Ectopic Expression of the Drosophila Homeotic Gene Proboscipedia Under Antennapedia P1 Control Causes Dominant Thoracic

Home , Antennapedia, Homeotic gene

Ectopic Expression of the Drosophila Homeotic Geneproboscipedia Under Antennapedia P1 Control Causes Dominant Thoracic Defects

David L. Cribbs,’ Angela M. Pattatucci,2 Mary Anne Pultz’ and Thomas C. Kaufman Howard Hughes Medical Institute, Institutefor Cellular and MolecularBiology, and Programs an Genetics and Cellular, Molecular and Developmental Biology, Department of Biology, Indiana University, Bloomington 47405 Manuscript received May 13, 1992 Accepted for publication July 17, 1992

ABSTRACT A deletion mutation in theAntennapedia Complex of Drosophilamelanogaster, Df(3R)SCBXL2, induces both dominant and recessive loss-of-function phenotypes. The deletion is associated with diminished function of proboscipedia (pb), a homeoticgene required for mouthpartsformation. Df(3R)SCBXL2also has associated dominant thoracic defects related to diminished expression of the homeotic Antennapedia (Antp) gene copy on the homologous chromosome. This is shown to be a consequence of ectopic pb expression in the thorax. Newly juxtaposed Antp sequences provide thepb gene on the deletion bearing chromosomewith a second promoter, Antp P1, in addition to its own. Ectopic pb protein expression occurs under Antp PI control, by alternate splicing, and results in diminished accumulationofAntp protein in the imaginal disc cellswhere Antp P1 is normally expressed. The analysis of this mutant chromosome thus demonstrates thatpb protein is capable of participating in the negative regulationof a more posteriorly expressedhomeotic gene, as well as servinga homeotic “selector” function in the head.

HE fruit fly Drosophila melanogaster is composed ROLL et al. 1986). In contrast in the head it appears T of a series of related but distinct segments or that the homeoticgenes are expressed in nonoverlap- parasegments. These reiterated pattern elements, the ping regions and that these exclusive domains do not outcome of a series of tightly coupled events in early resultfrom negative cross regulatoryinteractions embryogenesis [see AKAM 987)(1 and INGHAM (1 988) (MAHAFFEY,DIEDERICH and KAUFMAN1989; KAUF- for reviews], definethe domains of action of the MAN, SEEGERand OLSEN 1990). homeotic genes. The diversification of structures dis- The homeotic proboscipedia (fib) gene of the ANT- tinguishing one segment from another is believed to C is required to make adult mouthparts. Inits absence reflect homeotic gene regulatory functions directed a transformation of the labial palps to prothoracic legs toward “downstream” or “realisator” genes (GARCIA- occurs (BRIDGESand DOBZHANSKY1933; KAUFMAN BELLIDO1977; LEWIS1978; GOULDet al. 1990). In 1978; PULTZet al. 1988). A distinctly different adult Drosophila there are two major clusters of homeotic transformation of the labial palps to antennal aristae genes, the Bithorax Complex (BX-C) and the Anten- is seen in mutants which retain partial pb function. napedia Complex (ANT-C), which together control Consistent with the adult mutant phenotype, larval the segmental identity of most of the fly body [for expression of pb protein is detected in cells of the reviews, see PEIFER,KARCH and BENDER (1987), DUN- labial imaginal discswhich formthe adult labium CAN (1987), MAHAFFEYand KAUFMAN(1988), and (RANDAZZO,CRIBBS and KAUFMAN199 1). Embryonic KAUFMAN,SEEGER and OLSEN (1990)l.The genes of expression of pb protein is also observed in the labial the BX-C are required for specification of posterior and maxillary lobes of the gnathocephalon. These two thoracic and abdominal segments, while those of the segments are thelikely site of origin of the progenitor ANT-C are required in the anterior thorax and the cells of theadult head which are affected in pb- head. In the trunk, homeotic genes are expressed in animals (PULTZet al. 1988; JURGENS et al. 1986). overlapping domains and may interact among them- However, no embryonic or larval cuticular phenotype selves at thetranscriptional level to alter one another’s has been associated with the pb- condition (PULTZet pattern of expression (STRUHL1982; HAFEN,LEVINE al. 1988). and GEHRING 1984; STRUHL andWHITE 1985; CAR- The Antennapedia (Antp) gene of the ANT-C, in contrast, is required for mesothoracic identity both in ’ Present address: Centre de Biologie du Dkveloppement, Bitiment 4B3, 1 18 route de Narbonne, 3 1062 Toulouse Cedex,France. embryonic/larval and adult development (WAKIMOTO ‘ Present address: Laboratory of Biochemistry, Building 37, Room 4A13, and KAUFMAN 1981;STRUHL 1981; ABBOTTand National Institutes of Health, Bethesda, Maryland 20892. ’ Present address: Department of Biology, Western Washington Univer- KAUFMAN1986; MARTINEZ-ARIAS 1986). The Antp sity, Bellingharn, Washington 98225. protein is apparently sufficient to specify mesothoracic

