Development 113, 257-271 (1991) 257 Printed in Great Britain © The Company of Biologists Limited 1991

Rescue and regulation of : a homeotic gene of the Antennapedia Complex

FILIPPO M. RANDAZZO, DAVID L. CRIBBS* and THOMAS C. KAUFMAN

Howard Hughes Medical Institute, Institute for Cellular and Molecular Biology, and Programs in Genetics and Cellular, Molecular, and , Department of Biology, Indiana University, Bloomington, Indiana, U.S.A.

* Present address: Centre de Recherche de Biochimie et Genetique Cellulaires, 118, route de Narbonne 31062 Toulouse Cedex, France

Summary

The extraordinary positional conservation of the hom- minigene expresses pb protein in only a subset of pb's eotic genes within the Antennapedia and the Bithorax normal domains of expression. Therefore, the biological Complexes (ANT-C and BX-C) in Drosophila melanogas- significance of the excluded expression pattern elements ter and the murine Hox and human HOX clusters of remains unclear except to note they appear unnecessary genes can be interpreted as a reflection of functional for specifying normal labial identity. Additionally, by necessity. The homeotic gene proboscipedia (pb) resides using reporter gene constructs inserted into the Dros- within the ANT-C, and its sequence is related to that of ophila genome and by comparing /^-associated genomic Hox-1.5. We show that two independent pb minigene sequences from two divergent species, we have located P-element insertion lines completely rescue the labial m-acting regulatory elements required for pb expression palp-to-first leg homeotic transformation caused by pb in embryos and larvae. null ; thus, a homeotic gene of the ANT-C can properly carry out its homeotic function outside of the Key words: proboscipedia, homeotic gene, Antennapedia complex. Despite the complete rescue of the null, the Complex, rescue, regulation, sequence conservation.

Introduction head, thorax and abdomen. The homeotic genes then interpret this positional information and act as ontogen- Determining how the three-dimensional architecture of etic switches to specify differing segmental identities an organism results from information encoded in the (Garcfa-Bellido, 1977; Lewis, 1978). linear DNA molecule is a primary goal of developmen- Eight of these homeotic genes in Drosophila cluster tal geneticists. The process of and into two separate complexes, the Antennapedia Com- in defines a para- plex (ANT-C) and the Bithorax Complex (BX-C) digm that addresses aspects of this question. The (Kaufman et al. 1980, 1989; Lewis, 1978). The BX-C segmented body-plan of Drosophila derives from the contains three homeotic genes that specify thoracic and functions of a complex hierarchy of coordinately abdominal segment identity: Ultrabithorax (Ubx), expressed genes. Enumerating the primary metameric abdominal A (abdA) and Abdominal B (AbdB) (Lewis, pattern of the embryonic germ band involves a 1978; Sanchez-Herrero et al. 1985; Peifer et al. 1987). repertoire of genes classified into four groups according The ANT-C includes five homeotic genes which specify to their function: (a) the coordinate genes, (b) the gap head and thoracic segment identity: Antennapedia genes, (c) the pair rule genes, and (d) the segment (Antp), Sex combs reduced (Scr), Deformed (Dfd), polarity genes (Niisslein-Volhard and Wieschaus, 1980; proboscipedia (pb) and labial (lab) (Denell et al. 1981; Niisslein-Volhard et al. 1987). The products of the Struhl, 1981; Abbott and Kaufman, 1986; Mahaffey and coordinate genes are laid down by the mother and set Kaufman, 1987; Merrill etal. 1987; Regulski etal. 1987; up the axial polarity of the organism. The gap genes Pultz et al. 1988; Diederich et al. 1989). A number of divide the embryo into sets of contiguous segments, non-homeotic genes also reside within the ANT-C, whereas the pair rule genes divide the embryo into including the pair rule gene fushi tarazu (ftz), the alternating segments or parasegments. Finally, the gene bicoid (bed) which encodes a polarity of pattern elements within each segment are set important in the specification of anterior- up by the segment polarity genes. The actions of each posterior polarity, and the zygotic gene zerkniillt (zen) class occur as a cascade of dependent events and taken together specify the proper number of segments in the which is required for the specification of dorsal structures (Wakimoto etal. 1984; Driever and Niisslein- 258 F. M. Randazzo, D. L. Cribbs and T. C. Kaufman

