Development 114, 877-886 (1992)Printed in Great Britain © The Company of Biologists Limited 1992

877

Function of an Ultrabithorax minigene in imaginal cells

JAMES CASTELLI-GAIR2, JURG MULLER1 and MARIANN BIENZ1

1 Zoological Institute, University of Zurich, Wintenhurerstrasse 190, 8057 Zurich, Switzerland2Centro de Biologla Molecular, Universidad Autdnoma de Madrid, Canto Blanco, 28049 Madrid, Spain

Summary

An Ultrabithorax (Ubx) minigene constructed from threekey Ubx control regions is capable of supportingdevelopment of Ubx null mutants throughout larval lifeand beyond to pharate flies, thereby rescuing the larvallethality due to the homeotic mutation. The cuticle ofthese flies shows that the minigene provides at leastpartial Ubx function in each of the four compartmentswhose morphogenetic pathways are determined by Ubx.We analyse /J-galactosidase patterns in imaginal discsconferred by each individual Ubx control region. Fromthe comparison of these patterns with Ubx expression in

Cbx mutants, we infer that long-range repressor el-ements in the chromosomal Ubx gene play an importantrole in the generation of Ubx expression patterns inimaginal discs. Expression and function of our Ubxminigenes indicate that Ubx control regions are capableof functioning properly out of context and detached fromtheir normal chromosomal location within the homeoticgene complex.

Key words: Drosophila homeotic gene, imaginal discs,long-range repression.

Introduction

The Ultrabithorax (Ubx) gene belongs to the group ofhomeotic genes in Drosophila whose function it is tocontrol the morphogenesis of various position-specificstructures in the embryo, the larva and the adult(Lewis, 1963, 1978; S£nchez-Herrero et al., 1985a;Teugels and Ghysen, 1985; Hooper, 1986; Bienz andTremml, 1988). It is one of three genes located withinthe bithorax complex (S&nchez-Herrero et al., 1985b)and it extends over more than 130 kb (Bender et al.,1983). Ubx transcripts first appear in the early embryowithin a domain restricted along the anteroposterioraxis (Akam and Martfnez-Arias, 1985). Although thepattern of Ubx expression becomes modified sub-sequently, its limits along the axis are maintainedthroughout development (Akam, 1983; White andWilcox, 1984, 1985a; Beachy et al., 1985).

The main realm of Ubx function comprises paraseg-ments (ps) 5 and 6 in the epidermis (Lewis, 1963, 1978;Morata and Garcfa-Bellido, 1976; Morata and Ker-ridge, 1981; Struhl, 1981, 1984; Hayes et al., 1984;SSnchez-Herrero et al., 1985a; Casanova et al., 1985)and in the nervous system (Teugels and Ghysen, 1985;Weinzierl et al., 1987). Minor effects of Ubx mutationcan be observed also in more posterior segments of thelarval epidermis (Lewis, 1978; Bender et al., 1983;Struhl, 1984). Internally, Ubx exerts a control functionin abdominal segments 1-5 in the larval somaticmesoderm (Hooper, 1986), in ps7 in the embryonicvisceral mesoderm (Bienz and Tremml, 1988) and,indirectly, in the endoderm (Immergliick et al., 1990).

Absence of Ubx function is not lethal for individualcells, though animals lacking Ubx function usually dieas young larvae (Lewis, 1978), perhaps due to cumulat-ive defects in different germ layers. However, clones ofhomozygous Ubx~ cells survive till adulthood and formadult cuticular structures (Lewis, 1963; Morata andGarcia-Bellido, 1976; Morata and Kerridge, 1981;Miflana and Garcfa-Bellido, 1982). From studies ofthese Ubx~ clones, it was concluded that, in theabsence of Ubx function, adult cuticular structuresbelonging to ps5 and 6 (T2p-T3a-T3p-Ala) weretransformed into those of ps4 (Tlp-T2a; reviewed byS^nchez-Herrero et al., 1985a; T and A, thoracic andabdominal segments; a and p, anterior and posteriorcompartments). As a rule, Ubx function is requiredcontinuously throughout development (Morata andGarcfa-Bellido, 1976), except for T2p and, to someextent, T3p in which Ubx function becomes dispensableafter an early embryonic stage (Morata and Kerridge,1981; Casanova et al., 1985). This early Ubx require-ment in T2p and T3p was termed postprothorax (ppx+)function (Morata and Kerridge, 1981).

