Development 110. 915-925 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

915

Isolation of an abdominal-A gene from the locust Schistocerca gregaria

and its expression during early embryogenesis

GUY TEAR1, MICHAEL AKAM1 and ALFONSO MARTINEZ-ARIAS2

^Department of Genetics, Cambridge University, Downing Street, Cambridge, CB2 3EH, UK2Department of Zoology, Cambridge University, Downing Street, Cambridge CB2 3EJ, UK

Summary

Using sequence homology to Drosophila homeobox-containing genes, we have cloned a homologue ofabdominal-A from the locust Schistocerca gregaria. TheSchistocerca clone encodes a stretch of 78 amino acidsincluding the homeodomain and its flanking regionsidentical to the corresponding region of abdominal-A.

We have shown by in situ hybridization that this geneis transcribed and have used an antibody raised againstits protein product to examine the expression ofabdominal-A during early Schistocerca embryogenesis.Schistocerca is a short germ insect. Although thesegmented body plan is very similar to that ofDrosophila, the segments are generated sequentially by aprocess of growth, not simultaneously by subdivision of

a syncytial blastoderm. In both organisms, abdominal-Ais expressed throughout the abdomen from a sharpanterior boundary located within the first abdominalsegment (Al). The initial activation of the genes in thetwo species differs. Schistocerca initiates expression in asmall group of cells in the anterior of A2, shortly afterthis segment is defined by the appearance of engrailedprotein. This contrasts with the appearance of abdomi-nal-A expression in Drosophila, which appears simul-taneously throughout the entire abdomen.

Key words: Schistocerca, abdominal-A, homeotic gene,short germ insect, embryogenesis.

Introduction

Insects are characterized by a segmented body sub-divided into a head, a set of mouthparts, a thorax, anabdomen and a terminal region (Snodgrass, 1935;Anderson, 1973). This body plan is obscured in thepostembryonic stages of some insect groups, but it isclearly displayed in the embryonic germ band of allinsects. Such a conserved body plan suggests anunderlying conservation in the genes that define themetameric pattern, and in those that impose regionalidentity on the different segments.

The array of segments in the germ band can beformed in one of three general ways (Sander, 1976;Krause, 1939): (i) by subdivision of an epithelial sheetthat occupies the whole of the egg (long germ bandinsects); (ii) by growth from a primordium that occupiesa very small portion of the egg (short germ bandinsects), or (iii) by some combination of theseprocesses, whereby the anterior segments are generatedby subdivision, but the posterior segments are gener-ated by growth (intermediate germ band insects). Thesedifferent modes by which the germ band may begenerated suggest variation in the mechanisms thatestablish the patterns of gene activity in the germ band(Sander, 1983, 1988). By comparing aspects of geneexpression during segmentation of the locust Schisto-

cerca with the equivalent stages of development inDrosophila, we hope to learn something of theevolutionary changes that distinguish pattern gener-ation in short and long germ insects (Sander, 1988; Tearetal. 1988).

In Drosophila localized maternal cues co-ordinatethe partitioning of the blastoderm into metameric unitsthrough the concerted action of gap and pair-rule geneproducts. These establish the initial metameric activityof some of the segment polarity genes which theninteract to maintain and elaborate the repeatingsegment pattern (Ingham, 1988; Martinez-Arias, 1989).At the same time the gap and pair-rule genes direct thelocalized activation of the homeotic genes, therebyestablishing differences that will endow segments withunique identities. This patterning process occurs almostsimultaneously throughout the entire body axis, afterthe rapid cycles of nuclear cleavage that generate thesyncytial blastoderm, but before the completion ofcellularization and the onset of gastrulation (Howard,1988; Akam, 1987).

This situation contrasts with that of the short germband insects, e.g. Schistocerca gregaria, which generatesegments after cellularization, during a phase ofpolarized growth. In these insects, the embryonicprimordium is restricted to a small group of cells thatform as a disc near the posterior tip of the egg.

916 G. Tear, M. Akam and A. Martinez-Arias

Gastrulation begins during the disc stage and thegastrulation furrow extends into the posterior regions ofthe embryo as it elongates. The first visible signs ofsegmentation appear in the thorax, three days afterfertilization. During the next day, the gnathal segmentsand then the abdominal segments appear, the latterforming one at a time in an anterior-to-posteriorprogression (Roonwal, 1936; Krause, 1938).

Cell ablation studies during the elongating disc stagehave demonstrated that, in the early embryo, differentregions of the adult are represented in relativeproportions that differ markedly to those existing in themature germ band (Sander, 1976; Krause, 1953). In themature germ band, the abdomen occupies a muchlarger region than the thorax, whereas the relative sizesof these primordia appear to be reversed at the earlierblastoderm stage. Understanding this transition re-quires molecular markers for the processes of gener-ation and specification of metameres in wild-type andexperimental embryos.

In Drosophila, the posterior of every segment isdefined by the continuous expression of the geneengrailed (Kornberg et al. 1985; Dinardo et al. 1985). Ahomologue of the Drosophila engrailed gene has beenidentified in the locust (Patel et al. 1989a), where itsexpression is also restricted to the posterior part ofevery segment. The expression of engrailed in theemerging abdominal segments follows an anteropos-terior sequence which precedes visible segmentation(Patel et al. 19896). This suggests that at least one of thegenes that define the segment pattern in Drosophila hasa very similar role in the locust. It also confirms theconclusion from earlier experiments of UV and X-rayablations (Sander, 1976), ligations and heat shocks(Mee and French, 1986; Mee, 1986) that segmentpatterning takes place sequentially, and only shortlybefore the first overt signs of segmentation are visible.

