessive pypal lethal alleles, recessive viable alleles, a dominant viable allele that also acts as a recessive pupal lethal (Gull), and a ,r’evertant of Gull, Gw, which has lost the characteristics as$ociated with the dominant phenotype but which retains rs’dessive lethality. These mutations include spontaneous, d&genic, chemically induced, and X-ray-induced alteratibns.

Of these different fat alleles, the most interesting is Gull, a SpOntaneOUS allele originally isolated by Mohr (1923). ln the present study, we have shown that this spontaneous dominant mutation is associated with the insertion of a 412 transposable element in the 33rd (the 2nd to last) cadherin domain in the extracellular region of the predicted fat protein. This transposon is likely to cause premature termination of the normally initiated protein. If this is indeed the case, then the fat gene product is probably produced as a truncated, secreted protein in Gullmutants, since it retains the putative signal sequence and 32 out of 34 cadherin domains, but has lost the EGF-like repeats, the transmembrane domain, and the cytoplasmic domain. Such a secreted protein, which retains a large fraction of the extracellular portion of the molecule, could conceivably produce the dominant effects that are observed in the Gull mutant. ,However, two other alleles of fat, nh7 and fF, also contain alterations in the coding sequence that are predicted to produce truncated, secreted forms of the extracellular regionof thefatprotein, which areverysimilarto thatwhich may be produced in Gull mutants. Neither of these two alleles produces a dominant phenotype; both fV and ffZ44 are recessive lethal alleles of the fat locus (in’ addition, the fY44 allele is unique among all of the fat alleles in that it is associated with embryonic lethality in homozygotes, although the significance of this fact is unclear). The existence of two lethal recessive fat alleles encoding putative truncated forms of the fat protein very similar to that encoded by the dominant Gull allele suggests that the extracellular portion of the fat protein may not be causing the dominant effect associated with the Gull mutation. Rather, thedominance of the Gull mutation might arise from a transcript that is initiated from within’ the 412 transposon itself and translated beginning at one of the methionine codons that are present in the fat open reading frame 3’tothe site of the 412 insertion. Of the 19 methionine codons present in the fat open reading frame between the site of the 412 insertion and the predicted transmembrane domain, one of them, encoding amino acid 3970, is contained within a perfect eukaryotic translation consensus sequence (CCACCATG) (Kozak, 1984). If this methionine codon, which is contained within the first EGF-like repeat of the predicted fat protein, actually serves as a translation initiation codon, then a truncated fat protein containing the remaining EGF-like repeats as well as the transmembrane and cytoplasmic domains could be produced. The Go//allele behaves as an antimorphic(Muller, 1932) or dominant negative (Herskowitz, 1987) mutation, which lacks normal function and instead produces a product that appears to antagonize the function of the gene product encoded by other alleles. Many of the dominant visible characteristics of Gull mutants, including the divergent wings, closer wing crossveins, and bristle duplications on the head and thorax, can also be observed in some Of the fat homozygous recessive viable mutants. Moreover, the phenotype of Gull as a transheterozygote over a small deficiency for the region is the same as that of the other recessive lethal fat mutations, i.e., overgrown discs and

pupal lethality. These observations show that the Gull gene product lacks normal function and suggests that it may act as an antimorph. The antimorphic character of Gull is further shown by the fact that, when tested over the homozygous recessive viable allele fat’, Gull produces disc overgrowth and pupal lethality (with only about 3% viable adults; Mohr, 1929), whereas a deficiency of the region, recessive lethal alleles, or G” produce large percentages of viable adults when tested over the same fat’ allele (Bryant et al., 1988). We tested and confirmed the antimorphic character of Gull by showing that an extra wild-type copy of the fat gene markedly suppresses the dominant phenotype of Gull, resulting in fewer duplicated bristles, more normal wing crossveins, and a higher incidence of wings held in the wild-type position as opposed to the outstretched position. Thus, we conclude that the Gull mutant protein is acting in an antimorphic manner, such that in heterozygotes it produces a more extreme phenotype than a deficiency, and that additional wild-type copies of the fat gene can suppress the dominant effects of the Gull mutation. This conclusion is consistent with the idea that a truncated form of the fat protein in Gull heterozygotes is interfering with the normal functioning of the fatgene product that is generated by the wild-type copy of the fat gene present on the balancer chromosome. There are two alternative hypotheses regarding the form of the antimorphic Gull protein: the Gull protein is a secreted, truncated portion of the fat protein containing most of the extracellular cadherin domains, or, alternatively, the Gull protein is a truncated, membrane-bound portion of the fat protein containing the EGF-like repeats, transmembrane domain, and cytoplasmic domain. In the first case, it is possible that the 412 insertion in Gull leads to the production of a truncated, secreted fat protein in Gull mutants that is acting as an anti-adhesion molecule by binding to and neutralizing the normal fat receptor, which may very well be the fat protein itself, if indeed this cadherin-like protein functions ina homophilic fashion as do the other cadherins. An effect similar to the one proposed here for the Gull protein has previously been observed in cultured mammalian epithelial cells, where the addition of a purified 80 kd extracellular fragment of cell CAM 120/80 (homologous to E-cadherin) was able to disrupt cell-cell adhesion (Wheelock et al., 1987). A soluble variant of GMPI 40, a cell adhesion molecule of platelets and endothelial cells, also acts as an antiadhesion molecule for some cell types (Gamble et al., 1990). The existence of two other lethal recessive alleles of fat, fth’ and ftsz44,which both contain lesions that should produce a secreted form of the fat protein that is very similar to the predicted protein that may be produced in Gull mutants, argues against the extracellular portion of the truncated fat protein causing the antimorphic, dtiminant phenotype associated with the Gull mutation. It is formally possible that some sequences within the 412 element could cause either the RNA or the truncated protein encoded in Gull mutants to be more stable than the productsofthefi+lorffZ~alleles. It seemsmorelikely, however, that the antimorphic nature of the Gull allele arises from

