Development 103, 289-298 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

289

Localization of transcripts from the wingless gene in whole Drosophila

embryos

NICHOLAS E. BAKER

MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UKand Department of Biochemistry*, University of California, Berkeley, CA94720 USA

* Author's present address

Summary

In situ hybridization has been used to detect transcrip-tion in whole Drosophila embryos. Improved resultshave been obtained by incubating the hybridizedembryos in liquid emulsion prior to autoradiographicexposure. This technique has been used to map thedistribution of transcripts from the segment-polaritygene wingless (wg), which is transcribed in a stripe ineach segment of the trunk region. By the extendedgermband stage additional areas of transcription inthe head and caudal regions define a total of 21domains, comprising the foregut, six regions in the

head, three thoracic and ten abdominal segments, andthe hindgut. At the end of the extended germbandstage, the pattern of wg transcription is no longeruniform in the dorsoventral axis: wg transcripts areabsent from the lateral epidermis. This pattern of wgtranscription is discussed with particular regard to thesegmentation of the terminalia. Also it is suggestedthat the dorsoventral reorganization could be relatedto the specification of the imaginal disc primordia.

Key words: in situ hybridization, Drosophila embryowhole mounts, wingless, segmentation, pattern formation.

Introduction

Insects are a classic example of metameric metazoanswhose bodies comprise a series of homologous seg-ments. In Drosophila the overt segmentation of thetrunk region of the embryo depends on the action ofsegmentation genes. Mutations in these genes alterthe number or polarity of segments formed (Niisslein-Volhard & Wieschaus, 1980). A hierarchy of segmen-tation genes divides the central portion of the embryointo successively smaller units, culminating in theexpression of some genes in a specific part of everysegment (reviewed in Akam, 1987). One such geneexpressed in every segment is wingless (wg), which istranscribed in the posteriormost cells of each para-segment (Baker, 1987). The wg gene is thought tocontrol the proper organization of each segmentthrough an intercellular signalling mechanism (Nuss-lein-Volhard & Wieschaus, 1980; Baker, 1987, 1988a;Rijsewijk et al. 1987; Cabrera et al. 1987; Martinez-Arias et al. 1988).

Segmentation is best understood in the trunkregion, where fourteen segments are recognized be-

tween the mandibular and the eighth abdominalsegments. The head and caudal regions are alsosegmented, but their organization is not clear inDrosophila embryos (Poulson, 1950; Turner &Mahowald, 1979; Campos-Ortega & Hartenstein,1985; Martinez-Arias & Lawrence, 1985). However,comparative studies have suggested that all insectshave a similar underlying body plan (reviewed inAnderson, 1973). On this basis the head anterior ofthe mandibular segment is expected to contain in-tercalary, antennal and labral segments; the mostanterior part of the embryo invaginates to form theforegut. Posterior to A8 the number of furtherabdominal segments varies in different insects, themaximum number attained apparently being twelve.The variation is thought to be due to the loss or fusionof various of these caudal segments in differentgroups (reviewed in Matsuda, 1976). Evolutionarily,the head segments are probably most ancient. Thetrunk segments could have evolved by the serialreduplication of a pre-existing unit. This hypothesis isconsistent with the molecular mechanisms involved information of the trunk segments, which do not seem

290 N. E. Baker

to apply to the head (reviewed in Akam, 1987; Scott& Carroll, 1987). Thus the trunk segments are only apart of insect segmentation. If head segmentation ismore ancient, then it may be more pertinent tounderstanding the origins of segmentation and therelationship of insect segmentation to that of othermetameric organisms. For example, it appears thatgenes involved in head development may have closercounterparts in vertebrates than other segmentationand homeotic genes (Regulski et al. 1987; Joyner &Martin, 1987; Rijsewijk et al. 1987).

