compartmental organization of the drosophilagenital...

205Development 124, 205-218 (1997)Printed in Great Britain © The Company of Biologists Limited 1997DEV8366

Compartmental organization of the Drosophila genital imaginal discs

Elizabeth H. Chen1 and Bruce S. Baker2

1Department of Developmental Biology and 2Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA

We have investigated the anterior and posterior compart-mental organization of the genital imaginal disc. Unlike thethoracic discs, the genital disc is a compound disc consistingof three primordia – the female genital, male genital, and analprimordia. Here we provide evidence that each primordiumis divided into anterior and posterior compartments. Genesthat are known to be expressed in compartment-specificmanners in other discs (engrailed, hedgehog, patched,decapentaplegic, wingless and cubitus interruptus) areexpressed in analogous patterns in each primordium of thegenital disc. Specifically, engrailed and cubitus interruptus areexpressed in complementary domains, while patched,decapentaplegic and wingless are expressed along the borderbetween the two domains. Mitotic clones induced at thebeginning of the second larval instar do not cross theboundary between the engrailed-expressing and cubitus inter-

ruptus-expressing domains, indicating that these domains aretrue genetic compartments. Furthermore, we examined thephenotypes of mutant clones of the cAMP-dependent proteinkinase A and engrailed-invected, genes that are known to playcompartment-specific functions in other discs. These experi-ments demonstrate that the anterior/posterior patterningfunctions of these genes are conserved in the genital disc. Theadult clonal phenotypes of protein kinase A and engrailed-invected mutants also provide a more detailed map of theadult genitalia and analia with respect to theanterior/posterior compartmental subdivision. Our resultslead us to propose a new model to describe the anterior andposterior compartmental organization of the genital disc.

Key words: Drosophila, genital discs, compartments,anterior/posterior patterning, protein kinase A, engrailed

SUMMARY

INTRODUCTION

Imaginal discs are sac-like clusters of primordial cells in thelarvae that are set aside during embryogenesis, proliferateduring larval stages, and give rise to the adult cuticular struc-tures during metamorphosis (reviewed by Cohen, 1993).Genetic studies of the thoracic wing and leg discs haverevealed that the discs are divided into anterior and posterior(A/P) compartments (Garcia-Bellido et al., 1973). Each com-partment represents a functionally distinct developmental unit.Cells in one compartment do not mix with those of the other.The compartment boundary, therefore, serves as a line of celllineage restriction. Interactions between anterior and posteriorcells which occur at the compartment boundary are believed toorganize and pattern the entire disc (Meinhardt, 1983).

The genital imaginal disc gives rise to the adult terminalia,including the genitalia and the analia. Most imaginal discs areessentially two-dimensional, with folds in the plane of the discepithelium (reviewed by Fristrom and Fristrom, 1993). Incontrast, the genital disc epithelium is folded into a three-dimen-sional structure with distinct dorsal and ventral epithelia, whichgive rise to different adult structures, as shown on the three-dimensional fate maps constructed by Ehrensperger and Epper(Ehrensperger, 1972; Epper, 1980, 1983) (Fig. 1a-c,e-g). Thederivatives of the female genital disc include the female genitalia,the 8th tergite, the hind gut, and the dorsal and ventral anal plates(Fig. 1d), while the derivatives of the male genital disc includethe male genitalia, the hind gut, and the left and right anal plates(Fig. 1h) (reviewed by Bryant, 1978; Lauge, 1982).

In addition to its three-dimensional structure, the genital dischas several unique properties which set it apart from the otherimaginal discs (reviewed by Bryant, 1978; Lauge, 1982). First,it is the only single, unpaired imaginal disc. Second, it is theonly imaginal disc which shows strong sexual dimorphism.This dimorphism, manifested most clearly in the adult struc-tures, becomes evident in histological sections before thesecond larval molt and is quite pronounced in the third instardiscs. Finally, the embryonic origin of the genital disc isdifferent from that of the other discs. While the thoracic wingand leg discs are each derived from a single embryonicsegment (Bate and Martinez Arias, 1991), the three primordia(the female genital, the male genital and the anal primordia)that make up the genital disc originate from within threeembryonic tail segments, abdominal segments 8, 9, and 10,respectively (Dübendorfer and Nöthiger, 1982; Epper, 1980;Epper and Nöthiger, 1982; Nöthiger et al., 1977; Schüpbach etal., 1978). Cells from these three primordia are presumed tofuse together during late embryogenesis and form a trans-versely elongated cluster of genital disc precursor cellsbetween the A8 denticle belt and the larval anal pads, whichwill later invaginate to develop into either a female or a malegenital disc (Hartenstein and Jan, 1992). In a female larva, thefemale genital primordium will be activated, the male pri-mordium will be repressed, and the anal primordium will adoptthe female fate. Thus, the mature third instar female genitaldisc is composed of a well developed female genital pri-mordium, a repressed male genital primordium, and a femaleanal primordium. The opposite is true for the mature male

206 E. H. Chen and B. S. Baker

Fig. 1. Fate maps and adult derivatives of the female (A) and male(B) genital discs (modified after Ehrensperger, 1972, and Epper,1983). The fate maps of the ventral epithelia of a female and a malegenital disc are shown in a and e respectively, and the fate maps ofthe dorsal epithelia are shown in b and f. c and g show parasagittalsections of a female and a male genital disc. The planes of theparasagittal sections are indicated with an arrow in a or e. Anterior ofall the discs is up. D, dorsal epithelium; V, ventral epithelium; L,lumen. d and h show the adult derivatives of a female or a malegenital disc. The female genital disc is composed of the femalegenital primordium (fgp), the repressed male primordium (rmp), andthe anal primordium (ap). The fgp gives rise to the 8th tergite (t-8)and both the external and the internal genitalia, including the vaginalplates (vp) with three types of bristles on them (the long bristle (lb),the thorn bristle (thb), and the sensilla trichodea (s)), the dorsal andventral vulva (dvu and vvu), the uterus (u), the seminal receptacle(sr), the parovaria (pov), the spermathecae (spt), and the oviduct(od). These female genital structures are mapped to the thick ventralepithelium, except for the parovaria, which are mapped to the dorsalepithelium. The rmp does not give rise to any adult structures, and islocated at the thickened anterior and lateral regions of the dorsalepithelium. The ap gives rise to the dorsal and ventral anal plates(dap and vap), as well as the hind gut (not shown). The anal platesare derived from the thickened posterior part of the dorsalepithelium. The male genital disc is composed of the male genitalprimordium (mgp), the repressed female primordium (rfp), and theanal primordium (ap). The mgp gives rise to both the external andinternal genitalia, including the phragma (ph), the genital arch (ga),the lateral plates (lp), the claspers (cl), the penis apparatus (pa), thesperm pump (sp), the ejaculatory duct (ed), the paragonia (pg), andthe vas deferens (vd). These male genital structures are mapped tothe anterior lobe and the ventral lateral regions of the disc. The rfpdoes not give rise to any adult structures, and is located at thethickened posterior part of the ventral epithelium. The ap gives riseto the left and right anal plates (ap in (h)), as well as the hind gut (notshown). The anal plates are derived from the thickened posteriorregions of the dorsal epithelium.

genital disc (Belote and Baker, 1982; Epper, 1980; Epper andBryant, 1983; Epper and Nöthiger, 1982; Nöthiger et al., 1977;Schüpbach et al., 1978; Wieschaus and Nöthiger, 1982).

Although there is a clear compartmental boundary betweenthe genital and anal primordia, the A/P compartmental organ-ization of the genital disc has been unclear. Two models havebeen proposed to explain how the genital disc is divided intoanterior and posterior compartments. Based on the phenotypesof the engrailed (en) mutant clones in the adult terminalia,Lawrence and Struhl (1982) suggested that the female genitalprimordium is of anterior origin, whereas the male genital andanal primordia are of posterior origin. Based on the phenotypesof heteroallelic combinations of en mutations, Epper andSánchez (1983) suggested that all three terminal primordiahave anterior and posterior compartments, and that the femaleand male genital primordia are mostly made of posterior com-partment, with a very small anterior compartment.

Recent work on the thoracic wing disc has provided sub-stantial information about the genes involved in the A/P com-partmental organization. Genes that are involved in the A/Ppatterning of the wing disc include: (1) engrailed (en), whichencodes a homeobox-containing transcription factor expressedin the posterior compartment (Kornberg et al., 1985); (2)hedgehog (hh), whose protein product is a secreted moleculealso expressed in the posterior compartment (Lee et al., 1992;

Tabata et al., 1992); (3) decapentaplegic (dpp), a member ofthe transforming growth factor β superfamily of signalingmolecules (reviewed by Kingsley, 1994), which is expressedat the anterior side along the A/P compartment border, forseveral cell widths (these cells will be referred to as bordercells hereafter) (Raftery et al., 1991); (4) cubitus interruptus(ci), which encodes a Zn-finger containing protein, expressedin the anterior compartment (Eaton and Kornberg, 1990); and(5) patched (ptc), which encodes a putative transmembraneprotein (Hooper and Scott, 1989; Nakano et al., 1989), alsoexpressed in the anterior compartment, but is expressed mostpredominantly in the border cells (Phillips et al., 1990).