Volhard, 1988a, 19886; Rushlow era/. 1987). The ANT- the same linear order and relative transcription C also includes two transcription units with no direction on the chromosome as the BX-C and ANT-C genetically discernible function - the IgA homology- homeotic genes. Moreover, the spatial expression containing gene amalgam (ama) and the zerc-pattern- domains in the CNS reflect the linear array of the genes like gene z2 - and a cluster of eight transcripts which in mammals as in insects. This extraordinary conser- show sequence similarity to cuticle genes (Seeger et al. vation of linkage, linear array, and spatial expression 1988; Rushlow etal. 1987; Pultz et al. 1988; Fechtel etal. pattern suggests the possibility that there is some 1988; Pultz, 1988). necessary functional coupling of the genes of the ANT- Mutations in the homeotic loci result in the replace- C, BX-C, HOM-C and Hox complexes. ment of one or several segmentally derived structures Because of the substantial size and complexity of the by those associated with another metamere. Null homeotic genes, rescue of homeotic function has in proboscipedia mutations, for example, cause a distipro- practice been difficult. However, mutations in some of boscis-to-leg transformation while hypomorphic pb the smaller non-homeotic genes in the ANT-C - bed, alleles lead to a distiproboscis-to-antennal transform- zen and/fz - have been rescued by P-element mediated ation (Bridges and Dobzhansky, 1933; Kaufman, 1978). germline transformation (Berleth et al. 1988; Rushlow No other homeotic gene displays a dosage-dependent et al. 1987; Hiromi et al. 1985). We set out to examine transformation to one of two differing tissues. Ad- the expression and function of a proboscipedia mini- ditionally, loss of function pb alleles result in a size gene with several goals in mind: (1) to prove that the reduction of the maxillary palps, which has been identified pb DNA corresponds to the genetically interpreted as a transformation toward antennal ident- defined pb gene; (2) to determine whether a homeotic ity (Kaufman, 1978). The pb gene product is not gene that normally is located in the ANT-C must reside required for adult viability and its absence does not there to function properly; and (3) to determine the result in any visible changes in the embryo, an unusual relationship between the expression of pb protein in characteristic for a homeotic gene. As with all other specific tissues to its function in those tissues. In this homeotic genes, pb presumably determines segment paper, we present our progress toward these goals by identity by controlling the expression of a number of demonstrating that the pb homeotic may be 'downstream' or 'realisator' genes which either directly rescued by a pb gene located outside the ANT-C. or indirectly control the differentiation of tissues Additionally, through the use of pb/lacZ reporter (Garcfa-Bellido, 1977). fusion genes transformed into Drosophila and DNA Homeotic proteins are localized in the cell nucleus, sequence comparison between D. melanogaster and D. consistent with their inferred gene-regulatory func- pseudoobscura, we have located m-acting regulatory tions. These proteins possess a highly conserved 60 elements necessary for pb expression. amino acid sequence called the homeodomain (McGin- nis etal. 1984; Scott and Weiner, 1984), which has DNA Materials and methods binding properties (Desplan et al. 1985; Miiller et al. 1988). Additionally, tissue culture experiments demon- pb minigene construction strate that Ultrabithorax and Antennapedia proteins, The minigene was constructed by inserting a restriction both of which are -containing, can activate fragment containing pb upstream, promoter and RNA leader and/or inactivate transcription of target genes in vivo in sequences into an existing hsp70 promoter/'pb gene fusion a manner generally compatible with previous genetic construct (Cribbs, Randazzo and Kaufman, in prep.). The analysis (Krasnow et al. 1989; Winslow et al. 1989). hsp70 promoter//?£> gene fusion construct consists of the mini- Thus, homeotic genes encode DNA binding proteins white gene (an EcoKl fragment) which is a derivative of Bm that are presumably capable of activating and/or A-W (Pirrotta et al. 1985) inserted into the Xhol site of inactivating transcription of other target genes. pUChsneo vector (Steller and Pirrotta, 1985) from which the neo sequences have been deleted. Genomic pb sequences Molecular homologues to the homeotic genes of the were inserted into this vector such that the hsp70 RNA leader ANT-C and the BX-C are found in a variety of other was fused to the downstream half of the pb exon-1 RNA species. Results from a chromosome walk in D. leader. In order to make the minigene, a pb genomic pseudoobscura (Randazzo, Seeger, Huss and Kaufman, restriction fragment containing 7.3 kb of upstream sequence in prep.) indicate that homologues to the ANT-C genes and the upstream half of the exon-1 extending to an Ncol lie in the same linear order on the chromosome as in D. restriction site was inserted into the heat shock/pb vector at a melanogaster. Furthermore, genetic analysis of the red unique Xbal site created by insertion of a linker at the hsp70 flour beetle Tribolium castaneum demonstrates a RNA leader/pfe exon-1 RNA leader junction. These manipu- closely ordered and similarly aligned set of ANT-C-like lations lead to a net insertion of 27 nt (nucleotides) between and BX-C-like genes referred to as the HOM-C CCATG and G of the Ncol site in the first exon of pb. The (Beeman et al. 1989). Considerably more remarkable, inserted sequence reads as follows: GTCTAGAGCGG- the murine Hox and human complexes CCGCGGATCCGGCATG. contain clusters of genes with homeobox sequences Additionally, as a result of the manipulations described strikingly similar to those of the homeotic genes of the above, the pb minigene contains 191 nt of hsp70 5' flanking sequences and the first 92 nt of the hsp70 RNA leader. These BX-C and the ANT-C (Duboule and Dolle", 1989; sequences are 7.3 kb and 5.3 kb upstream of the pb and z2 Graham et al. 1989; Kappen et al. 1989; Boncinelli et al. transcription start sites, respectively. Flies of the genotype 1988). The genes of the Hox complexes are arranged in P[w+, pb]\/ P[vv+, pb]l; pbs/pb5 are completely rescued in Rescue and regulation of proboscipedia 259 terms of the homeotic transformation when grown at 18°C. A lacking the minigene, had white eyes. Presence of the Sb stock of P[w+, pb]l homozygous flies was grown at 29°C to phenotype is indicative of the pb5 heterozygous class of flies. see if mild heat shock has any dominant p£>-associated effects pb5 null stocks carrying either the P[vv+, pb] 2 insertion or the on the . When hsp70 promoter/pb gene fusion lines P[M<+, pb]l insertion have been propagated for numerous are grown at 29°C, thickening of aristae, transformation of generations. Hundreds of flies have been observed and all antennae to maxillary palps and increased pupal lethality have displayed a completely rescued homeotic phenotype. are observed (Cribbs, Randazzo and Kaufman, in Flies were stored in 70% ethanol before preparation for prep.). These phenotypes are inferred to be due to ectopic SEM. Specimen preparation and SEM methods have been expression of pb protein. None of these changes are observed described by others (Merrill et al. 1987). in P[w+, pb\\ homozygous flies when cultured at 29°C. Therefore, it appears that the hsp70 sequences in the Antibody preparation construct do not detectably affect the pb portion of the A /3-galactosidase/pb fusion construct was made using a minigene. pWR590-l /S-galactosidase fusion cassette (Guo et al. 1984), which incorporated the 5al pb cDNA (Cribbs, Pultz, Johnson pb/lacZ reporter gene fusion construction and Kaufman, in prep.). The fusion protein was expressed in The pbG2 construct was made by first removing the ninaE JM101 E. coli cells, extracted by standard Laemmli tech- sequences from pDM66A (kindly provided by D. Mismer) as niques (Mahaffey and Kaufman, 1987), isolated on a standard a Kpnl/BamHl fragment, blunt-ending with T4 DNA 8% SDS-PAGE gel, and then injected into rabbits. A polymerase, and then blunt end ligating the vector to recreate pATHll/pfe 5al cDNA fusion construct was used to make a the BamHI site. A 7.8 kb fragment, beginning at the BamHl TrpE/pb fusion protein (Dieckmann and Tzagoloff, 1985). site 7.3 kb upstream of the pb transcription initiation site and The fusion protein was coupled to Affi-Gel 10 (Bio-Rad) in ending at an Ncol site within the first exon of pb which was order to make an affinity chromatography column. Finally, modified into a BamHl site using linkers, was cloned into the the terminal bleed was purified over the TrpE/pb column BamHl site of the modified pDM66A vector. This plasmid twice and then stored at 4°C. was then cut with Noil to separate the pHSS7 sequences from the pb/lacZ cassette that was inserted into the Noil site of a Antibody staining CoSpeR Notl transformation vector (kindly provided by J. Collection, fixation, and staining of embryos were performed Tamkun) which was derived from the original CaSpeR vector using standard techniques (Mahaffey and Kaufman, 1987). (Pirrotta, 1988). The secondary antibody used for the staining of wild-type and The pbGl construct was made by inserting pb sequences V[w+, pb]; pb5/pb5 embryos was EAI grade affinity purified beginning at the BamHl site that lies downstream of the ATG polyclonal goat anti-rabbit antibodies conjugated to horse- translation start site in the second pb exon to the BamHl site radish peroxidase (HRP) used at a 1:150 dilution (Bio-Rad). 7.3 kb upstream of the pb transcription start site into the Embryos from the pbGl, pbG2, and pbGl/intron stocks plus pCaSpeR-i3gal vector (Thummel et al. 1988). Next, an a PI/}1"1", ftz/lacA] positive control (Hiromi et al. 1985) and a oligonucleotide fragment with BamHl sticky ends was w/w negative control stock which lacks a /3-galactosidase inserted into the downstream BamHl site. This manipulation containing construct were collected, fixed, and then immuno- resulted in the in-frame fusion of pb coding sequences to lacZ stained using rabbit polyclonal antibodies directed against the coding sequences. The pCaSpeR-^gal vector contains /3-galactosidase protein and goat anti-rabbit HRP conjugated P-element ends and the mini-white gene as the visible secondary antibodies. The positive control exhibited the selectable marker. The pbGl/intron construct was made by expected /irz-like pattern of expression plus hindgut staining. inserting a 1.6 EcoRl fragment containing the pb intron-2 The hindgut staining pattern is a characteristic of this J3- conserved elements from D. melanogaster into an EcoRl galactosidase antiserum. restriction site in the z2 transcript of the pbGl construct. Third instar larvae were stained according to the techniques of Angela Pattatucci (Pattatucci and Kaufman, 1991). FAB' Culture of Drosophila goat anti-rabbit HRP conjugate (1:50 dilution) was the Flies were cultured at 25 °C on standard Drosophila media secondary antibody used for the third instar larval staining supplemented with baker's yeast. In(3R)MAP17 is a V-type (Protos Immuno Research). Stained embryos and imaginal position effect pb that is suppressed at 29°C (Pultz, disks were photographed using a Zeiss Axiophot with 1988). Therefore, homozygous In(3R)MAP17 flies were Nomarski optics except for Figs 5D-G which were photo- cultured at 29 °C. graphed under normal light optics and Figs 4A-D which were photographed under Nomarski optics using a Zeiss Photo- P-element transformation scope III. Standard P-element mediated germline transformation tech- niques were utilized for obtaining independent insertion lines DNA sequencing of the pb minigene and both the pbGl and pbG2 constructs The restriction fragments were subcloned into either mplS, (Robertson et al. 1988). All reported transformants derived mpl9, mWB238, or mWB239 vectors (Barnes et al. 1983). independently from separate injections, except that pbG2 line Single stranded template was prepared using standard B' was obtained by jumping pbG2 line A' onto another techniques as described in the Sequenase® Version 2.0 kit chromosome using the P[ry+, A2-3](99B) element (Robert- (U.S. Biochemical Corp.). The sequencing reactions were son et al. 1988). performed using such a kit. Rescue of pb Results Flies of the genotype w; P[iv+, pb]/+; Ki pb5 pP/ TM3 Sb were inbred and the four expected classes of progeny were scored. Homozygous pb5 flies were scored by both the A pb minigene can completely rescue the pb homeotic presence of Ki/Ki phenotype and the absence of Sb. Animals phenotype carrying the minigene had yellow eyes and their siblings, Because the length of DNA contained within pb's 260 F. M. Randazzo, D. L. Cribbs and T. C. Kaufman genetic boundaries approximates to the technical limits the central 25 kb of the gene and consequently an of P-element germline transformation, a minigene alternatively spliced 15 bp microexon, positioned in the rather than a full sized transgene was utilized (Fig. 1). middle of the 25 kb intron, which was found in most pb The choice of the pb sequences included in the cDNAs generated by PCR amplification (Cribbs, Pultz, minigene was based on previous molecular and genetic Johnson and Kaufman, in prep.). The final minigene analysis (Pultz et al: 1988; Cribbs, Pultz, Johnson and thus includes all those sequences corresponding to pb Kaufman, in prep.). Searches for cDNAs representa- mRNA, as defined by northern blot, SI nuclease tive of pb mRNA in standard libraries to date have not protection, and DNA sequence analysis with the single been successful (Cribbs, Pultz, Johnson and Kaufman, exception of the 15 bp microexon. Closely associated in prep.). Therefore, three restriction fragments of exon flanking sequences are also included (Pultz et al. genomic DNA encompassing sequences inferred to be 1988; Cribbs, Pultz, Johnson and Kaufman, in prep.). relevant for pb function were used to construct the The minigene also contains a 27 bp oligonucleotide minigene. insertion in the middle of the pb 5' RNA leader and The proximal and distal genetic boundaries of pb lie some hsp70 promoter sequences that lie 7.3 kb between the In(3R)MAPl 7 breakpoint and the In(3R)- upstream of th&pb transcription start site (see Materials lab*76 breakpoint, respectively (Pultz et al. 1988). Both and methods for details). of these mutations fully complement pb deficiencies Introduction of this construct into the Drosophila and, therefore, define the maximum size of the pb gene. genome via P-element mediated germline transform- The proximal breakpoint of In(3R)MAP17 is approxi- ation resulted in the recovery of three independent mately 7 kb upstream of the pb transcription start site insertion lines: P[w+, pb]l, a homozygous viable and the minigene includes all of the 5' sequences insertion located on the second chromosome; P[w+, defined by In(3R)MAP17. The genetically defined 3' pb]2, a recessive semi-lethal also located on the second boundary of pb' is In(3R)laba16, which breaks in the chromosome (we interpret the semi-lethality as being labial transcription unit approximately 34 kb down- due to a disruption of the chromosomal locus of stream of the most 3' detectable pb sequences (Pultz et insertion); and P[w+, pb]3, a male semi-lethal located al. 1988). The construction excludes all of the sequences on the X chromosome. downstream of the first _polyadenylation site of pb The following experiments demonstrated that the pb (Cribbs, Pultz, Johnson and Kaufman, in prep.). The minigene lines 1 and 2 are functionally competent. transgene excludes most of the large intron composing Stocks were constructed bearing one of the three pb

pb 5' end pb 3' end

H-box w/////. ln(3R)MAP17

2Kb

Fig. 1. The diagram represents the molecular map of the pb region in D. melanogaster. Darkened boxes depict protein coding sequences, whereas white boxes delineate noncoding mRNA sequences. Asterisk(s) designate homeobox sequences. The darkened stippled bar below the molecular map represents the inversion In(3R)MAP17 which defines the proximal boundary of the pb gene; the adjacent cross hatches illustrate the range of uncertainty with respect to the breakpoint. The two black bars directly above the molecular map overlie the pb genomic sequences contained within the minigene. The linear diagram above outlines the entire minigene construct. It consists of two genomic fragments containing the 5' and 3' clusters of pb exons. The construct excludes most of the large intron and the alternatively spliced microexon. The transgene therefore includes the z2 pseudogene. The pb minigene also contains the mini-white gene as a visible marker, P-element ends, and pUC8 sequences (see Methods). As a consequence of its derivation from an hsp70 promoter//?£> transgene, the minigene construct contains part of the hsp70 promoter at a location 7.3 kb upstream of the pb transcription start site and a 27nt adapter sequence inserted in the middle of the pb RNA leader (see Methods). Rescue and regulation of proboscipedia 261