Since segmentation gene expression precedes mor-phological segmentation, it can be useful in mappingsegmental primordia during early development (e.g.Hafen et al. 1984; Kornberg et al. 1985; DiNardo et al.1985; Ingham et al. 1985a,b; Carroll & Scott, 1985;Martinez-Arias & Lawrence, 1985). This paperfurther describes the accumulation of transcripts fromthe wg gene during embryogenesis. Previous descrip-tions of wg expression have been based on detectionof transcripts in tissue sections (Baker, 1987; Rijsew-ijk et al. 1987; Ingham et al. 1988). These studiesshowed that wg is also transcribed outside the trunk,in the head and caudal regions. The striped pattern oftranscription of the trunk region is clearly revealed bysagittal and parasagittal sections, but patterns in thedorsoventral axis and in the head are more difficult toreconstruct. This information can be obtained morereadily by in situ hybridization to whole-mountedembryos. In this paper, an improved procedure hasbeen used to detect wg transcription in wholeembryos, leading to a more detailed description of wgtranscription outside the trunk region. This is dis-cussed with respect to the putative segmentation ofthese regions. In addition, it is reported that thepattern of wg transcription does not remain constantthroughout embryogenesis. Midway through the ex-tended germband stage, marked changes in wgexpression occur in the dorsoventral axis of allsegments.

Materials and methods

Parts of existing hybridization protocols (Cox et al. 1984;Kornberg et al. 1985; Ingham et al. 1985a) were combined inthe detection of transcripts in whole embryos. Embryoswere dechorionated with generic bleach (1:1 with H2O),washed in H2O, and prefixed [three parts 4 % paraformal-dehyde in PBS (130mM-NaCl, lOmM-sodium phosphatepH 6-8), freshly made, 1 part n-heptane] for 15 min over icewith frequent agitation, then removed from the vitellinemembrane as previously described (Karr & Alberts, 1986).Postfixation and rehydration were accomplished by 5 minwashes in each of the following: (1) 90% aqueousmethanol, 50mM-EGTA pH7-0; (2) 7:3 MeOH/EGTA:PBS + 4% paraformaldehyde; (3) 5:5 MeOH/EGTA: PBS + 4 % paraformaldehyde; (4) 3:7 MeOH/

EGTA:PBS + 4 % paraformaldehyde; (5) PBS +4 % para-formaldehyde; (6) PBS. Embryos were either hybridizeddirectly or dehydrated through alcohol to 70% ethanol,30 % PBS and stored at -20°C, but storage for more than afew days was found to be detrimental. Before hybridizing,embryos were transferred from PBS to 0-2 M-HCI (20 min),washed five times in PBS and digested with pronase(0-15mgml"1 in 50mM-Tris pH7-5, 5mM-EDTA, for7-5 min at room temperature). Proteolysis was stopped withglycine (added to 2mgml"1, then washed in 2mgml~'glycine in Tris/EDTA) and embryos washed five times inPBS. Hybridization was overnight at 53°C in the bufferdescribed by Ingham et al. (1985a), using 35S-labelledantisense-RNA probes derived from cDNA sequencesusing the T3 promoter of pwg-cUa (Baker, 1987) or the T7promoter of Tl(2)en (a gift of Dr M. Weir). Probes werereduced to a mean (mass average) length of 100-200 bpusing base hydrolysis (Cox et al. 1984). Embryos werewashed in ten changes of a buffer comprising 50 % forma-mide, 0-06M-NaCl, 5mM-Tris pH7-4, 5mM-sodiumphosphate pH6-8, 5mM-EDTA, 1 x Denhardt's Solution,14 mM-/3-mercaptoethanol at 53°C over a 24 h period, thenwashed once in NTE (NTE = 0-5M-NaCl, 10mM-TrispH8-0,1 mM-EDTA), incubated with RNase A (20 fig ml"1

in NTE, 30 min, 37°C) and washed twice with NTE at 37°Cand twice with PBS at 45 °C. In the experiment shown inFig. 1A, embryos were then dried onto slides and autora-diographed as described by Kornberg et al. (1985) forimaginal discs. For the other experiments, this procedurewas replaced with the following: an approximately equalvolume of molten Kodak NTB2 emulsion was added to thefinal PBS wash and the embryos incubated in this mixturefor 20 min with occasional shaking. The emulsion/embryomixture was spread dropwise on gelatin-subbed slides andallowed to air-dry. After 21 days exposure at 4°C, slideswere developed in Kodak D19 (2min 15s, 15°C), stoppedin 2 % acetic acid (30 s, 15°C) and fixed in Kodak fixer (1 h),washed extensively in H2O, air-dried and viewed byNomarski-interference microscopy.

