The EMBO Journal vol.8 no. 8 pp.2381 - 2386, 1 989

The spatial and temporal expression pattern of sevenlessis exclusively controlled by gene-internal elements

Konrad Basler, Peter Siegrist and Ernst Hafen

Zoologisches Institut, Universitat Zuirich, CH-8057 Zurich, Switzerland

Communicated by M.Bienz

The sevenless gene controls the fate of a single photo-receptor cell type in the developing eye of Drosophila.Its RNA and protein accumulate transiently in a sub-population of cells in the developing eye imaginal disc.We have used P-element transformation with differentminigenes and marker gene constructs to identify cis-acting regulatory regions required for the complexsevenless expression pattern. Our results indicate that theupstream region of the sevenless gene is devoid of anydetectable regulatory elements and that sequences located3' to the transcription start site are sufficient to promotethe sevenless expression pattern. These gene-internalsequences function in both orientations on heterologouspromoters, also when placed at the 3' end of a lacZreporter gene.Key words: gene expression/internal enhancer/lacZ reportergenelminigeneslsevenless

as the hsp70 promoter or the P-element promoter to producethe sevenless specific expression pattern. This fragment hasthe properties of an enhancer since it can act in bothorientations and is independent of its position with respectto the start site of transcription.

ResultsWe have previously shown that a 16.3 kb genomic EcoRIrestriction fragment containing 950 bp of 5' flankingsequences can rescue the sevenless mutant phenotype whenintroduced into the germline by P-element-mediated trans-formation (Hafen et al., 1987). The spatial and temporalexpression pattern of the sevenless gene contained in thistransformed fragment is indistinguishable from the patternof the endogenous gene (Basler and Hafen, 1988b). Thisindicates that the 16.3 kb fragment contains all the cis-actingsequences necessary for correct expression and function ofthe sevenless gene. In an attempt to increase the transfor-mation frequency by reducing the size of the fragmentcontaining the sevenless gene we constructed severalsevenless minigenes and assayed their function in vivo.

Introduction

The sevenless gene of Drosophila is required for the correctdevelopment of one of the eight photoreceptor cell types (R7)in the compound eye (for review, see Ready, 1989; Baslerand Hafen, 1988a). It encodes an integral membrane proteinwith a tyrosine kinase domain (Hafen et al., 1987; Baslerand Hafen, 1988b; Bowtell et al., 1988). Its structure andsimilarities to growth factor receptors suggested that thesevenless protein is a receptor for a positional signal requiredfor R7 determination. Expression of the sevenless gene isregulated in a complex spatial and temporal manner.

Sevenless RNA and protein are almost exclusively expressedin a subpopulation of ommatidial precursor cells (Tomlinsonet al., 1987). The sevenless protein accumulates transientlyon photoreceptor cells R3, R4 and R7 and at a later stagealso in the four cone cells. Photoreceptor cells R2, R5 andR8 never exhibit detectable levels of sevenless protein.Although the restricted expression pattern of sevenless is notessential for sevenless function (Basler and Hafen, 1989),it reflects the first known biochemical difference betweendifferent photoreceptor cells. The sevenless regulatorysequences might therefore provide a useful tool for theectopic expression of other genes involved in eyedevelopment.

Here we describe the identification of cis-acting sequenceswithin the sevenless gene that are necessary and sufficientto produce this specific expression pattern. Interestingly thesesequences are exclusively located downstream of thetranscription initiation site. An internal restriction fragmentof the sevenless gene can act on heterologous promoters such