i tut

Fig. 2. (A) Left: scanning electron micrograph (SEM) of a Ki/Ki fly head preparation (this is wild-type lorpb). Right: close up view of the mouthparts. The white arrowhead with the black center points to a pseudotracheal row (p) of the anterior labellum. (B) Left: SEM preparation of a Ki pbs/Ki pb5 head, pb5 is a null allele. The proboscis is transformed into prothoracic legs. Right: close up of the transformed mouthparts. (C) Left: SEM preparation of the head of a rescued animal. The genotype is P[*v+, pb]l/+; Ki pb5/Ki pb5. Right: a close up view of the mouthparts. minigene lines in a pb5 homozygous (pb-nu\\) back- labellum (labial palps) which contains six pseudotra- ground (see Materials and methods for details). Fig. 2A cheal rows on each side. The dorsal half of each shows a fly with wild-type mouthparts. This structure pseudotrachea is smooth while the ventral half is has been described previously in detail (Hodgkin and convoluted. The posterior end of the labellum contains Bryant, 1978). The mouthparts include the anterior a characteristic set of bristles. Fig. 2B illustrates a 262 F. M. Randazzo, D. L. Cribbs and T. C. Kaufman

typical pb5 homozygous animal possessing prothoracic P[tv+, pb]3 transgene may have been damaged during legs in place of the labial palps. Flies carrying one P[w+, the original insertion event, resulting in reduced pb /?6]l-bearing chromosome in a pfr^homozygous back- function and the consequent partial rescue of the ground display completely normal mouthparts (Fig. 2C, mutant phenotype. However, the most likely cause of compare to 2A). The six pseudotracheal rows on each the apparent misregulation is a position effect in the labella form perfectly, the bristle patterns develop original line which can be ameliorated by transposition normally, and no indication of tissue transformation is to a new site. observed. Likewise, the P[tv+, pb]2 insertion line completely rescues the pb homeotic phenotype. Typical The pb transgene protein distribution pattern is a pb5/pb5 adults also display a reduced maxillary palp subset of the normal pb pattern in embryos as well as phenotype. Presence of one copy of either the P[w+, in third instar larvae + pb]2 chromosome or the P[w , pb]l chromosome in the In order to determine whether the pb transgene is pb-nu\\ background rescues the overall maxillary palp properly regulated, its derived embryonic and larval morphology, although in some animals the maxillary protein localization pattern was examined. The pb palps remain bent at the base. transgene protein exhibited a wild-type pattern of The third insertion line, P[w+, pb]3, shows only expression in the labial, maxillary and mandibular lobes partial rescue of the pb homeotic phenotype. Unlike of embryos and in the labial disks of third instar larvae. the ectopic leg phenotype seen in pb null animals, On the other hand, we found that aspects of pb's neural hypomorphic alleles of pb result in a transformation of and mesodermal pattern in embryos and pb's neural distiproboscis to antennae (compare Figs 2B and 3A). pattern in larvae were either significantly downregu- Fig. 3B exemplifies the effect of one P[w+, pb]3 lated or absent. chromosome in a pb5 null background. The observed Embryos from a pb5 /TM3 stock were probed with phenotype clearly resembles that produced by pb the pb antisera to determine whether pb5/pb5 embryos hypomorphic alleles. In addition to the above effects, expressed detectable amounts of pb protein. Twenty- flies carrying the P[TV+, pb]3 insertion chromosome five percent of the embryos were expected to possess exhibit thickened aristae, a male associated wings-held- the pb''/pb5 genotype and approximately 25 % (9 of 42) straight-out phenotype, and male semi-lethality. The lacked any pb protein staining while all of the remaining presence of thickened aristae also is observed also in a embryos (33 of 42) exhibited the wild-type pb staining number of hsp70 promoter//?b gene fusion lines and has pattern (mid-stage 10-stage 16 embryos); thus, it been interpreted in those cases to be a result of appears that the pb5 allele is a protein null, at least with misexpression of pb protein in the antennal anlagen respect to the antiserum used in this analysis. (Cribbs, Randazzo and Kaufman, in prep.). Thus, the + Embryos obtained from a V[w+, pb]l/ P[w+, pb]l; P[W , pb]3 insertion may be misexpressing the pb pb5/pb5 and a P[w+, pb]2/ +; pb5/TM6B stock were transgene protein in the antennal anlagen, resulting in probed with the pb polyclonal antibodies in order to the thickened aristae phenotype. Subsequent transpo- determine the pb transgenic protein distribution pat- sition of the P[w, pb]3 transgene onto another tern. Because the pb5 allele does not encode a protein chromosome resulted in simultaneous reversion of the product detectable with thispb antiserum, the observed three above mentioned phenotypes. This transposed pattern exclusively reflects pb protein accumulation minigene, however, still only partially rescues the resulting from expression of the transgene. The wild- homeotic phenotype. Therefore, we suspect that the type embryonic pattern of pb expression has been A

Fig. 3. (A) Scanning electron micrograph (SEM) preparation of the head of a Ki pb5/Ki pb4 adult animal focusing in on the mouthparts. pb is a null allele while pb4 is a hypomorphic allele. The transformation of mouthparts is to aristae (a). (B) SEM showing the mouthparts of a typical partially rescued adult animal. The genotype of this female is P[w+, pb]3/+; Ki pb5/ Ki pb5. Rescue and regulation of proboscipedia 263

described previously (Pultz et al. 1988). In wild-type ever, these results as well as the observed supraesopha- embryos, pb protein is not detectable until the geal and subesophageal ganglia expression are consist- beginning of stage 10 (approx. 4h 20min) (embryonic ent with the results seen in larvae (see below). stages established by Campos-Ortega and Hartenstein, Third instar larvae homozygous for either P[w+, 5 5 1985). During stage 10 (4h 20min-5h 20min), the pb]l;pb or P[H>+, pb]2; pb were stained with antibody protein appears abruptly in a set of mesodermal cells directed against pb protein. Internal controls consisted that reside anterior to the maxillary lobes and span of pb5/pb3 larvae (negative control) which are null for transversely across the embryo as a triangular belt pb protein and white/white (w/w) larvae (positive (Fig. 4A). The protein derived from the transgene, on control) which are wild-type for pb. The midgut, the other hand, seems to accumulate at significantly including the proventriculus, and the hindgut were lower levels in those mesodermal cells. Furthermore, removed from both the positive and negative control the F[w+, pb]l and P[w+, pb]2 transgene-derived larvae but not from the experimental larvae before the proteins subsequently become undetectable, while in fixation step. Therefore, after immunostaining, both wild-type animals the level of protein accumulation is the positive and negative controls as a group could be maintained (compare Fig. 4B with 4A). identified independently from the experimental group The start of stage 10 (4h 20min) is marked by the of larvae found in the same reaction tube. beginning of the stomodeal invagination which occurs In wild-type larvae, the labial disks and the tracheal ventrally near the anterior end of the embryo. The epithelial cells show pb protein accumulation; the stomodeum gives rise to the larval mouth. During stage observed punctate staining pattern is characteristic of 10 in wild-type embryos just after the onset of the the nuclear localization of homeotic proteins (Fig. 5A). mesodermal pb expression, the pb protein begins to The labial disk and tracheal epithelial cells stain gradually accumulate in the labial and maxillary lobes strongly in both the P[w+, pb]l; pb5 (Fig. 5C) and the and a small set of ectodermal cells in the mandibular P[w+, pb]2; pb5 homozygote lines, but fail to stain in lobe all of which will eventually contribute to parts of the pb null negative control larvae (Fig. 5B). The labial the larval head structure. Protein from the pb transgene disks are the anlagen of the adult distiproboscis or expressed by the P[»v+, pb]l line, however, becomes mouthparts (Bryant, 1978). Consistent with the hom- detectable in the labial and maxillary lobes and 5 or 6 eotic transformation of mouthparts to first thoracic (Tl) ectodermal cells of each mandibular lobe slightly earlier legs in pb null adults, the labial disks are transformed to in stage 10 when the stomodeal invagination is shorter resemble leg disks in pb null larvae (Fig. 5B). The (data not shown). expression of pb protein by the minigene in the labial Wild type embryos accumulate pb protein in a small disks is consistent with the observed adult rescue, for subset of ventral nerve chord cells in a metamerically the absence of pb expression in the mouthparts anlagen arranged fashion. Normal pb expression in the ventral results in a visible homeotic transformation. nerve chord first becomes detectable after head In third instar larvae, pb protein accumulates in the involution starts at the beginning of stage 14 (10 h brain and the ventral ganglion (Figs 5D,G). A clustered 20min). During stage 15 (11 h 20min-13h) the labial subset of cells in both the supraesophageal and lobes migrate anterioventrally and fuse on the ventral subesophageal ganglia of the brain hemispheres shows midline, prior to their involution. At this time the pb protein accumulation. In the ventral ganglion, pb number of ventral nerve chord cells with detectable pb protein accumulates in well over 200 nuclei. The pattern expression, positioned close to the ventral midline, has seems metameric only in the anterior portion of the increased. After the labial lobes involute and the cells ventral ganglion. In the posterior portion, one large of the maxillary lobes meet near the anterior pole of the area is devoid of pb protein accumulation, while in the stage 16 embryo (13-16h), additional ventral nerve remaining areas pb seems to be localized in closely chord cells accumulate detectable amounts of pb paired sets of cells. In two rescued lines, P[w+, pb\l/ protein more laterally from the median. In contrast to ?[w+, pb]l; pb5/pb5 (Fig. 5F) and P[w+, pb]2/P[w+, the wild-type pattern, pfo protein expression in homo- pb]2; pb /pb third instar larvae fail to detectably zygous P[H>+, pb]l; pb embryos was not detected in the accumulate pb protein in the ventral ganglion except for ventral nerve chord at any time during embryonic 5-8 cells in the most anterior region. These results are development (compare Fig. 4C with 4D). Elsewhere, in consistent with the absence of ventral nerve chord the central nervous system, a small number of cells in staining in embryos. On the other hand, pb protein does the supraesophageal and subesophageal ganglia express accumulate in what appears to be the same cells of the detectable amounts of pb protein in both rescued and supraesophageal and subesophageal ganglia where pb is wild-type embryos during stage 15 (data not shown). normally expressed. The levels of protein accumu- Thus, the pb transgene is properly regulated in the lation, however, are greatly reduced (compare Figs 5D brain at stage 15, but not in the ventral nerve chord of and 5F). the P[w, pb]l line. We cannot independently confirm In order to determine whether the transgene protein the ventral nerve chord expression defect in the P[w, expression was under proper negative regulatory pb]2 transgene line and formally eliminate the possi- + controls, we examined third instar larvae of the bility of a position effect, since +/+; pb/pb embryos + + s following genotypes: • (1) P[w , pb]l/ P[w , pb]l; cannot be reliably differentiated from P[w s, pb]2/+; 5 5 + + 5 5 Pb /pb , (2) P[w , pb]2/ P[w , pb}2; Pb /pb , and (3) pb/pb embryos at that time in development. How- P[n>+, pb]3/ P[w+, pb]3. Ectopic expression of trans- 264 b. M. Randazzo, D. L. Cribbs and T. C. Kaufman