The sectioned material has been described previously(Baker, 1987), except for Fig. 3D, which was provided byDr P. Ingham.

Results

Whole-mount embryo in situAlthough most in situ hybridization studies have usedtissue sections, Kornberg et al. (1985) described theuse of in situ hybridization to detect transcripts fromthe engrailed (en) gene in whole imaginal discs. In aninitial experiment, a wg probe was hybridized towhole embryos following a similar protocol (seeMaterials and methods), and others have also de-scribed similar experiments (St. Johnston & Gelbart,1987; Baumgartner et al. 1987). An example is illus-trated in Fig. 1A; autoradiography revealed thestriped pattern of wg transcription, but the mor-phology was poor and uneven coating with emulsionmade the signal irreproducible. Both these problems

Wingless transcripts in whole embryos 291

were offset by a straightforward modification of theprocedure. Hybridized embryos were transferred di-rectly into liquid emulsion after washing, and theemulsion/embryo mixture allowed to air dry ontomicroscope slides. This results in more even-coatingwith emulsion than if specimens are first dried ontoslides before dipping, and morphology is improvedwhen the ethanol dehydration prior to drying iseliminated (Fig. 1). A series of embryos hybridizedfollowing the new method is shown in Fig. 2. It isimportant to mention shortcomings and sources ofartefact that remain in the technique. (1) Signal isfrequently stronger around the edge of the specimenand weaker over the highest point. This bias insensitivity could be due to the thickness of theemulsion coat, which is probably thinner over the topof the embryo. This artefact is circumvented byexamining embryos mounted in different orien-tations. (2) Silver grains are usually present wherethere are deep grooves on the surface of the speci-men, such as the cephalic furrow or segment bound-aries. This could be caused by incomplete washing ofprobe from these grooves. In the present exper-iments, this has not caused difficulties; rather, theinterpretation of hybridization signals is made easierby this highlighting of morphological features. (3)Both wg and en transcripts are localized predomi-nantly in external parts of the embryo. Although wgtranscripts are detected internally in the anal regionand hindgut (Fig. 2K), this signal is weaker thanmight be expected on the basis of sectioned material(see Fig. 3). Therefore, it is not certain how readilytranscripts in internal tissues will be detected by thismethod.

wg transcripts in whole embryosPrevious studies of wg transcription have concen-trated on the trunk region of the embryo (Baker,1987; Rijsewijk et al. 1987; Ingham et al. 1988). Incombination with sectioned material (Fig. 3), thewholemounts shown in Fig. 2 have provided a morecomplete picture of wg transcription in the wholeembryo. Details are described below, and summar-ized in Fig. 4.

The onset of wg transcriptionwg transcripts in parasegments 0—13 are first detectedduring the cellular blastoderm stage. The stripesappear first at the anterior, and in even-numberedparasegments before odd. At this stage, signal seemsstronger ventrally than dorsally (see Fig. 2C,D).Expression of the segmentation genes engrailed andgooseberry also develops with pair-rule modulation inthe trunk region (Weir & Kornberg, 1985; DiNardo etal. 1985; Cot6 et al. 1987; Baumgartner et al. 1987).However, wg is unique amongst these in that the