In an attempt to reconstruct the embryonic Ubxexpression pattern, we identified three key controlregions in remote areas of the Ubx gene which, uponcombination, are capable of directing a Ubx-like /3-galactosidase (^-gal) expression pattern in transformedembryos (Miiller and Bienz, 1991). We found that thesecontrol regions, if linked to a Ubx cDNA in a minigeneconstruct, confer Ubx function in the larval epidermis.Here, we ask whether these control regions are alsocapable of supporting /3-gal expression in imaginal disc

878 /. Castelli-Gair, J. Mailer and M. Bienz

cells. We test whether the same minigene can provideUbx function in the adult epidermis.

Materials and methods

PlasmidsThe basic /3-gal and Ubx minigene constructs used fortransformation were previously described (Bienz et al., 1988;Miiller and Bienz, 1991; Mliller, 1991). Details of maps areavailable on request.

Fly strainsen; ry42 flies were used for P-element transformation, andtransformant lines were obtained as described (Bienz et al.,1988). In 2 of the 11 newly isolated BPuA lines (T2 and T8),the minigene transposon is inserted in the chromosomal Ubxgene, disrupting endogenous Ubx function (to be describedelsewhere). The U12 transposon is inserted near the fushitarazu gene (D. Yen and J. M., unpublished results);however, none of the other transposons analysed is insertednear a homeotic gene complex (U81 maps to the thirdchromosome at a distance from the Ubx gene of at least 50Morgans; T7 and T10 map to the second chromosome).

Recombinant chromosomes bearing a minigene transposonand the Ubx1 mutation were constructed (using a ry Ubx1

recombinant in which at least 3/4 of the original Ubx1

chromosome was replaced), and balanced strains wereestablished by standard genetic procedures. These werecrossed with balanced strains containing various Ubx mu-tations (see text), and non-balancer offspring flies {Ubxhomozygotes or transheterozygotes bearing a minigene) wereanalysed. Control crosses were done with a Ub^fTMl strainwhose original Ubx1 chromosome was "cleaned up" byrecombination (J. C.-G., unpublished). All Ubx mutationshave been described (Lewis, 1963, 1982).

To determine the lethality of Ubx1 mutants, crosses weredone, as described above, and homozygous Ubx larvae,recognisable by their extra spiracles (Lewis, 1978), werecounted at various stages. We could not find any of thesehomozygous Ubx~ larvae which survived beyond the earlyfirst instar. Homozygous Ubx larvae bearing a minigene canbe recognised by their denticle belt pattern (Milller andBienz, 1991).

Staining of imaginal discs and analysis of adult cuticleInverted anterior halves of third instar larvae (typicallyhomozygous for their transposon insert) were prepared inRinger's solution, rinsed in phosphate-buffered saline (PBS)and fixed for 2 minutes in 1% glutaraldehyde in PBS. Afterthorough rinsing in PBS, they were incubated in /3-gal stainingsolution (Bienz et al., 1988) for two hours. Stained discs wereseparated from the larval remains, dehydrated, passedthrough methylsalicylate and mounted as described forembryos (Bienz et al., 1988). The location of /3-gal staining inposterior parts of discs was confirmed by /3-gal staining oflarvae obtained from a cross between ABP transformantswith an engrailed//3-gal transformant line (in the latter, /3-galstaining is restricted to posterior compartments; Hama et al.,1990). Staining with a monoclonal antibody against Ubxprotein (White and Wilcox, 1984) was done as described(CasteUi-Gair et al., 1990). Analysis of adult cuticle was doneby following standard procedures (Morata and Garcfa-Bellido, 1976).

Results

In this study, we use the same Ubx fragments from thePBX, ABX and BXD control regions whose maps andregulatory properties we have described (Miiller andBienz, 1991). Briefly, the PBX and BXD fragments arederived from remote regions upstream, the ABXfragment from an intronic region downstream of theUbx transcription start site, as sketched out in Fig. 1. Ofthese fragments, PBX and ABX direct an early patternof /3-gal expression which is restricted to the Ubxdomain along the anteroposterior axis of the embryoand strongest anteriorly within ps6 and ps5, respect-ively. The BXD fragment is activated at a laterembryonic stage to direct a pattern extending from