The fat Tumor 866

Suppressor

Gene

of Drosophila

the translation of a transcript that is initiated from within the 412 element and translated beginning with one of the methionine codons present within the fat coding sequence following the 412 insertion. Deletion analysis of vertebrate cadherins has demonstrated that the cytoplasmic domain is required in order for cadherins to mediate intercellular adhesion (Nagafuchi and Takeichi, 1988). A truncated portion of the fat protein that contains the cytoplasmic domain but no extracellular cadherin domains might interfere with normal fat protein function by competing with the cyto,plasmic domain of the wild-type fat gene product. Such competition might include binding to cytoplasmic proteins that normally associate with the cytoplasmic domain of the fat gene product. It will be interesting to test these two possibilities using fly transformation methods in order to determine which truncated variant of the fat gene product can lead to a similar antimorphic phenotype in vivo. Experimental

Procedures

Standard Procedures Unless otherwise indicated, all molecular biological techniques were performed essentially as described by Maniatis et al. (1962) and the references therein. PCR PCR primers that would amplify one entire cadherin domain were designed, based on the published sequences corresponding to the cadherin domains of vertebrate N-, E-, and P-cadherin and L-CAM (see Hatta et al., 1966 for alignment). For one primer, completely degenerate oligonucleotides were designed that encoded amino acids EWV IPPI, found in the first cadherin domain of chick N-cadherin and L-CAM and mouse E-and P-cadherin. For the other primer, completely degenerate oligonucleotides were designed that were complementary to the nucleotides that encode some of the amino acids thought to be involved in calcium binding in vertebrate cadherins (Ringwald et al., 1987;Ozawaetal., 1990)TheconsensussequenceDgNDN :P,which represents the amino acids found in the first cadherin domain of chick N-cadherin and L-CAM and mouse E-and P-cadherin, as well as some ofthe amino acids found in the corresponding second, third, and fourth cadherin domains, was used to design the second primer. To reduce the level of overall degeneracy, each primer was constructed in four pools of lesser degeneracy, and all 16 possible combinations of oligonucleotide primers were tested. Primers were annealed to 1 pg of Drosophila melanogaster genomic DNA in a 50 PI reaction volume, and amplified with Taq polymerase according to the manufacturer’s specifications (Perkin Elmer/Cetus). Following 35 cycles of primer hy br/dization atGOOC(1 min),extension at72%(1 min), and denaturation at 94°C (45 s)l reaction products were analyzed on 2% agarose gels. One pair of primers (GAATGGGTXATXCCXCCXAT and GGXCGBT TAGTCBTTCATkTC, X = A, C, G, T) produced a band of the expected size, which was ligated into Bluescript SK+ (Stratagene). Following transformation of Escherichia coli JM63 cells, the inserts of several independent clones were sequenced; this analysis revealed that two d’ifferent cadherin domains had been amplified and cloned (see Results). eDNA Clones One of the cadherin domains amplified from Drosophila genomic DNA was used to screen a cDNA library constructed from 9-12 hr Drosophifa’embryos (Zinn et al., 1986), yielding a 5 kb cDNA. Fragments from the 5’ and 3’ ends of this cDNA were used to screen the same library again, as well as a cDNA library constructed from Drosophila eye imaginal discs (provided by A. Cowman and G. M. Rubin) and a randomly primed’cDNA library constructed from 8-21 hr Drosophila embryos (provided by Y. Jan) in order to obtain overlapping clones. This prob’ess was repeated, together with in situ hybridizations of newly isolated cDNAs to polytene chromosomes, until almost the entire 15.4 kb open reading frame was obtained, as determined by sequencing.

