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Developmental Biology

Molecular basis of Transabdominal-a sexually dimorphic mutant of

the

bithorax

complex of

Drosophila

(cisregulation/macronutation/homeotic/Abdominal B/evolution) SUSAN E.

CELNIKER

AND E. B. LEWIS

BiologyDivision,California Institute of Technology, Pasadena, CA 91125

Contributed byE.B. Lewis, November5, 1992

ABSTRACT Transabdominal (Tab)is adominant

gain-of-functionmutationthat resultsin islandsof sexuallydimorphic

abdominal cuticlein the dorsal thorax ofthe adultfly. This

phenotype hascomplete penetranceandconstant expressivity,

and we show that it results from ectopic expression of ABD-BII, one oftwoproteins derived from the Abdominal B (Abd-B)

domain of the bithorax complex (BX-C) and one that is normallyexpressedonly in terminal portionsoftheabdomen. In Tab/+ animals ABD-BII is ectopically expressed in the

relevantimaginal "wing" disc asthreeislands ofcells whose location on the fate map correspondsto thethreeislands of

transformed cuticle in each half of theadult thorax. Tab is

associatedwith aninseparable inversion bringingsequencesin

90E next to sequences in the transcription unit encoding

ABD-BHin89E.That90Esequences driveectopicexpression

ofABD-BIIisindicatedby ourfindingthatsuchsequences in aP-elementtransformantexpress the reportergene's product

(Ji-galactosidase)

in the same three islandsofwing disc cells.On

morphological grounds, the transformedislands in the adult thoraxcorrespondtosubsets ofmuscleattachment cells.

Ec-topic expression of a homeodomain protein thus creates a

uniqueandinvariantpatternofsexualdimorphism.

Conspicuous sexual dimorphism in wild-type Drosophila melanogasterislargely confinedtothe posterior abdomen,

where the male hassolidly blacktergitesonthefifth abdom-inal

(AS)

andA6segmentsand lackstergitesonA7andA8,

while thefemale has only black-banded tergites on A5-A8 inclusive. In Tab/+ animals, the same strikingly dimorphic

patternofpigmentationalsooccurs onthedorsal thorax(T2)

in theformof islands ofabdominal-likecuticle thataresolidly

black pigmented in themale andonlypartiallybanded with black in the female(Fig. 1).

In wild type, the ability to differentiate properly the

abdominalbodysegmentsdependsontheexpressionof the

homeoticgenesof the bithorax complex(BX-C) (1).

Devel-opment of A5 and the more posterior segments of the abdomen is specified by the Abd-B domain, one of three domains of the BX-C-the other two being Ultrabithorax (Ubx) and abdominal A(abd-A)(ref. 2; reviewed in ref. 3).

The Abd-B domain encodes two proteins, ABD-BI and ABD-BII (4).They share thesamehomeodomain,a60-amino acid helix-turn-helix motifthat mediates DNA-binding in-teractions and is also found in other proteins that regulate development(reviewed in ref. 5). Inwild-type embryos, the ABD-Bproteinshavespatial distributions thatarerestricted

to the posterior body regions (4). Later in development Abd-B isexpressed in the imaginal disc that gives risetothe adultinternaland externalgenitalia(6).

Todetermine whatrole the Abd-B domain plays inbringing about theTab/+ phenotype,wehavestudied thepatternof

protein and transcriptexpressionin thelarvalwing discs and

inembryosofTab/+ animals.Hereweprovide evidencethat ABD-BIIis abletodirect differentiation ofabdominaltissues in T2 in the absence of otherabdominalgenes oftheBX-C.

MATERIALS AND

METHODS

Materials. We were provided with gifts of materials as

follows: a monoclonal anti-UBX (Ultrabithorax) antibody wasfromR.Mannand D. Hogness (Department of

Biochem-istry, StanfordUniversity; originally made byR.White and

M. Wilcox), monoclonal anti-ABD-A (abdominal A) anti-body was from I. Duncan (Department ofBiology, Wash-ingtonUniversity, St. Louis), and the B14 P-elementstrain was from R. Kolodkin and C. Goodman (Department of Molecular and Cellular Biology, University of California,

Berkeley).