Genetics 132: 699-71 1 (November, 1992) 700 L. D. Cribbs et al. identity; a conclusion drawn from genetic and molec- otherwise noted. Mutant strains and nomenclature are as in ular analyses of dominant gain-of-function Antp mu- LINDSLEYand ZIMM (1992).The Df(3R)SCBxL2chromosome was supplied by C. NUSSLEIN-VOLHARDand was recovered tations which transform head tissues to mesothorax in her laboratory as a deletion of the bicoid (bcd) locus of (e.g., see DENELL1973; HAZELRIGG and KAUFMAN the ANT-C. This deletion also removes the Antp, fushi 1983; FRISCHER, HACEN and GARBER1986; tarazu (ftz),Sex combs reduced (Scr),Deformed (Dfd),Amalgam SCHNEUWLY,KUROIWA and GEHRING1987; (AMA), and zerknullt (zen) loci as well as showing a partial SCHNEUWLY,KLEMENZ and GEHRING 1987).Antp also and variegating loss-of-function phenotype for the pb locus. In addition to the recessive lethal effects of the deletion it contributes to adult development of the prothorax, is associated with a dominant phenotype most readily seen notably the humeral callus and the mesonotum (AB- as a deletion of bristles and cuticle in the region of the BOTT and KAUFMAN1986). Two distinct promoters, humeral callus. This dominant effect closely resembles re- P1 and P2, direct Antp expression (LAUCHONet al. cessive loss-of-function phenotypes associated with certain 1986; SCHNEUWLYet al. 1986; STROEHER,JORGENSON alleles of the Antp locus (ABBOTTand KAUFMAN1986). Geneticreversion analysis: Males of genotype and GARBER 1986;JORGENSON and GARBER1987). Df(3R)SCBXL2/TM3,Sbwere placed overnight (about 16 hr) Whereas P2 is necessary from embryogenesis onwards in vials containing tissues wetted with 10% sucrose solution/ and is required in the ventral thorax, theP 1promoter 6 mM diepoxybutane (DEB) (OLSENand GREEN1982), or is required for adult viability and for proper devel- with ethyl methanesulfonate (EMS) using standard condi- opment of dorsal thoracic structures (WAKIMOTOand tions (BACHERand LEWIS1968). Males were then removed to fresh vials, allowed several hours to recover, then mated KAUFMAN1981; STRUHL 1981; ABBOTTand KAUF- with virgin pb‘”pp females. The weak pbI3 allele in combi- MAN 1986). nation with Df(3R)SCBXLZonly rarely gives a phenotype Loci homologous to the homeotic genes of the BX- which is visible under a dissecting microscope. F, progeny C and the ANT-C are united in a single complex in carrying the deficiency chromosome (i.e., Sb+)were screened various species including the flour beetle Tribolium for reversion of the humeral defects, and for labial defects indicating the introduction of a pb lesionon the castaneum, mice and humans (BEEMAN 1987; BEEMAN Df(3R)SCBXL2chromosome. The humeral defects associated et al. 1989; DUBOULEand DOLL^? 1989;GRAHAM, withthis chromosome were thoughtto becaused by a PAPALOPULUand KRUMLAUF1989; ACAMPORAet al. negative regulation of the Antp locus resident on the ho- 1989; KAPPEN, SCHUGHARTand RUDDLE 1989).The mologous chromosome. Thus “reversion” of the dominant protein products of the homeotic genes are seen to be phenotype should be associated with the inactivation of the entity on the deletion chromosome causing the shut down. potent developmental effectors through their known Candidates expressing either phenotype were recrossed to or inferred activities as transcriptional regulators.The pb” Pp to confirm the introduction of a new pb lesion and/ extraordinary conservationof the homeotic genecom- or a reversion and then balanced over TM3,Sb. The DEB- plexes seems likely to reflect a necessity to maintain a induced alleleswere first tested for complementation of fine balance of the homeotic genes’ functions and lethal labial mutations, the next most proximal member of the ANT-C, and stronger alleles of fib. The DEB induced hence their spatial, temporal and quantitative expres- alleles were also mapped by genomic Southern blots. The sion for proper development. A knowledge of what mutation frequency observed with DEB for pb was about interactions may comprise homeotic gene regulation, 0.02%and EMS was similarly efficient; hence the expected and how such interactions may be modulated, is thus frequency of chromosomes doubly hit at pb and any other central to our understanding of developmental con- second site should be on the order of 4 X Gene dosagetests: To vary the dosage of Antp+, an trol. additional copyof the ANT-C was introduced via the Y This paperdeals with the regulation of Antp expres- chromosome using Dp(Y;3)ANT-C+(a Y chromosome-linked sion by the pb locus of the ANT-C. We have analyzed translocation of the entire ANT-C, duplicated for polytene a deficiency mutation in which the Antp P1 promoter region 83DE-84D; obtained by R. DENELL,Kansas State is placed upstream of the homeobox genes pb and University). Males carrying the Dp(Y;3)ANT-C+and balan- 22. cer third chromosomes were crossed to Df(3R)SCBXL2/ This chromosomal rearrangement generates overlap- TM3,Sb females. The male progeny of this cross carrying ping chimeric transcription units in which two hom- the duplication andthe deletion (X/Dp(Y;3)ANT-C+ eotic gene promoters, pb and Antp P1, respectively ;Df(3R)SCBXL2/TM3)were collected after scoring (see below) directnormal pb expression in the head as well as and crossed to AntpW’*/TM3,Sbfemales. Males of the genotype Dp(Y;3)ANT-C+; Df(3R)SCBXL2/ANT-C+(with two cop- ectopic expression in a subset of the Antp domain in ies of Antp+) were compared to sibling females of genotype the thorax. The misdirected expression of pb protein Df(3R)SCBXLZ/ANT-C’carrying a single co of the ANT- is associated with adominant Antp loss-of-function C, or toDp(Y;3)Antp+; Df(3R)SCBxL2/Antp”~Ymales with two phenotype caused by a loss of Antp expression. Thus intact copies of all ANT-C genes except Antp+, which in this pb protein is apparently capable of modulating Antp genotype is present in one copy on the Y-linked duplication. expression, in addition to serving as a homeotic selec- GenomicSouthern analyses: Genomic DNAwas prepared as described by SCOTTet al. (1983). For genomic tor in its normal domain of expression. Southern blots, 5 pg of digested DNA were fractionated by size using electrophoresis through 0.8% agarose gels, par- MATERIALS AND METHODS tially depurinated in the gels by treatment with 0.1 M HCI, Stocks andculture conditions:Flies were raised on stand- denatured with alkali/high salt, neutralized and transferred ard cornmeal-molasses-brewer’s yeast medium at 25” unless to Nytran (Schleicher and Schuell) filter membranes. DNA pb Expression Under Antp Control 701 probes were prepared by oligonucleotide priming of puri- isolated and 3‘ end-labeled with [a-”P]dATP plus [(U-’~P] fied restriction fragments (FEINBERGand VOGELSTEIN1983) dGTP, and the strands separated by polyacrylamidegel or by nick translation of recombinant X phage. Hybridiza- electrophoresis (4% acrylamide, 0.08% bis-acrylamide, with tions were in high salt (MANIATIS,FRITSCH and SAMBROOK Tris-borate buffer; as in MANIATIS, FRITSCHand SAMBROOK 1982). 1982). Each hybridization contained 80,000 Cerenkov cpm Cloning of thefusion point of thedeficiency: The of single-stranded probe. Hybridizations and digestions with interpretation of the chromosome structure obtained by nuclease S 1 were as described above. genomic Southern mapping was confirmed by cloning the Antibody staining: Embryos were prepared and stained -15-kb fusion BamHI restriction fragment (seen in Figure as described in MAHAFFEYand KAUFMAN(1 987) and PULTZ 3, probes A and B). Genomic DNA from Df(3R)SCBXLZ/ et al. (1988). The rabbit polyclonal antibody usedin the TM3,Sb adults was digested with BamHI, ligated into the experiments shown here was raised against a lacZ/pb exon9 bacteriophage X vector EMBL3, and the fusion fragment fusion protein (GUOet al. 1984; CRIBBSet al. 1992), then was identified by probing duplicate filters with sequences purified by affinity chromatography on a column carrying from the first exon of Antp (4.0-kb EcoRI fragment located trpE/E9 protein (as described by DRIEVERand NUSSLEIN- at +200 kb; SCOTT et al. 1983)and the 3.9-kb EcoRI VOLHARD1988). Antibody staining of imaginal discswas as fragment which contains most of z2 exon 2 (PULTZet al. described in DIEDERICH,PATTATUCCI and KAUFMAN(1 99 1). 