sevenless minigenes do not rescue the mutantphenotypeThe sevenless minigenes were constructed by replacing thegenomic sequence encompassing the first seven introns bythe corresponding cDNA fragment. 5' and 3' flankingsequences were as in the 16 kb genomic fragment (Figure1). Two different cDNA clones were used for the minigeneconstructions. Bowtell et al. (1988) reported a cDNA clonein which the first untranslated exon was 608 bp longer thanin the several independently isolated clones that we havedescribed (Basler and Hafen, 1988b). This size differencecould indicate an alternative splicing of the sevenless mRNA,however it does not affect the protein coding region. To testwhether this difference was functionally significant, theminigene constructs lacking the first intron were alwaysconstructed with both types of cDNAs. We will refer to thetwo types ofcDNAs as type A (short exon) and type B (longexon). The minigenes were cloned into the pW8 transfor-mation vector and were introduced into the germline ofwhite'18, sevenlessd2 mutant hosts. Several independenttransformed lines were established for each construct. Thecorrect expression of the minigene constructs was assayedboth by their ability to rescue the sevenless mutant phenotypeand by immunohistochemical staining of eye imaginal discswith an antiserum against the sevenless protein.The transformed lines containing either a type A or type

B minigene lacking the first seven introns did not rescue thesevenless phenotype. Only one of the seven lines containingthe type B minigene exhibited partial rescue of the sevenlessmutant phenotype with mosaic eyes consisting of both wild-type and mutant ommatidia (data not shown). The partialrescue in this line was due to position effect of integration

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Fig. 1. Structure of the sevenless gene constructs used for germ-line transformation and their ability to rescue the sevenless mutant phenotype. At thetop of the figure a restriction map indicates the organization of the sevenless transcript and the origin of fragments used for the constructs illustratedbelow. At the right, the analysis of the transformed lines is summarized. Bars represent exons and lines represent introns or flanking sequences. Thedifferently spliced first exon (type B) is dashed, the coding region shaded, the transmembrane regions (TM) filled and the tyrosine kinase domainhatched. Arrow-hatched and stippled bars indicate the gene-internal restriction fragments used to locate the regulatory sequences responsible for wild-type sevenless expression.

since subsequent mobilization of the insert to other placesin the genome also resulted in lines that did not rescue.Staining of eye imaginal discs of several of the transformedlines with an antiserum against the sevenless protein indicated

that no detectable amount of sevenless protein is made inthese lines (Figure 2C). These results suggest that theintronic sequences which have been deleted in the minigeneconstructs are essential for correct expression of the sevenless

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K.Basler, P.Siegrist and E.Hafen

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Fig. 2. Expression pattern of sevenless protein (A-C) and ,B-galactosidase (D) in eye imaginal discs of different transformed lines.(A - C) Immunohistochemical detection of sevenless protein synthesized from the endogenous wild-type gene (A) or from integrated sevenlessminigenes (B,C). In the lines carrying the internal fragment upstream of the hsp-sev cDNA (B) the sevenless protein accumulates in the sametemporal and spatial distribution in the developing ommatidial clusters as in wild-type (A). In the enlargements at the right of (A) and (B) the changein staining pattern of progressively older clusters (top to bottom) is shown. The intensity difference of the staining is due to the different number ofgene copies (homozygous versus heterozygous). No sevenless protein is detectable in lines transformed with sevenless minigenes that are missingintrons 2-7: the eye disc stained in (C) is from a line containing the sev minigene with first intron (see Figure 1). (D) The internal sevenlessfragment can also direct tissue-specific transcription of a lacZ reporter gene from a downstream position: eye discs (ed) of a P-lacZ-sevtransformant stained with Xgal exhibit accumulation of /3-galactosidase in ommatidial clusters posterior to the morphogenetic furrow. No staining isobserved in other imaginal discs [e.g. the antennal disc (ad) or the leg discs (Id)]. The inset shows the first cells expressing 0-galactosidase at a10-fold higher magnification; they correspond to the precursor cells R3 and R4, that are known to be the first cells expressing high levels ofsevenless protein during the ommatidial assembly. Scale bar is 50 tm.

protein. Assuming that the regulatory sequences would belocated in the first intron, we constructed a minigenecontaining the first intron (Figure 1). In none of the fourtransformed lines obtained with this construct did we observerescue of the mutant phenotype. Hence the sequences withinthe first intron are not sufficient to produce any sevenlessexpression pattern. The inability to rescue the sevenlessphenotype by minigene constructs involving the type BcDNA has also been reported by Bowtell et al. (1988).

