ultrabithorax and engrailed drosophila for segmentation...

Development 102, 325-338 (1988)Printed in Great Britain © The Company of Biologists Limited 1988

325

Ultrabithorax and engrailed expression in Drosophila embryos mutant

for segmentation genes of the pair-rule class

ALFONSO MARTINEZ-ARIAS1* and ROBERT A. H. WHITE2

'MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK2Department of Anatomy, Downing Street, Cambridge, CB2 3DY, UK.

* Present address: Department of Zoology, Downing Street, Cambridge, CB2 3EJ, UK

Summary

Mutations in the pair-rule class of segmentation genescause pattern deletions with a double segment period-icity in the Drosophila embryo. This phenotype is, inpart, due to cell death. Using molecular probes forengrailed (en) and Ultrabithorax (Ubx) expression asmarkers for the body plan, we have studied the

phenotype of pair-rule mutants prior to cell death. Allthese mutants alter the expression of en and Ubx; theirmolecular phenotypes suggest a pathway wherebypair-rule gene functions construct metamerie units.

Key words: pair-rule loci, parasegments, Ultrabithorax,engrailed, Drosophila, mutant, pattern formation.

Introduction

A conspicuous feature of all insects is their metamer-ism, which allows a number of developmental andevolutionary strategies to operate (Snodgrass, 1938;Lewis, 1963; Raff & Kaufman, 1983). In the embryo,larva and imago of Drosophila, metamerie units havetwo invariant properties: their number and theirindividual phenotypes. The first of these properties isunder the control of the segmentation genes (Niiss-lein-Volhard & Wieschaus, 1980; Niisslein-Volhard etal. 1982); the second is the responsibility of homeoticgenes (Lewis, 1963,1978; Garcia-Bellido, 1975; Kauf-mann & Abbot, 1984).

During embryogenesis, the first morphological signof metamerism is the appearance of the parasegmen-tal grooves shortly after gastrulation (Martinez-Arias& Lawrence, 1985). However, before parasegmentsare visible, the transient expression of pair-rule genes(Hafen et al. 1984; Ingham et al. 1985a; Carroll &Scott, 1985; Kilchherr et al. 1986; Harding etal. 1986;MacDonald et al. 1986) indicates that the metameriz-ation of the embryo is achieved early in the blasto-derm stage. Homeotic genes (Levine et al. 1983;Akam & Martinez-Arias, 1985; Ingham & Martinez-Arias, 1986; Martinez-Arias et al. 1987) and some

segment polarity genes (Weir & Kornberg, 1985;Baker, 1987; Cote et al. 1987) are also expressed inthe blastoderm but, in contrast to pair-rule genes,their expression remains relatively stable throughoutdevelopment and provides a measure of the fate mapof the Drosophila embryo.

The epistatic relationships between the differentsegmentation genes (Carroll & Scott, 1986; Hardinget al. 1986; Howard & Ingham, 1986; Ingham, Ish-Horowicz & Howard, 1986; Ingham & Martinez-Arias, 1986) suggest that the pair-rule loci play acritical role in the establishment of the blastodermfate map. To explore these relationships further, wehave used probes for the expression of two genes,engrailed {en) and Ultrabithorax (Ubx) to assay theeffect on the body plan of mutations at the pair-rulesegmentation loci.

Our results support previous observations (Akam,1985; Akam & Martinez-Arias, 1985; Howard &Ingham, 1986; Ingham et al. 1986; Ingham & Mar-tinez-Arias, 1986) suggesting that the function of thepair-rule loci is to set up a transient prepattern (Stern,1968) which will be interpreted by segment polarityand homeotic genes. This prepattern depends on afunctional hierarchy (Howard & Ingham, 1986;

326 A. Martinez-Arias and R. A. H. White

Ingham & Martinez-Arias, 1986) with combinatorialinteractions acting at each of its levels.

Materials and methods

StocksThe wild-type flies used in this study were from a Canton Sstock. To generate mutant embryos, mutant stocks werecrossed to Canton S and the heterozygous progeny wereused to collect embryos; one fourth of them were mutant.The different mutations and alleles used in this study are asfollows: engrailed, enw and enlK (Niisslein-Volhard et al.1984), Df(2R)enB (Eberlein & Russell, 1983); even skipped,eve3 21" and Df(2R)eve1 ^ (Nusslein-Volhard et al. 1984,1985); fushi tarazu, ftz9H34 and Df(3R)4Scb (Jurgens et al.1984); hairy, h[K and DfQVjh02 (Ingham etal. 1985ft); oddskipped, oddiUD36 (Nusslein-Volhard et al. 1984, 1985); oddpaired, opa5Hsn (Jurgens et al. 1984); paired, prd2*517

(Nusslein-Volhard etal. 1984, 1985); runt, runXAOb, runYC128

(Wieschaus et al. 1984).Mutant embryos were identified by their unusual patterns

of en and Ubx expression and always amounted to, approxi-mately, one fourth of the progeny.