1988). Restriction mapping places the proximal breakpoint within a 0.5-kb interval about 1 kb 5’ of the z2 cap site RESULTS (RUSHLOWet al. 1987). The breakpoint in the P1 region was positioned with a corresponding precision. The Df(3R)SCeL2chromosome is associated with Northern analyses: RNA for Northern and S1 nuclease both dominant and recessive defects: The mutant protection analyses were prepared by the methods of SPRA- chromosome Df(3R)SCBxL2 (kindly provided by C. DLING and MAHOWALD(1979). Oligo-dT cellulose chromatography, glyoxal gel electrophoresis and blotting of RNA NUSSLEIN-VOLHARD)carries a deletion removingmost to Nytran membranes were done as described by MANIATIS, of the ANT-C. This chromosomefails to complement FRITSCHand SAMBROOK(1982) and HAGENBUCHLEet al. mutations in loci from Antp through zerkniillt (zen) (1984). (see Figure 3). It only partially complements null p6 S1 protection analyses: Nuclease S1 protection analyses mutations, and confers a recessive variegating loss-of- were done as described in CRIBBSet al. (1992). The specific probes used here are indicated in the corresponding figure function phenotype (PULTZet al. 1988). The proximal legends. breakpoint of the Df(3R)SCBXL2deficiency was previ- Pl/z2: To test for “hybrid” mRNAs fused between the ously shown to fall near the5’ end of the p6 transcrip- first exon (El) of Antp P1 and the second exon (E2) of r2, a tion unit. The variegating loss-of-function phenotype plasmid was constructed which contained Antp P1 sequences likely reflects defective transcriptional regulation of from the XbaI site at position 710 to the BamHI site at position 2 137 in El (coordinates as in LAUGHONet al. l’986) p6, due to removal of ®ulatory elements and/or ligated to the 5‘ endof a 200-bp BglIIIEcoRI fragment from to position effects exerted by the newly juxtaposed exon 2 of z2 (spans amino acids 124-188, coordinates as in sequences (PULTZet al. 1988). RUSHLOWet al. 1987) cloned in the polylinker of pGEM2 The dominantdevelopmental defects associated (ProMega). The 260-bp XhoI/EcoRI probe extendsfrom the with the Df(3R)SCBXL2chromosome are the focus of XhoI site in Antp P1 sequences (position 2071; LAUGHONet al. 1986)to the EcoRI site in z2 (above). This chimeric this work. This chromosome is associated with defects fragment was end-labeled with [(U-’~P]~ATPplus [(U-’~P] in a numberof structures dependent onAntp function dGTP using the Klenow fragment of DNA polymerase I, (ABBOTTand KAUFMAN1986). Defects of the humeral and the strands were separated on a preparative 5% poly- callus are observed with nearly full penetrance, nota- acrylamide/O. 1% bis-acrylamidegel (as in MANIATIS, FRITSCHand SAMBROOK1982). To minimize the back- bly the frequent malformation or absence of one or ground protection due to contaminating noncoding strand, both of the large bristles normally present there (com- the purified strands were self-annealed overnight at 65O in pare Figure 1, b to a). Additionally, the wingsof 1 X SSC, then repurified as before to separate double- animals heterozygous for thedeficiency are often held stranded sequences from contaminating noncoding strand. abnormally, away from the body and drooping. This The probe (20,000 Cerenkov cpm) was hybridized over- condition of the wings, as well as the humeral pheno- night at 44” (50% formamide, 0.8 M NaCI, 20 mMHEPES, pH 6.4) to 20 pg RNA, then digested with S1 nuclease (200 type, can be further aggravated by the presence of units/ml, Boehringer Mannheim), as in HAGENBUCHLEet al. the TM6B balancer chromosome carryingthe Humeral (1984) and FAVALORO,TREISMAN and KAMEN(1 980). Pro- (Hu) mutation of the Antp gene. The dominant phe- tection products were separated by size usingelectrophoresis notype of Hu (supernumerary bristles on thehumeral on 6% polyacrylamide sequencing gels, then detected by autoradiography. callus) is enhanced when Hu is combined with loss-of- Pl/pb: To test alternate splicing from the Df(3R)SCBXL2 function Antp alleles. However the phenotype of chromosome between Antp P1 El and pb E2, a plasmid was TM6B, Hu/Df(3R)SCBXLZflies is more extreme than constructed (in the vector pGEM2) with Antp P1 sequences that of TM6B,Hu/Antp-. Other dominant effects as- as above fused to a 1.55-kb BamHI/EcoRI fragment extend- sociated with the Df(3R)SCBXL2chromosome are pupal ing from the middle of pb E2 into the second intron. The coding strand probe joins 65 nucleotides of Antp P1 se- lethality, a failure of pupae to evert their anterior quence, beginning from the XhoI site at position 2071 spiracles and adultleg defects[usually fusions of prox- (LAUGHONet al. 1986) to 735 nucleotides of pb sequence. imal structures (in -1-1095 of adults varying with An 800-bp XhoI fragment from the above chimera was genetic background)]. 702 D. L. Cribbs et al. We emphasizethat the formation of allof the aforementioned adult or pupal structures depends on functions of the Antp gene, andparticularly on the P 1 promoter (ABBOTTand KAUFMAN1986). Thus the array of defects associated with Df(3R)SCBXL2resem- bled a dominant, partial loss-of-function phenotype. Gene dosage experiments were done to address the nature of the effect exerted by the deficiency chromosome on Antp expression. Animals of the genotype Df(3R)SCBXL2/+possess a single functional copy of Antp and exhibit the dominant phenotypes described above. These dominant defects were largely amelio- rated by the presence of a second copy of the entire ANT-C, but not if the entire ANT-Cexcept for Antp was present in two copies. Therefore the dominant defects depend on the dosage of Antp’ but not on the other loci in the ANT-C. This result suggests that the Df(3R)SCBXL2chromosome is antimorphic with re- spect to Antp function, a resultwhich was unexpected since Df(3R)SCBXL2deletes all Antp proteincoding sequences on this chromosome (see below). pb protein is ectopically expressed in the thorax of embryos carrying theDf(3R)SCBxL2 chromosome: The unusual variegating pb loss-of-function phenotype and the dominantthoracic defectsassociated with Df(3R)SCBXL2led us to examine pb expression in embryos carrying this chromosome, using antisera directed against pb protein. Ectopic accumulation of pb protein was observed in the thorax of embryos carrying the Df(3R)SCBXL2chromosome, in a pattern that strongly resembles the normal Antp expression pattern (Figure 2). This ectopic expression is localized to the ventral nerve cord in posterior prothorax (pT1) through posterior metathorax (pT3) (Figure 2, D and A). Normal Antp protein expression in the thorax is first observed at the beginning of germ band retrac- tion (-7 hr 30 min; CARROLLet al. 1986; WIRZ, FE~SLER and GEHRINC1986) (stages as inCAMPOS- ORTECAand HARTENSTEIN1985) and precedes ectopic expression of pb protein by as much as 56 hr (data not shown). Still, the striking similarity of the Antp and pb patterns at approximately the end of ventralnerve cord contraction (stage 16; -15 hr) indicates that pb expression has come at least partially