Molecular and genetic studies have also revealed a regula-tory hierarchy that controls the A/P patterning of the wing disc.In the anterior compartment, PTC represses the expression ofdpp and ptc itself (Capdevila et al., 1994; Tabata and Kornberg,1994). HH is secreted from the posterior compartment into theborder cells and antagonizes the inhibitory activity of PTC onthe expression of dpp and ptc, which results in the expressionof dpp and higher expression level of ptc in the border cells(Lee et al., 1992; Tabata and Kornberg, 1994). Recently, thecAMP-dependent protein kinase A catalytic subunit gene(DC0, referred to as Pka in this paper) has been shown to berequired for patterning the anterior compartment of the thoracicdiscs (Jiang and Struhl, 1995; Lepage et al., 1995; Li et al.,

207Genital disc compartmental organization

1995; Pan and Rubin, 1995). Pka mutant clones in the anteriorcompartment cause ectopic expression of dpp and ptc andresult in adult pattern duplications and/or local overgrowth.However, Pka mutant clones in the posterior compartment donot show any abnormalities. In the posterior compartment, en,the posterior selector gene, activates the expression of hh, andrepresses the expression of ci, ptc and dpp (Eaton andKornberg, 1990; Sanicola et al., 1995; Schwartz et al., 1995;Tabata et al., 1995; Zecca et al., 1995). It has been recentlyshown that invected (inv), a gene that contains a homeodomainalmost identical to that of en (Coleman et al., 1987), con-tributes a non-essential and partially redundant function to en(Tabata et al., 1995). en-inv mutant clones in the posteriorcompartment cause loss of expression of hh, and ectopicexpression of ci, dpp, and ptc, which result in posterior toanterior transformations in adult flies. However, en-inv mutantclones in the anterior compartment are normal. Thus, Pka hasan anterior compartment-specific function in the wing disc,while en-inv have a posterior compartment-specific function.

In the leg disc, the relationship between domains of en, hh,ptc, and ci expression is analogous to those in the wing disc(reviewed by Perrimon, 1995). However, dpp is predomi-nantly expressed in the dorsal half of the border cells,whereas wingless (wg), a member of the Wnt family ofsecreted molecules (reviewed by Nusse and Varmus, 1992),is expressed in the ventral half of the border cells (Williamset al., 1993). Correspondingly, Pka mutant clones causeectopic expression of dpp in the dorsal anterior region, andectopic expression of wg in the ventral anterior region of theleg disc (Jiang and Struhl, 1995; Lepage et al., 1995; Li etal., 1995).

As part of a long term study to elucidate imaginal disc devel-opment, and to understand how information from the sex deter-mination hierarchy is integrated with input from other hier-archies to control developmental fates, we have undertakenmolecular and genetic analyses to resolve the controversy overthe A/P compartmental organization of the genital disc. Weused genes which have A/P compartment-specific expressionpatterns and functions in wing and leg discs as molecularmarkers to localize the anterior and posterior domains in thegenital disc. We show that the relationship between domainsof en, hh, ptc, dpp, wg, and ci expression in the genital disc isanalogous to those in the wing and leg discs, which suggeststhat the genital disc is also divided into anterior and posteriorcompartments. Moreover, mitotic clones induced at thebeginning of the second instar in the genital disc do not crossthe boundary between the en- expressing and ci-expressingcells, demonstrating that these two cell populations belong totwo true genetic compartments. We also find that the spatialexpression patterns of these genes are restricted to subsets ofthe cells that make up each of the three primordia of the genitaldisc, suggesting that each of the three primordia is composedof anterior and posterior compartments. Moreover, clonalanalyses with Pka and en-inv mutants, which are known to playcompartment-specific roles in the A/P patterning of other discs,show that the functions of these genes are conserved in thegenital disc. The adult clonal phenotypes of Pka and en-invmutants also provide a more detailed map of A/P compart-mental divisions within the adult genitalia and analia. Basedon these results we propose a new model for the A/P com-partmental organization of the genital disc.

MATERIALS AND METHODS

Fly stocksThe enhancer trap and promoter lacZ lines used for antibody stainingwere as follows: Xho25 (en; Hama et al., 1990), P30 (hh; Lee et al.,1992), AT90 ptc (ptc; kindly provided by C. Goodman), BS3.0 (dpp;Blackman et al., 1991), P999 (wg; kindly provided by T. Tabata andT. Kornberg), 7.1-ci (ci; Schwartz et al., 1995).

Stocks used for Pka clonal analysis (Pan and Rubin, 1995) were:(1) y,w; PkaB3 P[ry+; hs-neo; FRT]40A; BS3.0/T(2:3) SM6;TM6B,Tb. (2) y,w; PkaB3 P999 P[ry+; hs-neo; FRT]40A/CyO. (3)y,w; PkaB3 P[ry+; hs-neo; FRT]40A AT90 ptc/T(2:3) SM6; TM6B,Tb.The control stock used was y,w; P[ry+; hs-neo; FRT]40A; ry.

Stocks used for en-inv clonal analysis were: (1) y,w; P[ry+; hs-neo;FRT]42D DfenE/CyO. (2) y,w; BS 3.0 P[ry+; hs-neo; FRT]42DDfenE/CyO. (3) y,w; P[ry+; hs-neo; FRT]42D DfenE; P30/T(2:3)SM6; TM6B,Tb. The control stock used was y,w; P[ry+; hs-neo;FRT]42D; ry.

For making mitotic clones and marking the posterior compartmentof the genital disc, P[ry+; hs-neo; FRT]40A; P30/T(2:3) SM6;TM6B,Tb was used.

The following lines were used to introduce the flipase into the het-erozygous flies and mark the clones in adults or discs: (1) y, w,hsFLP1; P[ry+; y+]25F, P[ry+; hs-neo; FRT]40A. (2)w, hsFLP1;P[mini-w+; hs-NM]31E, P[ry+; hs-neo; FRT]40A. (3) y, w, hsFLP1;P[ry+; hs-neo; FRT]42D, P[ry+; y+]44B.

Two types of wg mutants were used to examine the terminalia phe-notypes (kindly provided by K. Cadigan and R. Nusse). One was aheteroallelic combination of wgCX3/wgCX4. The other was a wg tem-perature sensitive mutant, wgIL.

Generation of mitotic clones in the genital discMitotic clones of wild type, Pka mutant, and en-inv mutant alleleswere generated by the FLP/FRT system as described by Golic (1991)and Xu and Rubin (1993). FLP-mediated recombination was inducedby heat shocking second instar larvae at 38oC for one hour. Adultclones were identified by the y marker. Clones in genital discs wereidentified by using either antibodies against the ubiquitouslyexpressed NOTCH-MYC epitope (for wild-type and Pka clones), orantibodies against EN protein (for en-inv clones).

Whole-mount immunostaining of the genital discDouble and triple immunofluorescence labeling of genital discs weredone following the protocol of Xu and Rubin (1993). The primary anti-bodies used were: rabbit anti-β-galactosidase (β-gal) (Cappel), mouseanti-EN (Patel et al., 1989), rat anti-WG (gift from K. Cadigan and R.Nusse), mouse anti-MYC (gift from S. Carroll), rat anti-CI (gift from R.Holmgren), rabbit anti-DPP (gift from M. Hoffman), rabbit anti-HH (giftfrom T. Tabata), mouse anti-PTC (gift from I. Guerrero). The secondaryantibodies used were from Jackson Immunologicals, including goatFITC anti-rabbit, goat Cy5 anti-rabbit, goat FITC anti-rat, goat Cy3 anti-mouse, goat rhodamine anti-mouse, goat Cy3 anti-rat.

Abdominal cuticle preparationsAdult abdomens were dissected, boiled in 10% NaOH for 10 minutes,and washed in water four times. The cuticles of the abdomens werethen washed in isopropanol 3-4 times and mounted in Gary Struhl’sMagic Mount for inspection under a compound microscope.

Internal genitalia dissectionsThe adult terminalia were dissected and fixed in 4% glutaraldehydefor 20 minutes. The internal genitalia were then scored using a dis-secting microscope.

MicroscopyConfocal images were collected using a Bio-Rad MRC-1024 system.Combined confocal images were made using Adobe Photoshopsoftware.