In a parallel set of experiments sevenless minigenes wereconstructed under the control of the hsp70 promoter. Full-length type A and type B cDNAs encompassing all 12 exonswere assembled from overlapping cDNAs and the last exonwas fused to the 3' genomic region to provide the endo-genous termination/processing sequences. These cDNA/genomic hybrids were fused to the hsp70 promoter in thehsp70 leader sequence (Figure 1). No rescue of the mutantphenotype was observed when the lines were raised withouta heat shock. However, repeated induction of the type A

hsp-sev construct by 30 min heat shocks at 37°C every 4 hduring third instar and pupal development resulted incomplete rescue of the mutant phenotype (Basler and Hafen,1989). In contrast, all three lines containing the type Bhsp - sev construct could not be rescued by this inductionprotocol. These results indicate that at least the type A cDNAis fully functional and that its ubiquitous expression resultsin the correct specification of R7 development. It is possiblethat the long untranslated leader sequence in the type BcDNA results in less efficient translation or abnormalsplicing. Since this type of cDNA was isolated only once(Bowtell et al., 1988) it most likely represents an aberrantlyspliced sevenless transcript.

A gene-internal fragment is sufficient for correctsevenless expressionTo test whether the regulatory elements located 3' of thetranscription start site are sufficient to confer the wild-typesevenless expression pattern on the hsp -sev construct, we

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K.Basler, P.Siegrist and E.Hafen

Fig. 3. Rescue of the sevenless mutant phenotype: gene-internal sequences promote sevenless transcription from the hsp-sev cDNA gene resulting ina correct formation of R7 photoreceptor cells. The rhabdomere pattem of two transformed lines are shown using the optical neutralization technique.(A) A transformant carrying the hsp-sev cDNA exhibits an irregular rhabdomere pattem caused by the absence of R7 photoreceptor cells. (B) Atransformant containing the internal sevenless fragment upstream of the hsp-sev cDNA (construct 202) displays a wild-type rhabdomere pattern.

have cloned a genomic EcoRV fragment containing the firsthalf of the transcription unit, but not including the transcrip-tion start site, in front of the hsp -sev construct in bothorientations (constructs 201 and 202, see Figure 1). Whereasthe hsp -sev construct without heat induction does not rescuethe mutant phenotype, the constructs containing the sevenlessinternal sequences upstream of the hsp70 promoter exhibitedcomplete rescue of the sevenless phenotype (Figure 3).Mutant rescue was observed irrespective of the orientationof the internal sevenless fragment. Furthermore, the spatialand temporal expression pattern of sevenless proteinproduced by these constructs is indistinguishable from thedistribution of the endogenous sevenless protein (Figure 2Aand B). The sequential expression of sevenless protein indifferent ommatidial cells is revealed by the progressivelychanging staining pattern in the ommatidial clusters (enlarge-ments in Figure 2A and B). In a similar construct a shorterinternal XhoI fragment containing only sequences from thesecond intron was not sufficient to direct expression of thesevenless gene (constructs 203 and 204, see Figure 1).However, the 3' adjacent XhoI restriction fragment wascapable of conferring mutant rescue as well as wild-typeexpression of sevenless (construct 224, see Figure 1). Thisgenomic fragment is 1.2 kb in length and contains the thirdand fourth introns as well as the 3' end of intron 2. Theseresults indicate that the expression of the sevenless gene iscontrolled exclusively by gene-internal sequences containedin the 1.2 kb XAoI fragment. Furthermore, the gene-internalsequences can confer their regulatory function on theheterologous hsp7O promoter.