chaetae of the humeral callus (left), and closed arrows the anterior notopleural (right, bottom)and presutural (right, top) bristles. The spiracle (sp) is also indicated. This thoracic bristle pattern is the same as that seen in normal individuals. (b)Dorsal thorax of a Df(3R)SCBXL2/fibf3p’ individual. The expressivity of the dominant defects seen for this fly is extreme, and approximates the Antp PI null condition. Note that both of the macrochaetae of the humeral callus are absent (no arrows) and that the spiracular opening (sp) is malformed. (c) Dorsal thorax of revertant Df(3R)SCBXLZ-Drl/pbf’ p’. Drl has superimposed a deletion which removes 22, pb and lab FIGURE1 .-Dominant phenotype associated with the from the original deficiency chromosome, and eliminates all aspects Df(3R)SCBXLZchromosome. (a) Shown is a portion of the dorsal of the dominant defects, restoring the humeral callus (arrows, left) thorax of a pbf3p’ fly, with open arrows indicating the two macro and the spiracle (sp) to wild type. pb Expression Under Antp Control 703

FIGURE2.-Misexpression of pb protein in embryos carrying theDf(3R)SCBXL2 chromosome occurs in a pattern which closely approximates the normal Antp pattern. (A) Oregon R (wild type) stained with antidntp monoclonal; (B) Df(3R)SCBXLZ/TM3,Sbstained with anti-Antp monoclonal; (C) Oregon R (wild type) stained with anti-pb polyclonal; (D) Df(3R)SCBxLZ/TM3,Sbstained with anti-pb polyclonal. Panels A and B display embryos from the ventral side in order to more clearly show the accumulation of Antp protein in the ventral nerve chord (vnc). In panels C and D the animals are displayed laterally to show the anterior extent of pb expression and the more "patchy" accumulation of pb protein in the vnc. The apparent fading of Antp protein accumulation in the vnc of the animal in B relative to A is accounted for by the flatter preparation and thus uniform focal plane for the entire structure in A and the more advanced age of the animal in B. All embryos shown are about stage 16, or -15 hr of development (CAMPOS-ORTEGAand HARTENSTEIN1985), during the period of vnc contraction. The large arrows show the approximate position of parasegments 5 and 6 in the vnc, the position of predominant Antp protein accumulation in this structure. The small arrows show the position of the caudal neuromeres in which Antp protein is not detected. Note that pb protein is found in a pattern resembling Antp accumulation in deletion bearing animals (compare A and C with D) and that Antp protein accumulation in ps5,6 is not apparently affected by pb ectopic expression (compare A and B). under the controlof Antp regulatory elements. Drl, removes all remaining ANT-C sequences and is Ectopic fib expression is causative for the domi- no longer associated with any dominant defects (Fig- nant defects:Since the ectopic pb expression observed ure IC). This demonstrates that the genetic cause of inDf(3R)SCBXL2-bearing embryos resembles Antp the defects resides within or very near the partially expression (CARROLLet al. 1986; WIRZ,FESSLER and deleted ANT-C of the SCBXL2chromosome. The two GEHRING1986) we thought it likely that misexpres- most informative revertant lines obtained were Dr2 sion of pb could account for the dominant thoracic and Erl, induced with DEB and EMS, respectively. defects associated with the mutant chromosome. This Dr2 is phenotypically pb-null and has reverted to wild possibility was tested by two types of genetic screens. type as is the case for Drl. However, itshows no First, we screenedfor mutagenized Df(3R)SCBXL2 discernible deletion in the pb/z2 region and still pro- chromosomes on which pb gene function had been duces detectable pb protein in the thorax of embryos lessened or abolished, as identified by a pb loss-of- (data not shown). The Erl lesion is associated with a functionphenotype in thehead. We then asked hypomorphic allele of pb which also largely amelio- whether the dominant thoracic defects were simulta- rates the dominant effects. It should be noted how- neously eliminated.Alternatively, we screened di- ever, that the "reversion" associated with this chro- rectly for reversion of the dominant defects of the mosome isless completethan that for the pb-null humerusand then asked whetherrevertants were alleles, again supportingthe hypothesis that fib is simultaneously pb-. Similar results were obtained by causative for the dominant defects. The number of both approaches, asthe threepb mutants isolated were simultaneously pb- - revertant lines isolated (most revertant for dominant thoracic defects, and the two pertinently the Dr2 andErl lines, which are apparent revertants selected directly were fib-. point mutations; see also MATERIALS AND METHODS) Four DEB-induced pb mutations and one mutation makes it unlikely that a gene other than pb [for ex- induced by EMS were obtained from mutagenesis of ample the homeobox gene z2, which is not removed the Df3R)SCBXL2chromosome; each mutation issi- in the Df3R)SCBXL2chromosome] can be implicated multaneously revertant for all aspects of the dominant as a cause of thedominant defects. We therefore pupal and adult defects. The first revertant isolated, conclude that ectopic expression of pb directed by the 704 D. L. Cribbs et al. Df(3R)SCBXL2chromosome is causative forthe ob- Df(3R)SCBXL2/TM3embryos (0-24 hr postoviposi- served thoracic defects. That is, in some fashion, pb tion) for Northern blot analysis. The