RESULTS

Expression of hh, en, ci, ptc, dpp, and wg in thegenital discTo determine whether the six known A/P patterning genes

Fig. 3. Expression patterns of hh, en, ci, dpp, wg and ptc inthe male genital disc. In each panel, anterior of the disc isup, and an optical section through the dorsal epithelium ofthe disc is shown. (A,B) Complementary expressionpatterns of hh (red) and ci (green) in the male disc. Thefocal plane is either on the male genital primordium (A) orthe anal primordium (B) of the same disc. The expression ofhh in the male genital primordium is located on the dorsalepithelium of the anterior lobe (also see Fig. 4F). Theexpression of hh-lacZ was revealed by anti-β-gal antibody.ci expression was detected by anti-CI antibody. (C,D)Similar expression of hh (green) and en (red) in the maledisc. The expression of hh-lacZ was revealed by anti-β-galantibody (C). en expression pattern was detected by anti-ENantibody (D). (E-G) dpp and wg are expressed along theborder between en-expressing and ci-expressing domains ina complementary manner in the male disc. E and F showdouble labeling with anti-EN antibody (red) and anti-β-galantibody (green) using a dpp-lacZ line (E) or a wg-lacZ line(F). G shows triple labeling with anti-EN antibody (red),anti-WG antibody (green), and anti-β-gal antibody (blue)using a dpp-lacZ line. (H) ptc is expressed at a high levelalong the entire border between en-expressing and ci-expressing domains. en expression was revealed by anti-ENantibody (red) and ptc expression revealed by anti-β-galantibody (green) using a ptc-lacZ line.

Fig. 2. Expression patterns of hh, en, ci,dpp, wg and ptc in the female genitaldisc. Anterior of the discs is up in eachpanel, and an optical section througheither the dorsal or the ventral epitheliumof the disc is shown. (A,B) Thecomplementary expression patterns of hh(red) and ci (green) on the ventral (A)and dorsal (B) epithelia of the femaledisc. hh expression pattern wasvisualized by anti-β-gal antibody usingthe hh-lacZ line. ci expression wasdetected by anti-CI antibody. (C,D) hh(green) and en (red) are expressed insimilar patterns in the female disc. hhexpression pattern (C) was visualized asin A and B. en expression (D) wasdetected by anti-EN antibody. (E-G) dppand wg are expressed in complementarypatterns along the border between en-expressing and ci-expressing domains inthe female disc. E and F show doublelabeling with anti-EN antibody (red) and anti-β-gal antibody (green) usinwith anti-EN antibody (red), anti-WG antibody (green), and anti-β-gal alevel along the entire border between en-expressing and ci-expressing doexpression was revealed by anti-EN antibody (red) and ptc expression re

function in the genital disc as they do in the thoracic discs, weexamined the relative domains of expression of these genes byperforming double and triple fluorescent labeling experiments.Since both female and male genital discs are bilaterally sym-metrical, the expression patterns of these genes are bilaterally

g a dpp-lacZ line (E) or a wg-lacZ line (F). G shows triple labelingntibody (blue) using a dpp-lacZ line. (H,I) ptc is expressed at a highmains on the ventral (H) and dorsal (I) epithelia of the female disc. envealed by anti-β-gal antibody (green) using a ptc-lacZ line.


sentations of the expression patterns of hh, en, ci, dpp, wg and ptc in thel discs. Anterior of the discs is up in all panels. The expression patternsose from several optical sections through the ventral epithelium (A,D) or,E). (A-C) The expression patterns of en and hh (red), ci (light green), and ptc (dark green) in the female disc. (A) a ventral view (compared a dorsal view (compared with Fig. 2B,I), and (C) a reconstructed female disc. The plane of the parasagittal section is indicated withe the disc is bilaterally symmetrical, the expression patterns of dpp, wg

on one side of the discs in A and B. In C, the expression of ptc, but notthe border between en-expressing and ci-expressing cells. Although ptcvel throughout the anterior compartment, only the high level expressionThe uncolored portion on the dorsal epithelium in C represents the thinn the rmp and the ap. ap: anal primordium; d: dorsal epithelium; fgp:um; rmp: repressed male primordium; v: ventral epithelium. (D-F) Thehese genes in the male disc. (D) A ventral view (data not shown), (E) awith Fig. 3), and (F) a reconstructed parasagittal view of the male disc.gittal section is indicated with arrows in D and E. The gene expression A-C. The uncolored portions on the ventral and dorsal epithelia in Flial sheaths between different primordia. mgp: male genital primordium;

rimordium. The translation of the expression patterns onto the fate mapsring the domains of gene expression to the positions of differentimordia have unique morphologies such as lobes or thickenings on the1), which allows one to place the gene expression onto a givenusly. However, within each primordium, there are not obvioush cells that will form different adult structures (except for a group of will give rise to the paragonia and the vas deferens). Therefore, then a primordium outline the domains of gene expression relative to theordium.

symmetrical as well. In the female genital disc, hh is differen-tially expressed in dorsal and ventral disc epithelia, while ci isexpressed in a pattern complementary to that of hh (Fig. 2A,B).In the male genital disc, hh is also expressed in both dorsal andventral disc epithelia. hh expression in the ventral anterior lobeis confined to the dorsal surface of the lobe, which can beobserved from the dorsal view of the disc. Here too, ci isexpressed in a pattern complementary to that of hh (Fig. 3A,B).There is no overlap between these two cell populations at theborder. The en gene has a expression pattern similar to that ofhh in both female and male genital discs (Figs 2C,D, 3C,D).The complementary patterns of en(hh) and ci expression areanalogous to the situation in the wing and leg discs, whereen(hh) define the posterior compartment and ci the anteriorcompartment. In both female and male genital discs, ptc isexpressed at a high level along the entire border betweenen(hh)- and ci-expressing cells. Moreover, these cells overlapwith ci-, but not en(hh)-, expressing cells at the border (Figs2H,I, 3H). Again, this is very similar tothe expression pattern of ptc in the wingand leg discs. Interestingly, dpp and wgare also expressed in non-overlappingstripes along the border between en(hh)-and ci-expressing domains in female andmale discs (Figs 2E-G, 3E-G). Forexample, on the anterior lobe of the maledisc, wg is expressed in the medianregion, while dpp is expressed in themore lateral positions along the border.This is analogous to what is seen in theleg disc, where dpp and wg are expressedin non-overlapping dorsal and ventralsectors along the A/P compartmentborder. Taken together, these resultssuggest that the genital disc, like thethoracic discs, is divided into anterior andposterior compartments.

A comparison of en(hh) and ciexpression patterns with the known fatemaps of the genital discs reveals that boththe genital and the anal primordia containcomplementary en(hh)- and ci-expressingregions (Fig. 4). For example, in the malegenital disc, the en(hh)-expressing regionson the anterior lobe of the disc correspondto part of the male genital primordium,whereas the en(hh)-expressing regions onthe dorsal posterior lobes of the disc cor-respond to part of the male anal pri-mordium (Fig. 4E,F). Complementaryen(hh) and ci expression patterns are alsoobserved in the female genital and analprimordia of the female disc (Fig. 4A-C).en(hh) and ci are also expressed in non-overlapping cells in the repressed maleand female primordia, respectively, of thefemale and male genital discs (Fig. 4A-D,F). These results suggest that each pri-mordium of the genital disc may bedivided into anterior and posterior com-partments.

Fig. 4. Schematic reprefemale and male genitashown here represent ththe dorsal epithelium (Bdpp (blue), wg (yellow)with Fig. 2A,C-H), (B)parasagittal view of thearrows in A and B. Sincand ptc are only showndpp or wg, is shown at is expressed at a low leat the border is shown. epithelial sheath betweefemale genital primordiexpression patterns of tdorsal view (compared The plane of the parasapatterns are shown as inrepresent the thin epitherfp: repressed female pwas achieved by compaprimordia. The three prepithelia (also see Fig. primordium unambiguolandmarks to distinguiscells in the mgp, whichboundaries drawn withiwhole area of that prim

Mitotic clones in the genital disc do not cross theborder between en(hh)- and ci-expressing cells In order to test directly whether the genital disc is composedof anterior and posterior compartments, which are defined byci- and en(hh)-expressing domains respectively, we mademitotic clones in the genital disc and asked if these clonescould cross the border between the two domains. The mitoticclones were induced using the FRT-FLP system. Specifically,flies homozygous for an FRT and heterozygous for a ubiqui-tously expressed MYC epitope on the FRT-carrying chromo-some arm were heat-shocked at the beginning of the secondinstar, when cell division normally resumes in the genital disc(Madhavan and Schneiderman, 1977). Clones were scored inthe mature third instar discs. Homozygous Myc clones wereidentified by their elevated level of MYC epitope expression(above the heterozygous background), and their twin spots bythe absence of MYC epitope expression. In 210 mosaic discsexamined, over 20 large clones were found that abutted the


Fig. 5. Mitotic clones induced at second instar stage do not cross theborder between en(hh)- and ci-expressing domains. Anterior of thediscs is up in all panels. Mitotic clones were induced by the FRT-FLP system. The clones were marked by the elevated level of MYCeitope expression (red), and their twin spots marked by the absenceof MYC epitope expression. The postulated posterior compartmentwas marked by hh-lacZ expression revealed by anti-β-gal antibody(green). The postulated ci-expressing anterior compartment wasmarked by the absence of hh-lacZ expression. In a total number of210 mosaic discs examined, 25 large clones were found abutting theputative compartment borders within the female genital primordium(8/25), the male genital primordium (9/25), the female analprimordium (3/25), and the male anal primordium (5/25). Singleclones that abutted at least one-third of the length of a putativeborder were considered as large clones. In each of the two examplesshown here, three images are included, the first of MYC staining(A,D), the second of β-gal staining (B,E), and the third ofsuperimposed MYC and β-gal staining (C,F). A-C show a cloneinduced within the hh-non-expressing domain in the male genitalprimordium. This clone (arrowhead) grew along the border betweenhh-expressing and non-expressing domains, and did not cross theborder defined by hh expression (arrow). Its twin spot also resided inthe hh-non-expressing region (asterisk). D-F show a clone within thehh-expressing domain in the female genital primordium. This clone(arrowhead) also grew along the border between hh-expressing andnon-expressing domains, and did not cross the border defined by hhexpression (arrow). Its twin spot was also in the hh-expressingdomain (asterisk).

border between hh-expressing and non-expressing cells. Noneof these clones crossed the border (Fig. 5).