In situ hybridizationsThe protocols used have been described before (Akam &Martinez-Arias, 1985; Ingham et al. 1985a). The engrailedprobe corresponds to a genomic fragment spanning thehomeobox (Fjose etal. 1985).

Antibody stainingThroughout this study, we used the monoclonal antibodyFT.3.38 (White & Wilcox, 1984) to detect Ubx proteinproducts. Labelling of embryos for immunofluorescence(White & Lehmann, 1986) or immunoperoxidase (Ingham& Martinez-Arias, 1986) has been described before.

Results

Phenotypes and embryogenesis of pair-rule mutantsPair-rule loci were identified as a class of mutants withsegmentation defects, which, in the Drosophila em-bryo, delete pattern elements every other metamericrepeat (Nusslein-Volhard & Wieschaus, 1980). Thecuticular phenotypes of these mutants have beendescribed before in detail; our observations basicallyagree with those already published (Nusslein-Volhardetal. 1982; Gergen etal. 1986) and are summarized inFig. 1. The deletions in fushi tarazu (ftz) mutants(Wakimoto et al. 1981) closely correspond to evennumbered-parasegments and, in hypomorphic evenskipped {eve) alleles, odd numbered parasegments areeliminated. The total absence of eve results in acompletely aperiodic pattern where thoracic andabdominal regions can still be distinguished (Niiss-lein-Volhard et al. 1985). The mutant odd skipped(odd) removes a part of each even numbered paraseg-ment. The precise deletion frames in odd paired

PS ftz eve h run prd opa odd SG

Y YT3

Al

A2

Fig. 1. Summary of pattern deletions in the pair-rulemutants utilized in this study. The pattern deletions arepresented with respect to parasegments (PS) andsegments (SG), and are drawn for the third thoracicsegment (T3), and first and second abdominal segments(Al, A2). The dots indicate denticles and the Keihnorgans are sketched in T3. The hatching represents theapproximate regions deleted in every mutant. In the caseof eve, the cross hatching indicates the additionaldeletions produced by the deficiency Df(2R)evel 27.

(opa), hairy (h) and paired (prd) have been denned indouble mutant combinations with Ubx (Niisslein-Volhard et al. 1985; Howard & Ingham, 1986 andunpublished observations) and do not coincide(Fig. 1). Finally, the mutant runt (run) combinesproperties of pair-rule and segment-polarity mu-tations (Nusslein-Volhard & Wieschaus, 1980;Gergen & Wieschaus, 1985), deleting alternatemetameric units from the pattern and giving rise tomirror-image duplications in the remaining units. Inall pair-rule mutants, the boundaries of the patterndeletions are variable and allele dependent.

Analysis of the embryonic development of thesemutants indicates that cell death contributes to thefinal phenotype (Ingham et al. 1985a; Ingham &Martinez-Arias, 1986 and unpublished observations);in all cases, it begins in the mesoderm at about 5i-6 hand then extends into the ectoderm (data not shown).We have examined the morphology of mutant em-bryos 1 or 2h after gastrulation, before the onset ofmajor cell death (Figs 4, 5 and summary in Fig. 7). Atthis stage, the metamerism of the wild-type embryo isevident in the parasegmental grooves. Mutant em-bryos also exhibit grooves delimiting morphologicalunits. On the basis of morphology, these mutants canbe divided into two classes: those that make half thenumber of morphological units, each being approxi-mately twice the wild-type parasegmental size (ftz,eve, prd, opa and h) and those that make a normalnumber of these units, each of variable size (run,odd). The complete absence of eve abolishes most ofthe metameric boundaries, although some very shal-low grooves can still be visible in certain preparationsdefining units as in eve hypomorphs (Fig. 5).

Segmentation in Drosophila 327

Phenotype and embryogenesis of engrailed mutantsThe segment polarity gene en is responsible for theidentity (Morata & Lawrence, 1975) and structuralintegrity (Niisslein-Volhard & Wieschaus, 1980;Kornberg, 1981) of posterior (P) compartments in theembryo and the adult. We have studied the develop-ment of embryos mutant for three alleles of en: twocytologically normal, enlK and en10 (Niisslein-Vol-hard et al. 1984), and a deficiency for the locus,Df{2R)enB (Eberlein & Russell, 1983). In all threecases, parasegmental grooves form. However, whilein the case of enlK and enlu we cannot detect any grossmorphological abnormality until germ band shorten-ing, Df(2R)enB embryos show abnormal morphology2h after gastrulation (Fig. 3B, D). In Df(2R)enB

homozygotes, the parasegmental grooves are irregu-lar and occasionally some of them are missing, thegnathal segments are malformed and the germ band isnot fully extended. After germ band shortening,massive cell death is observed in the ectoderm andthe mesoderm of embryos homozygous for the threealleles, and this is probably responsible for the finalsmall size and metameric fusion of cuticle. In en10 andeniK embryos soon after shortening of the germ band,we observe a pairwise fusion of metameres as de-scribed earlier for other en alleles (Kornberg, 1981);at this time, Df(2R)enB shows no morphological signsof metamerism in the ectoderm (unpublished obser-vations).