results, as shown protein expressed in the thoracic imaginal tissue in- in Figure 3, probe C, indicatethat one orperhaps two terferes with the normal function of the Antp gene on novel transcripts containing P1 first exon sequences the homologous chromosome. Interestingly, there are are present in the RNA from mutant animals. One of no detectable phenotypic consequenceson larval mor- these, marked Pl/22, hybridizes to Antp P1 exon 1- phology despite the clearly discernable ectopic accu- specific as well as to 22 (22 exons 1 and 2)-specific mulation of pb protein in the embryo (Figure2). probes (data not shown), and is of the size predicted Structure of the ANT-C on the Df13R)SCpLzchro- for a fusion Pl/22 mRNA (-1,900 nucleotides, in- mosome: Inorder to understand how ectopic pb cluding -200 nucleotides in the polyA tail; LAUCHON expression causes thedominant Antp defects, the et al. 1986; RUSHLOWet al. 1987). These blots also structure of this interval of the ANT-C was character- allowed the detection of a second novel band (Pllpb?; ized. Genomic Southern mappingof the Df(3R)SCBXL2 Figure3, probe C) containing Antp P1 first exon chromosome was performed to localize the endpoints sequences which suggested the presence of a hybrid of the deficiency. The structure of the Df(3R)SCBXL2 Pl/pb mRNA. This possibility was tested in the ex- chromosome is shown in Figure 3. The proximal periments described below. breakpoint, localized previously to the 4.0-kb EcoRI In order to test more rigorously for the existence fragment containing the 22 cap site and homeobox of hybrid mRNAs containingsequences from theAntp (PULTZet al. 1988; RUSHLOWet al. 1987) was con- P1 first exonfused to sequences fromthe second firmed. The distal breakpoint resides about 8 kb exons of 22 or fib, S 1 nuclease protection experiments proximal (3’) to the first P1 promoter-specific exon were performed. Chimeric end-labeled probes were of Antp mRNA (Figure 3). The deficiency thus re- prepared that contained sequences from P1 and22 or moves -240 kb from the ANT-C, of which -92 kb P1 and pb, joined in the predicted 5‘ to 3’ configu- are from theAntp P1 transcription unit.The pb RNA- ration. Exonic Antp P1 El and either 22 E2 or pb E2 coding sequences and -3 kb of 5”flanking sequence sequences were fused at convenient restriction sites remain intactin the deficiency chromosome (the struc- rather than at predicted splice junctions. The assay ture of the pb gene is described elsewhere; CRIBBSet used here relies on base-stacking forces to maximize al. 1992). The region thus retainsall pb and 22 coding the formation of a nearly continuous duplex between sequences plus the lone Antp P1 exon. the radiolabeled DNA strand and the looped-out chi- All three genes are normally transcribed from cen- meric cognate mRNA, rendering the probe resistant tromere-distal to proximal. The P1 promoter on the at least in part toS 1 nuclease digestion. In theabsence Df(3R)SCBXL2chromosome has the potential to ex- of a “hybrid” mRNA, only protection corresponding press the P1 first exon with its splice donor,and to Antp P1-derivedtranscripts should be seen; the produce a transcript in which this exon can bejoined detection of a“hybrid,” mutant-specific protection with the downstream splice acceptor sites of the sec- product may be considered proof that Antp P1 and ond exons of 22 and/or pb. Though the predicted 22 either 22 or pb sequences reside in the same mRNA protein is initiated in the first exon of this gene, the molecule. first three nucleotides of the second 22 exon are an Sequences of the P1 first exon were fused to thepb ATG triplet in frame with the 22 homeodomain second exon (where the pb open reading frame initi- (RUSHLOWet al. 1987). Hence a Pl/22 fusion mRNA ates; CRIBBSet al. 1992). The 800-nucleotide probe could plausibly direct the synthesis of a shortened 22 used begins within the transcribed P1 sequences and homeodomain protein under Antp control. However, extends past the end of pb E2 (Figure 4). The pre- the genetic reversion analysis described above indi- dicted S1 protectionproducts are a65-nucleotide cates that such a protein, if it exists, is not the cause fragment (derived from the normal Antp P1 and/or of the dominant phenotype associated with the dele- Pl/22 transcripts) and a mutant-specific product of tion bearing chromosome. The predicted pb protein 240 nucleotides ending at the 3’ terminus of the pb is initiated in the second pb exon (CRIBBSet al. 1992) second exon. These expectations were fully met (Fig- and a fusion Ant$ P l/pb mRNA is expected to encode ure 4). A similar experiment was performed using a a normal pb protein that is subject to regulation by chimeric P /221 probe (MATERIALS AND METHODS) and the remaining P1 promoter elements. thepredicted protection products were again ob- Overlappingtranscription of Antp, x2 and pb tained (data not shown). Thus, transcripts directed by fromthe Df13R)SCpL2chromosome: To examine the Antp P1 promoter and initiated at the first Antp the possibility that transcripts initiated from the Antp exon on the deletion bearing chromosomecan extend P1 promoter yield chimeric Antp Pl/22 or Antp P1/ through both the 22 and pb genes. Subsequently, by pb mRNAs by alternate splicing, poly(A+) RNA was alternate splicing, elements of the three normally dis- preparedfrom wild type (Oregon-R)and tinct genes are joined to generate at least two novel pb Expression Under Antp Control 705