This result demonstrates that there is indeed a clonalboundary between the en(hh)- and ci-expressing cells in thegenital disc. We conclude that the genital disc has been sub-divided into anterior and posterior compartments by thebeginning of the second instar, if not earlier. To be consistentwith the nomenclature in the wing and leg discs, we will referto cells expressing en(hh) as posterior cells, and cells express-ing ci as anterior cells in the genital disc.

Pka activity is required to repress ptc, dpp and wgexpression in the anterior compartment of thegenital discGiven that the relative domains of expression of the A/P pat-terning genes in the genital disc are similar to those in the legdisc, and that there is a clonal boundary between en(hh)- andci-expressing cells, we tested whether the genetic interactionsamong these genes are conserved in the genital disc. Previousstudies in thoracic discs have shown that in the anterior com-partment, Pka activity represses the expression of genes thatare normally expressed at the A/P compartment border,including dpp, wg and ptc. We generated Pka mutant clones inthe genital disc and examined the expression of ptc, dpp, andwg by using lacZ lines that faithfully represent their endoge-nous expression patterns (Blackman et al., 1991; Chen andBaker, unpublished observation).

As shown above, ptc, dpp and wg are expressed along the A/Pcompartment border of the genital disc. While ptc is expressedalong the entire length of the A/P border, dpp and wg areexpressed in non-overlapping stripes along the border. In Pkamosaic discs, we observed that ptc was ectopically expressed inall the Pka mutant clones located in the anterior compartment ofthe genital disc (Fig. 6A,B). Likewise, dpp and wg were ectopi-cally expressed in Pka mutant clones in the anterior compart-ment (Fig. 6C-F). Interestingly, the region in which dpp wasectopically expressed was distinct from the region in which wgwas ectopically expressed. This is analogous to the situation inthe leg disc where dorsal anterior Pka mutant clones express dpp,and ventral anterior clones express wg. We did not observeectopic dpp, wg or ptc expression in Pka mutant clones in theposterior compartment of the genital disc (data not shown).Taken together, these results show that in the genital disc, Pkafunctions in the anterior compartment to repress the expressionof genes that are normally expressed along the A/P compartmentborder. Thus, as in the thoracic discs, Pka exerts an anterior com-partment-specific function in the genital disc.

en and inv are required in the posteriorcompartment in the genital disc to repress dpp andactivate hhPrevious studies have shown that in the posterior compartmentof the wing disc, en can activate the expression of hh, andrepress the expression of dpp, ptc and ci. The invected (inv)gene encodes a protein that has a function partially redundantto that of en. Therefore, the en-inv double mutant has a moresevere mutant phenotype than en mutant alone (Tabata et al.,1995). Homozygous en-inv mutant clones in the posterior com-partment of the wing disc cause ectopic expression of dpp, ptcand ci, and the loss of hh expression. We have made en-invmutant clones in the genital disc and examined the expression


Fig. 6. Cell-autonomous ectopic expression of ptc, dppand wg in Pka mutant clones in the anteriorcompartment of the genital disc. Anterior of the discsis up in all panels. All discs shown here are femalediscs. Pka mutant clones were induced by the FRT-FLP system. The Pka mutant clones were marked bythe absence of MYC epitope expression that wasrevealed by anti-MYC antibody (red). The expressionof the reporter genes, ptc-lacZ, dpp-lacZ and wg-lacZwere detected by anti-β-gal antibody (green). Twoimages of each disc are shown, one of MYC staining(B,D,F), the other of superimposed MYC staining andβ-gal staining (A,C,E). (A,B) ptc (green) is normallyexpressed along the A/P compartment border of thegenital disc (arrows). In Pka mutant clones in theanterior compartment (arrowheads), ptc wasectopically expressed. (C,D) dpp (green) is normallyexpressed along the lateral portion of the A/Pcompartment border in the genital primordium(arrows). Note that dpp was ectopically expressed inPka mutant clones in the anterior compartment(arrowheads). (E,F) wg is normally expressed in themedian portion of the A/P compartment border in thegenital primordium (arrows). Note that wg wasectopically expressed in a Pka mutant clone located inthe median region of the disc (arrowhead), but not in aPka mutant clone at a more lateral position (asterisk).

repression of hh expression in en-inv mutant clones in the posteriorisc. Anterior of the discs is up in all panels. en-inv mutant clones weretem. The clones were marked by the lack of en expression that was

y (red). The expression of the reporter genes, dpp-lacZ and hh-lacZantibody (green). Two images of each disc are shown, one of ENf superimposed EN staining and β-gal staining (A,C) or β-gal stainingmally expressed along the lateral portion of the A/P compartmentote that dpp was ectopically expressed in a en-inv mutant clone in the disc. (C,D) dpp (green) is normally expressed along part of the A/P

enital and the anal primordia of the male disc. Note that dpp wasnv mutant clones in the posterior compartment in both genital and anals normally expressed in the posterior compartment as en. Note the lossutant clones in the posterior compartment of a female disc.

of dpp and hh. As in the wing disc, dpp was ectopicallyexpressed in the en-inv mutant clones in the posterior com-partment (Fig. 7A-D). However,ectopic dpp expression was neverobserved in the anterior compart-ment. As in the wing disc, en-invmutant clones in the posteriorcompartment resulted in the lossof hh expression (Fig. 7E,F). Thus,en-inv function in the posteriorcompartment of the genital disc tonegatively regulate dpp and posi-tively regulate hh.

Pka mutant clones result inpattern duplications in adultterminaliaSince our previous results demon-strated that the genital disc isdivided into anterior and posteriorcompartments, and the interac-tions among the A/P patterninggenes are likely to be conserved inthe genital disc, genes that havecompartment-specific functions,such as Pka and en-inv, can beused in genetic mosaic analyses tolocalize the boundary between theanterior and posterior compart-ments in the adult structures.Previous studies have shown thatPka mutant clones can causepattern duplications and/or local

Fig. 7. Activation of dpp andcompartment of the genital dinduced by the FRT-FLP sysrevealed by anti-EN antibodwere detected by anti-β-gal staining (B,D,F), the other o(E). (A,B) dpp (green) is norborder of the female disc. Nposterior compartment of thecompartment border in the gectopically expressed in en-iprimordia. (E,F) hh (green) iof hh expression in en-inv m

overgrowth in the anterior, but not posterior, structures in adultwing and leg. We reasoned that the adult terminalia structures


Fig. 8. Effects of Pka and en-invmutations on the adult femaleterminalia. The Pka and en-invmutant clones were induced by theFRT-FLP system, and marked byy. (A,B) Morphology of the wild-type female external terminalia. Aventral view is shown in A and adorsal view is shown in B. Thebilateral symmetrical externalterminalia consist of the vaginalplates (vp), the 8th tergites (t-8),and the dorsal (dap) and ventral(vap) anal plates (ap). Each vaginalplate is bordered by 11-16 thornbristles, one long bristle, and threesensilla trichodea (not in focus).The dorsal anal plate has a mean of18 bristles of which two are large.The ventral anal plate has a meanof 19 bristles and four of them arelarge. (C,D) Pka mutant clonesinduce pattern duplications in adultfemale external terminalia.Duplicated vaginal plates are shown in C. The ectopic yellow thorn bristle (arrowhead) reorganized the surrounding wild-type tissues to form acomplete third row of thorn bristles (arrow). Local overgrowth of the anal plate is shown in D. The two yellow long bristles (arrowhead)reorganized the surrounding wild-type tissues to form the extra ‘bump’ on the dorsal anal plate (arrow). Duplications on the ventral anal platewere also observed (not shown). (E,F) Pka mutant clones cause pattern duplications in adult female internal genitalia. Wild-type spermathecae(spt) inside a female abdomen are shown in E. Most wild-type females have two spermathecae. Rarely, some females have three. Duplicatedspermathecae in a Pka mosaic animal are shown in F. Although spermathecae are not marked by y, this mosaic female has four spermathecae,which have never been observed in wild-type females. (G,H,I) en-inv mutant clones cause posterior to anterior transformations in the adultfemale terminalia. In G, en-inv mutant clones in structures that belong to the anterior compartment, e.g. vaginal thorn bristles (arrowhead),appeared normal in morphology. In H, ectopic anterior structures, e.g. vaginal thorn bristles (arrowhead) were induced by en-inv mutant clones.In I, long bristles were induced by an en-inv mutant clone on the dorsal part of the 8th tergite, which is devoid of bristles in wild-type females(arrowhead).