The pattern of en expression in pair-rule mutantsThe expression of engrailed, in 14 evenly spacedstripes spanning the germ band (Fig. 2A), provides ameasure of metamerism in the animal and thus it is arepresentation of this aspect of the blastoderm fatemap (Kornberg et al. 1985; Weir & Kornberg, 1985;DiNardo et al. 1985; Howard & Ingham, 1986).Previous studies have shown that engrailed expressionis altered in some pair-rule loci (Howard & Ingham,1986; Ingham & Martinez-Arias, 1986; MacDonald etal. 1986). Here we have looked at the expression of enin other mutants of this class and show that all of themalter the pattern of engrailed products (Fig. 2; sum-marized in Fig. 7).

In some of the mutants, only half of the stripes arepresent. In ftz" embryos, these are the odd-num-bered ones (Howard & Ingham, 1986) and in prd~embryos, the even-numbered ones (Fig. 2B); in bothcases, the remaining stripes appear wild type and arespaced at double metameric intervals. In opaT em-bryos, the pattern is very reminiscent of that of ftz~,but there are vestiges of the even-numbered stripesparticularly in the mesodermal and neural regions(Ingham & Martinez-Arias, 1986; Fig. 2D). In h"',half of the stripes are present, they are broadenedand spaced at large intervals; occasionally, thinner

stripes are interspersed between the broad ones(Howard & Ingham, 1986).

The other mutants display rearrangements ratherthan losses of stripes. In odd~ embryos, every otherstripe is broader than wild type (Fig. 2F). In run'embryos, there are seven pairs of stripes; at timessome of the pairs fuse (Fig. 2E). In eve", the patterndepends on the allele used: eve32111 produces pairingand deletion of stripes in homo- or hemizygouscondition (Fig. 2C). In Df(2R)eve121 embryos, no enstripes are detectable in the main region of the germband, although they remain in the gnathal append-ages (MacDonald et al. 1986).

We have also looked at the expression of engrailedin the different en alleles used in this study. WhileDf(2R)enB embryos do not produce any detectableengrailed RNA or protein product, eniK and enw doproduce protein or proteins, which are distributedabnormally (DiNardo, personal communication andunpublished observations).

The pattern of Ubx expression in the wild typeThe Ubx products can be detected for the first time40-60 min after gastrulation when they appear as ablock of antigen in the area of parasegment 6 (PS6),with little or no label in PS7-12. Later, the completepattern emerges and by the time of stomodeal invagi-nation (approx. 6h), the pattern of Ubx products(RNA and proteins) is clearly modulated betweendifferent metameres as well as within metameres(Akam & Martinez-Arias, 1985; White & Wilcox,1985; White & Lehmann, 1986; Figs 3A,C, 6A). Wehave focused our study on the distribution of theantigen in the extended germ band, before cell deathbegins in the ectoderm of the mutants. The wild-typefeatures we have used as a reference are (a) PS6displays high levels of antigen with relatively littlevariation in intensity across the parasegment; (b)PS7-12 show a marked intraparasegmental gradientof expression; (c) in parasegments 6-12, the pre-sumptive posterior compartment is less labelled thanthe anterior one and (d) PS5 and PS13 have hetero-geneous and unique patterns of antigenic distri-bution.

en regulates Ubx in posterior compartmentsA conspicuous feature of the Ubx pattern in theextended germ band is the low level of Ubx ex-pression in the P compartments of parasegments 6-12(Akam & Martinez-Arias, 1985; White & Lehmann,1986). This area of low expression coincides, pre-cisely, with the expression of en (S. Carroll, S. DiNardo, P. O'Farrell & Matthew P. Scott, personalcommunication) and, for this reason, it was of inter-est to study the expression of Ubx in en~ mutants. Ineither en10, enlK or the transheterozygote, there is


fi f i t i1

Fig. 2. Dark-field photographs of the expression of engrailed RNA one hour after gastrulation in wild type and differentpair-rule mutants. (A) Wild type. The number of each engrailed stripe also indicates the corresponding parasegment;p, premandibular; a, anus; h, hindgut. (B) Homozygous prd2 4517 embryo. Only half of the stripes are present. On thebasis of their position along the anteroposterior axis, they probably correspond to the wild-type even-numbered stripes.(C) Hemizygous eve embryo: eve3 2171/Df(2R)evel 21. An almost wild-type number of stripes is present (arrowheads),however, they are of variable intensity and many have an abnormal spacing. (D) Homozygous opaiwn embryo. Theodd-numbered stripes are present and there are also vestiges of the even-numbered ones. (E) Heterozygous

runXA06'/runYC128 embryo. Notice that the stripes have been paired and, in most cases in this section, fused. The

position of stripes 5 and 6 (arrows) is indicated. (F) Homozygous odd111036 embryo. Notice how the even numberedstripes (arrowheads) are enlarged. All in situs were performed with a 35S-labelled engrailed probe and the exposures arebetween 10 and 15 days for probes of comparable specific activity.