(+193) Barn (-57) Barn (+201) breakpoint subclone fusion phage (3.5kb EcoRI) I , (15kb Barn) ' M-SCB bkpt

Pb AntpP1 I -*

probe A

probeC / \ \

l5Kb L 4800 w Antp P1 Pl/pb? -4200 3400 w ..'* I,-c*

? 4 PVz2 3.2 Kb -1900

'U *

FIGURE3.-Structure of the ANT-C in the Df3R)SCBXL2chromosome. The Df(3R)SCBXL2chromosome fails to complement mutations in genes from zen, bcd, Dfd, Scr,jlz and Antp, but partially complements pb alleles (PULTZet al. 1988) and complements lethal alleles of 1(3)84Ba (the next locus distal to Antp). The breakpoints of the deficiency were located by genomic Southern blot analysis shownat the bottom of the figure (probes A and B). Genomic blot data for the breakpoint localization were confirmed by cloning the 15-kb BamHl fusion fragment in bacteriophage X (MATERIALSAND METHODS)and then performing restriction analysis on the subcloned 3.5-kb EcoRI restriction fragment indicated (pb/r2 sequences are stippled, Antp P1 sequences are cross-hatched). The position of the breakpoint was delimited to within 0.6 kb by the presence within the 3.5-kb EcoRl fragment of a SmaI restriction site from pb/z2 (circled), coupled with the absence of a P~tlsite normally located 0.6 kb upstream of the Smal site. The correspondence between the transcription map of the fusion region and the cloned genomic DNA is indicated by vertical dotted lines. For the genomic blots shown at the bottom, lanes 1 and 3 of each set contain 5 pg of DNA from Df(3R)DC-5 ri PpITM3,Sb flies (DC-5 is a deletion which removes all complementation units from twt proximal of labial through minimally1(3)84B1 and thus the entire ANT-C; D.L. CRIBBS,unpublished observation). Bands seenin lanes 1 and 3 therefore derive exclusively from the TM3, Sb chromosome. Lanes 2 and 4 contain 5 pg of DNA from flies of genotype DJ3R)SCBXL2/TM3,Sb.Bands detected in lanes 2 and 4 which are not present in lanes 1 and 3 derive from the Df(3R)SCBXL2chromosome. Df(3R)SCBXL2-specificbands detected by both Antp P1 and pb/z2 region probes reflect the presence of fusion fragments that span the deficiency breakpoints. DNA was digested with BamHI (lanes 1 and 2 of each set), or with Sal1 (lanes 3 and 4). (Probe A) The proximal breakpoint was described previously (PULTZet al. 1988) and is confirmed here (the probe used was a 2.0-kb EcoRI/PstI fragment spanning coordinates -51 to -53, indicated by the lightly stippled box; see PULTZet al. (1988) for description of coordinates). (Probe B) The distal breakpoint was located by probing with X phage A577 (cross hatched box), carrying sequences from the Antp Plregion which span the P1 promoter and first exon of Antp (SCOTTet al. 1983; LAUCHONet al. 1986). The location of the breakpoints was further confirmed, as described above, by cloning and mapping of the 15-kb fusion BamHI fragment (lanes A2 and B2). The exon structure surrounding the deletion breakpoints is shown in the middle of the figure, with pb, 12 and the lone Antp P1 exon all transcribed in the same direction. The positions of the protein coding sequences are indicated by open boxes while the nontranslated exonic intervals are black. Note that first exons of Antp P1 and pb are entirely noncoding and that the open reading frame of fib initiates in exon 2. (Probe C) For Northern blot analysis of mutant derived RNA, poly(A+) RNA was prepared from wild type (Oregon R) or Df13R)SCBXL2/TM3,Sb embryos (0-24 hr). Shown is a gel track containing 10 pg of mutant poly(A+)RNA that was transferred to a Nitran membrane and probed with a 1.8-kb BamHl/EcoRI fragment containing most of Antp P1 and El (probe C; coordinates 2137 to 3959, LAUGHONet al. 1986). Similar hybridizations were performed using z2 and pb-specific probes. As indicated in (probe C) (arrows, left), two bands correspond to normal Antp P1 transcripts of about 4,800 and 3,400nucleotides. These are products of the TM3,Sb chromosome. A novel RNA class of about 1,900 nt was detected with both Antp P1 and z2-specific sequences (labelled Pl/z2). This band is presumed to be a product of the deletion bearing chromosome and toresult from alternate splicing. Additionally a second novel band of about 4,200 nucleotides was sometimes detected (labeled Pllpb?).

"hybrid" transcripts,the Pl/z2 and Pl/pbspecies (Fig- by this deletion (ie., a partial loss-of-function). More- ure 5). Additionally, the DJ3R)SCBXL2chromosome over, pb protein is detected inDf(3R)SCBXL2/pb""" must be capable of directing the synthesis of normal embryos, demonstrating that the remaining approxi- pb mRNA and protein under normal pb control. This mately 3 kb of pb 5' flanking sequences are sufficient is inferred from the pb mutant phenotype conferred todirect spatially correct pb expression though in 706 D. L. Cribbs et al.

4 R enr., end-label, strandsep.

8OOnt Probe

4 SIprotection

240nt Pllpb

Protection products <

Pl (or P11zZ)

FIGURE4.-Demonstration of alternate splicing from the Df(3R)SCBX”’chromosome between the first exon (El) of Antp PI and the second exon (E2) of pb. The probe, prepared as described in MATERIALS AND METHODS, contains 65 nucleotides (nt) of Antp P1 sequence and 735 nt of pb sequence, and fuses P1 El to pb E2 as indicated. Each hybridization contained 80,000 Cerenkov cpm of single-stranded probe. Hybridizations and digestions with nuclease S1 were as described in MATERIALS AND METHODS, and only the results obtained with the coding strand probe are shown. Lanes shown are: (1) Probe only, approximately 20 Cerenkov cpm: (2) probe hybridized to 20 pg of Escherichia coli tRNA: (3) probe hybridized to 20 pg of wild-type (Oregon R) embryonic (0-24 hr) poly(A+) RNA; and (4) probe hybridized to 20 pg embryonic (0-24 hr) poly(A+) RNA from a Df(3R)SCBx’-’/TM3,Sb stock. The predicted classes of protection products are shown to the left, and the corresponding protection products indicated (arrows) to the right of the autoradiograph. The upper band (240 nt) is specific to the mutant RNA track and corresponds to Antp Pl/pb E2 fusion mRNA.

Pb Antp P1 domain protein itself might interfere with the function or accumulation of Antp protein in the imaginal discs, perhaps at the level of transcriptional control. If pb protein negatively regulates Antp transcription,

22 122 P1 Pllpb whether directly or indirectly, then Antp protein lev- b3mPb1-11 I_ ‘79 els should be reduced compared to wild type in cells FIGURE5,”Summary of expression from the pb region of the expressing pb under the control of the Antp P1 pro- ANT-C onthe Df(3R)SCBX”chromosome. The structureshown on moter. To test this prediction, imaginal tissues derived top indicates the novel juxtaposition of the Antp, 22 and pb tran- from Df(3R)SCBXL2-bearingor from normal third in- scription start sites on the deletion chromosome. Dark motifs within starlarvae were stained with monoclonal antisera boxes indicate the position of transcribed but non coding portions directed against Antp protein (CONDIE,MUSTARD and of the three loci (Antp exon 1 is black). Light motifs within boxes show the positions of the two remaining open reading frames in 22 BROWER199 1) (Figure 6, A and B) or with polyclonal (stippled fill) and pb (diagonal line fill). Above the indicated exons anti-pb sera (Figure 6, C and D). Cells associated with are shown the normal and postulated alternate splicing events: while the dorsal prothoracic and wing discs give rise to the below, the four possible mature RNA products are indicated. Note humeral callus, anterior spiracles and anteriormeson- that the two novel products shown at [heright result in the splicing of only leader sequence in the case of Pl/fb and thefusion of Antp otum (ABBOT”and KAUFMAN 1986), all of which are P1 leader into protein coding sequences in the case of Pl/22. perturbed in Df(3R)SCBXL2/+bearing adults. Staining experiments show that pb protein is present in the diminished quantities (PULTZ 1988). We have not dorsal prothoracic discs of deficiency-bearing hetero- tested directly for the presence of normal 22 tran- zygotes but is not detectable in wild-type discs (com- scripts. However, assuming that all of the necessary pare Figure 6, C and D). Conversely, the level of Antp cis-acting regulatory elements for this locus are pres- protein present in the same discs is markedly reduced ent, thedeletion chromosome doeshave the potential in the mutant larvae. It is also possible to detect pb to encode such a product. protein in the wing discs in regions where the level of Accumulation of Antp protein is reduced in ima- Antp protein is diminished (data not shown). Thus ginaltissues of Df(3R)SCBXL2-bearinganimals: ectopic pb expression correlates to reduced accumu- Expression of pb protein in the embryonic thorax was lation of Antp protein in the imaginal tissues which correlated with a dominant Antp loss-of-function phe- give rise to the affected adult structures. Further, the notype. This suggested that the nuclear pb homeo- diminished accumulation of Antp protein is correlated pb Expression Under Antp Control 707