Table 1. Pka mutant clones cause duplications in adult internal genitalia

A. Female Genotypes Total External‡ u spt pov sr od

Wild type (hs)† 76 0 0 3 (3.9)* 0 0 00**

Pka− (no hs)†† 144 0 0 12 (4.2)* 0 0 00**

Pka− (hs) 265 104 (39.2) 19 (7.2) 37 (14.0)* 0 7 (2.6) 5 (1.9)11 (4.2)**

B. MaleGenotypes Total External‡ sp ed pg vd

Wild type (hs)† 53 0 0 0 0 0

Pka− (no hs)†† 118 0 0 0 0 0

Pka− (hs) 182 51(28.0) 5 (2.7) 7 (3.8) 10 (5.5) 4 (2.2)

Mitotic clones were induced by the FRT-FLP system as described in Materials and Methods. Numbers under each category (except for ‘Total’) are the numberof flies that showed duplications and/or local overgrowth in different terminalia structures. Numbers in parentheses are the percentages of these abnormal flies.The abbreviations used here are the same as in Fig. 1.

†Control experiment using a Pka+ chromosome carrying the same FRT site with clonal induction. ††Control experiment using a Pka− chromosome without clonal induction. ‡The external terminalia phenotypes were scored as an indicator of the approximate clonal frequency. Since y marks the cuticular structures instead of the

internal soft tissues, only the apparent internal genitalia duplications and/or overgrowth could be scored. This could explain the lower frequencies ofabnormalities observed in the internal genitalia compared with that of the external terminalia.

*Number of females that had three spermathecae. Note that a low frequency of wild-type females had three, instead of two, spermathecae. This frequency wassignificantly increased in Pka mosaic flies.

**Number of females that had four spermathecae. Note that four spermathecae were only observed in Pka mosaic flies.


Fig. 9. Effects of Pka, en-inv and wgmutations on the adult maleterminalia. The Pka and en-invmutant clones were induced by theFRT-FLP system, and marked by y.(A) Morphology of the wild-typemale external terminalia. As in thefemales, the male terminalia arebilaterally symmetrical. The externalterminalia consist of the genital arch(ga), the lateral plates (lp), theclaspers (cl), the complex penisapparatus (pa), and the left and rightanal plates (ap). The genital archcarries about 15 peripheral bristles(not in focus). Each clasper bears anaverage of 25 thorn-like bristles(clasper teeth), and a long bristle atthe end. The lateral plates each carryabout 15 long thin bristles. Each ofthe anal plates is covered with anaverage of 36 thin bristles similar tothose of the lateral plates. (B-D) Pkamutant clones organize duplications of the genital arch, the lateral plate, the clasper, the penis apparatus and the anal plate. In B, the ectopicPka mutant clasper teeth (arrowhead) reorganized the surrounding wild-type tissues to form ectopic lateral plate and genital arch (arrow). Amore severe phenotype is shown in C, in which ectopic Pka mutant clasper teeth (arrowhead) induced a new set of genitalia (top half of thepanel) in addition to the normal set of genitalia (bottom half of the panel). An extra smaller anal plate was also induced between the two sets ofgenitalia (arrow). In D, a Pka mutant clone on the left anal plate (arrowhead) induced excessive growth of the anal plate, resulting in an extraportion of the anal plate between the endogenous ones. (E-G) en-inv mutant clones cause posterior to anterior transformations in the adult maleterminalia. In E, en-inv mutant clones in structures that belong to the anterior compartment, e.g. the clasper teeth (arrowhead), appeared normalin morphology. In F, ectopic anterior structures, e.g. lateral plate bristles (arrowhead), were induced by en-inv mutant clones inside theabdomen. In G, an en-inv mutant clone (arrowhead) caused the formation of an abnormally shaped anal plate. (H) The phenotypes in theexternal genitalia of the wgCX3/wgCX4 flies. Note that the number of clasper teeth was greatly reduced (arrowhead), the two lateral plates werefused into one with reduced number of bristles (arrow), and there is only a very rudimentary penis apparatus (asterisk).

showing pattern duplications and/or local overgrowth associ-ated with Pka mutant clones are likely to belong to the anteriorcompartment, whereas structures not affected by Pka mutantclones are likely to belong to the posterior compartment.

We made Pka mutant clones by using the FRT-FLP system.These clones, in adult flies, were marked by y. We firstexamined the external terminalia of Pka mosaic animals. Infemales, Pka mutant clones were associated with duplicationsand/or local overgrowth in the vaginal thorn bristles, vulva,both the dorsal and ventral anal plates (Fig. 8C,D), and thesensillae in the ventral part of the 8th tergite (data not shown).Pka mutant clones often induced the overgrowth of the sur-rounding wild-type tissues. In males, duplications and/or localovergrowth were also frequently observed in most parts of theexternal terminalia, including the clasper teeth, the lateral platebristles, the penis apparatus, and both the left and right analplates (Fig. 9B-D).

The internal genitalia of Pka mosaic flies were alsoexamined. In females, duplications were found in most partsof the internal genitalia, including the uterus, the oviduct, theseminal receptacles, and the spermathecae. In some flies, fourspermathecae were observed instead of the two, or rarely three,found in wild type (Fig. 8E,F). However, duplicated parovariawere not observed (Table 1A). In males, most parts of theinternal genitalia were also found duplicated in Pka mosaicanimals, including the sperm pump, the ejaculatory duct, theparagonium, and the vas deferens (Table 1B).

Taken together, these data show that Pka mutant clones can

induce duplications and/or local overgrowth in many parts ofthe female, male and anal structures, suggesting that each ofthe three primordia of the genital disc has an anterior com-partment. In contrast, a few structures, such as the parovariumand the dorsal part of the 8th tergite, never showed duplica-tions in Pka mosaic flies, suggesting that these structuresbelong to the posterior compartment. Our finding that most ofthe female and male genital structures showed duplicationsand/or local overgrowth associated with Pka mutant clonessuggests that most of the genital primordia are of anterior,rather than posterior, origin. This is consistent with the obser-vation that ci is expressed in larger regions of each genital discprimordium than is en (Fig. 4).

en-inv mutant clones cause posterior to anteriortransformations in adult terminaliaPrevious studies in the wing disc have shown that en functionsas the posterior selector gene. Homozygous en-inv mutant clonesin the anterior compartment appear normal, whereas the mutantclones in the posterior compartment can cause posterior toanterior transformations. In order to further test which adult struc-tures belong to the anterior or the posterior compartment, we havemade en-inv mutant clones in the genital disc and examined theadult terminalia phenotypes in the en-inv mosaic flies. In females,most en-inv mutant clones appear normal. These clones arelocated within the anterior structures defined by Pka mosaicanalysis, such as the vaginal thorn bristles (Fig. 8G). The regionsthat showed abnormalities in en-inv mosaic flies are much smaller


s of wg on the adult internal genitalia. (A) Wild-type adult male external terminalia include the anal plates (ap), the external genitalia

enis apparatus (pa). The internal genitalia consist of the sperm pumpatory duct (ed), the paragonia (pg), and the vas deferens (vd). The hindn in all panels. (B) Wild-type adult female terminalia. The externaln here are the anal plates (ap) and the vaginal plate (vp). The internal

ures shown are the uterus (u), the seminal receptacle (sr), thespt), and the oviduct (od). (C) The terminalia structures ofale flies. The internal genitalia were completely deleted. The penis

e external genitalia structures were also partially deleted. See Fig. 9Hed external genital phenotypes. (D) The terminalia structures ofemale flies. The internal genitalia were also completely deleted. Thel structures were generally normal.

than the areas that were affected by Pka mutant clones. en-invmutant vaginal thorn bristles were found in the region betweenthe ventral anal plate and the dorsal vaginal plate which is, inwild type, naked cuticle (Fig. 8H). In addition, en-inv mutantbristles were observed on the dorsal part of the 8th tergite, whichis normally devoid of bristles (Fig. 8I). These regions were notaffected by Pka mutant clones and were likely to be of posteriororigin. We interpret the appearance of these bristles as posteriorto anterior transformations, which is analogous to what happensto en-inv mutant clones in the wing discs.

Similar results were obtained in males. Most of the en-invmutant clones appear normal. For example, no defects wereassociated with en-inv mutant clones in the clasper teeth, thelateral plate bristles or the genital arch, which have beendefined as anterior structures by Pka clonal analysis (Fig. 9E).However, inside the abdomen and near the penis apparatus,where no cuticular structures are found in wild-type flies,ectopic anterior structures, such as clasper teeth and lateralplate bristles, were found associated with en-inv mutantclones (Fig. 9F). These ectopic anterior structures were alsolikely to result from posterior to anterior transformation. Inaddition, the anal plates were malformed in en-inv mutantanimals (Fig. 9G), suggesting that at least some cells in theanal primordium are of posterior origin.