little disruption of the metameric pattern before germband shortening and there is a conspicuous, thoughnot dramatic, increase of antigenic intensity in Pcompartments (data not shown). This increase be-comes dramatic in the total absence of engrailedproduct, (Figs 3B,D,F, 6C), and suggests that the lowlevels of Ubx expression in P compartments are duedirectly or indirectly to en activity.

The pattern of Ubx in pair-rule mutantsIn order to present these results, we shall use tworelated but different terms; we shall refer to 'paraseg-ments' as those metameric units delimited by theparasegmental grooves (Martinez-Arias & Lawrence,1985), and we shall refer to 'Ubx metameres' (White& Wilcox, 1985) as recognizable repeat units of Ubxexpression. In the wild type, both coincide (Martinez-


3A B

W T r

ftD

i

Fig. 3. Expression of Ubx protein products in wild-type and Df(2R)enB embryos in the extended germ band, viewedwith Nomarski optics. All embryos stained with the anti Ubx monoclonal antibody FP.3.38 (White & Wilcox, 1984) andvisualized with peroxidase labelling. (A) Superficial view of a wild-type embryo; parasegments are indicated. Notice theintraparasegmental modulation of antigenic activity, which is maximal at the posterior edge of the parasegment.(B) Df(2R)enB embryo at a similar stage. Notice that the modulation has disappeared from PS6 and is less marked,though still present, in the more posterior parasegments where arrowheads indicate the posterior edges. Occasionally,two parasegments are fused; 8 and 9 in this embryo. (C) Medial view of the same embryo as A; parasegmental grooves(arrowheads) are clearly visible. (D) Medial view of the same embryo as B; some grooves (arrowheads) are present.Notice, also the intrametameric modulation of Ubx expression. (E) Ventral view of a wild-type embryo at a similar stageas A-D. The main features of Ubx expression in PS5, PS6 and PS7 are clearly visible (see text). (F) Ventral view of aDf(2R)enB at a comparable stage. Notice the absence of modulation of Ubx expression in PS6 and the irregularities inits anterior boundary (compare with E). (See Note added in proof.)

Arias & Lawrence, 1985; Akam & Martinez-Arias,1985; White & Wilcox, 1985) but in the mutants thisneed not be the case. Using the Ubx metamerecorresponding to the wild-type PS6 as a criterion, it ispossible to divide the pair-rule mutants into two mainclasses. In class I, the morphological extent of thismetamere is not changed; in class II, it is changed. In

addition, class I can be further subdivided into thosemutants with ectopic parasegments (la) and thosewith half the number of parasegmental units (Ib).

Class la includes the mutant odd (Figs 4E,F, 6C).In this mutant, there is an extra parasegmentalgroove in every other Ubx metamere; the paraseg-ments thus defined are not of equal size and there is


B

8

Fig. 4. Expression of Ubx proteins in class I pair-rule mutant embryos, viewed with Nomarski optics. (A,B)Homozygous prd2 4517 embryo. (A) Medial view; notice the large parasegments and the composite nature of thePS6 + PS7. (B) Ventral view of a similar embryo; PS5 is labelled in its wild-type position. (C,D) Homozygous opa5Hvl

embryo. (C) Medial view showing the composite nature of the PS5 + PS6 unit and the intrametameric modulation of themore posterior ones; compare with A. (D) Ventral view of a similar embryo to C. (E,F) Homozygous odd"1036

embryo. Medial (E) and lateral (F) views showing the abnormalities every other parasegment. Notice the almost normalappearance of the pattern, although clear morphological abnormalities are visible in the even numbered parasegments.

no strict correlation between Ubx metameres andparasegment boundaries. For example, the first Ubxmetamere has an extra parasegmental groove in themiddle. In these embryos the first, third, fifth andseventh Ubx metameres have the regions of low Ubxexpression enlarged, probably reflecting the exten-sion of en expression in those regions (see above).

In class Ib mutants, the extent of the first Ubxmetamere is not altered and there are half thenumber of parasegmental units. The mutants prd(Figs 4A,B, 6H) and opa (Figs 4C,D, 6F) belong to

this class. In these mutants, the most noticeabledefects are due to the loss of en product in even {opa)or odd (prd)-numbered parasegments and the conse-quent increase of Ubx expression in these regions.Other alterations of the Ubx pattern are due tosecondary effects caused by the absence of thesegment polarity gene wingless (A. Martinez-Arias,N. Baker & P. Ingham, in preparation).