In Drosophila, the activity of the homeotic genes inrestricted spatial domains defines the identity ofdifferent regions of the body (Lewis, 1978; Akam,1987). Thus these genes provide molecular markers todetermine when, in relation to segmentation, regionalidentity is specified. In this paper, we report theisolation of the locust homologue for one of theDrosophila homeotic genes, abdominal-A. Genetic andmolecular analysis of this gene in Drosophila suggestthat it is largely responsible for the identity of the

abdominal segments (Lewis, 1978; Sanchez-Herrero etal. 1985; Tiong et al. 1985; Karch et al. 1990). Using anantibody raised against the coding region of the locustgene, we show that its product is expressed in theembryo in a wide domain comprising the whole of theabdominal region, comparable to that defined by theDrosophila abdominal-A gene. The generation of thispattern follows an anteroposterior sequence, compat-ible with the known features of locust embryogenesisbut which highlights the different developmentalstrategies evolved by long and short germ insects.

Materials and methods

AnimalsSchistocerca gregaria (Forskal) eggs were laid in moist sandand collected over defined periods at 26°C. Those requiredfor experiments were removed from the sand and cultured onmoist filter paper in a Petri dish at 26°C.

Construction and screening of libraryThe Schistocerca gregaria genomic library was constructed bysize fractionating a Sau3A partial digest of adult testis DNAon a sucrose gradient and ligating the 9-23 kb fraction into theBamHl site of AEMBL3. The library contained 2xlO6

recombinant plaque forming units (pfu). The Drosophilaprobes used were: engrailed, a 400bp BamHI-Aval fragmentof pS799-7 (Fjose et al. 1985); even-skipped, a 580 bpHindlll-Accl fragment of p48-X1.4 (Macdonald et al. 1986);Antennapedia, a 400bp BamHl-EcoRl fragment of plD203a(gift of I. Dawson). These were labelled with a--32P-dATP bynick-translation to a specific activity of 108-109 disintsmin"1

jUg"1. All hybridizations were carried out at reducedstringency in 5xSSPE, 0.5% SDS, 5xDenhardts, 43%formamide and 100 /xgrnP1 sheared single-stranded salmontestis DNA at 37°C followed by washing at 50°C in 2xSSPE,0.1% SDS. (McGinnis et al. 1984).

SequencingRegions of interest were sequenced by the dideoxy chaintermination method (Sanger etal. 1977) from double-strandedtemplates with Sequenase™ enzyme (USB Corporation,Ohio) using forward and reverse primers. Sequence analysiswas performed using ANALYSEQ (Staden, 1984), FASTN(Lipman and Pearson, 1985) and the University of WisconsinGenetics Computing Group package (Devereux et al. 1984).

Generation of antibodyA 790 bp Apal-Pstl fragment from the Schistocerca abdomi-nal-A clone AG610 (Fig. 1) was subcloned into Bluescript

Left arm

o, X X II si

homeoboxhomology

in situprobe

Right arm

j= 1 kilobase

Fig. 1. Restriction map of the abdominal-A genomic clone from Schistocerca, AG610, showing position of homeoboxhomology and the region used as a probe for in situ hybridization.

abdominal-A in Schistocerca 917

(Stratagene, La Jolla. California) to generate pAP3. Usingthe Kpnl and Sad sites from the polylinker the fragment wascloned into Bluescribe (Stratagene, La Jolla, California).BamWX digestion of this subclone released an 810 bp fragmentcontaining the entire open reading frame of the genomic exonplus 14 nucleotides from the polylinker at the 5' end. Thisfragment was subcloned into pGEX2T (Amrad Corporation,Melbourne, Australia) to yield the plasmid, pFUSl.

A second fusion was constructed by subcloning the 640 bpBglll-Pstl fragment from AG610 into the BamHl-Pstl sites ofpUR292 (gift from R.Weinzierl, Department of Anatomy,Cambridge) to yield the resulting plasmid, pF4.

The region of fusion of the coding regions was sequenced tocheck that the open reading frames were continuous and inthe correct frame.

E. coli strain JM101 carrying pFUSl or pF4 were grown tolate log phase and then induced with 1 mM IPTG for 2-3 h.The cultures were harvested and resuspended in 2x SDS gelloading buffer (100mM Tris-HCl, pH6.8, 4% SDS, 20%glycerol, 10% /3-mercaptoethanol, 0.1 % bromophenol blue).Samples were then loaded onto SDS-polyacrylamide slab gelsas described by Hames (1981) for analysis.

Protein bands were visualized on 10% preparative SDS-polyacrylamide gels by incubation in 0.25 M KG at 0°C for20 min. The band containing the fusion protein was excisedand the protein eluted in SDS running buffer (0.1% SDS,25 mM Tris, 192 mM glycine) using a Biotrap elution chamber(Schleicher and Schuell); electrophoresis was at 200 V for2-4 h. The eluted protein was dialyzed against PBS (130 mMNaCl, 7mM Na2HPO4, 3mM NaH2PO4) overnight, snapfrozen in liquid nitrogen and stored at -70°C.

Rabbits were immunized subcutaneously at multiple siteswith an initial inoculum of 200 ug of fusion protein expressedfrom the pFUSl construct, in complete Freund's adjuvant,followed by boosts of 100-250 fig in incomplete Freund'sadjuvant every two to four weeks. Activity against theABDOMINAL-A (ABD-A) protein was first observed afterten weeks. All immunizations were carried out commerciallyby ABC Ltd, Cambridge.