1A

B

Fig. 1. Comparison of whole-mount methods.Whole embryos were used for in situ hybridization as

described in Materials and methods. In A, embryos weredehydrated and stuck to gelatin-subbed slides prior todipping in emulsion (Kornberg et al. 1985). Silver grainsindicate the striped pattern of wg transcription at theextended germband stage. However, morphology of theembryos is poor and signal is irreproducible both betweenembryos and in different regions of the same embryo,presumably due to variation in the thickness of theemulsion coat. In this, as in all subsequent figures,anterior is to the left, dorsal uppermost. B. An extendedgermband-stage embryo which was incubated in liquidemulsion before being allowed to air dry onto the slide(see Methods and methods). Note the improvement inmorphology. In C, an enlarged photograph of an embryosimilar to B reveals the outline of individual cells in thedorsal head. Bar, 100(im.

earliest expression is outside the trunk region. First toappear are transcripts at the anterior tip of theembryo and in a posterior ring, which occupy, re-spectively, the primordia of the foregut and of thehindgut and anal region. The next step is the appear-ance of blocks of wg transcription on either side of thedorsal head (Fig. 2C), then followed by the stripes ofthe trunk region (Baker, 1987; Ingham et al. 1988).

292 N. E. Baker

Thus when gastrulation begins regions of wg tran-scription are spread over most of the embryo, exceptfor the endodermal regions (the primordia of theanterior and posterior midguts) and the germline (thepole cells).

Development of the segmented germbandAfter gastrulation, the movements of germband ex-tension occur and the segmentation of the trunkregion becomes morphologically apparent (Poulson,1950; Campos-Onega & Hartenstein, 1985; Marti-

It* i *

Wingless transcripts in whole embryos 293

nez-Arias & Lawrence, 1985). During this period thedistribution of wg transcripts continues to elaborate.By the end of the rapid phase of germband extension,two small patches of wg transcription have appearedon either side of the dorsal head, behind the foregutsignal and anterior to the large dorsal patches (Fig.2E,I). As the stomodeum invaginates and cells in themost anterior wg domain move into the foregut, thesetwo patches fuse at the dorsal midline and moveanteriorly to occupy the most dorsal part of theclypeolabral lobe. In some embryos at this stage,another two small patches of wg transcription areweakly detected on the ventral lateral head, justabove parasegment 0 (Fig. 2J). These could corre-spond to the - 1 signal previously observed in sectionsof the extended germband stage (Baker, 1987). Thistranscription is also sometimes detectable at blasto-

derm (Fig. 2D).During germband extension, the pattern of wg

transcription also becomes further resolved in thecaudal region behind parasegment 13. wg tanscriptsin parasegment 14 are first detected at the end of therapid phase of germband extension (stage 9, 4h afterfertilization according to Campos-Ortega & Harten-stein, 1985; Fig. 3A,B). Posterior to this, signalextends over the whole of the proctodeum andhindgut, as in the blastoderm. By the time thestomodeum invaginates (stage 10; ~5 h after fertiliz-ation; Fig. 3C) the anus and the hindgut haveresolved into separate wg domains. The signal in thehindgut is at the boundary with the posterior midgutand is later found in the malpighian tubules, whichare the most posterior ectodermal structures (Fig.3G,H; Lawrence & Johnston, 1986).

Fig. 2. wg transcripts in whole-mount embryos.(A,B) Embryos hybridized with an en probe. At the

blastoderm stage en transcripts are initially restricted toparasegment 2 (Weir & Kornberg, 1985; DiNardo et al.1985). (B) A stage-11 embryo (—6-7 h after fertilization),illustrating en transcription now in 15 parasegments, inthe clypeolabral lobe (cf) close to the stomodeum, in thegnathal lobes and more anteriorly in the intercalarysegment (open arrow). Note that in the trunk region entranscripts extend completely around the epidermis fromventral to dorsal.