•51 <-47 -10-15 -28-34

flposition In kb fromtranscription start

I /I /

+H-I5 Ft R X 3 V R R Ft

genomicsubclones

ABX BXD PBX

B

B

Fig. 1. Map of Ubx control regions and plasmids. (A)Maps of PBX, ABX and BXD fragments (genomicsubclones; B, BamHI; P, Pstl; R, £coRI; S, Sa/I; V,£coRV; X, Xhol; all sites shown in the case of B, R, S)and their position with respect to the Ubx transcriptionstart site (transcription from right to left). Fragments usedin our constructs are hatched; in the case of ABX andBXD, these correspond to the minimal fragmentsconferring embryonic expression (Muller and Bienz, 1991).The imaginal PBX pattern is due to sequences downstreamof the BamHI site within the PBX fragment (the 5' end ofthe genomic subclone 3104; see text), whereas sequencesupstream of this BamHI site do not direct any expressionin imaginal discs (although they contain the minimal PBXfragment conferring embryonic expression; Miiller andBienz, 1991). (B) Maps of the two types of minigenes(ABP and, underneath, BPuA; Xb, Xbal; K, Kpnl; otherrestriction sites as in Fig. 1A; all sites shown except forXhol). Open boxes, Ubx coding region; thick lines, Ubxcontrol regions (see Fig. 1A); thin lines, Ubx sequencesflanking the Ubx coding region (transcription start site anddirection of transcription indicated by arrow). Orientationof fragments same as in Ubx gene.

Ultrabithorax minigene in imaginal cells 879

head to tail which resembles the Ubx expression patternin abdominal segments.

Most transformants used for this analysis have beenisolated previously (including the minigene-bearingtransformant lines U12 and U81; Miiller and Bienz,1991). In addition, we constructed a second type ofminigene in which the ABX fragment is placeddownstream of the coding region (BPuA; Fig. 1). Weisolated 11 individual lines of which 5 showed a strongChr'-like phenotype (Fig. 3D), like the U12 and U81lines, a phenotype that we presumed to be caused byminigene-derived ectopic Ubx+ activity (Miiller andBienz, 1991). We took this phenotype as an indicationof high minigene efficacy and chose two of these lines(T7 and T10) for further analysis.

Imaginal disc patternsWe stained imaginal discs from late third instar larvaeof PBX, ABX and BXD transformants for £-galactivity. We found that neither the Ubx proximalpromoter by itself (not shown) nor the BXD fragmentlinked to the Ubx proximal promoter (Fig. 2A,B) iscapable of directing any substantial /3-gal expression inimaginal discs. In contrast, both the ABX (Fig. 2C-F)and the PBX fragment (Fig. 2G-K) linked to the Ubxproximal promoter confer patterns of strong /3-galexpression in individual imaginal discs as follows.

/3-gal expression in ABX transformants is restrictedto the dorsal discs, the wing (Fig. 2C) and the halteredisc (Fig. 2D); neither of the leg discs (Fig. 2E,F) norany other disc show /3-gal staining. Staining in the wingand the haltere disc is very strong in their centers, their"pouch" regions, which give rise to the most distalderivatives of these discs, the wing blade and the halterecapitellum (reviewed by Bryant, 1978). In some but notall transformant lines, we also see strong yS-gal ex-pression in the stalk region of the wing disc (Fig. 2C)and, very occasionally, a strong /5-gal spot in the stalkregion of the haltere disc, the regions giving rise to themeso- and metanotum. Expression in the "pouch"regions of the two discs is regularly observed andequally strong in virtually all ABX lines, although theextent of the "pouch" expression varies slightly (insome lines restricted to the most central regions; cf. Fig.3A). We shall refer to this expression in the wing andhaltere "pouches" as the imaginal ABX pattern. Weobserve it independently of the type of ABX construct,i.e. whether the latter contains the large genomic 3.0 kbor the minimal 0.6 kb ABX fragment, and whether theminimal fragment is located upstream or downstream ofthe fusion gene (B/3A transformants; cf. Miiller andBienz, 1991).

The imaginal ABX pattern shows very little resem-blance to Ubx protein expression in imaginal discs(White and Wilcox, 1984, 1985a). However, thestaining in the wing "pouch" is reminiscent of theectopic Ubx expression pattern in wing discs of Hm anda>x*"mutants (Fig. 3A,E; Cabrera et al., 1985;GonzSlez-Gaitdn et al., 1990). In Hm (Bender et al.,1983) and probably in Cbx*41 mutants (Gonz&lez-Gait&n et al., 1990), most of the upstream flanking

region of the Ubx gene is lacking and, thus, the mutantUbx expression patterns may reveal the intrinsic activityof control regions downstream of the promoter (e.g. ofthe ABX fragment), a notion strongly supported by ourimaginal ABX pattern.