Take together, these results are in agreement with our Pkaclonal analysis of the A/P compartmental division in theadults. The fact that en-inv clones affected structures derivedfrom all three primordia demonstrates thateach primordium has a posterior compart-ment, contradicting a previous proposal(Lawrence and Struhl, 1982) that the femalegenital primordium is purely of anteriororigin. Our finding that en-inv clones onlyaffected a small part of the female and malegenitalia suggests that the posterior compart-ment only occupies a small portion of eachgenital primordium, and is inconsistent witha previous suggestion (Epper and Sanchez,1983) that the genital primordia are largely ofposterior origin. In terms of the genital struc-tures that are affected by both Pka and en-invmutant clones, such as the penis apparatus inmales, and the 8th tergite in females, wesuggest that the A/P compartment boundarypasses through these structures.

wg plays an essential role in patteringthe genital discPrevious studies using wgts alleles or wg het-eroallelic mutants failed to reveal anyspecific function of wg in genital disc devel-opment (Baker, 1988a,b). In these studies,only the external genitalia phenotypes wereexamined. Since we have observed theexpression of wg along part of the A/P com-partment border in the genital disc, as well asthe ectopic expression of wg in the Pkamutant clones, we reasoned that wg activitymight play a role in patterning the genitaldisc.

Comparison of the wg expression pattern

Fig. 10. Effectterminalia. The(eg), and the p(sp), the ejaculgut is not showstructures showgenitalia structspermathecae (wgCX3/wgCX4 mapparatus in thfor more detailwgCX3/wgCX4 fexternal genita

with the fate maps of the genital discs suggests that the wgexpression domains in both the female and male genital discscorrespond to the internal, but not the external, genitalia. Wetherefore examined both the internal and the external genitaliaphenotypes of wg mutants. The wg mutant allele we used wasa heteroallelic mutant combination between wgCX3 and wgCX4,which specifically lacks wg activity during imaginal develop-ment (Baker, 1987, 1988b). When the internal genitalia of thepharate adults were examined, all internal structures weredeleted in both females and males (Fig. 10). In addition, themale external genitalia showed deletions in certain structures,such as the clasper teeth, the lateral plate bristles and the penisapparatus (Fig. 9H). The female external genitalia weregenerally unaffected (Fig. 10D). Similar results were obtainedby reducing wg activity at the end of the second instar using awgts allele (data not shown). We note that the structures deletedin the internal genitalia correspond to larger regions than thewg expression domains in the third instar disc, which mayreflect a cell non-autonomous function of this signalingmolecule.

DISCUSSION

The genital disc is divided into anterior andposterior compartmentsOur studies of the expression patterns of genes known to play


Fig. 11. Models of the anteriorand posterior compartmentalorganization of the genital disc.The three boxes represent thethree primordia of the genitaldisc: the female genitalprimordium (f), the malegenital primordium (m), andthe anal primordium (a). Theanterior compartment ismarked by green, and theposterior compartment ismarked by red. (A) The modelproposed by Lawrence andStruhl (1982), based on theadult phenotypes of en mutantclones. The female primordium is of anterior origin. The maleprimordium is likely to be of posterior origin. The anal primordiumis of posterior origin. (B) The model proposed by Epper and Sánchez(1983), based on the adult phenotypes of en heteroalleliccombination animals, e.g. en1/en3. Each primordium has anterior andposterior compartments. Both female and male primordia arecomposed mainly of posterior compartments, in addition to verysmall anterior compartments. (C) The new model, based on thisstudy. Each of the three primordia is composed of its own anteriorand posterior compartments. In addition, each primordium has alarger anterior compartment and a smaller posterior compartment.

roles in the A/P patterning of the thoracic discs providemolecular evidence that the genital disc is divided into anteriorand posterior compartments. As in the thoracic discs, en(hh)and ci are expressed in complementary domains in the genitaldisc. ptc is expressed in a stripe of cells within the ci expressiondomain abutting the en-expressing cells. wg and dpp areexpressed in non-overlapping subregions along the borderbetween the en(hh)-expressing and ci-expressing domains.This is strikingly similar to the situation in the leg disc, wherewg and dpp are expressed in complementary subregions alongthe A/P compartment border. These results are largely consis-tent with those of Freeland and Kuhn (1996) who character-ized en, hh, dpp, wg, and ci expression in the genital disc atthe light microscopic level. In addition, our results from theclonal analysis demonstrate that the en(hh)-expressing and ci-expressing domains are true genetic compartments, sincemitotic clones induced in the genital disc do not cross theborder between the two domains. We refer to cells expressingen and hh as posterior cells, and to cells expressing ci asanterior cells.

It is not clear, however, when the genital disc begins to bedivided into anterior and posterior compartments. Since themitotic clones were induced at the beginning of the secondinstar stage, we believe that the two compartments havealready been established by this time. Genetic mosaic analysishas shown that the A/P compartment boundary in the thoracicdiscs already exists in the blastoderm stage embryo (Lawrenceand Morata, 1977; Steiner, 1976; Wieschaus and Gehring,1976). It has been proposed that the primordia of the two com-partments originate on opposite sides of the parasegmentboundary in the embryo, and that en specifies the identity ofthe posterior compartment in both the primordia and maturediscs (reviewed by Cohen, 1993). However, the onset of theA/P compartment division in the genital disc may not be assimple, since en is not continuously expressed throughout thedevelopment of the genital disc. At the end of germ-band short-ening stage, the expression patterns of en in the tail region ofthe embryo (A8, A9 and A10) go through dramatic changes.en-expressing cells on the ventral ectoderm move dorsally,resulting in the juxtaposition and fusion of ‘anterior’ A8, A9,and A10 cells that lack en expression on the ventral epithelium(DiNardo et al., 1985; Kuhn et al., 1992). This is the stagewhen one can first detect histochemically the genital discprecursor cells (GDPC), which are a group of transverselyelongated cells located between the A8 denticle belt and theanal pads on the ventral ectoderm of the embryo. Not surpris-ingly, en is not expressed in the GDPC (Chen and Baker,unpublished observation). Thus, unlike the situation in thethoracic discs, there is a period of time in which en is notexpressed in the genital disc primordia. The later expression ofen in the genital disc likely results from the re-specification ofposterior cells among the fused ‘anterior’ cells, which probablyhappens between late embryogenesis and the beginning of thesecond instar. It is not clear at this point how this re-specifi-cation occurs.

Each primordium of the genital disc is composed ofanterior and posterior compartmentsA comparison of the expression patterns of en(hh) and ci withthe fate maps of the third instar genital disc provides insightinto the A/P compartmental organization of each of the three

primordia of the genital disc. In both the female and malegenital primordia, en(hh) and ci are expressed in complemen-tary subregions, suggesting that each primordium has anteriorand posterior compartments. Moreover, in the repressed maleand female primordia, en(hh) and ci are also expressed in non-overlapping cells. en(hh)-expressing regions are smaller thanthose of ci, indicating that the two genital primordia haverelatively smaller posterior compartments, and larger anteriorcompartments. Results from the Pka and en-inv clonal analysesare consistent with this conclusion. Duplications and/or localovergrowth were observed in Pka mosaic animals in moststructures of the female and male genitalia. In contrast,posterior to anterior transformations were found at a limitednumber of positions in en-inv mosaic flies, indicating that onlya few structures belong to the posterior compartment.

Our results also demonstrate that there are anterior andposterior compartments within the anal primordium. en(hh)and ci are expressed in complementary patterns in the analprimordia in both female and male discs. Consistent with theseexpression patterns, malformed anal plates were found in bothPka and en-inv mosaic animals. Moreover, en(hh) areexpressed in a smaller region than is ci in the anal primordiumof the disc, suggesting that the anal primordium also has a rela-tively larger anterior and a smaller posterior compartment.

Based on our studies, we propose that each of the threeprimordia that make up the genital disc are composed of ananterior and a posterior compartment, and that most of thegenital disc cells are of anterior origin (Fig. 11C). This modelis different from the two previously proposed models in tworegards. While Lawrence and Struhl’s model suggested that thefemale primordium is of anterior origin, and the male and analprimordia are made of posterior cells (Fig. 11A), our modelproposes that each primordium contains both anterior andposterior compartments. In addition, while Epper and


Sánchez’s model suggested that most of the genital primordialcells are of posterior origin (Fig. 11B), we propose that mostcells of each primordium are of anterior origin.