(C-K) wg transcription. (C) Early blastoderm in whichwg transcripts are restricted to the primordium of theforegut (fg), a ring around the posterior containing theprimordia of the hindgut and proctodeum and largedorsal patches (arrowhead). Ventrally, two anteriorstripes are clearly visible (solid circles), and others areappearing (arrowheads). (D) By the end of the cellularblastoderm stage, fourteen stripes of alternating intensityare established (black dots). These lie withinparasegments 0-13 (Baker, 1987; Ingham et al. 1988).Anterior to stripe 0, a small strip of expression isdetectable in some embryos (open arrow). The dorsalpatches project a thinner arm laterally. The signal in theforegut primordium has assumed a characteristic'crescent' shape, with two cusps encircling the ventralcells where the anterior midgut will invaginate.(E) Extended germband embryo (stage 9, ~4h afterfertilization). The transcription pattern of the gastrula hasbeen maintained, except that two additional regions oftranscription have appeared on either side of the dorsalmidline in the anterior head (filled arrow; see Fig. 21).Parasegment 0 lies mostly within the cephalic furrow.Posterior to psl3 the primordia for the hindgut andproctodeum have become internal. (F) By the beginningof germband shortening (early stage 12, —7-5 h afterfertilization, the anal region has reappeared at theposterior of the embryo {an), most of the foregut hasinvaginated and the clypeolabral (c/) and gnathal(md, mandibular; mx, maxillary; Ib, labial) lobes areapparent. In the thoracic and abdominal segments, the

stripes of wg transcripts no longer extend completelyaround the segments (compare en transcripts in Fig. 2B),but are restricted to the ventral region and also the dorsalpart of the stripe near the amnioserosal membrane.Dorsal and gnathal signals are unusually weak in thisembryo and these transcripts are better illustrated inFig. 2G,K. (G) Ventral view of an embryo of similar ageto F. In the trunk segments, wg transcripts are found onlyin ventral and dorsal parts of each segment, wg isexpressed in a small strip in each of the labial andmaxillary lobes (arrows). Especially in the thoracicsegments (tl, t2, t3), the ventral transcript domain isbroader away from the ventral midline (see also Fig. 2K).(H) Ventral view of an embryo after germbandshortening (stage 13), exhibiting a similar transcriptionpattern to that seen in Fig. 2F,G. Ventrally, wgtranscription in the trunk segments is limited by sharplateral boundaries. Silver grains close behind the wgstripes, especially in the thoracic segments, probablyreflect residual probe trapped in the deepening segmentalgrooves and do not represent specific hybridization.(I) Close up of the dorsal head of an extended germbandstage embryo like that shown in Fig. 2E. A pair of smalldomains of wg transcription (arrows) are apparentbetween the foregut signal and the large dorsal patches.(J). Close up of the ventrolateral head of an embryo afew minutes older than I. The foregut primordium isbeginning to enter the stomodeum (st). Small regions ofweak ventrolateral wg transcription are visible anterior tostripe 0. (K) Close up of the ventral trunk region of anembryo like those in Fig. 2F,G. wg transcription nolonger extends completely around the embryo in thedorsoventral axis. In the thoracic segments (tl, t2, t3),the ventral wg transcript domain has a dumbbell shape,thicker laterally than at the ventral midline (also visible inFig. 2G,H). Transcripts in the labial and maxillary lobesare confined to small lateral strips (arrows). The largepatches of silver grains beneath the maxillary and secondthoracic segments (and out of focus in this picture) are inthe anal region and hindgut respectively.

294 N. E. Baker

Fig. 3. wg transcripts in tissue sections.wg transcription is first detected in parasegment 14 at the end of the rapid phase of germband extension (stage 9, ~4h

after fertilization). In the bright-field micrograph shown in A, the positions of the cephalic furrow (filled arrowhead),anterior midgut imagination (open arrowhead) and primordium of the foregut (fg) are indicated. At this stage, acommon wg signal still extends over the primordia of the hindgut and anal region (hg). Note that wg transcripts are stillpresent in the mesoderm. (B) A dark-field micrograph of the same embryo. (C) wg transcripts are found separately inthe region of the anus and the hindgut by stage 10 (~5h after fertilization). In D, a similar embryo hybridized with anen probe shows that the juxtaposition of wg and en transcription in the trunk region may extend to the anal region andthe foregut. (E,F) Bright- and dark-field micrographs of an embryo beginning germband shortening (at a similar stageto Fig. 2F). wg transcription is no longer separately distinguishable in psl4; signal in the hindgut is at the boundary withthe posterior midgut. (G,H) Bright- and dark-field micrographs of a horizontal section through a slightly older embryo,illustrating the location of the wg hindgut transcription in the forming diverticulae of the malpighian tubules, (mt,malpighian tubules; pmg, posterior midgut). (I) After germband shortening (this embryo is comparable to the wholemount illustrated in Fig. 2H), wg transcripts are still found in the most posterior part of the hindgut and in a veryanterior part of the foregut, close to the anterior midgut.