We initially obtained only one transformant line withthe large genomic PBX fragment. Two further lineswere generated by "jump-starting" (Robertson et al.,1988); however, one of these lines showed /3-gal stainingin all discs, whereas the other showed none. We shallrefer to the /3-gal staining pattern in the initial line as theimaginal PBX pattern since we observe virtually thesame pattern in other transformants carrying part or thewhole of the large PBX fragment (see below). Theimaginal PBX pattern consists of strong /3-gal ex-pression in the wing and in the haltere discs (Fig. 2G,H)and of a comparatively low but significant level of /3-galexpression, evenly distributed, in all three types of legdiscs (Fig. 2H-K). There is also yS-gal expression in theeye and in the humeral disc (not shown). /3-gal stainingin the haltere disc is usually observed throughout thedisc (Fig. 2H), with occasional white patches in theanterior part. In the wing disc, /3-gal staining is confinedmostly to the posterior part, with an anterior expressionboundary forming a striking line across the disc (Fig.2G), a line reminiscent of the anteroposterior compart-ment boundary (Brower, 1986). The /3-gal patternvaries somewhat between individuals as we frequentlyobserve /3-gal patches in the anterior part (Fig. 3B). Onoccasions, the whole disc is stained except for a whiteline approximately following the anteroposterior com-partment boundary (Fig. 3C). Surprisingly, we do notsee the imaginal PBX pattern in transformants contain-ing the minimal PBX control region (between theBamHI and the Pstl site; see Fig. 1) which is sufficientto confer the embryonic PBX pattern (Miiller andBienz, 1991), but we find a very similar pattern inimaginal discs of transformants containing a moreproximal genomic fragment (3104; see Fig. 1) whichpartly overlaps the large PBX fragment. Thus, theregulatory sequences conferring the imaginal PBXpattern are closely linked to, but separable from (anddownstream of) the sequences conferring the embry-onic PBX pattern.

The imaginal PBX pattern resembles the Ubx proteinpattern in Cbx1 mutants, particularly within the wingdisc (Fig. 3; White and Akam, 1985; Cabrera et al.,1985; see also Castelli-Gair et al., 1990). Variablepatterns of Ubx protein expression are observedbetween individual Cbx1 mutants (Fig. 3F,G), verysimilar to the variable /S-gal patterns of PBX transfor-mant individuals (Fig. 3B,C). The Cbx1 mutantchromosome bears a duplicated copy of an upstreamgenomic fragment at an intronic location closer to anddownstream of the Ubx transcription start site (—1-13kb; Bender et al., 1983). The transposed piece ofgenomic DNA contains our large PBX fragment.Evidently, the pattern of ectopic Ubx protein ex-pression in Cbx1 wing discs reflects the intrinsiccapability of the PBX control region to direct thisparticular pattern of expression.

880 J. Castelli-Gair, J. Mutter and M. Bienz

B

• & • •

BXD

Fig. 2. /S-gal staining patterns in imaginal discs. Wing discs(first column), haltere and third leg discs (second column,haltere disc to the left; D, only haltere disc), second legdiscs (third column) and pairs of first leg discs (fourthcolumn), dissected from late third instar larvae of BXD(A,B), ABX (C-F), PBX (G-K) and ABP (L-O)transformants and stained for /3-gal acticvity. White asterisksindicate "pouch" staining in wing and haltere discs.Arrowhead, anterior expression boundaries, apparentlyfollowing anteroposterior compartment boundaries in secondleg and, partly, in wing discs. Note the /J-gal staining in allthe leg discs of PBX transformants which is slightlyincreased, but restricted to ps5 and 6 in leg discs of ABPtransformants. Anterior to the left.

ABX

PBX

ABP

Finally, we examined transformants of a triplecombination construct, bearing all three control regionsupstream of the Ubx proximal promoter (ABP; Miillerand Bienz, 1991). The majority of these ABP lines show

a /S-gal-staining pattern in the dorsal imaginal discswhich corresponds to a PBX pattern superimposed onan ABX pattern (Fig. 2L,M), best visible in the wingdisc where /J-gal staining in the "pouch" protrudes

Ultrabithorax minigene in imaginal cells 881

Fig. 3. Cbx expression patterns and phenotypes. Similarities between expression patterns in the wing disc from an ABXtransformant (A; a different line from the one in Fig. 2 is shown in which /3-gal staining is restricted to the center of the"pouch" and absent from the stalk region; see text) and from HmJ+ mutants (E; disc stained with Ubx antibody) as wellas between the variable PBX patterns (B,C; jS-gal staining) and the variable Ubx protein expression patterns in CbxJ/+mutant individuals (F,G). Wing phenotypes observed in U12 transformants (D; virtually the same phenotype is alsoapparent in U81, T7 and T10 transformants) and in Cbx1/+ mutants (H).

across the PBX expression boundary into the anteriorpart of the disc. Compared to the wing disc pattern inPBX transformants which extends variably into theanterior compartment, we note that )S-gal staining inABP transformants is more regularly restricted to theposterior part of the disc, reflecting some degree of non-additivity of patterns. More striking non-additivity isobserved in the leg discs: the first leg discs in ABPtransformants do not stain at all (Fig. 2O), and /3-galstaining in the second leg discs is restricted to theposterior half (Fig. 2N). There is moderately strong /S-gal staining in the whole of the third leg discs (Fig. 2M).As in the wing disc, the /S-gal expression boundaryin the second leg disc apparently follows the antero-posterior compartment boundary (cf. Steiner, 1976).There is virtually no yS-gal expression anterior to theanteroposterior compartment boundary in T2 discs.This boundary corresponds to the anterior boundaryof ps5 and thus to the anterior limit of Ubx ex-pression.