In order to reconcile these disparate findings, it is useful toconsider the following factors. The first model proposes thatthe anal primordium is purely of posterior origin based on thefact that the adult anal plates were affected by en mutantclones. However, if the anal plates contain both anterior andposterior compartments, as our results demonstrate, the analplates would still be affected by en mutations. Second, it hasbeen shown recently that inv contributes to the posteriorselector function of en, and that the en-inv double mutantcauses stronger posterior to anterior transformation phenotypesin the wing than do en mutants alone. Indeed, we have evidencethat en-inv mutant clones have stronger phenotypes than ensingle mutant clones in the genital disc. Accordingly, we usedan en-inv double mutant in our clonal analysis instead of ensingle mutants used in previous studies. Third, in both of theprevious studies, en mutations were found to cause deletionsin the genitalia, while we observed posterior to anterior trans-formations associated with en-inv mutant clones. A possibleorigin of these differences is the time at which en function wasremoved in these three studies. In the study by Lawrence andStruhl (1982) en mutant clones were induced at the first instarstage and in the study by Epper and Sánchez (1983) heteroal-lelic combinations of en mutations were used, while in ourstudies en-inv mutant clones were induced in second larvalinstar. It is conceivable that en has an early function that isdistinct from its compartment selector function. For example,en could also be involved in cell survival/proliferation beforethe second larval instar, such that the reduction of this earlyactivity would confound the interpretation of its compartmentselector function. In fact, an involvement of en in cellsurvival/proliferation has been documented in the wing(Hidalgo, 1994). Thus, one cannot solely rely on such deletionphenotypes as indications of the compartment selector functionfor en, nor use such phenotypes to infer the position of the A/Pcompartment boundary. Finally, while both previous modelswere based on the mutant phenotypes of a single gene, en, ourmodel is based on the mutant phenotypes of anterior compart-ment-specific gene Pka and posterior compartment-specificgenes en-inv, as well as expression patterns of six molecularmarkers. Moreover, the expression patterns of the molecularmarkers are consistent with the mutant phenotypes of the com-partment-specific genes. Such a multifaceted approach shouldreveal more reliably the compartmental organization of thegenital disc.

The A/P compartmental organization in the adultterminaliaUnlike the regularly patterned wing or leg, the adult termina-lia are composed of many different sexually dimorphic tissuesarranged in a more complex pattern. In theory, one coulddeduce the A/P compartment boundary in the terminalia bycomparing the expression patterns of compartment-specificgenes in the third instar disc to the fate maps. However, thelow resolution of the fate maps makes such a deductiondifficult. In this study, we have taken advantage of the clonalphenotypes of Pka and en-inv mutations to map the anteriorand posterior compartments in adult terminalia more precisely.We found that Pka mutant clones caused duplications and/or

local overgrowth in most of the terminalia structures, suggest-ing that these structures, or at least parts of them, belong to theanterior compartment. On the other hand, posterior to anteriortransformations caused by en-inv mutant clones were onlyobserved in a limited number of structures, indicating that fewregions belong to the posterior compartment. Moreover, a fewstructures, such as the penis apparatus in male and the 8thtergite in female terminalia, are affected by both Pka and en-inv mutant clones. We suggest that the latter structures arecomposed of both anterior and posterior compartments. Thus,the A/P compartment boundary in the adults does not simplylie between different structures. In some cases, the compart-ment boundary bisects certain structures.

The organizing activities of dpp and wg in A/Ppatterning in the genital discOur results from Pka and en-inv clonal analyses have demon-strated that the interactions among the A/P patterning genes inthe genital disc are analogous to those in the thoracic discs. Wehave shown that Pka is required to repress the downstreamtarget genes dpp, wg and ptc in the anterior compartment,whereas en and inv are required to activate hh expression andrepress dpp expression in the posterior compartment.

It has been shown that DPP controls growth and patterningof both the anterior and posterior compartments of the wingdisc (Basler and Struhl, 1994; Capdevila and Guerrero, 1994;Lecuit et al., 1996; Nellen et al., 1996; Tabata and Kornberg,1994; Zecca et al., 1995). It is also known that dppdisk mutantscause deletions in both female and male genital disc deriva-tives (Blackman et al., 1991). In this study, we have demon-strated that, as in the thoracic discs, dpp is expressed along partof the A/P compartment border of the genital disc, and isectopically expressed in Pka mutant clones in the anterior com-partment and in en-inv mutant clones in the posterior compart-ment. We suggest that dpp is also likely to be an importantorganizer of the A/P developmental field in the genital disc.

Our studies have also revealed a function for wg duringgenital disc development. It has been suggested that WG actsas a gradient morphogen to pattern the leg disc (Struhl andBasler, 1993). However, the function of wg in genital discdevelopment had not been uncovered previously, partlybecause only the external terminalia were examined (Baker,1988a,b). In this study, we have demonstrated that wg isexpressed along the A/P compartment border in the genitaldisc, in a pattern complementary to that of dpp. In addition, wgis ectopically expressed in Pka mutant clones. The expressiondomains of wg correspond to part of the regions that will giverise to the internal genitalia in both female and male discs.Consistent with its expression pattern, wg mutant flies showdeletions of the internal genitalia. The fact that the deletedtissues correspond to a larger region than the wg expressiondomain is consistent with the cell non-autonomous function ofwg.

We would like to thank K. Cadigan, J. Hooper, T. Kornberg, T.Laverty, K. Matthews, R. Nusse, D. Pan, G. Rubin, and T. Tabata forfly stocks, K. Cadigan, S. Carroll, C. Goodman, I. Guerrero, M.Hoffman, R. Holmgren, R. Nusse, and T. Tabata for antibodies, andR. Nöthiger for permitting us to modify the figures of the genital discfate maps and the adult derivatives. We would also like to thank S.Ahmad, A. Franke, E. Keisman for their help with computer work and


confocal microscopy, S. Ahmad, G. Bashaw, E. Keisman, H. Li, I.Marín, D. Pan, and S. Plump for their valuable comments on the man-uscript, and G. Bohm for preparing the fly food. E. Chen would espe-cially like to thank Duojia Pan for discussion and support during thework. This work was funded by an NIH grant to B. S. B.

REFERENCES

Baker, N. E. (1987). Molecular cloning of sequences from wingless, a segmentpolarity gene in Drosophila: the spatial distribution of a transcript inembryos. EMBO J. 6, 1765-1773.

Baker, N. E. (1988a). Embryonic and imaginal requirements for wingless, asegment-polarity gene in Drosophila. Dev. Biol. 125, 96-108.

Baker, N. E. (1988b). Transcription of the segment-polarity gene wingless inthe imaginal discs of Drosophila, and the phenotype of a pupal-lethal wgmutation. Development 102, 489-497.

Basler, K. and Struhl, G. (1994). Compartment boundaries and the control ofDrosophila limb pattern by the hedgehog protein. Nature 368, 208-215.

Bate, M. and Martinez Arias, A. (1991). The embryonic origin of imaginaldiscs in Drosophila. Development 112, 755-761.

Belote, J. M. and Baker, B. S. (1982). Sex determination in Drosophilamelanogaster: Analysis of transformer-2, a sex transforming locus. Proc.Nat. Acad. Sci. USA 79, 1568-1572.

Blackman, R. K., Sanicola, M., Raftery, L. A., Gillevet, T. and Gelbart, W.M. (1991). An extensive 3′ cis-regulatory region directs the imaginal diskexpression of decapentaplegic, a member of the TGF-β family inDrosophila. Development 111, 657-666.

Bryant, P. J. (1978). Pattern formation in iamginal discs. In The Genetics andBiology of Drosophila (ed. M. Ashburner and T. R. F. Wright), pp. 229-335.Academic Press Inc, London.

Capdevila, J., Estrada, M. P., Sanchez-Herrero, E. and Guerrero, I. (1994).The Drosophila segment polarity gene patched interacts withdecapentaplegic in wing development. EMBO J. 13, 71-82.

Capdevila, J. and Guerrero, I. (1994). Targeted expression of the signallingmolecule decapentaplegic induces pattern duplications and growthalterations in Drosophila wing. EMBO J. 13, 4459-4468.

Cohen, S. M. (1993). Imaginal disc development. In The Development ofDrosophila melanogaster (ed. M. Bate and A. Martinez Arias), pp. 747-842.Cold Spring Harbor Laboratory Press, New York.

Coleman, K. G., Poole, S. J., Weir, M. P., Soeller, W. C. and Kornberg, T.(1987). The invected gene of Drosophila: sequence analysis and expressionstudies reveal a close kinship to the engrailed gene. Genes Dev. 1, 19-28.

DiNardo, S., Kuner, J. M., Theis, J. and O’Farrell, P. H. (1985).Development of embryonic pattern in D. melanogaster as revealed byaccumulation of the nuclear engrailed protein. Cell 43, 59-69.

Dübendorfer, K. and Nöthiger, R. (1982). A clonal analysis of cell lineageand growth in the male and female genital discs of Drosophila melanogaster.Roux’s Arch. Dev. Biol. 191, 42-45.

Eaton, S. and Kornberg, T. B. (1990). Repression of ci-D in posteriorcompartments of Drosophila by engrailed. Genes Dev. 4, 1068-1077.

Ehrensperger, P. (1972). Transplantationsexperimente und entwicklungsbeo-bachtungen in situ zur bestimmung des dreidimensionalen anlageplanes dermännlichen genital-imaginalscheibe von Drosophila melanogaster.Diplomarbeit, Zoological Institute, University of Zurich, Switzerland.

Epper, F. (1980). The genital disc of Drosophila melanogaster: adevelopmental and genetic analysis of its sexual dimorphism. Ph. D. Thesis,Zoological Institute, University of Zurich, Switzerland.

Epper, F. (1983). Three-dimensional fate map of the female genital disc ofDrosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol. 192, 270-274.