gene derived pb protein was not detectable in these Fig. 4. Whole embryos stained with polyclonal rabbit lines (data not shown). We observed the pb protein antiserum specific for the pb protein and a secondary accumulation pattern in third instar larvae homozygous antibody conjugated to horseradish peroxidase (see for the In(3R)MAP17 mutation to determine if Methods). Embryos are oriented anterior to the left and posterior to the right. 20x magnification. (A) Frontal plane sequences upstream of the proximal breakpoint are of focus, wild-type embryo during the germ band retraction necessary for any portions of the pb pattern. We could stage showing nuclear staining in the labial lobes (lab), not detect any missing aspects from the wild-type maxillary lobes (), a few ectodermal cells of the pattern (data not shown). mandibular lobe (see arrowhead) and also in a triangular belt of mesoderm cells (msd). (B) Similarly staged homozygous P[w+, pb]2; pb5 embryo showing pb transgene Results from pb/ /3-galactosidase fusion transgene lines protein accumulation. Note the presence of staining in a demonstrate that sequences necessary for the pb subset of ectodermal cells of the mandibular lobes (see pattern of expression are located downstream of the arrowhead) and the absence of mesodermal staining. (C) Frontal plane of focus, stage 16 wild-type embryo pb translation start site expressing pb in individual ventral nerve chord cells (vnc) In order to determine the location of the ds-acting in a metameric pattern. (D) Similarly staged homozygous regulatory elements responsible for the pb expression P[iv+, pb]l; pb embryo showing complete absence of vnc pattern, pb/lacZ reporter gene constructs were made expression. and introduced into the Drosophila genome. The pbGl Fig. 5. Dissected portions of third instar larvae stained construct consists of 7.3 kb of sequence upstream of the with polyclonal rabbit antiserum specific for the pb protein pb transcription initiation site, exon-1, intron-1, and and a secondary antibody conjugated to horseradish two thirds of exon-2 including the ATG translation peroxidase (HRP). Top is anterior and bottom is posterior. initiation site (Fig. 6). The exon-2 coding sequences are 5A-C. Imaginal disks from third instar larvae stained in fused in-frame with the lacZ coding sequences to make the same reaction tube. 20x magnification. 5A. The a p6//?-galactosidase fusion protein. The pbG2 con- internal positive control (w/w) is wild-type for pb struct also contains 7.3 kb of sequence upstream of the expression in the labial disks (lab). The small arrowhead near the bottom right points to tracheal epithelial cell pb transcription initiation site (Fig. 6). In this case, staining. (B) Internal negative control - pb null (pbs/pb5). however, RNA leader sequences in the first exon of pb The labial disks are transformed into first thoracic leg disks are fused to the alcohol dehydrogenase (ADH) RNA (Tl) and there is no pb staining, only nonspecific leader. The ADH sequences also include an ATG background staining. This nonspecific staining occurs in all translation start site and ADH coding sequences which imaginal disks and is independent of the specific antibody are fused in-frame with lacZ sequences. Both the pbGl used (it is not specific to just pb antisera). The intensity of and the pbG2 constructs include precisely the same pb the nonspecific background staining is dependent on the 5' upstream sequences as the minigene. Six indepen- elapsed time of the HRP color reaction. This particular dent pbGl insertion lines and two pbG2 lines were batch (A-C) was exposed to the HRP reaction for a longer obtained via P-element mediated germline transform- period of time, hence the visible background staining. Batches of pb antisera-treated larvae that were exposed to ation. Embryos and third instar larvae carrying the the HRP reaction for shorter amounts of time showed different reporter gene insertions were immunostained proportionally less background. (C) Labial disks from a using polyclonal antibodies directed against /3-galactosi- rescued larva (homozygous P[w+, pb]\; pb5) stains strongly dase. for pb. The disks are not flattened on the slide as much as The results from the six pbGl transformant lines with the positive control (A). The labial disk on the right demonstrate that sequences upstream of the pb was torn during mounting. The small arrowhead near the bottom right shows staining in tracheal epithelial cells translation start site are not sufficient for the pb pattern somewhat out of the plane of focus. The boxed-in area is of expression in embryos and third instar larvae. In both shown magnified 4x in H to highlight the punctate nuclear embryos and larvae, all six of the pbGl transgene lines staining. (D-F) Central nervous system from third instar and both pbG2 lines show different patterns of larvae stained in the same reaction tube. The brain expression, or no expression at all, and none of the hemispheres (br) are above and the ventral ganglion (vg) is patterns are similar to that of pb (data not shown). below. (D) The internal positive control (w/w) is wild-type Thus, rather than displaying major components of or for pb expression. The lines point to pb expression in cells the full pb pattern of expression, both the pbGl and of the supraesophageal (spg) and subesophageal ganglia pbG2 reporter genes seem to be acting as 'enhancer (sbg). Cells staining in the sbg of the left brain lobe is out traps', i.e. the pb promoter sequences in the constructs of the focal plane. (E) Internal negative control - pb null (pb/pb). (F) Homozygous P[w, pb]l; pb. The upper are presumably being driven or repressed by the ex- arrowhead points to weak spg staining and the lower right acting regulatory elements of other genes located arrowhead points to weak sbg staining. The arrowhead on nearby the sites of insertion in the genome. the lower left points to the area where seven anterior vg Because the minigene rescues the homeotic pheno- cells show pb accumulation. (G) 40x magnification of wild- type and also expresses the normal pb protein pattern in type pb protein accumulation in the CNS. the embryonic and larval head, sequences necessary for this pattern must be present in the minigene but absent from these two reporter constructs (see Fig. 6). Thus, sequences included in the pbGl construct but not in important pb regulatory elements that are necessary for either the large intronic region absent from the this pattern must be located downstream of the pb minigene or downstream of the first pb poly (A) site.

Fig. 8. (A) pbGl/intron reporter gene line stained with antibodies directed against fi- galactosidase and secondary antibodies conjugated to horseradish peroxidase (HRP). 20x magnification. Embryo is oriented anterior to the left and posterior to the right. /3-galactosidase accumulates in the labial lobes (lab), the maxillary lobes (max) and a subset of ectodermal cells of the mandibular lobes (see arrowhead). Negative controls (w/w), i.e. animals without a pbGl/intron reporter gene, had the same nonspecific background staining levels as the areas outside lab and max (data not shown). (B,C) Dissected third instar larvae showing labial disks (lab) stained with antibodies directed against /3-galactosidase and secondary antibodies conjugated to HRP. Anterior is above and posterior is below. 20x magnification. (B) Internal negative control (w/w). (C) pbGl/intron reporter gene line stained in the same reaction tube as B. The HRP/chromogen reaction was not allowed to continue as long as with the embryo shown in A. Thus, nonspecific background staining levels are lower with the negative control and the staining intensity of C is concomitantly lower. Rescue and regulation of proboscipedia 265 elements conserved between D. melanogaster and D. pseudoobscura

H-box pbG(l )/intron pb/B-galactosidase protein fusion • pb exon-2/intron-2 fragment

pbGU) pb/B-galactosidase protein fusion

ADH RNA leader-AUG pb6(2) pb RNA leader/ADH/B-galactosidase fusion

2 Kb

4- •+- Fig. 6. The molecular map of the pb region in D. melanogaster is depicted as in Fig. 1. Aligned directly below are representations of the pbGl/intron, pbGl, and pbG2 constructs. The pbGl construct consists of sequences beginning 7.3 kb upstream of the pb transcription start site to downstream of the pb translation start site in the second exon fused in frame with lacZ. The pb sequences are drawn as in the pb molecular map and the lacZ sequences are designated by the stippled box labeled /3-gal. The pbGl/intron construct is identical to the pbGl construct except for the insertion into z2 of a 1.6 kb DNA fragment spanning most of exon-2 and the 5' most portion of intron-2. The area that the fragment encompasses is highlighted by the stippled area directly below the pb molecular map and the point of insertion is indicated by the funneling in of the stippled area into z2. The pbG2 construct fuses first exon pb RNA leader sequences with alcohol dehydrogenase (ADH) RNA leader sequences. The ADH sequences continue past the ADH translation start site (diagonally striped box) and then fuse in frame with lacZ coding sequences. The pb sequences included in the minigene are represented by the stippled boxes aligned directly above the corresponding sequences in the molecular map. The positions of the elements conserved between D. melanogaster and D. pseudoobscura are highlighted by the vertical dotted lines that run through the corresponding sequences in the appropriate constructs. The upstream element is short and therefore is bisected by one vertical dotted line; the intron-2 element is larger and resides between the two dotted lines which form a narrow rectangular box.