Immunohistochemistry. Wing discs were dissected from

third-instar larvae in lx TBS(0.1MTris/1.29MNaCl/0.05

MKCI/0.01MEGTA, pH

8.0)/0.1%

Triton X-100 and fixed for 5 min in2%EM-grade formaldehyde(Polysciences).The discswereseriallyincubated withamonoclonalanti-ABD-B antibody(1A2E9) (4), biotinylatedanti-immunoglobulin an-tibody (Bio-Rad), and avidin DH-biotinylated

anti-horseradish peroxidase H complex (Vector Laboratories).

Discswerethen stained with theperoxidasesubstrate diami-nobenzidine tetrahydrochloride (1

mg/ml)

(Polysciences)

and

0.01%

hydrogenperoxidase.

Embryos(3-15hrold)werefixed andproteinwaslocalized asdescribed (4).

InSitu

Hybridization

and Tissue liion. Wing discs

were dissected from third-instar larvae in 1x

phosphate-buffered saline (PBS) and fixed three times: first in 4% paraformaldehydeinlx PBS for 5 minatroomtemperature

and 15 min at 4°C, second in 4%

paraformaldehyde/lx

PBS/0.6%Triton X-100 (PBT) for 5 minat roomtemperature,

and third after treatment with proteinase K (10

mg/ml

in PBT)for3 min in4%

paraformaldehyde/0.2%

glutaraldehyde

in PBS for 15 min at room temperature. Hybridization

conditions were as described (7). Embryos were prepared

and hybridization conditions were as described (7).

Anti-sense single-stranded probeslabeled withdigoxigenin were generated byPCR.Transcripts encoding ABD-BI (also called

classAtranscripts) and ABD-BII(class B)weredetectedby

PCR-generated anti-sense probes 5013 and 5005,

respec-tively. Their position on the genomic map relative to the Abd-B cDNAs is shown inFig. 2.Probe5013wasgenerated byusinga1327-bpBssHII genomicrestriction fragment from

+157 on the BX-C walk and the primer 5'-TACTCGAGT-TCTGCCCC-3'. Probe

5005

was generated by using a 1145-bpSpe

I/Sca

Igenomic restriction fragment from +187 on the BX-C walk and the primer

5'-CCGCTGATTAAT-Abbreviations: BX-C, bithorax complex; T2, second thoracic

seg-ment;A5,etc., fifthabdominalsegment, etc.

Thepublicationcostsofthis articleweredefrayed in part by page charge

payment.This article must therefore beherebymarked"advertisement"

(2)

Proc. Natl. Acad. Sci. USA 90 (1993) 1567

FIG. 1. Sexual dimorphism in the thorax of Tab/+ male and female compared with wild type. (LefttoRight) Tab/+ male with threesolidlyblack-pigmented islands on each half of T2, wild-type male with uniformly tan-colored T2 (the wild-type female being identical), Tab/+ female with islands in T2 that are only black banded.

TCAAG-3'. Discs and embryos were photographed with Nomarski optics.

5-Bromo-4-chloro-3-indolyl

/3-D-galactoside

staining of discsfrom the B14 line was as described (13).

RESULTS

InTab/+wing discs, we detect ABD-B proteinprincipallyin three regions whose location in the fate map of the disc

correlatesremarkablywellwith the threeislands of abdom-inal-like cuticle in each half of the adult T2(Fig.3A). We have confirmed

by

insitu

hybridization

that suchdiscs express the

transcript encoding ABD-BII protein (Fig. 3B). We fail to detect

transcripts encoding

ABD-BIinthesediscs,nordowe

detect either UBX or ABD-A protein (data not

shown).

[ABD-BI

and ABD-BII are also known as Abd-B m and Abd-B r,

respectively (15).]