Large quantities of the fusion protein F4 were purified foruse on affinity-purification columns using APTG affinitychromatography exactly as described by Carroll and Laughon(1987).

Purification of antiseraTo remove any antibodies to bacterial proteins or glutathionetransferase, the serum was pretreated with an acetone powder(Harlow and Lane, 1988) prepared from E. coli JM101overproducing glutathione transferase from pGEX2T for 1 hat room temperature. The supernatant was passed over anAffigel 10 (Biorad) column bound with 200-400 fig of purifiedfusion protein. Bound antibodies were eluted with 0.1Mglycine-HCl, pH2.5 and neutralized with 0.1 volume 2 MTris-HCl, pH7.4. Bovine serum albumin (BSA) was addedto 1 % and the serum was dialysed against PBS. Affinity-purified serum was stored at 4°C, in the presence of 0.2 %sodium azide.

ImmunochemistrySchistocerca gregaria embryos were dissected from their eggcases under locust embryo saline (8.76gl~' NaCl, 0.22glKC1, 0.29gl"' CaCl2, 0.25gr' MgSO4, 1.15gr' TES). Theywere then fixed in 3.7% formaldehyde in PBS at 0°C for40-60min. The embryos were washed in PTX (PBS+0.3%Triton X-100) and then blocked in PBTX (PTX+0.1% BSAand 10 mM NaN3) at 4°C for 2-4h. Affinity-purified primaryantibody was diluted 1:500 in PBTX and incubated with the

embryos at 4°C overnight. The embryos were then washed inPBTX for 3h with 4-6 changes. The embryos weresubsequently incubated with secondary antibody, biotinylatedgoat anti-rabbit IgG (Vector Laboratories, Peterborough,England), diluted 1:400 in PBTX for 90min at 4°C thenwashed in PBS+0.1% Tween 20 (PT) for lh with 4-6changes at room temperature. The embryos were thenincubated with avidin-biotin complex (ABC Elite, VectorLaboratories) for 30min at room temperature and thenwashed through six changes of PT in 30 min and stained in thefollowing solution: 50u\ 6% nickel ammonium sulphate,6.25fA lOmgml"1 diaminobenzidine (Sigma), I u\ commer-cial hydrogen peroxide solution (6%; 20 vols availableoxygen, Boots, Nottingham) and PT to 500/d.

For double antibody stainings, the embryos were firststained with Mab 4D9 (Patel etal. 1989a) under the conditionsdescribed above using biotinylated horse anti-mouse IgG(Vector Laboratories) as secondary antibody. After visualiz-ation of the first antibody, the embryos were washedextensively with PBTX overnight and then treated exactly asabove except that nickel ammonium sulphate enhancement(Adams, 1981) was not used in the ensuing staining reaction.

Drosophila embryos were dechorionated with commercialbleach then washed with water and fixed in heptane saturatedwith 0.1M Pipes, pH6.95, lmM MgSO4, 2mM EGTA, 4%paraformaldehyde for 40min. The aqueous phase wasremoved and replaced with methanol. This mixture wasshaken vigorously to remove the embryonic vitelline mem-brane. The devitellinized embryos were washed with meth-anol, then PBTX and then treated as the Schistocercaembryos except that the primary antibody (anti-Drosophilaabdominal-A, supplied by J. Casonova) was used at a dilutionof 1:5000 and biotinylated rabbit anti-rat (Vector Labora-tories) was used as the secondary antibody.

All embryos were dehydrated, cleared in xylene andmounted in DPX-Permount (BDH Ltd. Poole, England).

In situ hybridization to embryo sectionsIn situ hybridization was performed essentially as describedfor Drosophila (Akam and Martinez-Arias, 1985; Sanchez-Herrero and Crosby, 1988). A 1.4kb EcoRl-Xbal fragmentfrom AG610 (Fig. 1) was random primed (Feinberg andVogelstein, 1983) in the presence of 35S-dATP and used asprobe.

Results

Cloning and identification of an abdominal-AhomologueWe constructed a genomic library from Schistocercagregaria containing 2xlO6 recombinant phage particleswith an average insert size of 16 kb. Assuming a haploidgenome size of 60xl08 bp (Wilmore and Brown, 1975)this represents 4 genome equivalents. A total of 5xlO5

phage were screened at reduced stringency withDrosophila homeobox probes derived from the genesengrailed, even-skipped and Antennapedia (see Ma-terials and methods). Plaques that hybridized withmore than one probe were selected for furthercharacterization. One of these phages, AG610, hybrid-ized with the three probes and was analyzed further.The region of homology, located within a 790 bpfragment of the clone (Fig. 1), was subcloned andsequenced on both strands.

918 G. Tear, M. Akam and A. Martinez-Arias

This clone contained a complete homeobox se-quence, 82% identical to the homeobox from abdomi-nal-A of Drosophila (Fig. 2). The conceptual trans-lation of this region generates a peptide that is identicalto the Drosophila ABD-A protein throughout thehomeodomain and for six aminoacids 5' and twelveaminoacids 3' to the homeodomain (Karch et al. 1990).This extensive conserved region serves to identify theSchistocerca fragment as a specific homologue of theDrosophila abdominal-A gene.