Changes in wg expression during the extendedgermband stageMidway through the extended germband stage, whenthe gnathal lobes have appeared (stage 11, ~5£-7£h),it is apparent that changes in wg expression haveoccurred in the dorsal-ventral axis. Most strikingly,wg transcripts become excluded from the lateral partof the epidermis of the thoracic and abdominalsegments and are restricted to the ventral and dorsalparts of the original stripes (Fig. 2F,G). In addition,the shape of this remaining wg domain is altered.Midventrally the region containing wg transcripts isnarrower than previously, but the lateral boundary ofthe domain is broader, so that the stripes have adumbbell appearance. This is more pronounced inthe anterior trunk segments (Fig. 2K). This change inwg transcription during the extended germband stage

is in contrast to the constancy of the pattern of enexpression in the trunk region, which seems to labelthe epidermal cells of posterior compartments con-stantly throughout development (Kornberg et al.1985; Fjose et al. 1985; DiNardo et al. 1985; compareFig. 2B with Fig. 2F).

In the labial and maxillary segments, transcripts arenow restricted to a strip across the lobes themselvesand do not extend to the ventral midline. The —1stripe moves close to the transcripts in the mandibularsegment and can no longer be found when germbandshortening begins. The dorsal patch becomes lessextensive and, as stomodeal invagination continues,signal in the clypeolabral lobe moves more ventrally.At the caudal end of the embryo, wg transcripts areno longer distinguishable in parasegment 14 when thegermband starts to retract, but transcripts are still

Wingless transcripts in whole embryos 295

found in the anal region and the hindgut even afterthe germband has shortened (Figs 2F, 3E,F,I). Thewg transcription pattern now seems to remain con-stant during germband shortening and dorsal closure(Fig. 2H).

The wg transcription pattern is summarized in Fig.4.

Discussion

Whole mount in situ hybridizations

Detection of transcripts in whole Drosophila embryos

Foregut

Labral

is a useful complement to results obtained withsectioned material. Although whole mounts offer lessresolution on a cell-by-cell basis, the whole pattern isbetter revealed in a single specimen. For example,the two small dorsal patches of wg transcription (Fig.21) had previously been missed in sections. Themethod should be useful for comparing the accumu-lation of transcripts with the distribution of proteinfor gene products against which antibodies have beenraised and analysing the expression of genes for whichantibodies are not available. The technique is notlimited to Drosophila embryos and has also been usedfor imaginal discs (Baker, 1988b, and unpublisheddata).

Expression of wg in the head and caudal regionsIn the trunk region, the wg gene is required for thedevelopment of every segment and wg transcriptsaccumulate in a specific portion of each segment ofthe extended-germband-stage embryo (Niisslein-Vol-hard & Wieschaus, 1980; Baker, 1987). This suggeststhat wg expression can be used as a molecular probefor segmentation, as has been the case with othersegmentation genes (e.g. Kornberg et al. 1985;DiNardo etal. 1985; Ingham et al. 1985a,b; Carroll &Scott, 1986). In situ hybridization has shown that bythe end of germband extension all the embryo except-ing the endoderm and germline (which are notthought to be segmented) is divided by 21 domains ofwg transcription. These may identify segments in thehead and caudal regions, as well as the trunk.Comparative studies suggest the head should containintercalary, antennal and labral segments anterior tothe three gnathal segments (Anderson, 1973; Rem-

Fig. 4. Summary diagram of wg transcript accumulationin embryos.