We conclude that ectopic

ex-pressionof ABD-BII isresponsible forthe Tab/+ transfor-mation in T2.

Thatenhancer-like elements in 90E drive ectopic expres-sion of ABD-B in Tab/+ discs is madelikely byresults of

studyingagerm-linetransformant (B14) havingaP-element

inserted in 90E. Inan otherwise wild-type background, the reporter genein this case, 3-galactosidase,isexpressedin the samethree islands ofwingdisc cells(Fig. 3C)that inTab/+

discs express ABD-B

protein

andtranscripts encoding ABD-BII.

In T2 of Tab/+ animals, the position of the

sexually

dimorphic

cuticlecoincides with the position ofsubsets of muscle attachment sites(Fig. 4). We presume that the cells in such sites secrete cuticle as well as having a muscle attachment function. If so, the cuticular transformation in T2 ofTab!+ is ahomeotic oneinwhichectopic expression of ABD-BII causes thoracic-type cuticle to be replaced

by

abdominal-like cuticle typical of the terminal half of the abdomen.

Indirectevidence suggests thatTab/+ animalsare

globally

expressing ABD-B protein and transcripts for ABD-BII in subsets of muscle attachment cells throughout the animal.

Thus, such expressionisseenin groups ofectodermal cells invirtuallyall segments of the Tab/+ embryo(Fig. 5). Ata

iab-9 5005 Probe

I.

+200kb 89E/90E P4

*-1

5C

Class

A

P3

FIG.2. Organizationofwild-typeandIn(3R)Tab genomicDNA andrepresentativehomeobox-containingcDNAsfrom the Abd-B domain (4, 8, 9).TheAbd-B domain extendsapproximately from+100to +200 kb of theBX-C walk(10).Theiab-S-iab-9regionsaredelineatedby

brackets(11).BoldfacedarrowpointstoTabbreakpoint.Tabis associated withasmallinversion withbreakpointsin 89E and 90E of thesalivary

glandchromosomes(6). Four classes of Abd-Btranscriptsaredesignated A,B,C,andy(12).Class Atranscriptsencode ABD-BI and class

B,C,andytranscriptsencodeABD-BII.Transcriptionstartsites for classes A and BaremarkedwithbentarrowsandaredesignatedP4 and

P3, respectively. Molecularbreakpointin 89Eislocated ina5' nontranslatedexonof class C and4315bpfromthetranscriptionstartsite P3 for class B. iab-5

+

iab-6

+

+100 Tab

iab-7 Abd-B iab-8

5013 Probe +1 50 Homeobox ABD-BI ABD-BII Homeobox rz z z z

Developmental Biology:

CeInikerandLewis

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1568 Developmental Biology: Celniker and Lewis

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\;.w.

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FIG. 3. Comparisonof ABD-BproteinandtranscriptexpressionpatternsinTab/+wing discs with thepatternof,-galactosidase expression in a disc fromaP-elementtransformant. (A) ABD-BproteininaTab/+disc. (B) RNAencodingABD-BIIin aTab/+disc. Since the ABD-B antibodyis directedagainstanepitopecommon toboth ABD-BI and ABD-BIIproteins,it hasbeen necessarytodetermine thedistributionof transcriptsencodingtheseproteinsin ordertoidentifytheectopically expressedone(s).(C)13-Galactosidase staininginadiscfromP-element transformant B14. (D) Diagram of the wingdisc in A superimposed onthe fate map of the wild-type disc [modifiedfrom Bryant (14)]; ABD-B-expressingregionsof the discareshownstippledand numbered. Location ofpresumptivebristleprimordiain thewing disc isshown bysolid circles. Dashed lines outline theregionof the disc thatgivesrisetothewingtissue. Two smallislands, numbered 4 and 5,ofABD-B protein expression fail to haveadiscernible effectonthecuticleof T2. NP,notopleuralbristle;PS, presutural bristle;SA,supraalarbristle; DC,dorsocentralbristle;SC, scutellarbristle;PA,postalarbristle.