The imaginal ABP pattern closely resembles thepattern of Ubx protein expression in imaginal discs(White and Wilcox, 1984, 1985b), although it differsfrom the latter in two main respects. Firstly, there isectopic /3-gal expression in the wing disc which probablycauses the Cta'-like phenotype of our minigenetransformants. Secondly, the levels of /3-gal staining in

the leg discs are somewhat low, suggesting that the^ABPconstruct lacks enhancer sequences required for ef-ficient expression in ventral discs.

Minigene function in individual imaginalcompartmentsSince an ABP minigene (all three fragments linked to aUbx cDNA; Miiller and Bienz, 1991) is capable ofconferring a Cbx1 phenotype through its activity in thewing disc (Fig. 3D), we wondered whether thisminigene might be sufficiently active in other imaginaldisc cells to rescue aspects of the adult Ubx phenotype.For this, we established balanced strains containing aminigene transposon (U12, U81, T7 or T10) and theUbx mutation (an antibody-negative null mutation;Weinzierl et al., 1987; see Material and Methods). Wefirst tested the function of these minigenes in abx, bx,pbx and bxd mutants. Flies carrying either of thesemutations in combination with Ubx1 are viable; how-ever, they show transformations of individual compart-ments in their epidermis: T3a is transformed into T2a inabx/Ubx or in bx/Ubx mutants, T3p is transformed intoT2p in pbx/Ubx mutants, whereas both T3p and Alaare transformed into T2p and T3a in bxd/Ubxmutants (reviewed by Sdnchez-Herrero et al., 1985a;the T2p>Tlp transformation caused by abx'/Ubx1 or

882 J. Castelli-Gair, J. Miiller and M. Bienz

bx3/Ubx1 mutation cannot be discerned in the aboveallelic combinations).

Homeotic transformations in the haltere disc deriva-tives (dorsal T3a or T3p) which are caused by abx1bx3

(Fig. 4A) or by pbx1 mutation (Fig. 4C) are mostly and,in the case of pbx1 mutation, virtually fully rescued byone copy of either of the minigenes (Fig. 4B,D; U12and U81 minigenes show slightly better rescue activitythan T7 and T10 minigenes). The rescue activity isparticularly strong in the distal region of T3, the

haltere, but there is also some rescue activity in theproximal region, the notum. This activity is particularlystriking in the case of the four-winged triple mutantabx1 bx3 pbx1 (Fig. 4E) whose second pair of wings arereverted towards normal halteres and whose duplicatedmesonotum is partly suppressed, due to minigenefunction (Fig. 4F). However, these flies have two pairsof T2 legs (not shown) and, thus, we do not observe fullrescue function of the minigene in the third legs. This issomewhat surprising, given that there is • /S-gal ex-

Fig. 4. Minigene function inindividual adult compartments.Adult phenotypes of differentmutant alleles (A,B,abx1bx3/UbxJ'; C,D, pbx'/Ubx1;E,F, abx1 bx3 pbx1; G,H,bxd'/Ubx1) in the absence (leftcolumn) or presence (rightcolumn) of a U12 minigene.(B) Partial rescue by theminigene of the T3a>T2atransformation, resulting in ahaltere-like structure (lackingmost wing tissue landmarks,except for the triple rowchaetae indicated byarrowhead) instead of a wing(arrowhead in A) and in thereduction of extra mesonotaltissue (marked by asterisks).(D) Virtually complete rescueof the T3p>T2ptransformation in the haltere(arrowhead indicates almostnormal haltere; compare towing-like structure in C,arrowhead), but very little ifany rescue of thistransformation in the posteriornotum (asterisks). (F) Highrescue activity of the minigene,as visualised by an almostcomplete reversal of thehaltere to wing transformation(arrowheads in E and F) aswell as by the reduction ofmesonotum tissue (asterisks inE and F). (H) Completesuppression of the extra pair ofAl legs (marked by blackarrow in G; white arrow in Hmarks the position where theAl legs normally arise in themutants). Numbers refer tosegmental identities of legs.The extent of minigene-dependent rescue variesbetween flies, although the Alleg suppression is observed inall individuals. For technicalreasons, the dorsal appendagesof T2 were removed in allcases except E.