Stage- and tissue-specific expression of a lacZreporter gene directed by a sevenless gene-internalfragmentTo test whether the gene-internal sequences are sufficientto confer the time and cell-type specific expression pattern

of sevenless on a heterologous reporter gene, we inserteda fragment encompassing introns 2-6 (Figure 1) at the 3'end of the bacterial lacZ gene in the vector P-lrB (Bellen,Wilson and Gehring, unpublished data). In this vector thelacZ gene is fused to the weak P-element promoter whichhas been shown to be susceptible to nearby enhancerelements (O'Kane and Gehring, 1987). In all transformedlines intense ,B-galactosidase activity was observed in cellsposterior to the morphogenetic furrow in the eye disc. Thestaining was confined to the eye disc and was associated withthe developing ommatidial clusters. Immediately posteriorto the furrow, pairs of cells, corresponding to the developingphotoreceptors R3 and R4, were intensely stained (Figure2D, inset). In these cells, high accumulation of sevenlessprotein is also first detected (Tomlinson et al., 1987). Inmore posterior regions of the disc additional cells are stained.This expression pattern corresponds essentially to the normalsevenless expression pattern although the correlation withthe distribution of sevenless protein in older ommatidialclusters is more difficult due to the different stability andsubcellular localization of the ,B-galactosidase and thesevenless protein. These results support the conclusion thatthis gene-internal fragment is sufficient to direct the normalsevenless expression pattern even when linked to a hetero-logous gene.

DiscussionThe main result of our analysis is that gene-internalsequences are necessary and sufficient for the complex spatialand temporal expression pattern of sevenless. Minigeneconstructs lacking sequences of the first seven introns werenon-functional and did not yield detectable levels of sevenlessprotein. The replacement of the sevenless 5' region by thehsp7O heat shock promoter caused heat shock-dependentrescue of the mutant phenotype. Furthermore, placing

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Gene-internal elements control sevenless expression

genomic sevenless sequences normally located 3' of thetranscription start site upstream of the hsp-sev cDNAproduced a wild-type sevenless expression pattern andrescued the mutant phenotype. These sequences can alsodirect the expression of a heterologous lacZ reporter gene.They have the characteristics of a transcriptional enhancersince they function in both orientations.The constructs tested permit several conclusions about the

location of regulatory sequences within the sevenless gene.The coding region itself appears not to contain any elementsthat promote sevenless expression. Furthermore, neither thefirst nor the second intron is able to contribute to thesevenless expression pattern, since minigenes containingsequences of either of the two introns fail to rescue and toexpress sevenless. However, a fragment encompassing thethird and fourth introns (as well as a small portion of thesecond intron) contains all the cis-acting elements requiredfor proper spatial and temporal expression of sevenless. Theremaining part of the structural gene appears to be devoidof any regulatory sequences.

In most genes control regions are located upstream of thetranscription start site. In the case of the.fiishi tarazu gene,for example, the expression pattern is controlled by multipleelements exclusively located upstream of the transcriptionstart site (Hiromi et al., 1985). However, there are examplesof genes where control regions are located on both sides ofthe transcription start site. The spatial and temporalexpression of the Ultrabithorax gene depends on elementslocated on either side of the transcription start site (Bienzet al., 1988). Another example is the 33-tubulin gene:expression in the somatic musculature and pharyngeal muscleis dependent on upstream sequences, whereas expression of33-tubulin in the visceral mesoderm appears to be controlledby a tissue-specific element located in an intron (Gasch etal., 1989). In the case of sevenless, however, there appearto be no regulatory sequences upstream of the transcriptionstart site. A putative general signal for transcription initiationcan be substituted by the hsp70 or the P-element promoter.Interestingly, in contrast to these promoters, sevenless seemsto lack a TATA box, which in the case of the hsp70 promoterhas been shown to be sufficient to respond to enhancerelements (Riddihough and Pelham, 1986). Instead, however,the transcription initiation site of the sevenless genecomprises a conserved heptanucleotide sequence (Hultmarket al., 1986; Basler and Hafen, 1988b) not found in the hsp7Oand P-element promoters (O'Hare and Rubin, 1983). Thiselement could serve a similar function as the 'initiator'element recently reported by Smale and Baltimore (1989).The first example of a cellular, tissue-specific enhancer