somewhat laterstage,muscles form betweenadjacentsetsof these cells, suggestingthatthecells are atleast involved in themuscle attachmentprocess,iftheyarenotindeedmuscle attachmentcells. Consistent with ABD-BIIbeing expressed

in subsets of ectodermal cells in the embryonic abdomen,

Tab/+ flies have on abdominal segments A2-A5 inclusive patches of cuticle thatarehomeoticallytransformed toward

segmentsposteriortoA5(6). DISCUSSION

Ourresults indicate that the ABD-BII protein shares with each of the other homeodomain proteins of the BX-C-namely,UBX,ABD-A, andABD-BI-theabilitytoprogram

development ofabdominal cuticle,presumably by turningon one ormoredownstreamtarget genes. SinceonlyABD-BII isexpressed in the wing discs of Tab/+ animals,yetT2has abdominalcharacteristics normally developed under control ofABD-BI,weconclude that ABD-BII shares withABD-BI

the ability to activate genes required for developing the sexually dimorphic pattern ofthe posterior abdomen. This

sharing of similar functions by homeotic gene products is known for the development of other structures, such as

formation of thelongitudinaltracheal trunks (1) and

suppres-sion of chitinized plates (21).

Insomecases,theBX-Cproteinsappear to haveunique,

aswellasshared, functions-e.g.,UBXunder control of the bithoraxoid (bxd) cis-regulatory region suppresses ventral

pits in the abdominal segments (1). Similarly, ABD-BII

appears tohaveatleastoneunique function-programming development ofgenitalia. Thus, animals lacking this

pro-tein-namely, homozygotes for theinfraabdominal9mutant iab-91065-occasionally survive as adults and lack internal

and externalgenitalia(11). Heterozygotes for Tab and such iab-9mutantsalsolackgenitalia(6),asisexpectedsince the Tab breakpoint in 89E interrupts the transcription unit en-codingABD-BII(Fig. 2). The iab-8D)4mutantofDuncan is

a396-bpdeletionspanningthepromoterof thetranscription

unitencoding ABD-BI (11, 12). Homozygotes for thismutant survive,although rarely,andpossessgenitalia,showingthat

ABD-BI proteinisnotrequiredfor theirdevelopment. Inspite oftheabilityof ABD-BIItopromotedevelopment

ofgenitalia in the terminal abdominal regions, no trace of suchdevelopment is visible inT2ofTab/+adults. However, for such development to occur it may be necessary for ABD-BIItobe expressed inconjunction with one or more proteinsthatareonlyexpressedin theterminalbodyregions

[see reviewsbyLipshitz(22) and St. Johnson and

Nusslein-Volhard(23)]. We wouldpredictthatprotein(s)of the latter

types would have to be ectopically expressed along with

ectopic expression of ABD-BII before genitalia could de-velop inT2ofTab/+ animals. [Two BX-Cmutants, tumor-oushead(tu-h)(24) andSGA-62(25),arereportedtoexpress

rudimentsofgenitalia

ectopically

butonlyin the headregion

and with incompletepenetranceandvariable

expressivity.]

Unlike most

gain-of-function

mutants, Tab has complete penetranceandconstantexpressivity.Thearchetypal exam-ple of a dominant, homeotic,

gain-of-function

mutant is

(4)

Proc. Nati.Acad. Sci. USA 90 (1993) 1569

B

FIG. 4. Comparison of location of islands of abdominal-like cuticle in dorsal T2 ofTab/+ with the location of subsets of thoracic muscle attachment sites in dorsal T2of wild type. (A)Tab/+ male thorax. (B) Diagram of T2 as shown in A. On the right half, the islands are numbered in agreement with Fig. 3D and small opencircles representsites of macrochetes; on the left half, thoracic muscle insertion sites immediately underlying the cuticle are enclosed in dotted lines [modified from Miller(16)]. DC,dorsocentral bristle; DL, dorsal longitudinal muscle; DV, dorsoventral muscle; NP, notopleural bristle; PA, postalar bristle; PS, presutural bristle; SA, supraalarbristle; SC, scutellar bristle; TT, tergotrochanteralmuscle.