5' to the homeobox, similarity between the Schisto-cerca and Drosophila sequences stops abruptly at aposition where the Drosophila abdominal-A gene hasan intron. At this position, the Schistocerca DNA has astrong match to a consensus splice acceptor sequence(Py4TTGCAG/G, H. D. Lipshitz, personal communi-cation, Breathnach and Chambon, 1981). It too is likelyto contain an intron at this position. 27 bases 3' to thehomeobox, the Drosophila abdominal-A gene has a70bp intron (Karch et al. 1990; G. Tear, unpublisheddata). This is not present in the Schistocerca clone. Thegenomic fragment includes sequences homologous tothe Drosophila cDNA sequence on either side of theintron (Fig. 2). This 3' intron is also absent in anabdominal-A homologue from Manduca (L. Nagy andL. Riddiford personal communication) suggesting thatDrosophila has gained an intron after its separationfrom these insects.

Further 3' to the homeobox the similarity betweenDrosophila abdominal-A and AG610 is reduced. Thelocust open reading frame continues to a stop codonninety-two amino acids beyond the end of the homeo-box. The only similarities in this region are a longpolyglutamine repeat, and a short stretch of aminoacids located near the carboxy termini of both proteins.

The genomic fragment containing this abdominal-Asequence was used to probe Schistocerca DNA onSouthern blots. At high stringency, the clone hybridizesto only a single fragment in the Schistocerca genome(data not shown). To test whether this genomicfragment is transcribed, we used it as a probe forhomologous transcripts in sections of locust embryos.Using embryos that had completed formation of thegerm band (45% of development), we found that theprobe hybridized to transcripts, but only within theabdominal region of the locust embryos, in a domainbetween abdominal segments 1 and 10 (A1-A10)(Fig. 3). The abdominal-A gene in Drosophila isexpressed in a similar region (Rowe, 1987; E. Sanchez-Herrero, unpublished data) suggesting that the locustfragment derives from a functional homologue of theDrosophila abdominal-A gene.

Fig. 2. Comparison of the Schistocerca gregaria genomicsequence from AG610 with cDNA sequence of Drosophilaabdominal-A from Karch et al. (1990). Positions of nucleicacid identity are marked by a vertical bar and amino acididentity by a shaded box. The homeobox is underlined andthe positions of introns in the Drosophila sequence areindicated bv arrows.

10 30 50tgtgtacatgtgtcgtgacgaactgtgtaacacatgtccgtttcgggcttttctgttgca

70 90 1 1 0

0 610

Mbd-A

G610

Mbd-A

0 610

Mbd-A

C 6 10

Mbd-A

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Mbd-A

0 610

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0 610

Mbd-A

0 610

mbd-X

a 6 ! o

•bd-A

0 610

• bd-A

G 6 10

abd-A

0 610

abd-A

ggGCCCAATGGCTGCCCACGGCGACGCGGCCGGCAGACCTACACGCGCTTCCAGACGCTGII I I I I I I I I I I I I I I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I IIGGCCCCAACGGCTGTCCACGAAGGCGCGGTCGCCAGACCTACACTCGCTTCCAGACCCTC

I ' 1090 1110I 1070Intron 6

1 3 0 1 5 0 170

GAGCTGGAGAAGGAATTCCACTTCAACCACTACCTGACGCGACGCCGGCGGATTGAAATCI I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I IGAACTGGAGAAGGAGTTTCACTTCAACCACTACTTAACTCGGCGAAGGCGCATCGAGATC

1130 1150 1170

1 9 0 2 1 0 230

GCCCACGCGCTCTGCCTCACGGAGCGCCAGATCAAGATCTGGTTCCAGAACCGCCGCATGII II II I I I I I I I I II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I

GCACATGCCCTCTGCCTGACCGAGCGACAGATCAAGATCTGGTTTCAGAACCGTCGCATG

.feialtt SAia iettCyifce uShrS l«AfgElsl lety's t le*ijjPh<SGtftaan Ajjgftrgkot-1 1 9 0 1 2 1 0 1 2 3 0

270 2 9 0250

AAGCTCAAGAAGGAGCTGCGCGCGGTCAAGGAGATAAACGAGCAGGCCCGCCGCGAGCGCI I I I I I I I II I I I I I II II I I I I I I I I I I I I I II I I I I I II I I I I I IIAAGCTGAAGAAGGAGTTACGAGCCGTCAAGGAAATAAATGAACAGGCGCGACGCGATCGA

-a Ly »CI aha uArgA lava? LysG luilaAsTiGl QEIJIA J.aA"cg Arg As pferg1250 1270 |l290

•""~*"""*"~~~^^^~ Intron 77 0 n t 3310 330 t350

GAGGAGCAGGACCGTTTGAAGCAGCAGCAGGAGAAGAAGCTGGAGCAGCAACAGCAGCAGI I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I IGAGGAGCAGGAGAAAATGAAGGCCCAGGAGACGATGAAATCCGCCCAGCAGAACAAGCAA

13301310 1350

370 390 410G1 n 31 ftSIttS l»GXB<Ji riUIWSAaCl W3ia<JlWR.nS lUClltSiSA 1 aP ro P r oG 1 nO J.J.CAGCAGCAACAACAGCAACAGCAGCAGCAGCAGCAGCAGCAGCAGGCGCCGCCGCAACAG

III II II I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I II I III IIGTGCAACAGCAGCAGCAACAGCAACAGCAGCAGCAACAGCAGCAGCAGCAACAGCACCAA

va l CXw31»01*8IA<UaCliitO J.««l oSiiiG-lsGlnG i OSIOSIHG lnGlnGinHisSiii1370 1390 1410

430 450 470ProProSIfcaftiHisHisThrlleSerft-UHisLeuHisAspGlnHisLysLeuGlytSuCCGCCGCAGCAGCACCACACCATCAGCCACCACCTGCACGACCAACACAAGCTGGGGCTG