(A) At the blastoderm stage, wg transcripts accumulatein fourteen stripes from the mandibular segmentposteriorly. More anterior domains are hypothesized tobelong to the intercalary and antennal segments.Transcripts have not yet appeared in the putative labralsegment. The wg transcripts also extend into the ventralblastoderm, which will give rise to the mesoderm. Onlythe endodermal and germline regions of the blastodermseem to lack wg transcription completely (see Poulson,1950 for fate map of the blastoderm stage). (B) When thegermband has extended, segmentation.becomes evidentmorphologically. The transcription domains observed atblastoderm are localized as shown, with the addition oftranscripts in the labral segment and in parasegment 14,and the resolution of the anal and hindgut signals. (C) Asthe germband begins to retract, the wg transcriptionpattern becomes modified. This is most apparent in thethoracic and abdominal segments, where transcripts arerestricted to the dorsal and ventral parts of the stripe.Also transcription in psl4 and the anal region are nolonger separate.

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pel, 1975); the caudal region extends up to a 12thabdominal segment in some insects, but this numberis often reduced by the loss of some and fusion ofothers (Matsuda, 1976). Below, these predictions arediscussed in light of the wg transcription pattern(summarized in Fig. 4) and also with respect to theengrailed and gooseberry genes, which seem to haverelated patterns of expression. The picture thatemerges, though not definitive, is a contributiontowards understanding the terminalia of the Dros-ophila embryo.

The intercalary segmentThe intercalary segment is thought to occupy a smallventrolateral region anterior to the mandibular seg-ment (Schoeller, 1964; Turner & Mahowald, 1979;Jiirgens et al. 1986). Transcripts from wg (Fig. 2D,J)and from en and gsb (Kornberg et al. 1985; DiNardoet al. 1985; Ingham et al. 19856; Baumgartner et al.1987) probably define parts of this segment (Fig. 4).

The antennal segmentFate-mapping studies suggest a dorsolateral positionfor the embryonic antennal segment (Anderson,1973; Struhl, 1981, 1983; Jiirgens, 1985; Jiirgens et al.1986). This is consistent with the position of adorsolateral arm of wg transcription (Fig. 4). en andBSH9 are expressed in a similar region (DiNardo etal. 1985; Baumgartner et al. 1987). However, theputative antennal wg domain is larger than in othersegments and also extends over much of the pro-cephalic lobe. In Drosophila, the procephalic lobe,most of which contributes to the brain, has beendescribed as unsegmented (Poulson, 1950), but itseems possible it actually belongs to a large antennalsegment. Consistent with this, gynandromorph analy-sis showed the eye-antennal imaginal disc is recruitedfrom a larger region of the embryo than are discs inother segments (Struhl, 1981). This region is anomal-ous in another respect, since it contains a furthersmall patch of dorsolateral cells that express the enand gsb genes (DiNardo et al. 1985; Baumgartner etal. 1987).

The labral segmentThe small paired dorsal regions of wg transcriptioncorrespond to the fate-map position of the labralsegment (Jiirgens, 1985; Jiirgens et al. 1986). Duringgermband extension, these two primordia jointogether and move anteriorly around the front of theembryo as the stomodeum invaginates until, bygermband shortening, labral wg expression is on theventral surface of the clypeolabral lobe (Technau &Campos-Ortega, 1985). Corresponding labral ex-pression of gooseberry has been described (Baum-gartner et al. 1987), but en expression seen in the

clypeolabral lobe is more anterior and cannot liewithin the labral segment (Kornberg et al. 1985;DiNardo et al. 1985; Ingham et al. 1985/?; Figs 2B,3D). A better candidate for labral en expression hasnow been detected in the dorsal head using a mono-clonal antibody (T. Kornberg, personal communi-cation).