DNA sequence comparison between D. melanogaster coding sequences, blocks of strongly conserved se- and D. pseudoobscura indicates the location of quences were observed both in the second pb intron, putative pb cis-acting regulatory elements slightly downstream of the second exon, and about In order to help pinpoint the location of the important 300 bp upstream of the pb mRNA 5' terminus in D. cw-acting regulatory elements of pb, DNA sequences of melanogaster (Fig. 6). two divergent species were compared. Because D. Fig. 7A shows DNA sequence in the exon-2/ intron-2 pseudoobscura is approx. 46 million years diverged boundary region of pb in both species. The end of the from D. melanogaster (Beverley and Wilson, 1984), second exon of pb in D. pseudoobscura was inferred, sequence conservation between these two species based on sequence identity to the second pb exon in D. should reflect functional relevance. melanogaster, which was defined by SI nuclease Lambda EMBL4 genomic clones with high sequence protection analysis and PCR-generated cDNAs homology to specific pb probes from D. melanogaster (Cribbs, Pultz, Johnson and Kaufman, in prep.). Third were obtained in a chromosome walk through the base degeneracy which preserves identity at the amino Antennapedia Complex in D. pseudoobscura (Ran- acid level is evident in 4 of the last 12 codons of exon-2. dazzo, Seeger, Huss and Kaufman, in prep.). Regions Numerous blocks of sequence identity reside down- with high sequence similarity in D. pseudoobscura were stream of the second exon of both species. Five localized further by Southern blot analysis, subcloned, elements ranging in size from 18-28 bp and three and then sequenced. The D. pseudoobscura DNA elements between 38 and 59 bp in length exhibit 100 % sequence was then compared with the existing pb sequence conservation between the two species. sequence from D. melanogaster. Outside of mRNA- Shorter blocks of 100% sequence conservation are 266 F. M. Randazzo, D. L. Cribbs and T. C. Kaufman

R K N N N N

D. mel !AAGACA|TCCpGCftAG^GCAGCfUiC^ACpVACAATp exon2 ends D. pSO JKJbbKKkkritfa

AG 3GT \ \GTCC2 CATGCACATTACACTGGGGGAAGCATAAGGTTTTTAATTTAGCGATCGACCATGT AG 3GT 3 ^GTCC? AGA

consensus splice donor site CAATTTGAACACCATTCTCTGAAGAATAGGCGATAATCTCATCAGTCCATCTCAAACTATTGAATCA