I I I I I I I I I I I I I I I I I I I I I I IICAGCAGCAACAACAGCCGCAGGACCACCACTCGATCATCGCACACAATCCAGGCCACTTGGlnGlneijS&laGlnProGlnAspHisrtlsSerllelleAlaHlsAsnProGlyHisiEU

1430 1450 1470

490GluLysAlaProProSJjAlaGAGAAGGCCCCCCCGGGCGCG

I I I IICACCACTCGGTGGTAGGTCAAAACGATCTCAAGCTCGGCCTTGGCATGGGCGTGGGCGTCHlsHlsSerValValClyGlnAsnAspLeuLysLeuGlyLeuGlyMetGlyValGlyVal

1490 1510 1530

GGAGTGGGCGGCATCGGGCCGGGCATCGGTGGCGGCTTGGGCGGCAATCTGGGCATGATGGlyValGlyGlylleGlyProGlylleGlyGlyGlyLeuGlyGlyAsnLeuGlyMetMet

1550 1570 1590

510 530

GACCTGCTCAAGGCGGTGGCCAAGGTGCCCACCTAG

I I I I I I I I I I I I I I I I I I I II I I I I IAGCGCCCTGGACAAGAGCAATCACGACCTGCTAAAGGCGGTCAGCAAAGTCAACTCCTAA

SerAlaLeuAspLysSerAsnHIaAaptaiiLeuty.jAiaValSertysyaJAsnSeriivd1610 1630 1650

550 570 590ATGCCGGAGCCACTCTGCGACCTGCCGCTGGACGAGCTGCTGCCCACGGCCACGGCTCCA

I I I I I I I II I I I I I I I IGCTGACAACCACCCATCGCCCAGCCGAGCCAACGAGCCAATCTTGCACCAAGTGGTGCAA

1670 1690 1710

610 630 650GCTCCAGCCTGGACCGGGTTCCCCTACTTCCAGTAGGGCCGCCGCTCTGGCCACGGCAGG

II I I I I I I I IATTCGCACTGGAGATCTGGAAGGGAATCGCATGCGTTGCTCTATGGAATTC

1730 1750 1770

abdominal-A in Schistocerca 919

Fig. 3. In situ hybridizationusing the 1.4 kb Xbal-EcoRlfragment indicated in Fig. 1 toa section of a Schistocercaembryo at approximately 45%development. Anterior is tothe left, the asterisk indicatesthe third thoracic segment (T3)and an arrowhead marks theanterior extent of expression.Scale bar: 250 ^m.

Production of antibodies against Schistocerca ABD-Aprotein

To examine in more detail the expression of theSchistocerca abdominal-A gene, we raised antibodiesagainst a fragment of the Schistocerca protein. Twofusion proteins were generated. In the first (FUS1), 158aminoacids of Schistocerca ABD-A were fused to theSchistosoma japonicum glutathione transferase (Smithand Johnson, 1988). In the second (F4), 106 aminoacidsof Schistocerca ABD-A were fused to E. coli j5-galactosidase (Carroll and Laughon, 1987). An anti-serum was raised against FUS1 and affinity purifiedagainst F4 to ensure that the purified serum containedantibodies specific for the Schistocerca portion of thefusions.

The affinity-purified serum preferentially recognizesone new band that is not seen using preimmune serumon protein extracts from Schistocerca embryos immobi-lized on a nitrocellulose membrane. The size of thisprotein, 42-45xlO3 Mr, is similar to that seen for theDrosophila abdominal-A product on Westerns(R.Weinzierl personal communication).

In both species, abdominal-A is expressed within theabdominal segments of the embryo. In Schistocerca, theexpression of abdominal-A extends in the epidermisfrom the posterior of the first abdominal segment to thetenth abdominal segment (Fig. 4E) whereas in Dros-ophila, abdominal-A is expressed between the posteriorof Al and the seventh abdominal segment(Fig. 4F,G,H). The fact that our serum recognizes anepitope in locust embryos that is expressed solely in theabdomen and not in more anterior regions suggests thatour antiserum is specific for the Schistocerca homologueof ABD-A.

Expression of abdominal-A in Schistocerca embryosIn Schistocerca embryos, abdominal-A expression isfirst observed at 20% of development, when thethoracic and gnathal segments are already visible asbulges in the germ band (Fig. 4A). In embryos at thisstage, there are no morphological signs of segmentation

in the abdomen. Initially, only a few cells expressabdominal-A. These cells are located laterally, a shortdistance posterior to the metathoracic segment. Oftenthey appear asymmetrically, a small patch of cellsstaining on one side, but only a few cells on the other(Fig. 4A,6A). Other similar embryos, presumablyslightly older, show larger laterally paired elongatedpatches of staining with cells near the centre of eachpatch showing strongest expression (Fig. 4B,6B). Theposition of these strongly expressing cells relative to T3,corresponds approximately to that of the first express-ing cells visible in earlier embryos. Thus it seems thatexpression is initiated in a small group of cells and thenextends to both anterior and posterior neighbours.