located within the gene was the immunoglobulin heavy chainenhancer (Banerji et al., 1983). In addition to this enhancer,upstream elements in the promoter-proximal region alsocontribute to the B cell-specific expression of the immuno-globulin heavy chain gene (Grosschedl and Baltimore, 1985).In sevenless, however, upstream regions appear devoid ofany regulatory sequences. It is possible that the internalregulatory sequences in sevenless are remnants of a morecomplex regulatory region consisting of multiple elementslocated on either side of the transcription start site. Theupstream elements might have been lost by recombinationin an event that did not affect the function of the sevenlessgene.What is the function of the complex spatial and temporal

regulation of the sevenless gene? We have previouslydemonstrated using the hsp-sev construct that ubiquitousexpression of sevenless during the entire embryonic, larvaland pupal development correctly specifies R7 developmentwithout affecting the development of other cells wheresevenless is normally not expressed (Basler and Hafen, 1989;Bowtell, et al., 1989). The only minimal requirement forcorrect R7 development appears to be that sevenless isexpressed at sufficient levels in the R7 precursor at the timewhen it becomes integrated into the ommatidial cluster. Theexpression pattern of a gene as determined by in situ localiza-tion of the transcript or of the protein is often taken asevidence for the spatial domains of gene function. Ourfindings indicate that in the case of sevenless the expressionpattern is merely compatible but not required in thatparticular form for correct function. It may therefore bemisleading to deduce functional properties from theexpression pattern of a gene.

Materials and methodsPlasmid constructionsFor the assembly of the various sevenless (sev) minigenes and hsp -sev con-structs the following DNA fragments served as starting material: (i) thegenomic 16.3 kb EcoRI fragment that contains the entire sev gene and thatwas sequenced completely in a previous study (Basler and Hafen, 1988b);(ii) the 6.3 kb cDNA clone cED3.1 covering most of the coding region(Hafen et al., 1987); (iii) the 2.0 kb cDNA cK5.1, one of 15 identical cDNAclones derived from the 5' region of the sev transcript (Basler and Hafen,1988b); (iv) cDNA clone 10 containing the differently spliced first exontype B (Bowtell et al., 1988); (v) a 350 bp fragment encompassing- 250 bp of the Drosophila hsp70 promoter and - 90 bp of the hsp70 leadersequence (Schneuwly et al., 1987); (vi) the P-element transformation vectorspW8 (Klemenz et al., 1987) bearing the selectable marker gene white andpDM30 (Mismer and Rubin, 1987) containing the marker gene rosy.Due to the considerable length of the sev transcript (8.2 kb) numerous

successive cloning steps were required to assemble the differentcDNA/genomic hybrid genes. Most intermediate constructions were preparedin pBluescript (Stratagene) or derivatives of it that were altered to lackendogenous polylinker sites to facilitate subsequent cloning steps. A detailedprotocol of the plasmid construction strategy is available on request. Thefinal plasmids used for germline transformation were of the followingstructure [nucleotide numbers are from the genomic sev sequence in Baslerand Hafen (1988b)].

Genomic sev EcoRifragment. This fragment (position -964 to 15 379)was initially used in transformation vector pW8 (Basler and Hafen, 1988b)but, due to the low transformation frequency of this construct, the 16.3 kbEcoRI fragment was inserted into the transformation vector pDM30 for thegeneration of additional transformed lines. We observed an - 5-fold increasein transformation efficiency. All minigene constructs are in the transfor-mation vector pW8 unless otherwise indicated.

sev minigene type A. The region from pos. +33 (SnaBI site) to pos. 12 259(ClaI) of the genomic sev EcoRI fragment is replaced with the correspondingcDNA sequences of cK5.1 and cED3. 1.

sev minigene type B. The region from pos. +507 (SspI) to pos. 12 259(ClaI) of the genomic sev EcoRI fragment is replaced with the correspondingcDNA sequences of cDNA clone 10 and cED3. 1; it differs from the sevminigene type A in the length of the first exon (715 as opposed to 107 bp).