.ATCAAATAAAAAGTCAGCTATTTTTATAGACAGTTTTCCATATCAGTTTAGACCGGAATCAATTGG

GTTCTCTTGTCCCGGCCATTTTTTCTTGCCGTGTACTGGCTG3TGAAATGCGAAACTCTCATTAATC 3TGAAATGCGAAACTCTCATTAATC

3AAATCAATGCAATTTACCTTGCP TTWA. \TGAGGCS CAGCATG 3:GGCGCCTTC 3CCA Fig. 7. (A) DNA sequence ATCAATGCAATTTACCTTGCaACPlcGkTG A 5AGACG :AGCATG :GGCGCCTTC comparison of the exon-2/ intron-2 regions of CTTCCCGTCCAGAAAAACAGGGCCAGAACTCTATGTCCATACTGCCCGATCCCGATCCCACTCCCAA proboscipedia in D. TCTAGAACGGGGCACCCTTTTCTTTCCTT melanogaster and D. TCCTXCTGCMCCTTTTACCACGXACTCGATCA :GCAC pseudoobscura. The D. TCCirTTCCTC'IlCCT'lfccGTTTcbCACTCGATCAbAGACACAC^ GCACrCGTCTGGTCTCGTCTGGG melanogaster sequence is above and the D. pseudoobscura sequence is aligned directly CGAG, T XGCTCCTTTGATGGACAGCT. TGTGCCGGCAAAG ? P T 3C 3 rc... t below. The exonic sequences A;GGCTCCTTTGATGGACAGCI TG£ TGTGCCGGCAAAG Pf TWSm imi 3C 3 are boxed-in as are all of the r2 rl intronic sequences with A MAMM&i5 f i c Wt r m :GAG \GTAGAG :GGACCA lAGCIl GfcAAAT' complete sequence identity. GAG 3CAGACCAGTCCT:GGACCA \GTAGAGlAGCrlG 3AAATG The first box at the top of the figure consists of the last 12 codons of exon-2. The vertical :ATTTATTTCCCTTCG ;TTCGACTCGAGTCGA \TGTGTCGATTTCATTTGCGCAAGAAAT AAGAA \TGTGTCGATTTCATTTGCGCAAGAAAT A1ACGAA dotted lines separate codons. CATTTATTTCCCTTCG\ The asterisks below designate third base degeneracy in the AAGCGACGAGCAGAGTACAAAATATAAAAAAAAAAATACAGAAAAATACGCCTGGACCAGGGATCGA codons. Amino acids are TACTCGTAAGAAAAAAACCAAGAAAAGATCGAGACA designated by their single r3 letter abbreviations above the GC CAGCCGCATTTAAAATAAATGAGGTATCAAGGTTGTCCGACCATACG TTTTCCTGTCC ;AC TTGC: codon sequences. The rest of CAGCCGCATTTAAAATAAATGAGGTATCAAGGTTGTCCGACCATACG fTTTCCTGTCC =VTG ^T' the figure consists of intronic sequences. For alignment purposes, the dots represent CCAAAACCCGAGTCCCTTTC ICTCj. .AGAGCGATTGCA a ; iV&M sequences absent in the • ATGCCACTCCACACCACACCACACTCCGCTCTCTCTCTCTCIlCTcfa;G d?VGAGCGATTGCA j: respective species. Directly repeated sequences are KRJ A ET TTTTCCTGTCC XCTTTTT r'GCpTTCGATCG overlined by arrows. The Mi A PT rTTTCCTGTCC :CCTTTT1 \TTtTTTCirGCTCCCTCAATC stippled area highlights a long but interrupted direct repeat named rl, while r2 is a directly \CTGCGGATGTCGAGGGAATGCCGC 5TTGC AC. ;TTGCAA TTGCAf SCCTGTGCGCCGCTGGC 3AAAAGGCAGGGGCACTCAGCACCG ;TTGC GCTTGCAA3 rTGCAfTTGCAAGACAGGCAGGG repeated sequence that is CAG contained within rl and is also found in a third location GATGTTGTAGCACAGAAAATACGAATTTCATGTCTGCTGC between the two rl repeats. CCTGGGGATAGGTTTTGTGTAGCACATAAAATGTTTCGCGGCTGTTGTTGCTGCTGCTGCTGC^ An 11 bp directly repeated ACCATTTTAAA:CAG sequence is referred to as r3, ACCATTTTAAA 3CTT while two additional repeats are designated as r4 and r5. (B) Upstream DNA sequence comparison between D. B CCCATAGCCGAAAGACCAAAAAGCCGGAGAGCGAGAAACTCGCTACTGTGGCTTTTTCTTTTCATTT melanogaster and D. CTTTCTGCTGCCTCTGCTGCACCTCTTCTTTATTCCTTGTTTCTTCTGCCAGTTCTTCTTCTGCTGC pseudoobscura. Boxed-in areas show complete DNA sequence GCG S AATGTGTAAATAA 3 UUVAAC : ^AATAAATTCCGTCACAAGCAACACGTTC CCG identity. This'conserved GCGjAATGTGTAAATAA ^AAAAC 3 ^AATAAATTCCGTCACAAGCAACACGTTC!VAC element lies approximately 300 bp upstream of the pb 5' TTGGAAGTGGAAAAAA D. mel mRNA terminus in D. AAAAAAAAACGCACAA D. pSO melanogaster. Rescue and regulation of proboscipedia 267 .clustered around these elements. Between the two pattern of expression. It is highly unlikely that the species, sequences separating these conserved elements upstream conserved element is involved in z2 rather differ in both nucleotide sequence and in length. We than pb regulation; even though the 11.5 kb distance also have cloned the putative pb homologue from D. between zen and pb is approximately conserved virilis, a species estimated to be ~62 million years between D. melanogaster and D. pseudoobscura, diverged from D. melanogaster (Beverley and Wilson, preliminary evidence indicates that z2 coding sequences 1984). We have sequenced a portion of the inferred are not present in D. pseudoobscura between zen and intron-2/exon-2 region in D. virilis and preliminary pb (Randazzo, Seeger, Huss, and Kaufman, in prep.). results show a substantial amount of sequence identity with the conserved intron-2 region in D. melanogaster The region containing the conserved intronic elements and D. pseudoobscura including 100 % conservation of is necessary for pb expression in the embryonic and the 'rl' motif (see below). larval head A number of directly repeated sequences reside We next set out to determine whether the conserved within these conserved stretches. Included within two element region in the second intron was necessary for of:the conserved blocks is a directly repeated 32bp pb expression in the embryonic and larval head. A motif, ANCTGTTATTTNNGNTCATTTGTNANAA- 1.6 kb EcoRl restriction fragment containing the TNTT, referred to as 'rl' (stippled and indicated by conserved intronic elements was inserted into the pbGl arrows in Fig. 7A). A seven nucleotide motif consisting lacZ reporter gene construct 1 kb upstream of the pb of the sequence TCATTTG, and referred to as 'r2', is transcription start site (Fig. 6). The fragment was located within the rl repeat and also is found at a third inserted into z2 in the same relative orientation as its location between the two rl repeats (indicated by original position in pb. Two independent lines were arrows in Fig. 7A). Additionally, an r2 sequence is obtained by P-element transformation in Drosophila. [3- found in the opposite orientation approximately 150 bp galactosidase protein accumulation was monitored in downstream of the exon-1 splice donor site in D. both embryos and third instar larvae. Both reporter melanogaster (data not shown). The corresponding gene lines showed a pattern of /3-galactosidase accumu- region, however, has not yet been sequenced in D. lation in the embryonic head identical to that of pb pseudoobscura. An 11 bp directly repeated sequence, protein accumulation observed in rescued embryos TTTTCCTGTCC, is found in two locations and is (compare Figs 8A with 4B). The labial lobes, the designated as 'r.3' (indicated by arrows in Fig. 7A). Two maxillary lobes, and 5 or 6 ectodermal cells of the sets of conserved direct repeats, 'r4' and 'r5', overlap mandibular lobes show /3-galactosidase accumulation. one another and are located downstream of the second In older embryos of one of the independent pbGl/ r3 element (indicated by arrows in Fig. 7A). The intron lines, ectopic expression is observed in cells of sequences of r4 and r5 are similar; the r4 sequence is the central and peripheral nervous systems. As with GTTGC while the r5 sequence is TTGCAA. wild-type and rescued third instar larvae, both of the These conserved intron-2 sequences are contained independent pbGl/intron lines exhibit significant /?- within the minigene but are excluded from the pbGl galactosidase expression in the labial disks, as com- and pbG2 reporter gene constructs (Fig. 6). Because pared with the internal negative control (compare the minigene expression pattern resembles normal pb Figs 8C with 8B). No pb-\\ke pattern of expression is expression, whereas the /3-galactosidase reporter genes observed in the CNS. Additionally, larvae from both fail to reveal a pb-\\ke expression pattern in embryos lines exhibit ectopic expression in the distal most and third instar larvae, these blocks of conserved portion of the antennal and Tl, T2, and T3 leg disks. sequences are good candidates for ds-acting regulatory Taken together, the results from the embryonic and sequences necessary for the correct pattern of pb larval analyses of these transgenic lines demonstrate expression. that the restriction fragment containing the intron-2 conserved elements is necessary for pb expression in the Located 305 bp upstream of the pb transcription start head. At present we do not know why these reporter site and 696 bp downstream of the z2 poly(A) site in D. constructs also show ectopic expression of ^-galactosi- melanogaster is a highly AT rich 60 bp stretch sharing dase, a feature not observed in the minigene lines. 55 bp of sequence identity with D. pseudoobscura However, we suspect that the nervous system ex- (Fig. 7B). The distances from the second pb exon to the pression observed in the one line is best explained by conserved upstream sequence are similar in both position effects and that the ectopic disc expression in species, i.e. 2.6kb in D. melanogaster versus 1.85kb in both lines reflects the absence of a negative regulatory D. pseudoobscura, though we do not yet know the element in the reporter constructs. These possibilities precise size or location of exon-1 in D. pseudoobscura. are currently under investigation. The DNA sequences surrounding the conserved block diverge, presumably due to lack of functional con- straints. This upstream conserved element is included in the minigene and in both the pbGl and pbG2 reporter Discussion gene constructs (Fig. 6). The protein expression pat- terns of the minigene and of both these reporter genes Rescue of a homeotic gene indicate that while these sequences may be necessary Rescue of the proboscipedia gene with two independent they are not sufficient to direct any aspect of the pb insertion lines directly demonstrates that the DNA 268 F. M. Randazzo, D. L. Cribbs and T. C. Kaufman from 7.3 kb upstream to just downstream of the at least in terms of homeotic regulation and function proposed pb transcription unit is responsible for pb (we define homeotic function in terms of the visible homeotic function. Two transcription units reside cuticular transformations), that thepb gene works out within this interval, z2 and pb. The pattern of of its normal chromosomal context. Clearly, it is expression for z2 closely resembles that of zen possible to separate at least some of the homeotic genes (Rushlow et al. 1987), but genetic analysis has failed to from the presumed configuration of the ancestral gene ascribe any function to z2 (Pultz et al. 1988). Taken cluster and from the present ANT-C and BX-C together, previous genetic and molecular evidence structure in D. melanogaster and still have a viable fly. (Pultz et al. 1988) and the rescue results presented here Whether or not these flies can survive the selective demonstrate that pb function can indeed be attributed pressures imposed in a nonlaboratory environment is to the transcription unit previously identified as pb. unclear at this point. Immuno-staining of rescued embryos and third instar At least two possibilities exist for the evolutionary larvae with antibodies directed against pb protein shows selection of these homeotic gene complexes. One that the transgenic pb pattern of expression largely possible explanation is that genes within the cluster may mimics the normal endogenous pattern of ectodermal possess shared or interspersed ds-acting regulatory expression in the head region of both embryos and third elements. On the other hand, inversions in the ANT-C instar larvae. Additionally, ectopic expression is not that break within one gene fail to affect the other genes observed. These results seem to exclude the possibility in the complex and therefore suggest that the homeotic that the observed rescue of the homeotic phenotype is genes of the ANT-C may not share regulatory elements mediated by unregulated, global expression of the (Abbott and Kaufman, 1986; Pultz et al. 1988; transgenic pb protein. Diederich et al. 1989). To date, rescue of other homeotic genes of the ANT- A second possible explanation for the selection C and the BX-C with minigenes that utilize a throughout evolution against the dispersion of these nonheterologous promoter and regulatory elements has homeotic genes is the detrimental consequences of not been reported. However, it should be noted that position-effects on these genes, since inversion events labial, another homeotic gene in the ANT-C, has been within the complex could influence the transcription of partially rescued using a minigene, i.e. rescue of the the nearby homeotic genes by bringing in new embryonic but not adult phenotype (Chouinard and sequences that (1) contain enhancer-like elements Kaufman, in prep.). Since by homeotic standards pb and/or (2) change the local chromatin structure. While and labial have relatively small ds-acting regulatory ectopic expression of some genes in Drosophila fails to regions (Pultz et al. 1988; Diederich et al. 1989), it discernibly affect the organism (Bonner et al. 1984; would appear that difficulties in performing rescue of Basler and Hafen, 1989a), ectopic expression of the homeotic genes stem in part (if not primarily) from the homeotic genes Antp, Ubx and pb have been found to problems associated with the great size and complexity cause highly deleterious effects on the organism of these genes and not necessarily from residential (Gibson and Gehring, 1988; Mann and Hogness, 1990; requirements in a specific chromosomal domain. Gonzlez-Reyes et al. 1990; Cribbs, Randazzo and Kaufman, in prep.). Thus, the homeotic genes of the What is the functional significance of the ANT-C ANT-C may reside within the complex as 'prisoners' complex structure? whose escape, in many cases, may cause detrimental The homeobox-containing genes of the ANT-C and the effects on the organism as a whole. The minigene line BX-C presumably arose through duplication and that partially rescues may be a case in point, for its divergence events prior to the ancestral split between viability is severely compromised. In addition, Go mini- the deuterostomes and protostomes during the late Pre- gene transformants may have failed to survive (given Cambrian period. But why have these genes remained the low efficiency of recovery compared with the both clustered and in the same linear order for over 500 reporter gene constructs), although this type of million years of evolution? The ANT-C and the BX-C negative evidence is difficult to quantitate and remains in Drosophila melanogaster presumably arose from an distressingly anecdotal in character. event that split a larger single ancesteral complex. Additionally, genetic manipulations using deficiencies and translocations have demonstrated that Ubx, abd-A Putative elements missing from the minigene result in and Abd-B can be separated without compromising the loss of the full pb expression pattern function under laboratory conditions (Struhl, 1984; We were able to show, in animals null for endogenous Tiong et al. 1987). Moreover, Antp amd Ubx reporter pb, that levels of transgene-derived pb protein accumu- gene constructs inserted into chromosomal locations lation in the supraesophageal and subesophageal outside of the homeotic gene complexes have been ganglion seem normal in embryos and are significantly shown to closely mimic the patterns of expression of reduced in larvae. It is possible that expression is their respective genes (Boulet and Scott, 1988; Irvine et initiated in the embryo but is not maintained through- al. 1991). The two pb minigene transgenic lines that out larval development. Additionally, transgene- completely rescue the homeotic phenotype are second derived pb protein expression, in pb~ animals, is chromosome insertions, whereas the ANT-C resides on initiated but not maintained in the mesodermal cells of the third chromosome. Thus, our results demonstrate, the embryo where pb normally accumulates. Therefore, Rescue and regulation of proboscipedia 269 tissue specific maintenance elements may be missing phenomenon and not a function of the minigene per se - from the minigene. In pb~ animals, detectable ventral P[w+, pb]2 is a semilethal insertion nerve chord expression of pb in embryos and detectable ventral ganglion expression of pb in third instar larva Regulation of pb expression are absent in animals carrying the minigene. Consistent Through the use of a pb minigene, pb/lacZ reporter with all of these results, thepfr reporter gene constructs, gene fusions, and DNA sequence comparison between which contain only a subset of the total pb DNA two divergent species we have been able to locate sequences used to construct the minigene, fail to sequences necessary for much of pb's complex pattern demonstrate the ability to initiate or maintain a pfo-like of expression. This includes pb protein expression in pattern of expression in the mesoderm or nervous the labial, maxillary and mandibular lobes, subesopha- system. Missing c/s-acting regulatory elements, the geal ganglia, and supraesophageal ganglia of the missing microexon, or changes in genome context could embryo and in the labial disks, subesophageal ganglia, explain the defects in nonhomeotic expression. The and supraesophageal ganglia of the larvae. Regulatory presence of the 15 bp alternatively spliced microexon, elements necessary for this expression lie just down- an exon missing from the minigene, may be necessary stream of the second pb exon. This region contains a for pb protein stability in the CNS and mesoderm or for number of long blocks of DNA sequence that are the accumulation of the pb transcript in those cells. conserved between D. melanogaster and D. pseudoob- Analysis of the Ubx gene products indicates that scura, species approx. 46 million years diverged. alternate splice products are differentially localized in Present within these conserved sequences are a number the epidermis and mesoderm versus the CNS (Kornfeld of directly repeated elements. Some eucaryotic et al. 1989). At this point we cannot formally rule out enhancers include directly repeated sequences. The the possibility that these results are due to the minigene SV40 virus, for example, contains two directly repeated being outside of the context of the ANT-C. However, 72 bp enhancer elements (Moreau et al. 1981). The because the minigene is significantly truncated as intron-2 direct repeats therefore may represent compared with the endogenous pb gene and because enhancer elements responsible for pb localization in the the remaining aspects of transgene derived pb ex- embryonic and larval head region. There also is a pression are normal, we believe the defects in minigene possibility that rather than acting alone, these con- expression are due to missing cw-acting control el- served elements require the coordinate action of other ements or the missing microexon and not to changes in sequences to bring about proper pb distribution in the chromosome context. These putative missing cw-acting head. One candidate for such a sequence is the control elements would be located either in the major conserved element located upstream of the pb tran- intron or downstream of the first pb poly(A) site but not scription start site. This sequence is included in the upstream of the proximal In(3R)MAP17 breakpoint, minigene and also in the pb/lacZ reporter gene fusion because embryos (Pultz, 1988) and larvae homozygous constructs. Both the upstream conserved element and for this inversion seem to show neither reduced levels many of the intron-2 conserved blocks of sequence, nor missing aspects of the pb protein accumulation especially the direct repeats, are AT rich. Sequences pattern. Furthermore, because a clear role for pb that bind homeoproteins have been shown to be AT expression in the mesoderm and nervous system does rich (for review see Biggin and Tjian, 1989). Thus, these not exist, the possibility remains that pb expression in conserved repeats may interact with homeobox-con- these tissues is controlled fortuitously by long range taining trans-regulators. elements. A correlation exists between defective neural ex- Possible significance of the putative internal regulatory pression and changed behavior. Rescued homozygous elements P[w+, pb]l; pb5 and P[w+, pb]2; pb5 adults do not It has been hypothesized that the large introns of the readily attempt escape on removal of the plug from the homeotic genes are necessary for the proper timing of culture vessel but rather tend to stay on or near the translation initiation, i.e. longer primary transcripts bottom surfaces. These animals also do not fly properly. require more time for their transcription and thus a Homozygous P[w+, pb]l and wild-type control animals, greater lag time results between transcription initiation however, promptly escape when given an opportunity and translation (Kornfeld et al. 1989). Absence of the and commonly migrate to the top of the bottle. large pb intron, however, does not effect pb's homeotic Unfortunately, drawing comparisons with pb-nul\ adult function. We hypothesize that the presence of large controls seems inappropriate because the mouth-to-leg introns may function as a means for distancing internal transformation prevents the animal from eating and enhancer elements from the promoters of other nearby thus affects behavior via malnutrition. We are in the homeotic genes, thus preventing cis interactions be- process of testing the possibility of a cause and effect tween genes. A number of smaller Drosophila genes relationship between the neural expression and the possess regulatory elements within their introns (Basler behavioral phenotype. Additionally the homozygous et al. 19896; Gasch et al. 1989). Most of the homeotic P[w+, pb]l; pb5 and ?[w+, pb]2; pb5 stocks fail to genes of the ANT-C and the BX-C possess uncharacter- propagate well, the latter much more so than the istically large transcription units as compared with other former. The significant differences between these two Drosophila genes. For example, pb, Scr, Ubx, and Antp lines, however, indicates this is a position dependent contain transcription units of 33 kb, 65 kb, 77 kb, and 270 F. M. Randazzo, D. L. Cribbs and T. C. Kaufman