Ultrabithorax minigene in imaginal cells 883

pression in the third leg discs of ABP transformants(Fig. 2M). It is possible that the level of expression inthe third leg discs is too low, due to lack of enhancersequences required for high levels of ventral discexpression.

In addition to affecting the haltere disc derivatives(T3p>T2p), bxd^Ubx1 mutant flies show an Ala>T3atransformation, having an additional pair of legs (Fig.4G), and lacking an Ala tergite. If these flies carry oneof our Ubx minigenes, their T3p>T2p transformation isrescued (not shown) and their extra pair of legs iscompletely suppressed (Fig. 4H). However, they stilllack the Ala tergite and, thus, the minigenes appar-ently lack sequences mediating Ubx expression necess-ary for tergite development.

Rescue of Ubx null mutationThe recombinant strains containing both a Ubx1

mutation and a Ubx minigene allowed us to test therescue activity of this gene in Ubx1 homozygotes.Homozygous Ubx1 larvae, obtained from a controlcross, died shortly after eclosion from the embryonicmembranes (see Materials and methods; cf. also Lewis,1978; Frayne and Sato, 1991). However, we find thatone copy of a Ubx minigene can support developmentof Ubx homozygotes throughout larval life: a relativelylow, but significant fraction of these homozygotes(more individuals in the case of T7 and T10, comparedto U12 and U81) survive to the end of pupation andsecrete adult epidermis which is fully differentiated.The morphological features of these pharate adults canbe examined. Rescue of the larval lethality of Ubx nullmutation demonstrates that our Ubx minigenes arecapable of providing all Ubx function required for larvaland pupal viability.

We peeled from their pupal case Ubx1 homozygousor Ubx1/Ubx195 pharate flies containing one of ourminigenes and prepared their cuticle for microscopicanalysis (Fig. 5; there was no difference in rescueactivity in the two cases of allelic Ubx combinations). Inagreement with our previous results, the T3>T2transformations of the haltere derivatives are partiallyrescued by the minigenes (Fig. 5A,C,E), particularly inthe distal region, and the fourth pair of legs iscompletely suppressed (Fig. 5D,F). Again, the T3>T2transformation in the legs is not rescued (Fig. 5D,F),and the Ala tergite is also lacking (Fig. 5C,E).However, we find that the ppx phenotype (theT2p>Tlp transformation) in the legs is fully rescued(Fig. 5B,D,F), showing that our minigenes provideppx+ function.

Discussion

We have analysed expression and function of two typesof Ubx minigenes in imaginal disc cells. These mini-genes contain the same three Ubx control fragmentswhich, upon combination, confer a Ubx-like expressionpattern in the embryo as well as Ubx function in thelarval epidermis (Miiller and Bienz, 1991). We now find

that two of the three fragments also direct distinctpatterns of expression in imaginal discs, and that theircombination results in a pattern resembling Ubxexpression in these discs. Both types of Ubx minigenesare capable of providing all Ubx function needed forlarval viablity and pupal development as well as partialUbx function in the adult epidermis throughout the Ubxdomain. Certain control elements required for adultUbx function are still missing from these minigenes,which is not surprising as the initial screen for controlsequences was based entirely on their activity in theembryo. Nevertheless, at least some of the controlsequences conferring adult Ubx function are evidentlyclosely linked in the chromosomal Ubx gene to thoseconferring larval Ubx function.

Evidence for long-range repressor elementsIt has been proposed that the gain-of-function pheno-type of the Cbx1 mutation is due to ectopic Ubxexpression in the wing disc (Lewis, 1982), a predictionborne out by Ubx antibody staining results (White andAkam, 1985; Cabrera et al., 1985; Gonzalez-Gaitan etal., 1990). There remained the question as to why, upontransposition, an upstream ds-regulatory elementexpected to be active in ps6 and conferring pbx functionin T3p (Bender et al., 1983) should be activatedectopically in ps5 in Cbx1 mutants. To explain this,Peifer et al. (1987) proposed in their "open forbusiness" model the existence of parasegment-specificchromosomal domains which need to be "opened up"in a parasegment-specific way for enhancers locatedwithin these domains to become active. According tothis model, enhancers from the pbx region are activeectopically in ps5 in Cbx1 mutants, because of their newlocation within a chromosomal domain which is"opened up" in ps5.