Two regions could not be obtained from the cDNA libraries; the sequence of these regions, corresponding to the bases encoding amino acids 1-142 and 486-1278 in Figure 1, was obtained from genomic clones (see below). Genomic Clones The wild-type recombinant phage were isolated from an Oregon-RC library in lambda DASH I (Stratagene) generously provided by D. Woods. Overlapping genomic clones were obtained from a wild-type library in both directions (from the initial cDNA clone) using 32P-labeled RNA probes synthesized from the ends of the Drosophila inserts with T3 or T7 RNA polymerase. A total of 80 kb of genomic DNA, covering the entire fat coding region, was cloned. For the mutant allele libraries, genomic DNA was isolated from ds SG bpr/CyRoior G”/CyRoiflies, partially digested with Sau3A, and size selected on agarosegels(Maniatiset al., 1982). DNAoftheappropriate size was cloned into the lambda DASH II vector (Stratagene) and packaged using the Gigapack (Stratagene) reagents accordilng to the manufacturers instructions. Amplification of ft”’ and fP” DNA Oligonucleotide primers corresponding to the region of the fat gene that was found to contain restriction fragments that were altered in the rV/CyRoi and ffZm/CyRoi balanced stocks in the PCR. The DNA produced in the PCR was subcloned into Bluescript SK+(Stratagene), and duced in the PCR was subcloned into Bluescript SK+ (Stratagene), and multiple clones that were obtained were sequenced. Fior both mutant stocks, two classes of clones were identified following sequencing: those containing the wild-type sequence, presumably from the CyRoi chromosome, and an altered sequence that we inferred was from the fat mutant chromosome. Sequencing of cDNA and Genomic Clones Subclones of cDNA or genomic clones were sonicated and cloned into Ml3 following standard procedures (Maniatis et al., 1982). Singlestranded templates obtained from the Ml3 transfectants were sequenced using the dideoxy method @anger et al., 1977). Some fragments were subcloned into Bluescript (Stratagene) aind sequenced using the dideoxy method after obtaining single-stranlded templates following superinfection of E. coli transformants with the R406 helper phage (Stratagene), and/or sequenced directlymoff of the doublestranded plasmid DNA using the Sequenase reagents and protocol (United States Biochemical). Sequence datawere assembled and analyzed using the lntelligenetics software package, and homology searches in the GenBank data base were performed with the blastn and blastp programs (Karlin and Altshul, 1990; Altschul et al., 1990). In Situ Hybridization 35S-labeled single-stranded RNA probes were transcriblsd from a 2 kb cDNA subclone (corresponding to the nucleotides encoding amino acids 2333 to 3014 in Figure 1) in Bluescript using T3 or ‘l-7 polymerase (Stratagene). Probes were hybridized to sections of (O-20 hr) Drosophila embryos or third instar larvae that had been fixed, embedded in paraffin, and processed according to lngham et al. (1985). Chromosomal localizations were obtained by hybridizing biotinylated DNA probes to Drosophila salivary gland chromosomes, as described by Langer-Safer et al. (1982). G&netic Analysis The original Gulland far alleles were described by Mohr (1923, 1929). Al( other fat alleles were generated by Szidonya and Reuter (1966) and Bryant et al. (1986). The Gullmutation present in thedsSG bpr/CyRoi stock was reisolated on several different chromosomes in order to eliminate some of the accompanying mutations present oh the original chromosome. To determine if an extra copy of the wild..type fat gene could suppress the dominant Gull phenotype, virgin females from one of these reisolated stocks, Gpr/CyO, were mated to Df(2EJsc’~z/bprcn px sp; Tp(2,7) y ed cl dp males, which carry a transposition of the fat gene onto the X chromosome. The phenotype of Cy’prl-females from this cross was compared with the phenotype of Cy+pr females in the Ff generation; the former contain one wild-type copy of the fat gene in addition to the Gull mutation, while the latter contain two wild-type

copies of the fat gene in addition to the Gull mutation. Each fly was scored for the presence and number of extra thoracic bristles and the position in which the wings were held. Following this, individual flies were incubated in a solution of 3 parts 70% ethanol to 1 part glycerol for several days, after which the wings were dissected, mounted on microscope slides, and photographed. Measurements of total wing length and the distance between wing crossveins were made from photographic prints of the wings; no significant variation in total wing length was observed. Acknowledgments We thank J. Allison for use of his PCR machine, C. Klambt and L. Garcia-Alonso for helpful discussions, A. Nose and H. Clark for critical reading of the manuscript, H. Clark for allowing us to refer to her work in progress on the other cadherin-like gene, and Babak Kasravi for technical assistance. This work was supported by an American Cancer Society Postdoctoral Fellowship to P. A. M., a predoctoral fellowship from the UCI Corporate-University Partners Program to P. O., grants from the Monsanto Company and the American Cancer Society to P. J. B., and support from the Howard Hughes Medical Institute to C. S. G., who is an HHMI Investigator. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

June

14, 1991; revised

September

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