sev minigene with first intron. The region from pos. 2814 (Salt) to pos.12 259 (ClaI) of the genomic sev EcoRI fragment is replaced with thecorresponding cDNA sequences of cK5.1 and cED3. 1.

hsp-sev cDNA type A. A complete sev cDNA segment (exon A type) isjoined in its 5' region to the hsp7O promoter/leader fragment at pos. +33(SnaBI) and in the last exon fused to the corresponding 3' genomicregion [pos. 14 503 (EagI) to 15 379 (EcoRI)] to provide the endogenoustermination/processing sequences.

hsp-sev cDNA type B. A complete sev cDNA segment (exon B type) isin the 5' region joined to the hsp7O promoter/leader fragment at pos. 86

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K.Basler, P.Siegrist and E.Hafen

(EcoRV) and in the last exon fused to the corresponding 3' genomic region[pos. 14 503 (EagI) to 15 379 (EcoRI)].

Constructs 201 and 202 (internal fragment upstream of hsp-sev cDNA).A genomic EcoRV fragment (pos. 86-8332) was inserted at the 5' endof the hsp-sev cDNA type A in original and reverse orientation,respectively. The transformation vector was pDM30.

Constructs 203 and 204 (second intron upstream of hsp-sev cDNA). Agenomic XhoI fragment (pos. 3120-6347) was inserted at the 5' end ofthe hsp-sev cDNA type A in original and reverse orientation, respectively.The transformation vector was pDM30.

Construct 224 (inverted in the third andfourth introns upstream ofhsp -sevcDNA). A genomic XhoI fragment (6347 -7564) was inserted at the 5' endof the hsp-sev cDNA type A in reverse orientation.

P-lacZ-sev reporter construct. The genomic sev fragment [pos. 2883(XbaI) to 8332 (EcoRV)] was inserted into the XbaI site of the P-elementinsertion vector P-lrB (Bellen, Wilson and Gehring, unpublished data)downstream of the lacZ reporter gene in the original 5' to 3' orientation.P-lrB contains the rosy marker gene.

Germ-line transformationThe plasmid DNA was prepared for injection as described (Basler and Hafen,1988b) except that we used pUChspA2-3 (gift from Don Rio) as helperDNA for the constructs 201-204 and P-lacZ-sev. Embryos from thefollowing recipient strains were injected as described by Dudler and Travers(1984): w1118, sevd2 for pW8 derivatives, sevd2, ry506 for the pDM30 basedplasmids and ry506 for the P-lacZ-sev reporter construct. Transformantswere made homozygous or maintained over balancer chromosomes in asevd2 background except for the four P-lacZ-sev lines that were analyzedin a wild-type background.

For the mobilization of the type B minigene construct in the lineTsevIL33.2 that showed partial rescue, this strain was crossed to thejumpstarter A2-3(99B) as described by Robertson et al. (1988).The chromosomal site of integration was determined by in situ hybridiza-

tion (see Basler and Hafen, 1988b) of the following transformed lines:TsevIL33.2 (61D); hsp-sevA.ch21 (46E), hsp-sevA.ch4l (7D1 ,2),hsp-sevA.chSO (93B); hsp-sevB.41.2 (64F), hsp-sevB.91 (89B).

ReferencesBanerji,J., Olson,L. and Schaffner,W. (1983) Cell, 33, 729-740.Basler,K. and Hafen,E. (1988a) Trends Genet., 4, 74-79.Basler,K. and Hafen,E. (1988b) Cell, 54, 299-311.Basler,K. and Hafen,E. (1989) Science, 243, 931-934.Bienz,M., Saari,G., Tremml,G., Muiller,J., Zust,B. and Lawrence,P.A.

(1988) Cell, 53, 567-576.Bowtell,D.D.L., Simon,M.A. and Rubin,G.M. (1988) Genes Dev., 2,620-634.