Epper, F. and Bryant, P. J. (1983). Sex-specific control of growth anddifferentiation in the Drosophila genital disc, studied using a temperature-sensitive transformer-2 mutation. Dev. Biol. 100, 294-307.

Epper, F. and Nöthiger, R. (1982). Genetic and developmental evidence for arepressed genital primordium in Drosophila melanogaster. Dev. Biol. 94,163-175.

Epper, F. and Sánchez, L. (1983). Effects of engrailed in the genital disc ofDrosophila melanogaster. Dev. Biol. 100, 387-398.

Freeland, D. E. and Kuhn, D. T. (1996). Expression patterns ofdevelopmental genes reveal segment and parasegment organization of D.melanogaster genital discs. Mech. Dev. 56, 61-72.

Fristrom, D. and Fristrom, J. W. (1993). The metamorphic development ofthe adult epidermis. In The development of Drosophila melanogaster (ed. M.

Bate, A. Martinez Arias), pp. 843-898. Cold Spring Harbor Laboratory Press,New York.

Garcia-Bellido, A., Ripoll, P. and Morata, G. (1973). Developmentalcompartmentalization of the wing disc of Drosophila. Nature New Biol. 245,251-253.

Golic, K. G. (1991). Site-specific recombination between homologouschromosomes in Drosophila. Science 252, 958-961.

Hama, C., Ali, Z. and Kornberg, T. B. (1990). Region-specific recombinationand expression are directed by portions of the Drosophila engrailedpromoter. Genes Dev. 4, 1079-93.

Hartenstein, V. and Jan, Y. N. (1992). Studying Drosophila embryogenesiswith P-lacZ enhancer trap lines. Wilhelm Roux’s Arch. Dev. Biol. 201, 194-220.

Hidalgo, A. (1994). Three distinct roles for the engrailed gene in Drosophilawing development. Current Biology 4, 1087-1098.

Hooper, J. E. and Scott, M. P. (1989). The Drosophila patched gene encodesa putative membrane protein required for segmental patterning. Cell 59, 751-765.

Jiang, J. and Struhl, G. (1995). Protein kinase A and hedgehog signaling inDrosophila limb development. Cell 80, 563-572.

Kingsley, D. M. (1994). The TGF-β superfamily: new members, newreceptors, and new genetic tests of function in different organisms. GenesDev. 8, 133-146.

Kornberg, T., Siden, I., O’Farrell, P. and Simon, M. (1985). The engrailedlocus of Drosophila: in situ localization of transcripts reveals compartment-specific expression. Cell 40, 45-53.

Kuhn, D. T., Sawyer, M., Packert, G., Turenchalk, G., Mack, J. A., Sprey,T. E., Gustavson, E. and Kornberg, T. B. (1992). Development of the D.melanogaster caudal segments involves suppression of the ventral regions ofA8, A9 and A10. Development 116, 11-20.

Lauge, G. (1982). Development of the genitalia and analia. In Handbook ofDrosophila Development (ed. R. Ransom), pp. 237-264. Elsevier BiomedicalPress, Amsterdam.

Lawrence, P. A. and Morata, G. (1977). The early development ofmesothoracic compartments in Drosophila. An analysis of cell lineage andfate mapping and an assessment of methods. Dev. Biol. 56, 40-51.

Lawrence, P. A. and Struhl, G. (1982). Further studies of the engrailedphenotype in Drosophila. EMBO J. 1, 827-833.

Lecuit, T., Brook, W. J., Ng, M., Callega, M., Sun, H. and Cohen, S. M.(1996). Two distinct mechanisms for long-range patterning byDecapentaplegic in the Drosophila wing. Nature 381, 387-393.

Lee, J. J., von Kessler, D. P., Parks, S. and Beachy, P. A. (1992). Secretionand localized transcription suggest a role in positional signaling for productsof the segmentation gene hedgehog. Cell 71, 33-50.

Lepage, T., Cohen, S. M., Diaz-Benjumea, F. J. and Parkhurst, S. M.(1995). Signal transduction by cAMP-dependent protein kinase A inDrosophila limb patterning. Nature 373, 711-715.

Li, W., Ohlmeyer, J. T., Lane, M. E. and Kalderon, D. (1995). Function ofprotein kinase A in hedgehog signal transduction and Drosophila imaginaldisc development. Cell 80, 553-562.

Madhavan, M. M. and Schneiderman, H. A. (1977). Histological analysis ofthe dynamic growth of imaginal discs and histoblast nests during the larvaldevelopment of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol.183, 269-305.

Meinhardt, H. (1983). Cell determination boundaries as organizing regions forsecondary embryonic fields. Dev. Biol. 96, 375-385.

Nakano, Y., Guerrero, I., Hidalgo, A., Taylor, A., Whittle, J. R. S. andIngham, P. W. (1989). A protein with several possible membrane-spanningdomains encoded by the Drosophila segment polarity gene patched. Nature341, 508-513.

Nellen, D., Burke, R., Struhl, G. and Basler, K. (1996). Direct and long-rangeaction of a DPP morphogen gradient. Cell 85, 357-368.

Nöthiger, R., Dübendorfer, A. and Epper, F. (1977). Gynandromorphsreveal two separate primordia for male and female genitalia in Drosophilamelanogaster. Roux’s Arch. Dev. Biol. 181, 367-373.

Nusse, R. and Varmus, H. E. (1992). Wnt genes. Cell 69, 1073-1087.Pan, D. and Rubin, G. M. (1995). cAMP-dependent protein kinase and

hedgehog act antagonistically in regulating decapentaplegic transcription inDrosophila imaginal discs. Cell 80, 543-552.

Patel, N. H., Martin-Blanco, E., Coleman, K., Poole, S., Ellis, M. C.,Kornberg, T. B. and Goodman, C. S. (1989). Expression of engrailedproteins in arthropods, annelids, and chordates. Cell 58, 955-968.

Perrimon, N. (1995). Hedgehog and beyond. Cell 80, 517-20.Phillips, R. G., Roberts, I. J., Ingham, P. W. and Whittle, J. R. (1990). The


Drosophila segment polarity gene patched is involved in a position-signalling mechanism in imaginal discs. Development 110, 105-114.

Raftery, L. A., Sanicola, M., Blackman, R. K. and Gelbart, W. M. (1991).The relationship of decapentaplegic and engrailed expression in Drosophilaimaginal disks: do these genes mark the anterior-posterior compartmentboundary? Development 113, 27-33.

Sanicola, M., Sekelsky, J., Elson, S. and Gelbart, W. M. (1995). Drawing astripe in Drosophila imaginal disks: negative regulation of decapentaplegicand patched expression by engrailed. Genetics 139, 745-756.

Schüpbach, T., Wieschaus, E., and Nöthiger, R. (1978). The embryonicorganization of the genital disc studied in the genetic mosaics of Drosophilamelanogaster. Wilhelm Roux’s Arch. 185, 249-270.

Schwartz, C., Locke, J., Nishida, C. and Kornberg, T. B. (1995). Analysis ofcubitus interruptus regulation in Drosophila embryos and imaginal disks.Development 121, 1625-1635.

Steiner, E. (1976). Establishment of compartments in the developing legimaginal discs of Drosophila melanogaster. Wilhelm Roux’s Arch. Dev. Biol.180, 9-30.

Struhl, G. and Basler, K. (1993). Organizing activity of wingless protein inDrosophila. Cell 72, 527-540.

Tabata, T., Eaton, S. and Kornberg, T. B. (1992). The Drosophila hedgehoggene is expressed specifically in posterior compartment cells and is a target ofengrailed regulation. Genes Dev. 6, 2635-2645.

Tabata, T. and Kornberg, T. B. (1994). Hedgehog is a signaling protein witha key role in patterning Drosophila imaginal discs. Cell 76, 89-102.

Tabata, T., Schwartz, C., Gustavson, E., Ali, Z. and Kornberg, T. B. (1995).Creating a Drosophila wing de novo, the role of engrailed, and thecompartment border hypothesis. Development 121, 3359-3369.

Wieschaus, E. and Gehring, W. J. (1976). Clonal analysis of primordial cellsin the early embryo of Drosophila melanogaster. Dev. Biol. 50, 249-263.

Wieschaus, E. and Nöthiger, R. (1982). The role of the transformer genes inthe development of the genitalia and analia of Drosophila melanogaster.Dev. Biol. 90, 320-334.

Williams, J. A., Paddock, S. W. and Carroll, S. B. (1993). Pattern formationin a secondary field: a hierarchy of regulatory genes subdivides thedeveloping Drosophila wing disc into discrete subregions. Development 117,571-584.

Xu, T. and Rubin, G. M. (1993). Analysis of genetic mosaics in developingand adult Drosophila tissues. Development 117, 1223-1237.

Zecca, M., Basler, K. and Struhl, G. (1995). Sequential organizing activitiesof engrailed, hedgehog and decapentaplegic in the Drosophila wing.Development 121, 2265-2278.

(Accepted 3 October 1996)

compartmental organization of the drosophilagenital...

Documents