100 kb respectively (see Kaufman et al. 1989 and BEVERLEY, S. M. AND WILSON, A. C. (1984). Molecular Evolution Kornfeld et al. 1989). The Antp gene has been shown to in Drosophila and the Higher Diptera II. A Time Scale for Fly Evolution. ]. Mol. Evol. 21, 1-13. contain regulatory elements within its introns (Boulet BIGGIN, M. D. AND TJIAN, R. (1989). Transcription factors and the and Scott, 1988). Unpublished data on labial indicate control of Drosophila development. Trends Genet. 5, 377-383. that regulatory elements reside within its largest intron BONCINEbLI, E., SOMMA, R., ACAMPORA, D., PANNESE, M., (Chouinard and Kaufman, in prep.). Here we have D'ESPOSITO, M., FAIELLA, A. AND SIMEONE, A. (1988). presented evidence for c/s-acting regulatory elements Organization of human homeobox genes. Human Reproduction contained within the large pb intron. Long range cis 3, 880-886. BONNER, J. J., PARKS, C, PARKER-THORNBERG, J., MORTIN, M. A. interactions between genes within the ANT-C occur AND PELHAM, H. R. B. (1984). The Use of Promoter Fusions in when a deletion mutation juxtaposes fushi tarazu (ftz) Drosophila Genetics: Isolation of Mutations Affecting the Heat promoter sequences 20 kb upstream of the z2 promoter. Shock Response. Cell 37, 979-991. In this mutant, the ftz sequences drive the expression of BOULET, A. M. AND SCOTT, M. P. (1988). Control elements of the P2 promoter of the Antennapedia gene. Genes Dev. 2, z2 in a /fe-like striped pattern (Rushlow and Levine, 1600-1614. 1988). Thus, maximal separation of these m-acting BRIDGES, C. AND DOBZHANSKV, T. (1933). The mutant regulatory elements from other nearby homeotic 'proboscipedia' in.Drosophila melanogaster - A case of promoters may minimize long range regulatory influ- hereditary homoosis. Wilhelm Roux's Arch. Devi Biol. 127, ence over these promoters, including misexpression, 575-590. thereby creating a protective 'buffering effect'. BRYANT, P. J. (1978). in Imaginal Disks. In The Genetics and Biology of Drosophila Vol. 2c (ed. M. Ashburner Interestingly, buffering sequences have been recently and T. R. F. Wright). Academic Press: New York. identified in Drosophila (Kellum and Schedl, 1991). CAMPOS-ORTEGA, J. AND HARTENSTEIN, V. (1985). The Embryonic This hypothesis is consistent with the one forwarded Development of Drosophila melanogaster. Springer-Verlag: New above as to why these genes have remained clustered York. and in the same order over hundreds of millions of years DENELL, R. E., HUMMELS, K. R., WAKIMOTO, B. T. AND KAUFMAN, T. C. (1981). Developmental studies of lethality of evolution. associated with the Antennapedia gene complex in Drosophila melanogaster. Devi Biol. 81, 43-50. We thank Rudi Turner for the scanning electron mi- DESPLAN, C, THEIS, J. AND O'FARRELL, P. H. (1985). The croscopy work, Dwayne Johnson for sequencing, Marie Drosophila developmental gene, , encodes a sequence Mazzulla for the preparation of pb antibodies, Angela specific DNA binding activity. Nature 318, 630-635. Pattatucci for her expert advice on larval staining, John DIECKMANN, C. L. AND TZAGOLOFF, A. (1985). Assembly of the Tamkun, Carl Thummel and Drzislav Mizmer for the vectors, mitochondrial membrane system. J. biol. Chem. 260, 1513-1520. Mark Seeger and Kathy Huss for cloning in D. pseudoob- DIEDERICH, R. J., MERRILL, V. K. L., PULTZ, M. A. AND KAUFMAN, T. C. (1989). Isolation, structure, and expression of scura, and Jessica Kissinger for cloning in D. virilis. We are labial, a homeotic gene of the Antennapedia Complex involved grateful to Charles Langley for the D. pseudoobscura and in Drosophila head development. Genes Dev. 3, 399-414. Doug Cavener for the D. virilis genomic libraries, Scott DRIEVER, W. AND NUSSLEIN-VOLHARD, C. (1988a). A Gradient of Chouinard and Elizabeth Raff for critical reading and Chris bicoid Protein in Drosophila Embryos. Cell 54, 83-93. Sayre for proof reading of the manuscript. We also would like DRIEVER, W. AND NUSSLEIN-VOLHARD, C. (19886). The bicoid to thank Dee Verostko for her administrative assistantship. Protein Determines Position in the Drosophila Embryo in a This work was supported by a National Institutes of Health Concentration-Dependent Manner. Cell 54, 95-104. Predoctoral Fellowship (GM7757) to F.M.R. and an NIH DUBOULE, D. AND DOLLE, P. (1989). The structural and functional grant (GM24299) to T.C.K. T.C.K. is an investigator of the organization of the murine HOX gene family resembles that of Howard Hughes Medical Institute. Drosophila homeotic genes. EMBO J. 8, 1497-1505. FECHTEL, K., NATZLE, J. E., BROWN, E. E. AND FRISTROM, J. W. (1988). Prepupal Differentiation of Drosophila Imaginal Disks: References Identification of Four Genes Whose Transcripts Accumulate in Response to a Pulse of 20-Hydroxyecdysone. Genetics 120, ABBOTT, M. K. AND KAUFMAN, T. K. (1986). The relationship 465-474. between the functional complexity and the molecular GARcfA-BELLiDO, A. (1977). Homeotic and atavic mutations in organization of the Antennapedia locus of Drosophila insects. Am. Zool. 17, 613-630. melanogaster. Genetics 114, 919-942. GASCH, A., HINZ, U. AND RENKAWITZ-POHL, R. (1989). Intron BARNES, W. M., BEVANAND, M. AND SON, P. H. (1983). Kilo- and upstream sequences regulate expression of the Drosophila Sequencing: Creation of an Ordered Nest of Asymmetric /O-tubulin gene in the visceral and somatic musculature, Deletions across a Large Target Sequence Carried on Phage respectively. Proc. natn. Acad. Sci. U.S.A. 86, 3215-3218. M13. In Methods in Enzymology, vol. 101 (ed. R. Wu, L. GIBSON, G. AND GEHRING, W. J. (1988). Head and thoracic Grossman and K. Moldave). Academic Press: New York. transformation caused by ectopic expression of Antennapedia BASLER, K. AND HAFEN, E. (1989a). Ubiquitous Expression of during Drosophila development. Development 102, 657-675. sevenless: Position-Dependent Specification of Cell Fate. Science GONZALEZ-REYES, A., URQUIA, N., GEHRING, W. J., STRUHL, G. 243, 931-934. AND MORATA, G. (1990). Are cross-regulatory interactions BASLER, K., SIEGRIST, P. AND HAFEN, E. (1989ft). The spatial and between homeotic genes functionally significant? Nature 344. temporal expression pattern of sevenless is exclusively controlled 78-80. by gene-internal elements. EMBO J. 8, 2381-2386. GRAHAM, A., PAPALOPULU, N. AND KRUMLAUF, R. (1989). The BEEMAN, R. W., STUART, J. J., HAAS, M. S. AND DENELL, R. E. Murine and Drosophila Homeobox Gene Complexes Have (1989). Genetic Analysis of the Homeotic Gene Complex Common Features of Organization and Expression. Cell 57, (HOM-C) in the Beetle Tribolium castaneum. Devi Biol. 133. 367-378. 196-209. Guo, L. H., STEPIEN, P. P., BROUSSEAU, R., NARANG, S., THOMAS, BERLETH, T., BURRI, M., THOMA, G., BOPP, D., RICHSTEIN, S., D. Y. AND Wu, R. (1984). Synthesis of human insulin gene. FRIGERIO, G., NOLL, M. AND NUSSLEIN-VOLHARD, C. (1988). The VIII. Construction of expression vectors for fused proinsulin role of localization of bicoid RNA in organizing the anterior production in Escherichia coli. Gene 12, 251-254. pattern of the Drosophila embryo. EMBO J. 7, 1749-1756. HIROMI, Y., KUROIWA, A. AND GEHRING, W. J. (1985). Control Rescue and regulation of proboscipedia 271