Bowtell,D.D.L., Simon,M.A. and Rubin,G.M. (1989) Cell, 56, 931-936.Dudler,R. and Travers,A.A. (1984) Cell, 38, 391-398.Francescini,N. (1975) In Snyder,A.W. and Menzel,R. (eds), Photoreceptor

Optics. Springer-Verlag, Berlin, pp. 98-125.Gasch,A., Hinz,U. and Renkawitz-Pohl,R. (1989) Proc. Natl. Acad. Sci.

USA, in press.Grosschedl,R. and Baltimore,D. (1985) Cell, 41, 885-897.Hafen,E., Basler,K., Edstroem,J.E. and Rubin,G.M. (1987) Science, 236,55-63.

Hiromi,Y., Kuroiwa,A. and Gehring,W.J. (1985) Cell, 43, 603-613.Hultmark,D., Klemenz,R. and Gehring,W.J. (1986) Cell, 44, 429-438.Klemenz,R., Weber,U. and Gehring,W.J. (1987) Nucleic Acids Res., 15,

3947-3959.Mismer,D. and Rubin,G.M. (1987) Genetics, 116, 565-578.O'Hare,K. and Rubin,G.M. (1983) Cell, 34, 25-35.O'Kane,C.J. and Gehring,W.J. (1987) Proc. Natl. Acad. Sci. USA, 84,

9123 -9127.Ready,D.F. (1989) Trends Neurosci., 12, 102 -110.Riddihough,G. and Pelham,H.R.B. (1986) EMBO J., 5, 1653-1658.Robertson,H.M., Preston,C.R., Phillis,R.W., Johnson-Schlitz,D.M.,

Benz,W.K. and Engels,W.R. (1988) Genetics, 18, 461-470.Schneuwly,S., Klemenz,R. and Gehring,W.J. (1987) Nature, 325,

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Cell, 40, 805-817.Smale,S.T. and Baltimore,D. (1989) Cell, 57, 103-113.Tomiinson,A., Bowtell,D.D.L., Hafen,E. and Rubin,G.M. (1987) Cell,

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Received on March 29, 1989; revised on April 27, 1989

Analysis of transformed linesRescue of the sev mutant phenotype was assessed by examining thepseudopupils of adult flies of the appropriate transformed strain by the opticalneutralization technique (Franceschini, 1975). Heads were bisected by asagital cut with a razor blade, immobilized with brain juice and coveredwith oil to optically neutralize the cornea. Antidromic illumination of thecompound eye mounted on a goniometric microscope stage permitted thedetection of the presence or absence of the R7 cell in the entire retina.

Flies were raised at 25°C prior to analysis. The germline transformantsbearing an hsp70 promoter construct were analyzed in addition afterdeveloping under conditions of repeated heat shocks (30 min at 37°C every6 h in a programmable heat shock apparatus). Histological sections throughthe eye of some transformants were prepared as described previously (Baslerand Hafen, 1988b).For the analysis of the expression of sev protein in eye imaginal discs

we used the polyclonal goat antiserum g15-4 which was raised against abacterial protein corresponding to amino acids 45-1027 of the sev aminoacid sequence (M.Briggs, K.Basler, E.Hafen, unpublished). The immuno-histochemical staining reaction on the eye discs was performed as describedpreviously (Basler and Hafen, 1988b).

13-Galactosidase staining in imaginal discs was essentially as describedby Simon et al. (1985); the staining reaction was carried out at roomtemperature for <1 h. The discs were then dehydrated through a gradedseries of ethanol and mounted in DPX (BDH Chemicals, UK).

AcknowledgementsWe thank our colleagues M.Bienz, M.Aebi, A.Fritz and B.Dickson forhelpful discussions and comments on the manuscript, D.Yen for technicalassistance, and R.Wehner for providing the goniometric microscope stage.We also thank the students of the EWB Course, 1989, for injecting construct224. This work was supported by grants from the Swiss National ScienceFoundation and the Roche Research Foundation.

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