In class II mutant embryos, the extent of the first£/fct-metamere is altered and this is correlated with achange in the boundaries of parasegments. This class


5A B

7*

12

8

D

A 6

Fig. 5. Expression ot Ubx proteins in class 11 pair-rule mutant embryos, viewed with NomarsKi optics. {A) Ventrolateralview of a hemizygous/rz9H34/Df(3R)4 Scb embryo. At the posterior end of PS5* there is a cluster of cells with Ubxantigen. (B) Medial view of an embryo similar to A. There is no Ubx expression in PS5* and the other three PS* showa marked modulation of Ubx expression. (C,D) Embryos heterozygous for runXAU>>/runYC128. (C) Ventrolateral viewshowing the very narrow PS6 and the symmetric modulation of expression in PS7. (D) Lateral view of a similar embryo,(e) Ventrolateral view of an embryo homozygous for Df(2R)evelr>'. Notice the very shallow grooves (arrowheads) andthe very broad PS6*. Notice also the modulation in PS8*. (F) Lateral view of an embryo hemizygous for eve:evei-2lr'/Df(2R)eve{-27. Compare with E; grooves are clearly visible demarcating PS6*, PS8*, PS10*, PS12*.(G) Ventral view of a hemizygous h embryo; hlK/Df(3Qh122. Notice the very broad PS6*. (H) Medial view of a similarembryo: notice PS6* with more Ubx expression than the rest.


includes ftz, h, eve and run. In run , PS6 is severelyreduced, as are other even-numbered parasegments(Fig. 4C,D). By contrast, odd-numbered paraseg-ments are enlarged; they appear to be flanked by Pcompartments (determined by the en-dependent low

level of Ubx expression) and the Ubx antigenicdistribution across them is a mirror image with highlevel Ubx expression in the middle which decaytowards both ends (Fig. 5C,D). Embryos mutant forthe other members of this class have half the wild-


type number of parasegmental units; however, incontrast with prd and opa embryos, the morphologi-cal extent of the first Lfe-metamere is dramaticallyaltered. In eve~ and h~, it appears rather like thewild-type PS6 (Figs 4E-H, 6B,D) and we call it PS6*;in the more posterior parasegments, the Ubx ex-pression also suggests the existence of single meta-meric units, with the graded distribution expandedover the double parasegments. Although there aresome minor differences between them, eve~ and h~embryos both can be viewed as a chain of even-numbered* parasegments: PS2*; 4*; 6*; 8*; 10*; 12*and 14* (Fig. 5E-H). The asterisks stress that theyare different from the corresponding wild-type para-segments and contain some striking heterogeneitiesin the pattern of Ubx expression (see Fig. 6B,D). InDf(2R)eve embryos, although the morphologicalmetamerism disappears, PS6* can still be dis-tinguished from the rest by the high levels of Ubxantigen, and the Ubx metameres are similar to thosedescribed in the point mutants. The apparently neweffects are presumably due to the additive effect ofthe abolition of en and, probably, other segmentpolarity genes.

In ftz~, the pattern of the 5+6 parasegment is notsimple (Ingham & Martinez-Arias, 1986 and Figs5A,B, 6G); the label around the tracheal pit ispresent and the antigen in the region of PS6 is largelyabsent except in some cells located ventrally at theposterior edge of the double width parasegment(Figs 5A, 6G). With this exception, the 5+6 paraseg-ment resembles PS5, thus we call it PS5*, and the

Fig. 6. Expression of Ubx proteins in wild-type and pair-rule mutants in the extended germ band stage, viewedwith immunofluorescence. (A) Wild-type embryo showingthe main features of Ubx expression (see text).(B) Hemizygous eve327Tl'/Df(2R)evel ** embryo; noticethe striking demarcation of Ubx expression in PS6*.(C) Homozygous Df(2R)enB embryo; the levels of Ubxexpression are higher in the anterior part of allparasegments. Compare with A; considering the patternin detail, although the clearest effect of en is in thepresumptive P compartments, there are also alterations inthe pattern of the A compartment. (D) Hemizygous hK1

embryo. Notice the large parasegment with Ubxdistribution similar to PS6. (E) Homozygous oddulD36

embryo; the region of low expression is clearly enlargedin PS6 and PS8 (compare with PS7 in the same embryo).(F) Homozygous opir'ml embryo; the composite natureof PS6 + PS7 is obvious, although some irregularities canbe observed at the boundary between high and lowexpression. (G) Hemizygous ftz9m4/Df(3R)4 Scb. Thelabel in PS5* is obvious, as it is the almost wild-typeappearance of PS7* (compare to PS7 in A).(H) Homozygous prd2Ai l7 showing the almost wild-typepattern of Ubx expression.

embryo can be thought of as a chain of odd-num-bered* parasegments (Fig. 5; Ingham & Martinez-Arias, 1986).