To confirm the early expression in Schistocerca, andto locate the site of abdominal-A expression withrespect to the forming segments, we have double-stained embryos with our antibody and with Mab4D9,which recognises the locust Engrailed protein (Patel etal. 1989a). Fig. 5 shows the pattern of expression ofabdominal-A and engrailed in the Schistocerca embryoat 22 % of development. Abdominal-A is first expressedin cells that lie just anterior to, and sometimes overlap,the engrailed stripe that defines posterior A2 (A2p). Atthis stage, the A2p stripe of engrailed is well defined,and the A3p stripe is just appearing (Fig. 5A). Slightlylater the expression of abdominal-A extends anteriorlyto the engrailed stripe of Al and posteriorly beyond theA2 engrailed stripe (Fig. 5B,C). This pattern is verydifferent from that of abdominal-A in Drosophilaembryos where ABD-A protein is first observed in theposterior cells of each abdominal segment during stage9 (Campos-Ortega and Hartenstein, 1985) from pAl topA7; shortly afterwards, it is expressed throughout thisdomain with a strong intrasegmental modulation(Fig. 4G).

Initially the anterior margin of abdominal-A ' ex-pression in Schistocerca embryos is uneven. Whenexpression has reached the posterior of Al, but beforeany visible signs of abdominal segmentation, theanterior boundary straightens (Fig. 6). We do not know

920 G. Tear, M. Akam and A. Martinez-Arias

k,**

Fig. 4. Appearance of ABD-A protein during early development in Schistocerca and Drosophila embryos stained with anti-ABD-A antibodies. All Schistocerca embryos are shown at the same magnification, anterior is up in (A,B,C,D,E and H)and to the left in (F and G), the third thoracic segment is indicated by an asterisk. Photographs of abdominal-A staining inSchistocerca at (A) 22%, (B) 23%, (C) 26%, (D) 29%, (E) 31% of development and in Drosophila at (F) early stage 9,(G) late stage 9 and (H) after germ band shortening. Enlargements of parts of embryos A and B are shown in Fig. 6.Embryonic staging is as described in Bentley et al. (1979) or Campos-Ortega & Hartenstein, (1985). Expression ofabdominal-A is first seen in Schistocerca in a group of cells, indicated by an arrow, at about 22% of development (A),whereas in Drosophila the first expression occurs in cells in each of the abdominal segments (F). In Schistocerca expressionextends from the initially expressing cells anteriorly and posteriorly (B) and forms a defined anterior boundary (C).Expression concentrates at the lateral edges of the forming segments (D) and eventually extends to the tenth abdominalsegment (E). After the initial expression of abdominal-A in stripes in Drosophila, expression is activated throughout theexpression domain from parasegment 7 to the anterior of parasegment 13, marked in F and G, which is maintained aftergerm band shortening (H). Scale bar: 500/im (A,B,C,D and E) and 204/OTI (F,G and H).

whether this involves cell movement or changes inexpression. A similar sharpening of the anterior borderof the engrailed stripe also occurs and abdominal-Aexpression may be following the same cues. Once it isclearly defined, the anterior limit of abdominal-Aexpression coincides with the anterior margin of theengrailed stripe of A l . This boundary corresponds tothe anterior limit of parasegment 7 (Martinez-Arias andLawrence, 1985), and is therefore identical to theanterior limit of abdominal-A expression in Drosophila.Occasionally, ectopic cells expressing abdominal-A canbe seen anterior to a well-formed boundary (Fig. 6E),but such isolated cells are not seen in later stages, afterabdominal segmentation is visible. Similar ectopicallyexpressing cells can also be seen with Mab 4D9 whereexpression of engrailed anterior to the anterior limit ofthe engrailed stripe is observed (Patel et al. 19896).

At the time that ABD-A protein is first expressed,the unsegmented posterior region of the embryo is

growing rapidly. As it grows, detectable levels of ABD-A protein appear in more posteriorly located cells. Thedistance between these cells and the posterior tip of thegrowing germ band remains approximately constant,and the appearance of ABD-A protein follows aboutone segment behind the appearance of engrailedstripes. Thus, in cells throughout the abdomen, there isa slight delay between the first expression of engrailedand of abdominal-A.

When the germ band is just completed, (30% ofdevelopment) expression of abdominal-A extends as faras the posterior of the tenth abdominal segment(Fig. 7A). This posterior limit appears to be segmental,not parasegmental, as it coincides with the groovebetween A10 and All . For a short while, the level ofprotein in A10 is comparable with that in the moreanterior segments, but soon after the germ band iscomplete, it begins to fall. Segments Alp to A9 retainhigh levels of ABD-A protein, but in A10 ABD-A

Fig. 5. Expression ofabdominal-A and engrailed inSchistocerca embryos at •approximately 22% ofdevelopment. Cellsexpressing abdominal-A arestained brown and cellsexpressing engrailed arestained dark violet.(A) Initial expression ofabdominal-A in the anteriorof A2, which then extendsboth anteriorly andposteriorly (B). The finalanterior boundary ofabdominal-A expression (C)corresponds with the anteriorboundary of engrailed in Al.Scale bar: 50 fim.

abdominal-A in Schistocerca 921

Fig. 6. Detail of early stages of embryogenesis showing anterior extension of abdominal-A staining and maturation of theposterior Al boundary. Photograph A shows the same embryo as in Fig. 4A, revealing the bilateral asymmetry in theactivation of expression of abdominal-A, with fewer cells staining for expression on the right side. In B, expression extendsanterior and posterior to the initial cells. C shows the same embryo as Fig. 4B. In D, the anterior boundary has sharpened.Note the first appearance of staining in a line of cells near the ventral midline of the embryo. Photograph E shows anectopic abdominal-A expressing cell within Al, which is indicated by the arrowhead. Scale bar: 100fim (A,B,C & D) and80 fan (E).

protein falls to barely detectable levels by 40-42 % ofdevelopment (Fig. 7B).