Our results seem to argue against this view. The Ubxexpression pattern in Cbx1 mutant imaginal discsclosely resembles the /3-gal staining pattern conferredby the PBX fragment (which is contained within thetransposed DNA in Cbx1 mutants) and thus apparentlyreflects intrinsic PBX enhancer activity. This activity isovert in all our PBX-bearing transformants and there-fore does not seem to require any particular chromo-somal context (nor does it depend on the presence ofthe ABX control region). In the chromosomal Ubxgene, and also in a Ubx//3-gal construct containing 35 kbof Ubx upstream flanking sequence (Irvine et al., 1991),the intrinsic PBX activity in ps4 and ps5 is evidentlysuppressed. We suggest that there is an upstreamrepressor element in the Ubx gene, located between ourPBX fragment and the Ubx promoter, which acts tosuppress the PBX enhancer activity in ps4 and ps5.Whether this activity is suppressible by the upstreamrepressor may be determined by the position of the tworegulatory elements with respect to each other and tothe Ubx promoter, e.g. whether they are located on thesame or opposite sides of the promoter, or perhaps bytheir relative distance from the promoter (for adiscussion of distance affecting competition for pro-moter interaction between remote transcription factors,

884 /. Castelli-Gair, J. Miiller and M. Bienz

Fig. 5. Minigene rescue activity in Ubx mutations. Phenotypes of homozygous Ubx~ pharate adults (A-D, Ubx1

homozygotes; E,F, Ubx'/Ubx' ) in the presence of a U12 (A,B) or T7 (C-F) minigene. Dorsal (left column) and ventralaspects (right column) of the same flies are shown. Highest rescue activity of the minigene is visible in the halteres(arrowheads) and in the complete suppression of extra Al legs (white arrow in D and F). Full rescue of the ppx phenotypein the legs (B,D,F), apparent by the lack in T2p of long bristles typical for Tip (arrowheads in B). Numbers refer tosegmental identities of legs. The lack of an Ala tergite (asterisks in C and E) is not rescued by the minigene. Dorsal T2appendages (showing the Cbx1-\ike phenotype; cf. Fig. 3) were not removed in A and E.

see Hanscombe et al., 1991). We previously found asimilar repressor element within the PBX fragmentitself (mediating long-range repression anterior to ps6;Miiller and Bienz, 1991), but the latter is evidently onlyeffective in the embryo and not in imaginal cells.

Hm and Cbx1*1 mutants show ectopic Ubx proteinexpression in the wing disc "pouch" (White and Akam,1985; Cabrera et al., 1985; Gonzalez-Gaitan et al.,1990) which resembles the /3-gal expression patternconferred by the ABX fragment. It is likely that themutant ectopic Ubx expression reflects the intrinsicenhancer activity of the ABX fragment, and that thisintrinsic activity is normally suppressed by an upstreamrepressor element (which is uncoupled from the Ubxpromoter in the mutants). This repressor element maybe the same as the one mentioned above that suppressesthe PBX enhancer activity in ps4 and ps5.

We found that the imaginal ABP pattern does not

correspond to a simple superimposition of imaginalABX and PBX patterns: the anterior ABP expressionlimit to a large extent follows the anterior boundary ofps5, although the PBX fragment on its own mediatesexpression anteriorly to this limit, especially in the legdiscs. It thus appears that the ABX fragment contains along-range repressor element (suppressing PBXenhancer activity in the leg discs and perhaps in thewing discs anteriorly to ps5). Again, we found that theABX fragment does contain such a repressor elementwhich, in the embryo, mediates an anterior expressionboundary between ps4 and ps5 (Mtiller and Bienz,1991). Although the imaginal disc repressors may bedifferent from the embryonic repressors (one of whichis the hunchback protein; Zhang et al., 1991; Zhang andM.B., unpublished), their action may depend, directlyor indirectly, on the preceding action of embryonicrepressors.

Ultrabithorax minigene in imaginal cells 885

Ubx minigene function affecting imaginal discprimordiaTwo features of the adult Ubx phenotype are appar-ently fully rescued by our Ubx minigenes: the ppxphenotype (the T2p>Tlp transformation) and theformation of a fourth pair of legs. We propose, for thefollowing reasons, that rescue activity in these casesreflects minigene-dependent deployment of Ubx in theembryo, either within imaginal disc primordia orsuppressing the formation of these.