FIGURE6.-Distribution of Antp and pb proteins in imaginal discs of normal and Dj73R)SCHU larvae. Larvae of the desired genotypes were stained with primaryantisera directed against either Antp or pb proteins. Subsequent to treatment with the secondaryantibody and enzy- matic reactions to reveal the distribution of homeotic protein, the imaginal discswere dissected and mounted for photography. Shown are dorsal prothoracic discs,which give rise to the humeral callus of the adult. The discs are oriented such that the associated spiracles (located anteriorly inthe larva) are up and the tracheae (passing posteriorly) are down. (A) Wildtype (Oregon R) stained withanti-Ant# monoclonal; (B)Dj73R)SCExz/+ stained with anti- Ant# monoclonal; (C) wild type (Or- egon R) stained withanti-pb poly- CIOMI;(D) Dj73R)SCrru3/+ stained with anti-pb polyclonal. Note the in- verse relationship between accumulation of Antp and pb proteins.

with the ectopic “function” of pb protein, since in disadvantage to the product of the Ant$ locus. Taken larvae with the revertant chromosome Dr2 (carrying together, these various data show that pb protein is a fibnu1[mutation on the Df(3R)SCBxLzchromosome) capableof contributing to the negative regulation of Antp protein accumulation appears normal. The mu- Antp, and suggest that this may occur at the transcrip- tant phenotype observed in Df(3R)SCBxL2animals and tional level. its absencein the pb- revertants thus may be explained by these results. The most economicalexplanation for DISCUSSION this lessened accumulation of Antp protein is that pb pb has acquired a second homeotic promoter: We protein can directly or indirectly repress transcription present here the characterization of a chromosomal from the P1 promoter. Alternatively, pb protein or rearrangement which hasintegrated elements of three the product of a gene activated by pb may contribute distinct transcription units into one. The homeotic to reduced accumulation of Antp protein by compet- gene pb, with a single promoter and a single domain ing with it for binding to target DNA sequences and of expression in the head, has been converted into a thereby destabilizing it. This latter possibility seems gene with two promoters (pb and Antp P1) which is less likely especially if it is assumed that it is the pb expressed in two distinct regions of the fly body, the protein which directly competes with Antp protein. head and thorax. The important consequence for the The abundance of pb mRNA, and protein,appears to fly is that a new regulatory regime is imposed on Antp. be considerably less on a per cell basis than for other Inthe new arrangement, ectopic $6 expressionin homeotic loci of the ANT-C (our unpublished obser- imaginal tissue causes diminishedthoracic expression vations) and thuswould appear to be at a competitive of remaining copies of Antp. Most extant Antp domi- 708 D. L. Cribbs et al. nant gain-of-function mutations result from chromo- dence for the formationof z2/pb hybrid mRNA from some rearrangements in which the Antp P1 promoter Df(3)SCBxL2or from a normal chromosome(PULTZ et is replaced by other sequences, allowing Antp expres- al. 1988; RUSHLOWet al. 1987). Put from a slightly sionin the eye-antennal imaginal disc (e.g., different perspective, it is interesting that transcrip- SCHNEUWLY,KURIOWA GEHRINCand 1987; tion initiated at the P1 promoter can extend through FRISCHER,HACEN and GARBER 1986).Here we have both the 22 and pb loci but transcription initiated at encountered a different situation, where the super- the 22 promoter apparently does not extend over the imposition of Antp P1 control on another gene has much shorter interval between22 and pb. Perhaps this resulted indirectly in loss of Antp functions. Anumber is an indication of the existence of specific transcrip- of developmentally important Drosophila genes have tion factors which distinguish between early zygotic beendescribed whichpossess multiplepromoters, gene promoters such as 22 and those for later ex- such as Antp (LAUGHONet al. 1986; STROEHER,JOR- pressed homeotic genes such as Antp and pb. Consist- GENSEN and GARBER1986; SCHNEUWLYet al. 1986), ent with such a possibility, it is of interest to note that caudal (MACDONALDand STRUHL1986; MLODZIKand in embryos carrying the chromosome Df(3R)LZN, in GEHRINC 1987),and hunchback (TAUTZet al. 1987; which ftz and Sex combs reduced (Scr) 5‘ flanking se- BENDERet al. 1988). The deletion analyzed in this quences are juxtaposed to the 22 and pb genes, 22 work is an example of how gene fusion can result in RNA is detected in stripes asftzis normally (RUSHLOW the superimposition of two distinct regulatory para- and LEVINE1988), while pb protein accumulates in a digms. The acquisition or shuffling of elements nec- subset of cells in the foregutwhere normalScr expres- essary for regulation of gene expression is likely an sion occurs (pb expression in stripes and Zen accumu- important component of the evolution of regulatory lation in the foregut is not observed) (PULTZ1988). systems (for review, see SCHIBLERand SIERRA1987), Another potentially important variable is the time at and naturally occurring juxtapositions similar to the which the different promoters are expressed. A lim- one studied here may serve at least in part to explain ited supply of factorsrequired for the excision of the origin and existence of multiple independent pro- large (e.g., Antp and pb) but not small introns ut%,zen moters for a single gene. and 22) soon after the onset of zygotic transcription Mutant chimeric homeotic genesother than the one might suffice to explain the ability to make Pl/pb observed on theDf(3R)SCBXLZ chromosome have pre- mRNAs at 13-16 hr of embryogenesis but not z2/pb viously beendescribed in Drosophila. The Ultrabi- mRNAs at 2-4 hr. Lastly one can surmise that the thorax (Ubx) mutation C1 of the BX-C is associated transcriptional machinery which normally operates at with a deletion which fuses the abdominal-A (abd-A) the Antp P1 promoter is qualitatively different from and Ubx loci. In this case the result is a chimeric abd- that normally assembled at genes like 22. The P1 AIUbx transcriptionunit andprotein (ROWE and promoter drives transcriptionwhich normaliy extends AKAM 1988).Because the spatial expression domain over 100 kb of DNA and must traverse a segment of of abd-A overlaps that of Ubx, and the proteincontains the genome containingthe internal P2 promoter.The the Ubx homeobox, some Ubx functions are retained z2 promoter does not have to accomplish either feat. even in the absence of its normal control (CASANOVA, The nature of any proposed qualitative difference is SANCHEZ-HERREROand MORATA 1988; ROWE and obviously not addressed by the experiments or results AKAM 1988).The encoded chimeric protein gives no presented andwhich (if any) of the above speculations evidence of acquired new functions; rather, in this is relevant awaits further investigation. However these case the intriguing observation is that some functions results have pointed to some interesting differences normally attributedto either