Discussion

The pair-rule mutants of Drosophila (Niisslein-Vol-hard & Wieschaus, 1980) define a class of lociinvolved in the subdivision of the embryo into meta-meric units. The wild-type spatial expression of someof them displays rapidly evolving patterns whosetransient steady states during cellular blastodermoverlap but do not always coincide with the regionsaffected in the mutants (Jtz: Hafen et al. 1984; h:Ingham et al. 1985; prd: Kilchherr et al. 1986; eve:Harding et al. 1986, MacDonald et al. 1986). Inaddition, studies of gene expression in mutant back-grounds shows that the pair-rule class is functionallyheterogeneous (Howard & Ingham, 1986; Carroll &Scott, 1986; Ingham & Martinez-Arias, 1986) with atleast two clearly distinct functions represented by theprimary (e.g. h) and secondary (e.g. opa, ftz) pair-rule loci (Howard & Ingham, 1986; Ingham & Marti-nez-Arias, 1986). The latter are, probably, directlyinvolved in the activation of segment polarity andhomeotic genes, whereas the former are required forthe patterning of the secondary pair-rule gene prod-ucts. Thus, the different segmentation loci are part ofa hierarchical network which, possibly through pro-tein-DNA/RNA interactions, generates a transientprepattern to be read by segment polarity andhomeotic genes. The products of these genes trans-form the prepattern into a pattern and provide it witha metameric structure (segment polarity genes) and avaried identity (homeotic genes) in the Drosophilalarva.

We have looked at the effect of the pair-rule loci onen and Ubx expression in the early embryo. Ourresults provide further support for two levels offunction within this class and suggest some specificrole's for the different pair-rule loci.

Role of pair-rule loci in activating engrailedThe engrailed gene is transcribed and translatedduring gastrulation in 14 precisely defined rings ofcells (Kornberg et al. 1985; Weir & Kornberg, 1985;DiNardo etal. 1985; Howard & Ingham, 1986). In thewild type, this activation follows a very stereotypedpattern propagating from anterior to posterior, witheven-numbered bands being activated before odd-numbered ones (Weir & Kornberg, 1985). Previousresults have shown that two pair-rule loci, ftz and h,are involved in the onset of en expression (Howard &Ingham, 1986). Here, we extend these observationsand conclude that all pair-rule loci are involved in thisprocess. Nevertheless, according to their effects on en


UbxenPS 1

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Fig. 7. Summary of the patterns of en and L/fotexpression in pair-rule and engrailed mutant embryos.The idealized patterns of Ubx expression are two-dimensional interpretations of the patterns observed andshown in Figs 2-6; these patterns are reproducible andconstant for any particular genotype. Abbreviations as inother figures.

expression (summarized in Fig. 7) they can begrouped in two classes that define two levels at whichthey play a role: definition of the spatial position ofthe en stripes and activation of en transcription inthose positions.

The activating class is defined by those loci whoseabsence results in the elimination of some or all enstripes. Thus, in the complete absence of eve, thereare no engrailed stripes (MacDonald et al. 1986 andunpublished observations) which indicates that theproduct of this locus is an essential component of theactivating mechanism. Two other important com-ponents of this system are/fz and prd: since, in theabsence offtz, all even-numbered engrailed stripesare missing (Howard & Ingham, 1986), and, in theabsence of prd, all odd-numbered ones disappear.This observation also shows that there are twopathways of en activation and that ftz and prd mustoperate near the last step of each of them; these laststeps are, probably, independent. The absence oioparesults in a pattern of en expression similar to that offtz~ embryos, but with the difference that the even-numbered engrailed stripes are not completely absent

in opa embryos (Ingham & Martinez-Arias, 1986;Fig. 2D). Thus, whereas ftz, prd and eve are essentialfor the activation of some (ftz, prd) or all (eve) of thestripes, there is a partial requirement for opa in thesame process.

The pattern of engrailed expression in other pair-rule mutants suggests that their wild-type productsare involved in precisely locating, in space, theactivating function represented by ftz, opa and prd.Thus, for example, in the case of h~ animals, thestripes of en become reduced in number but enlargedin size (Howard & Ingham, 1986); suggesting that inthe absence of h, a spacing mechanism fails. Thisfailure could result in the juxtaposition of the acti-vating functions of odd- and even-numbered enstripes and thus lead to the production of broadstripes every two wild-type intervals. A similarphenomenon is illustrated by the engrailed patternsof odd~ and run~ embryos, where there are 14stripes, but with altered widths or positions (Figs2E,F, 7); in these mutants, one set of stripes (the evenor the odd) appears to be spatially fixed while theother set has its position altered. Some alleles of evealso produce this phenotype suggesting that eve isrequired for both the activation and the spatiallocalization of the en stripes. The observation that eveRNA (MacDonald et al. 1986; Harding et al. 1986)and protein (Frasch et al. 1987) are expressed by allnuclei of the blastoderm in rapidly evolving patternssupports multiple functions for this gene product inthe metamerization process.