The foregutThe most-anterior domain of wg expression is in theprimordium of the foregut. The foregut is part of theectoderm and, like typical segments, containsimaginal primordia which give rise to part of the adultat metamorphosis (Anderson, 1972). The foregutcould be considered the most anterior segmented partof the embryo. Possibly, en expression in the most-ventral part of the clypeolabral lobe (see above)corresponds to this metamere.

The caudal regionPosterior to parasegment 14, both the anal region andthe hindgut contain one domain each of wg and enexpression at the extended germband stage (Fig.3C,D). The straightforward interpretation is thatthere is one abdominal parasegment posterior topsl4, and that the hindgut should be considered afurther metameric unit. Each of these also forms apair of imaginal precursors which construct parts ofthe adult at metamorphosis (Anderson, 1972). How-ever, the anal region seems to be a composite unitderived from a larger number of ancestral metameres(Matsuda, 1976; Dubendorfer & Nothiger, 1982;Carroll & Scott, 1985). Vestiges of the primitivecondition are seen in the larval cuticular structures(Jurgens, 1987) and in the distribution of BSH9transcripts from the gsb locus (Baumgartner et al.1987). During germband shortening, the anal regionalso seems to assimilate parasegment 14 (Figs 2F,3E,F, 4; DiNardo et al. 1985).

Changes in wg expression in the dorsoventral axisIn contrast to previous conclusions, whole mounts insitu show that the pattern of wg transcription does notremain uniform in the dorsal—ventral axis. Although,until the middle of the extended germband stage, wgtranscription, like en, identifies cells at a particularanterior-posterior position in the segment, wg tran-scripts are subsequently lost from the lateral epider-mis of the trunk segments and the shape of theremaining wg domain altered. This result raisesdoubts about the idea that segment polarity genes actonly to label domains defined as rows of cells at theblastoderm stage. Apparently expression of theBHS9 transcript from gooseberry becomes modifiedsimilarly (Baumgartner et al. 1987).

Based on the timing and position of dorsoventral

Wingless transcripts in whole embryos 297

lineage restrictions in Drosophila and Oncopeltus, itdoes not seem likely that the lateral boundaries of wgtranscription are compartment boundaries (Law-rence, 1973; Garcia-Bellido et al. 1976; Steiner, 1976;Wright & Lawrence, 1981). So far the phenotype ofwg mutations has not suggested a difference in wgfunction laterally (Nusslein-Volhard & Wieschaus,1980; Nusslein-Volhard et al. 1984; Perrimon &Mahowald, 1987; Baker, 1988a). One speculation isthat the changes in wg transcription during theextended germband might reflect the formation ofimaginal discs in which the segmental field is pre-served whilst most cells become committed to thelarval epidermis (Anderson, 1963, 1972; Meinhardt,1983). This occurs at about the same time in embryo-genesis (Geigy, 1931; Wieschaus & Gehring, 1976).The leg discs form near the ventral-lateral boundaryof wg transcription (Turner & Mahowald, 1979).Mature leg discs contain wg transcripts only in ventralcells, as though they originate at the boundary region(Baker, 19886). Indeed, wg mutations can affect theformation of Keilin's organs (Baker, 1988a), whichsuggests they also affect disc formation (Keilin, 1915).

I thank Drs P. A. Lawrence and G. M. Rubin for spaceand materials, Drs A. Martinez-Arias and P. Ingham forteaching me about in situ hybridization and for encourage-ment, and Drs E. Wieschaus, V. Foe and, especially, M.Weir for interesting discussions and for communicatingunpublished results. The emulsion-incubation step wasinitially suggested by Dr H. Steller. I thank Drs M. Weir forthe T7(2)erc plasmid, T. Kornberg for permission to quoteunpublished work, G. Hess and R. Tjian for use of theirDrosophila population cage, and M. Weir and P. Inghamfor comments on the manuscript. Support was provided bya Training Fellowship from the Medical Research Council,postdoctoral fellowship DRG-901 from the DamonRunyon-Walter Winchell Cancer Fund and NTH grant GM32795 to Dr G. M. Rubin.

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(Accepted 23 February 1988)