The ppx+ function is a subfunction of Ubx which isrequired exclusively early in development to control themorphogenesis of T2p and in T3p in the adult (Morataand Kerridge, 1981; Casanova et al., 1985). In theabsence of ppx+ function, i.e. if Ubx product iseliminated within the first 6 hours of embryonicdevelopment, these two posterior compartments aretransformed into Tip, a transformation rescued by ourUbx minigenes. We note that the imaginal domainswithin which ppx+ function is required correspond tothe embryonic domains within which ABX- and PBX-mediated expression is highest (anterior part of ps5 andps6, corresponding to T2p and T3p; Miiller and Bienz,1991). Our evidence strongly suggests that this ex-pression anteriorly within ps5 and ps6 is due to directactivation of the ABX and PBX control regions by thesegmentation products even-skipped (eve) and fushitarazu (ftz), respectively (Miiller, 1991; Miiller andBienz, in preparation). It therefore appears likely thateve- and /iz-mediated Ubx expression in the earlyembryo confersppx+ function. Among the cells that weexpect to be supplied with this pulse of eve- and ftz-mediated Ubx expression are those forming the pro-spective posterior compartments of the T2 and T3imaginal disc primordia as the latter become visible atthis time, straddling parasegment boundaries (Cohen,1990; Cohen et al., 1991). This early Ubx protein mayfade later to become virtually undetectable in the wingdiscs (White and Wilcox, 1984, 1985b) as these are themost actively proliferating discs (Bryant, 1978).

A fourth pair of legs is formed in bxd mutants (Lewis,1963), apparently reflecting establishment of an ad-ditional pair of disc primordia in the embryo (Cohen etal., 1991). This extra pair of legs is completelysuppressed by our Ubx minigenes. The minigenes alsosuppress the extra pair of Keilin's organs (correspond-ing to rudimentary larval legs) due to Ubx mutation, anactivity which we ascribe to the BXD control element(Miiller, 1991). It therefore appears that BXD-me-diated Ubx expression may suppress the establishmentof larval and adult leg primordia in the embryo. Thelatter do not seem to be suppressed by PBX- and ABX-mediated Ubx expression (see above); however, thePBX and ABX control regions are active at differenttimes and levels as well as in different cells of theembryo than the BXD control region (Miiller andBienz, 1991).

Our Ubx minigenes are not capable of supportingdevelopment of Ala tergites which are missing in Ubxmutants or in bxd mutants lacking Ubx activity in ps6.These mutants lack abdominal histoblasts, the tergite

precursor cells, in Al at young larval stages; instead,they show buds of dorsal and ventral imaginal discs(Kerridge and Sang, 1981; Frayne and Sato, 1991).Although the Ubx minigenes apparently suppress thedisc primordia in Al (see above), they obviously do notsupply sufficient Ubx function to support developmentof abdominal histoblasts in Al. It is conceivable thatprimordial histoblasts never form in minigene transfor-mant embryos. We think it more likely that these form,but that they cannot develop and proliferate (cf. Frayneand Sato, 1991), due to insufficient minigene function atlater stages.

ConclusionHomeotic gene complexes are strikingly conservedbetween flies and mammals (Duboule and Dolle, 1989;Graham et al., 1989). It has been speculated that thisconservation reflects functional significance (cf. Akam,1989), though in the case of the bithorax complex,integrity of the complex is not necessary for bithoraxgene function (Struhl, 1984). Functional importance hasalso been attributed to the size of homeotic genes inDrosophila which are unusually large, apparently dueto the complexity and redundancy of cu-regulatoryelements (Bender et al., 1985; Simon et al., 1990; Irvineet al., 1991). The latter may be needed to generate thecomplex pattern of Ubx expression, some aspects ofwhich however are functionally insignificant (Gonzalez-Reyes and Morata, 1990).

Our results seem to indicate that there is no absoluterequirement for homeotic genes to be located withinhomeotic gene complexes: Ubx minigenes are capableof functioning, out of context, in a number of differentchromosomal locations to provide all Ubx functionsneeded for embryonic and larval life as well as partialUbx function for adult morphogenesis. The minigenesfunction to a perhaps surprising level, despite contain-ing few and comparatively short control regions. Theirfunctional shortcomings in certain imaginal cells (in thelegs and the Ala tergite) are probably due to lack of thecorresponding control elements which might be identi-fyable by appropriate searching. Finally, our minigenesdo not function reliably from individual to individual, asjudged by the varying rescue activities observed amongindividuals in any one transformed line. Thus, largecontrol regions and/or redundancy of control elementsmay be required in some way to ensure reliability of thecontrol process.

We thank Gines Morata, Ernesto Sdnchez-Herrero, JordiCasanova, Maria Paz Capdevila and Antonio Garcfa-Bellidofor comments on the manuscript. We also thank AntonioGarcfa-Bellido for support while this work was completed inhis laboratory, and Charo Hernandez and Ana Lopez for helpwith the cuticle preparations. This work was funded byEMBO (short-term fellowship awarded to J. C.-G.) and bythe Swiss National Science Foundation (grant nr. 31-26198.89to M. B.).

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