The patterns of expression for two of the genesinvolved in the activation of en (ftz and prd) areknown (Hafen et al. 1984; Carroll & Scott, 1985;Kilchherr et al. 1986) and they are, initially, four ormore cells wide, i.e. broader than the one-cell-widedomain in which en will be expressed; thus, some-thing other than the particular gene defines that stripeof cells in which en transcription is activated. In thecase of the even-numbered stripes, it is possible thatthe pattern of opa is offset from that of//z and, thus, acombination of both could define such row of cells.

Role of pair-rule loci in the activation of UbxIn the extended germ band, the pattern of Ubx ismodulated between different parasegments; two no-table features of this modulation are a rather homo-geneous distribution in PS6 and a conspicuous intra-parasegmental gradient of expression in PS7-12(Akam & Martinez-Arias, 1985; White & Lehmann,1986; see Results). The level and boundaries of Ubxexpression characterizing PS6 in the wild typedepend, critically, on one zygotic segmentation gene,ftz (Ingham & Martinez-Arias, 1986). The molecularbasis for the intraparasegmental gradient is unclear,but we have shown here that one component of this


1 2 3 Y 4 5 62n

-h, run, eve

2n+l

\///A WtKH [HUM bl opa •

odd •

— eve

prd

Fig. 8. Pathways for the activation of selector genes and engrailed during early embryogenesis. The expression of ftz ( • )is a key element in coordinating both processes (Ingham & Martinez-Arias, 1976). (a) First, the ftz gene is transcribedthroughout the prospective metameric germ band (Hafen et al. 1984). (b) Through the action of/;, run and eve (Howard& Ingham, 1986; Carroll & Scott, 1986), ftz expression is restricted to even-numbered parasegments (represented as 2non the right); the expression of ftz protein in these domains results (bl) in the selective activation of homeotic genes;Scr (•) in PS2, Antp (H) in PS4, and Ubx (II) in PS6. At the same time, engrailed (H) is activated through thecombinatorial activity of eve, opa and odd in the even-numbered and eve and prd in the odd-numbered parasegments.

system is the product of the en gene, which contrib-utes to the low levels of Ubx expression in P compart-ments.

Our data (summarized in Fig. 7) are consistent witha crucial role for ftz in the activation of Ubx in PS6.Indeed, since only mutations at ftz eliminate Ubxexpression in PS6 and none of the other mutationsstudied here causes comparable reductions in Ubxexpression, this supports a unique role for ftz in thisprocess. In the extended germ band, ftz~ embryoshave half the number of parasegmental units (Waki-moto et al. 1984) and our observations using molecu-lar probes (see also Ingham & Martinez-Arias, 1986),suggest that the identity of each parasegmental unit isnot composite, but resembles that of a single paraseg-mental unit. In the case of the 5+6 unit, it is more likethe 5 than the 6, and therefore we call it 5*. Theblastoderm fate map of ftz embryos has been changedand the odd-numbered-like parasegments have beenjuxtaposed; the germ band thus consists of the chainPS1*/PS3*/PS5*/PS7* etc., where the asterisk indi-cates that the broad parasegments are not exactly likePS1 or PS3 or PS5, although they resemble them.

Three mutants, h, run and eve, change the morpho-logical extent of the wild-type PS6. These threemutants are the only pair-rule loci known to affect theexpression of ftz (Howard & Ingham, 1986; Carroll &Scott, 1986; Harding et al. 1986) and, therefore, it islikely that their effects on Ubx are mediated throughftz. Although this is probably the case in h and runembryos, the effects of eve on Ubx expression aremore difficult to relate to a direct effect of thismutation on ftz.

Both h and run affect the width and stability of theftz pattern (Howard & Ingham, 1986; Carroll & Scott,1986) and, thus, Ubx expression in the PS6* of these

mutants can be interpreted as a direct correlate oftheir effect on ftz. For example, inh~ ,ftz is expressedin very broad bands at blastoderm. It has beensuggested that the role of ftz in establishing Ubxexpression in PS6 is to make available to the Ubxpromoter, a gap-gene product or a combination ofsuch products, which are broadly distributed aroundPS6 (Ingham & Martinez-Arias, 1986). In h~ em-bryos, the domain of availability for the gap-geneproducts would be enlarged through the enlarged ftzdomain and this would lead to the broader PS6*. Inthese embryos, the intrametameric modulation ofUbx posterior to PS6* reflects the effects of h on enand other segment polarity mutants and thus thecorresponding effects of these on the modulation ofUbx expression. Indeed, as in the wild type, a directcorrelation can be established between the low levelsof Ubx expression and the position and width of theen stripes in pair-rule mutants (Fig. 7). In the case ofrun, the width of PS6 can similarly be related to thepattern of ftz in the blastoderm of this mutant, wherethe third ftz stripe is narrow and weak (Carroll &Scott, 1986; P. Ingham, personal communication).Consequently, the gap-gene products are available tothe Ubx promoter in a smaller than wild-type domainand thus results in a more narrow PS6. Also, in thiscase, there is a correlation between the effects of runon en and other segment polarity loci and the modu-lation of Ubx expression, with low levels of Ubx in theputative regions of en expression.