Elements of the Drosophila Segmentation Gene fushi tarazu. PEIFER, M., KARCH, F. AND BENDER, W. (1987). The bithorax Cell 43, 603-613. complex: control of segmental identity. Genes Dev. 1, 891-898. HODGKIN, N. M. AND BRYANT, P. J. (1978). Scanning Electron PIRROTTA, V. (1988). Vectors for P-Mediated Transformation in Microscopy of the Adult of Drosophila melanogaster. In The Drosophila. In Vectors, A Survey of Molecular Cloning Vectors Genetics and Biology of Drosophila, vol. 2c, (ed. M. Ashburner and Their Uses, (ed. R. L. Rodriguez and D. T. Denhardt) and T. R. F. Wright) Academic Press: New York. Butterworths: Boston, pp. 437-456. IRVINE, K. D., HELFAND, S. L. AND HOGNESS, D. S. (1991). The PIRROTTA, V., STELLER, H. AND BOZZETTI, M. P. (1985). Multiple large upstream control region of the Drosophila homeotic gene upstream regulatory elements control the expression of the Ultrabithorax. Development 111, 407-424. Drosophila white gene. EMBO J. 4, 3501-3508. KAPPEN, C, SCHUGHART, K. AND RUDDLE, F. H. (1989). Two steps PULTZ, M. A. (1988). PhD thesis, Indiana University, in the evolution of Antennapedia-class vertebrate homeobox Bloomington. genes. Proc. natn. Acad. Sci. U.S.A. 86, 5459-5463. PULTZ, M. A., DIEDERICH, R. J., CRIBBS, D. L. AND KAUFMAN, T. KAUFMAN, T. C. (1978). Cytogenetic analysis of chromosome 3 in C. (1988). The proboscipedia locus of the Antennapedia Drosophila melanogaster. Isolation and characterization of four Complex: a molecular and genetic analysis. Genes Dev. 2, new alleles of the proboscipedia (pb) locus. Genetics 90, 901-920. 579-596. REGULSKI, M., MCGINNIS, N., CHADWICK, R. AND MCGINNIS, W. KAUFMAN, T. C, LEWIS, R. AND WAKIMOTO, B. (1980). (1987). Developmental and molecular analysis of Deformed; a Cytogenetic analysis of chromosome 3 in Drosophila homeotic gene controlling Drosophila head development. melanogaster. The homeotic gene complex in polytene EMBO J. 6, 767-777. chromosome interval 84A-B. Genetics 94, 115-133. ROBERTSON, H. M., PRESTON, C. R., PHILLIS, R. W., JOHNSON- KAUFMAN, T. C, SEEGER, M. A. AND OLSON, G. (1989). SCHLITZ, D. M., BENZ, W. K. AND ENGELS, W. R. (1988). A Molecular and Genetic Organization of the Antennapedia Gene Stable Genomic Source of P-element Transposase in Drosophila Complex of Drosophila melanogaster. Advances in Genetics 27, melanogaster. Genetics 118, 461-470. 309-362. RUSHLOW, C, DOYLE, H., HOEY, T. AND LEVINE, M. (1987). KELLUM, R. AND SCHEDL, P. (1991). A Position-Effect Assay for Molecular characterization of the zerkniilh region of the Boundaries of Higher Order Chromosomal Domains. Cell 64, Antennapedia gene complex in Drosophila. Genes Dev. 1, 941-950. 1268-1279. KORNFELD, K., SAINT, R. B., BEACHY, P. A., HARTE, P. J., RUSHLOW, C. AND LEVINE, M. (1988). Combinatorial expression of PEATTIE, D. A. AND HOGNESS, D. S. (1989). Structure and a ftz-zen fusion promoter suggests the occurrence of cis expression of a family of Ultrabithorax raRNAs generated by interactions between genes of the ANT-C. EMBO J. 7, alternative splicing and polyadenylation in Drosophila. Genes 3479-3485. Dev. 3, 243-258. SANCHEZ-HERRERO, E., VERN6S, I., MARCO, R. AND MORATA, G. KRASNOW, M. A., SAFFMAN, E. E., KORNFELD, K. AND HOGNESS, (1985). Genetic organization of Drosophila bithorax complex. D. S. (1989). Transcriptional Activation and Repression by Ultrabithorax Proteins in Cultured Drosophila Cells. Cell 57, Nature 313, 108-113. 1031-1043. SCOTT, M. P. AND WEINER, A. J. (1984). Structural Relationships LEWIS, E. B. (1978). A gene complex controlling segmentation in among genes that control development: sequence homology Drosophila. Nature 276, 565-570. between the Antennapedia, Ultrabithorax, and fushi tarazu loci MAHAFFEY, J. W. AND KAUFMAN, T. C. (1987). Distribution of the of Drosophila. Proc. natn. Acad. Sci. U.S.A. 81, 4115-4119. Sex combs reduced Gene Products in Drosophila melanogaster. SEEGER, M. A., HAFFLEY, L. AND KAUFMAN, T. C. (1988). Genetics 117, 51-60. Characterization of amalgam: A member of the Immunoglobulin MANN, R. S. AND HOGNESS, D. S. (1990). Functional Dissection of Superfamily from Drosophila. Cell 55, 589-600. Ultrabithorax Proteins in D. melanogaster. Cell 60, 597-610. STELLER, H. AND PIRROTTA, V. (1985). A transposable P vector MCGINNIS, W., GARBER, R. L., WIRZ, J., KUROIWA, A. AND that confers selectable G418 resistance to Drosophila larvae. GEHRING, W. J. (1984). A homologous protein-coding sequence EMBO J. 4, 167-171. in Drosophila homeotic genes and its conservation in other STRUHL, G. (1981). A homeotic mutation transforming leg to metazoans. Cell 37, 403-408. antenna in Drosophila. Nature 292, 635-638. MERRILL, V. K. L., TURNER, F. R. AND KAUFMAN, T. C. (1987). A STRUHL, G. (1984). Splitting the bithorax complex of Drosophila. Genetic and Developmental Analysis of Mutations in the Nature 308, 454-457. Deformed Locus in Drosophila melanogaster. Devi Biol. 122, THUMMEL, C. S., BOULET, A. M. AND LIPSHITZ, H. D. (1988). 379-395. Vectors for Drosophila P-element-mediated transformation and MOREAU, P., HEN, R., WASYLYK, B., EVERETT, R., GAUB, M. P. tissue culture transfection. Gene 74, 445-456. AND CHAMBON, P. (1981). The SV40 72 base pair repeat has a TIONG, S. Y. K., WHITTLE, J. R. S. AND GRIBBIN, M. C. (1987). striking effect on gene expression both in SV40 and other Chromosomal continuity in the abdominal region of the bithorax chimeric recombinants. Nucl. Acids Res. 9, 6047-6068. complex of Drosophila is not essential for its contribution to MULLER, M., AFFOLTER, M., LEUPIN, W., OTTING, G., WOTHRICH, metameric identity. Development 101, 135-142. K. AND GEHRING, W. J. (1988). Isolation and sequence-specific WAKIMOTO, B. T., TURNER, F. R. AND KAUFMAN, T. C. (1984). DNA binding of the Antennapedia homeodomain. EMBO J. 7, Defects in embryogenesis in mutants associated with the 4299-4304. Antennapedia gene complex of Drosophila melanogaster. Devi N0SSLEIN-VOLHARD, C, FROHNHOFER, H. G. AND LEHMANN, R. Biol. 102, 147-172. (1987). Determination of anteroposterior polarity in Drosophila. WINSLOW, G. M., HAYASHI, S., KRASNOW, M., HOGNESS, D. S. Science 238, 1675-1681. AND SCOTT, M. P. (1989). Transcriptional Activation by the NUSSLEIN-VOLHARD, C. AND WIESCHAUS, E. (1980). Mutations Antennapedia and fushi tarazu Proteins in Cultured Drosophila affecting segment number and polarity in Drosophila. Nature Cells. Cell 57, 1017-1030. 287, 795-801. PATTATUCCI, A. AND KAUFMAN, T. C. (1991). Antibody Staining of Imaginal Disks. In Dros. Info. Newsletter (electronic) vol. 1. (Accepted 4 June 1991)