Antennapedia (Antp). Althoughsomealleles have complete

penetrance, eventhemost extremeallele,Antpy", has vari-ableexpressivity; that is, only rarely are both antennae ofan

AntpYu/+ animaltransformed towardafullleg. In theBX-C, Haltere mimic (Hm) and Miscadastral pigmentation (Mcp) do have complete penetrance and constant expressivity and representstriking gain-of-function mutations. However, the gain of function extends onlytoonesegmentanterior to that

inwhich thefunction is normallyexpressed,whereas the Tab

transformation is expressed inanterior T2, which is 9

seg-ments anterior to A9 where ABD-BII is normally first

ex-pressed.

Evolutionary Considerations. Tab is remarkable among

dominanthomeotic mutantsinanumberofways. Itresults

in anextension ofsexually dimorphictraits into thedorsal

thorax of the adult, where it couldclearly playarole inthe

sexual behavior of theorganism and thereforemightunder some circumstances have a selectiveadvantage.

A

C~~~*<

Tab represents a dominant gain-of-function mutant that alters the cis regulation ofageneencodingABD-BII,oneof twoproteins of the Abd-Bdomain of the BX-C. In doing so, Tab doesnotappear toalter the ABD-BIIprotein itselfbut, instead, radically alters where that protein is expressed,at the same time eliminating its expression in the terminal abdomen. A macromutation will generally,asin thecaseof Tab, represent loss of an "old" genefunction and gain ofa

"new" one. Since the oldfunction will seldom be a dispens-ableone, such a mutant will tend to be lethal when homozy-gous and can be maintained in a population only over a

chromosome containing the wild-type allele. If aduplication

for the wild-type allele is already present in the species at the time the mutation to a new function arises, thenobviously the resultant mutant can immediately become viable as a ho-mozygote. Otherwise, a duplicationcontaining one copy of the mutant allele and onecopy of thewild-typeallelewould need toarise later andcould do sobyanumber of

mecha-B

D

Al

FIG.5. ABD-Bprotein and Abd-Btranscript expression in wild-type andTab/+heterozygous embryos that are 10 hr old. (A) Wild type. ABD-B proteins are expressed in parasegment 10 (PS10)-PS15 inclusive and in visceral mesoderm surrounding the hindgut (4, 15, 17). [Embryonic parasegments are one-half segment offset from the adult segments-e.g., PS10 comprises posterior A4 and anteriorAS(18).] (B) Tab/+. Superimposedonthe ABD-B proteinpattern in A, ectopic expression of that protein is observed in PS8 and PS9, the gnathal region thatgives rise to the pharynx and in a laterally localized pattern repeated in each abdominal segment. (C)Tab/+.Transcripts encodingABD-BI

arerestricted to the ectoderm of PS8-PS13. Since Tab was induced on a chromosome containing the double mutant Miscadastral pigmentation (Mcp) (1)andSuper-abdominal(Sap) (19),transcriptsencodingABD-BIshow anextension of their wild-type embryonic expression pattern (9, 20)intoPS8 and PS9. (D)Tab/+. Transcripts encodingABD-BIIfollow the sameABD-Bprotein expression in the gnathal region of the head andin restrictedregionsin theremainingsegmentsfrom PS4toPS15. Anterior istothe left.(A-C) Dorsal views. (D) Lateral. view.

Developmental Biology:

CeIniker and Lewis

(5)

nisms, suchas anillegitimate recombination eventbetween

themutant geneinonechromosome and the wild-type allele in the homologous chromosome.

Altering the cis regulation ofa gene, suchas occursin the Tabmutant,has been proposedas amechanism for produc-ing major changes in the structure and function of higher organisms (26). The Tab mutant is an unusually relevant example in which a change in cis regulation of a homeo-domain-containing proteindramatically alters cell fate and in doingso creates anunusualpattern of sexual dimorphism in theDrosophila thorax.