From its first appearance, the ABD-A protein isdifferentially expressed around the future dorsal-ventral axis of the embryo. In each segment, expressionis first seen in cells in the mediolateral portion of theembryo, underlying the mesoderm. Protein thenappears in more laterally located cells, just beforeconstrictions in the mesoderm demarcate the formingsegments (Roonwal, 1936; Krause, 1938). Shortly after

this the lateral edges of the ectoderm thicken to formthe characteristic epidermal bulges of the segments.Just prior to this thickening, the lateral edges of theepidermis begin to stain strongly for ABD-A protein.Highest levels of expression persist in these lateral budsas the segments arise. Later expression spreadsdorsally, and then extends completely around theventral epidermis.

By 31 % of development, the expression of abdomi-nal-A in the locust shows an intrasegmental modulation

1111

P '-

7A B

Fig. 7. Photographs ofSchistocerca embryos stainedfor abdominal-A expression at(A) 32% development and (B)42% development. Expressionextends to the posterior ofA10 in A but only as far asthe posterior of A9 in B.P, proctodeum. Scale bar:200 fim.

922 G. Tear, M. Akam and A. Martinez-Arias

similar to that of Drosophila with expression in theposterior of the segment stronger than that in theanterior (Fig. 4E).

Discussion

The conserved morphology of the mature germ bandled Seidel (1960) and later Sander (1983) to identify it asa phylotypic stage in insect development - a stageshowing markedly less variation between differentinsect orders than either earlier or later phases ofdevelopment. This conservation suggests that the genesthat define this stage may show conserved patterns ofactivity. However, the diversity in developmentalmechanisms used to generate the germ band indicatesthat the processes that establish these patterns maydiffer considerably between species.

The similarity of the mature germ band in Drosophilaand in the locust is now documented by two molecularmarkers, the engrailed gene product, (Patel etal. 1989b)and the ABD-A protein, examined here. For both ofthese genes, the pattern of expression in the maturegerm band of Schistocerca is similar to that seen inDrosophila, but the patterns of activation of the genesdiffer. In Drosophila their activation is dependent onthe spatial distributions of maternal and zygotic gap andpair-rule gene products during blastoderm formation.In Schistocerca it appears that their activation isdependent on cellular interactions that follow atemporal pattern in the postblastoderm embryo. Ourcloning of Schistocerca abdominal-A allows us tocompare the process of acquisition of segment identityin short and long germ insects.

Schistocerca gregaria possesses a close homologue ofDrosophila abdominal-AThe conservation between the locust and Drosophilahomologues of abdominal-A is 100% within andflanking the homeodomain. Such an extensive level ofsimilarity between a homeotic gene of Drosophila andthat of a different species has only been observed withinthe insects (Akam et al. 1988; Fleig et al. 1988) and assuch does indeed imply that those genes active in thegerm band are strongly conserved. This is confirmed bythe fact that the patterns of expression of homeoticgenes in the germ band of insects are very similar, e.g.Deformed in Apis (Fleig et al. 1988) and abdominal-Aand Sex combs reduced in Schistocerca (this report andDawson, Tear, Martinez-Arias and Akam in prep-aration). This conservation raises the question of howsuch highly conserved genes can give rise to species-specific segment identity.

Experiments involving the switching of homeoboxesbetween homeotic genes (Kuziora and McGinnis, 1989)have revealed that many of their regulatory propertiesare conferred by the homeodomain itself. Single aminoacid changes in the homeodomain can change its DNA-binding specificity (Treisman et al. 1989). The highconservation of the homeodomain between Schisto-cerca and Drosophila abdominal-A means that this

domain will be able to acquire the same detailed spatialconformation in both proteins. It is interesting thatalthough both proteins are expressed in comparabledomains in the germ band, each species developsunique segment specializations. This suggests that theABD-A protein must be using the same DNA-bindingactivity to activate similar sets of downstream genes inthese animals, but that during evolution these geneproducts have changed allowing the development ofspecific species differences. However, the ABD-Aproteins must also regulate those genes common toboth animals that promote the differentiation ofabdomen rather than thorax or gnathos; this constraintmight be an important element in the conservation ofthe homeodomain.

The activation of abdominal-A expression inSchistocerca

In Schistocerca, as in Drosophila, the expression ofabdominal-A characterizes the entire abdomen, fromthe parasegment border in the middle of the firstabdominal segment to the posterior of the overtlysegmented region (A7 in Drosophila, A10 in thelocust). In Drosophila, the development of the abdomi-nal segments occurs synchronously throughout thewhole abdomen (as evidenced by the pattern of the firstpostblasdoderm mitoses in the region (Foe, 1989)) andABD-A protein appears at the same time in eachsegment from Al to A7 (Karch et al. 1990, see Fig. 4F).By contrast, in the locust embryo the abdominalsegments develop in sequence, with each segment fromA2 to A10 reaching the same apparent stage ofdevelopment a few hours (1 % of development) laterthan its anterior neighbour. The expression of abdomi-nal-A first appears in the more anterior part of theabdomen, and spreads backward, appearing in eachsegment as it reaches approximately the same develop-mental stage, following the sequence of visible develop-ment.

The first expression of abdominal-A appears in cellswithin the anterior of A2, before any visible signs ofsegmentation in the abdomen, but shortly after themost anterior abdominal segments have been definedby the appearance of engrailed stripes. It does notappear that the initial expression of abdominal-A istightly linked to the process of segmentation. The firstcells to accumulate detectable levels of ABD-A proteinare not those that will abut the final anterior boundaryof expression. Neither do they appear in a preciserelationship to the nearest stripe of engrailed expressingcells; rather there appears to be some variability in thebehaviour of individual cells, as evidenced by theirregular shape and bilateral asymmetry frequently seenin the early patches of cells expressing abdominal-A.