The effects of eve on Ubx are more difficult to relateto a simple effect of these mutations on ftz. In eve~blastoderms, the pattern of ftz is similar to wild type,the main difference being its stability (Carroll &Scott, 1986; Harding et al. 1986). Thus, the extent ofPS6-like label in the extended germ band of eve~


embryos cannot be attributed, as in the case of runand A, to a change in the spatial extent of ftzexpression at blastoderm. In the extended germ bandof eve" embryos, the expression of Ubx in PS6resembles that of h~ embryos although compoundedwith striking heterogeneities (Fig. 6B). One possibleexplanation is that the eve product, by contrast withthe ftz product, inhibits the availability of the Ubxpromoter to the gap-gene products. Its absence,therefore, would lead to a spread of high-level Ubxexpression.

The redesign of the body plan that takes place inftz~, eve", run~ and h~ embryos does not take placein other mutant embryos. In them, the alterations ofUbx expression seem to be mediated primarily by theabsence of engrailed and other segment polarity loci.Although morphologically there are half the numberof parasegments, there are a wild-type number of Ubxmetameres, and only half of them show defects. Thisis most clearly seen in the effects of prd and opa onthe wild-type patterns of PS5 and PS6. Although, inboth cases, the embryo is malpartitioned, the firstUbx metamere is present as in wild type and anteriorto it there is label in a PS5 pattern. In these mutants,the most obvious defect is increased Ubx expressionin the presumptive P compartments affected by themutant. Additional small alterations in the Ubxpattern result from absence of other segment polarityproducts (A.M.A., unpublished observations). Insummary, the class I mutants do not affect theestablishment of Ubx expression, but its modulationvia segment polarity loci.

The function of pair-rule lociAlthough on the basis of the phenotypes the pair-ruleclass appears as a unified group of loci, molecularanalysis of various mutants has revealed an underly-ing heterogeneity (Howard & Ingham, 1986; Carroll& Scott, 1986; Harding et al. 1986; Ingham & Marti-nez-Arias, 1986; this work). There are currently twoviews as to the function and mode of action of theseloci: a combinatorial model (Gergen et al. 1986) and ahierarchical model (Howard & Ingham, 1986;Ingham et al. 1986; Ingham & Martinez-Arias, 1986).In the combinatorial model, the pattern deletionscharacteristic of the mutants reflect local require-ments for the different genes. Thus, the overlappingactivity of different pair-rule loci generates a per-iodic, one-cell wide, code of identities; pair-ruleproducts contributing to this code as an equivalencegroup. A different view suggests a hierarchy; differ-ent loci having different functions and the differentdeletions reflecting the different molecular functionsof the elements and not, directly, their spatial re-quirements. Our results indicate that elements ofboth models are involved and highlight the need for a

combinatorial component, particularly in the acti-vation of en by the secondary pair rule genes (seeFig. 8).

We believe that the function of the pair-rule loci isto establish parasegments (Ingham & Martinez-Arias, 1986). They do so in two steps (Fig. 8): first,generating a prepattern of primary pair-rule prod-ucts, like h and run, which serves to pattern thesecondary pair-rule products and ftz (Howard &Ingham, 1986; Ingham & Martinez-Arias, 1986). Thepattern of the latter leads to the definition of paraseg-ments and the endowment of unique identities to theeven-numbered ones. Interactions between second-ary pair rules, probably refine this pattern and serveto activate en and other segment polarity genes whichwill remain stably expressed in the epidermisthroughout embryogenesis.

We want to thank S. DiNardo for sharing unpublishedresults, P. Ingham and our colleagues at the MRC Labora-tory of Molecular Biology for discussions, in particular P.Lawrence for stimulating the experiments with engrailed.A.M.-A. was supported during part of this work by a long-term EMBO fellowship. R.A.H.W. is supported by theMRC and the Nuffield Foundation.

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(Accepted 9 October 1987)

* Note added in proof

To establish the registration of Ubx expression in en'embryos, we used double labelling for Ubx and y3-galactosidase in Df(2R)enB embryos, containing /?-galactosidase under ftz promoter control (Hiromi etal. 1985). We confirmed that the anterior boundary ofUbx expression is coincident with the anterior limit offtz expression marking the PS5/ boundary (unpub-lished observations).

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ultrabithorax and engrailed drosophila for segmentation...

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