We thank S. Sharma, J. Hardwick, and H. Park for technical assistance; A. Chopna and K. Vijayraghavan for bringing the em-bryonic expression pattern of the B14P-element insertion to our attention; and WelcomeBender, NancyBonini,Howard Lipshitz, and JoanneTopol for comments on an earlier draft of this paper. This work was supported by grants to E.B.L. from the National Institutes of Health (HD06331andGM40499)and from theLucilleP.Markey Charitable Trust.

1. Lewis, E. B. (1978) Nature(London)276, 565-570.

2. Sanchez-Herrero, E., Vernos, I., Marco, R. & Morata, G. (1985)Nature(London)313, 108-113.

3. Duncan,I. (1987) Annu. Rev. Genet. 21, 285-319.

4. Celniker, S.E., Keelan, D.J. & Lewis, E. B. (1989) Genes Dev.3, 1425-1437.

5. Scott,M.P.,Tamkun,J. W.&Harzell, G. W.(1989)Biochim. Biophys.Acta989, 25-48.

6. Celniker,S. E. &Lewis,E. B.(1987)Genes Dev. 1, 111-123. 7. Tautz, D.&Pfeifle,C.(1989) Chromosoma 98, 81-85.

8. DeLorenzi, M., Ali, N., Saari, G., Henry, C., Wilcox, M. & Bienz,M.(1988) EMBO J. 7, 3223-3231.

9. Kuziora,M. A.&McGinnis,W.(1988) EMBOJ. 7, 3233-3244. 10. Karch, F.,Weiffenbach,B.,Peifer,M.,Bender, W., Duncan, I., Celniker, S., Crosby,M.& Lewis,E.B.(1985)Cell43, 81-96. 11. Celniker, S. E., Sharma, S., Keelan, D.& Lewis, E. B. (1990)

EMBO J. 9, 4277-4286.

12. Zavortink, M. &Sakonju,S. (1989) Genes Dev. 3,1969-1981. 13. Bellen, H., O'Kane, C., Wilson, C., Grossniklaur, U., Pearson,

R. K. & Gehring, W. J. (1989) Genes Dev. 3, 1288-1300. 14. Bryant, P. J. (1975) J. Exp.Zool.193, 49-78.

15. DeLorenzi, M.&Bienz, M. (1990) Development 108, 323-329. 16. Miller, A.(1950) inBiologyofDrosophila, ed. Demerec, M.

(Wiley, New York), pp. 368-419.

17. Boulet,A.M., Lloyd,A. &Sakonju, S. (1991)Development 111,393-405.

18. Martinez-Arias,A. &Lawrence,P. A.(1985)Nature(London) 313,639-642.

19. Sakonju, S., Lewis, E. B.&Hogness, D. (1984) Genetics 107, s93.

20. Sanchez-Herrero, E. & Crosby, M. A. (1988) EMBO J. 7, 2163-2173.

21. Duncan,I. (1982)Genetics100,s20.

22. Lipshitz, H.(1991) Curr. Opin. CellBiol. 3, 966-975. 23. St.Johnston, D., Beuchle,D. &Nusslein-Volhard, C. (1991)

Cell66,51-63.

24. Kuhn, D., Woods, D. F. & Andrew, D. J. (1981) Genetics 99, 99-107.

25. Awad, A. A. M., Gausz, J., Gyurkovics, H. &Parducz, A. (1981)ActaBiol. Acad. Sci. Hung. 32, 219-228.

Figure

FIG. 2. Organization of wild-type and In(3R)Tab genomic DNA and representative homeobox-containing cDNAs from the Abd-B domain (4, 8, 9)
FIG. 3. Comparison of ABD-B protein and transcript expression patterns in Tab/+ wing discs with the pattern of ,-galactosidase expression in a disc from a P-element transformant
FIG. 5. ABD-B protein and Abd-B transcript expression in wild-type and Tab/+ heterozygous embryos that are 10 hr old

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