Clearly some property of the developing abdomenbecomes permissive for abdominal-A expression, andthis happens first in the anterior region of A2. Thatdecision seems to be made on a cell-by-cell basis, duringgrowth. There is no evidence that the abdominal regionof the embryo is 'predetermined' before cellularizationas it is in Drosophila. At present, we do not know what

abdominal-A in Schistocerca 923

signal might activate gene expression. In Drosophila,the choice between activating thoracic and abdominalhomeotic genes is defined by a spatial gradient in theconcentration of the gap protein Hunchback (Irish et al.1989; Hulskamp et al. 1989; Struhl, 1989). We couldinvoke a similar mechanism in Schistocerca, by as-suming that Hunchback protein levels are high in thecells of the embryo during thoracic and gnathalsegmentation, but fall rapidly in the cells of thedeveloping abdominal primordium. In addition, sincein Drosophila the expression of homeotic genes ismodulated by the activity of segment polarity genes(Martinez-Arias et al. 1988), whose products areinvolved in patterning within cellular fields, it is likelythey play an important role in the activation ofabdominal-A, once the cells have become permissivefor its expression.

Even though the segmentation machinery may nothave a role in the initial activation of abdominal-A inSchistocerca, it must have an important role in settingthe anterior limit of abdominal-A expression, for thisbecomes precisely 'parasegmental' respecting theequivalent of the A/P compartment boundary inDrosophila. The domain of abdominal-A expressionexpands anteriorly until it is coincident with theanterior boundary of an engrailed stripe. This anteriorexpansion may reflect an influence of cellular interac-tions on the ability of a cell to activate abdominal-A; foronce a cell expresses abdominal-A, its neighboursappear to follow suit soon after. At present we do notunderstand the mechanisms that act to limit thisexpansion of expression anteriorly.

The changing anterior boundary of abdominal-Aexpression in Schistocerca parallels in some respects theactivation of the vertebrate Hox genes in the CNS. Hox2.6 and 2.7 are first expressed in the posterior part ofthe neural tube, and expression then extends anteriorlyuntil it respects a sharp boundary coincident with thelimits of specific rhombomeres in the hindbrain(Wilkinson et al. 1989).

The limits of abdominal-A expression in SchistocercaIn both Drosophila and Schistocerca, abdominal-A is

expressed in most abdominal segments, but is notexpressed in the anterior compartment of the firstabdominal segment. In Drosophila the unique develop-ment of the first abdominal segment is under the controlof Ultrabithorax, the adjacent gene of the bithoraxcomplex. Ultrabithorax and abdominal-A are the twomost similar homeotic genes in Drosophila, both interms of protein sequence, extensively overlappingdomains of expression, and partially redundant func-tions (Akam et al. 1988). Ultrabithorax and abdominal-A are therefore candidates for the most recentlydiverged pair among the set of homeotic genes in theinsects. The observation that the Schistocerca abdomi-nal-A homologue is not expressed in anterior Alstrongly suggests that the functional distinction betweenUltrabithorax-\ike and abdominal-A-\\ke genes pre-dates the divergence of the Neopteran insect orders, (ca300My ago). Unique specialization of the first abdomi-

nal segment is clearly seen in early Schistocercaembryos. In common with many other lower insectorders (Anderson, 1973), but in contrast to Drosophila,a pair of limb buds develop in the first abdominalsegment. These give rise to the pleuropodia, specializedembryonic organs, which secrete a hatching enzymeand are lost at hatching (Slifer, 1937).

In the Drosophila embryo, the posterior limit ofabdominal-A expression extends to parasegment 13(i.e. A7p/A8a). The tail of the embryo (parasegment14 and the fused derivatives of more posterior segments(Jiirgens, 1987)) never express detectable ABD-Aprotein. Overt signs of segmentation are suppressed inthis tail region both in the embryo and the adult. It givesrise only to the male genitalia, (A9 or PS14), tospecialized sense organs of the posterior spiracle and toanal structures. In Schistocerca, the correspondingregion comprises fully formed segments. A9 generatesthe male genitalia, as in Drosophila; A10 is withoutappendages, and All gives rise to the anal cerci.Abdominal-A expression initially extends throughoutA9 and A10, but excludes All . This suggests to us thatthe differential development of the posterior abdomenin Schistocerca and Drosophila may in part depend onthe altered regulation of abdominal-A; in Drosophilawhen Abdominal-B is removed and abdominal-A isexpressed in parasegment 14, reduction of the posteriorsegments is suppressed, and the denticle belt of a typicalabdominal segment appears in A9 (Karch et al. 1990;Casanova et al. 1986).

We thank P. Lasko for his generous and patient assistanceand advice concerning fusion proteins and the production ofantisera, C. M. Bate and D. Shepherd for teaching us locustembryogenesis and together with I. Dawson and A. Bejsovecfor many helpful discussions. G.T. thanks M. Metzstein forhelp in producing Fig. 2 and C. Fox for technical assistance.This work was supported by a Medical Research Councilgrant to M.A. and G.T and a Wellcome Trust seniorfellowship to A.M.-A. The sequence data reported in thisarticle will appear in the EMBL/Genbank nucleotidesequence database with accession number X54674.

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(Accepted 15 August 1990)