Ubx orto abd-A are which do exist among these developmentally signifi- retained in the mutant. cant promoters andhave provided avenues forfuture Chromosomal rearrangements not associated with experimentation. deletionsprovide anothersort of plasticity tothe Ectopic pb protein diminishes normalAntp accu- genome. This is seen in a spectacular fashion for the mulation: The present work shows that pb protein is dominant Antp73bmutation; in these mutants ectopic capable of negative function(s) directed toward other expression of Antp protein in the eye antennal discs homeotic genes. The dominant thoracic defects due results from an inversion mutation that exchanges the to ectopic pb expression reflect the capacity of pb Antp P1 promoter for the 5’ end of a gene normally protein to negatively regulate Antp. This observation expressed in the head(FRISCHER, HACEN andGARBER poses anapparent paradox, since if pb negatively 1986; SCHNEUWLY,KURIOWA and GEHRINC 1987). regulates Antp P1then pb under Antp P1control The basis of differential mRNAaccumulation/ mightbe negatively autoregulatory. Assuming that processing: Curiously, though it is clearfrom the the down-regulation of Antp occurs through thebind- present work that an RNA polymerase can traverse ing of a protein to relevant targetsequences, a possible the sequences separating z2 and pb, there is no evi- resolution is that these targets reside, at least in part, Expression Underfib Expression Antfi Control 709 within sequences removed by the deficiency. Thus, guish critically whether or not regulation of Antp in pb protein need not participate in negative autoreg- the labial discs is a normal function ofpb. ulation of its newly acquired promoter (that lacks the The capacity of pb to negatively regulate Antp in target sequences), but might restrict its activities to the imaginal thorax might thus besimply an historical intact Antp copies elsewhere. relic, or an artifact of the pb homeodomain's func- Posteriorly directed regulation by pb: The data tional similarity to other such proteins. Negative reg- presented here indicate that regulation by ectopically ulation of Antp by pb in coexpressing cells might then expressed pb protein is directed toward a more pos- provide a powerful evolutionary incentive to establish teriorly expressed gene, Antp. In a study employing and maintain nonoverlapping expression domains by the conditional hsp70 promoter to direct ectopic and means other than and in addition to direct reciprocal overexpression of Antp and Ultrabithorax proteins, repression. Thus it could be that the two have differ- the notable result was that moreposteriorly expressed ent domains of expression due at least in part to the endogenous genes were apparently refractoryto reg- fortuitous capacity of pb to negatively regulate Antp ulation by those normally expressed more anteriorly and the negative consequences of their coexpression, (GONZALES-REYESet al. 1990). In double heat shock rather than to the present-day existence of anegative combinations, hs-Ubx was likewise epistatic to hs-Antp networkincluding pb which represses Antp in the (more anterior) in attributing segmental identity. In head. view of that work, it might be considered surprising An intriguing possibility which remains is that the that pb is capable of reducing Antp function and observed capacity of pb to regulate Antp in the ima- accumulation in the thorax. Perhaps the present case ginal discs appears to point to a different and alternate is a simple exception to a generally respected rule. relationship in the central nervous system. Spatially However, since the effect of pb on Antp accumulation restricted expression of homeotic proteins in the cen- in the thorax seems more severe in larval discs than tral nervous system is a reiterated theme amongthese in the embryo (compare Figure 2 and Figure 6) the genes from diverse species (DUBOULEand DOLL^ departure from the above rule of posterior primacy 1989; GRAHAM,PAPALOPULU and KRUMLAUF 1989; might rather emanate from differences between em- COSTAet al. 1988; KAUFMAN, SEEGERand OLSEN bryonic and larval developmentalcontrol. Indeed 1990). Additionally the homeotic genesof Drosophila such differences have been shown to exist for two have been shown to affect the development of the other members of the ANT-C, lab and Scr (CHOUI- central and peripheral nervous systems (THOMASand NARD and KAUFMAN 199 1; DIEDERICH,PATTATUCCI WYMAN1984; WEINZIERLet al. 1987; MANN and and KAUFMAN 1991; PATTATUCCIand KAUFMAN 1991). HOGNESS1990; HEUERand KAUFMAN1992). A small Negative regulation of Antennapedia by pb in a number of cells in the embryonic (Figure 2C; PULTZ normal fly? The analysis of Df(3)SCBxL2 presented et al. 1988) and larval (RANDAZZO,CRIBBS and KAUF- here shows that pb is capable of negative regulation MAN 1991) nervecords normally express pb. This of Antp in the thorax. We thereforeattempted to domain extends far posterior of the labial segment assess whether this reflectsnormal functions of pb along the anteroposterior axis into a region in which directed toward Antp in the head. This was done first normal Antp expression is found. The function(s) of by examining the spatial expression of Antp in pbnu" pb protein in these cells remains unknown. However, larvae, or in embryos lacking both pb and Sex combs the reduced motility both of pb- flies and of mini@+; reduced (Scr),the otherhomeotic protein known to be transgenic flies (with rescued mouthparts but lacking expressed normally in the labial discs (MAHAFFEYand expression in the nervous system; RANDAZZO,CRIBBS KAUFMAN 1987; RANDAZZO,CRIBBS and KAUFMAN and KAUFMAN 1991) supportsan important role. 1991). No ectopic Antp expression was observed in Moreover ectopic expression of pb into the normal either case. Hence, if regulation of Antp in the head domain ofAntpassociated with the Df(3R)SCBXL2chro- is a normal function of pb, it appears to involve a mosome does not cause repression of Antp expression network comprisingother as yet unidentified member in the embryonic CNS. Thus coexpression of these loci.As analternative approach, we examined the two genes at this stage and in this cell type is permitted contribution of pb to the regulation of Antp gain-of- and indicates that these two Ioci have a very different function alleles, reasoning that sequences recognized relationship in this spatiotemporal domain and that by DNA bindingregulatory proteins might be re- they may act in a concerted rather than antagonistic moved by the chromosomalrearrangement. How- fashion. The existence of such alternate regulatory ever, though one allele (Antp736)may be expressed in logic points out thepotential for cell- or tissue-specific the labial discs provided the level of pb function is cross-regulation of homeotic genes. Such mechanisms sufficiently reduced, the results obtained were equiv- might then serve toward the integration of pattern ocal. As a consequence, we remain unable to distin- among segments in the construction of adifferen- 710 D. L. 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