Regulation of Two Pair-Rule Stripes by a Single Enhancer in theDrosophilaEmbryo

DEVELOPMENTAL BIOLOGY 175, 314–324 (1996)ARTICLE NO. 0117

Regulation of Two Pair-Rule Stripes by a SingleEnhancer in the Drosophila Embryo

Stephen Small,* Adrienne Blair,†,1 and Michael Levine†*Department of Biology, New York University, 100 Washington Square East, New York,New York 10003; and †Department of Biology, Center for Molecular Genetics, Pacific Hall,9500 Gilman Drive, University of California, San Diego, La Jolla, California 92093-0347

Previous studies on the regulation of the segmentation gene even-skipped (eve) have centered on the transcription of stripe2. Here, we characterize another enhancer module contained within the complex eve promoter that directs expression ofstripes 3 and 7. This enhancer is Ç500 bp in length and maps Ç3.3 kb upstream of the transcription start site. The stripe3 / 7 enhancer appears to be regulated by one or more ubiquitously distributed activators, including components of a JAK-Stat pathway. The two-stripe pattern results via multiple tiers of repressors which delimit this ubiquitous activation. Thezinc finger repressor hunchback appears to be responsible for establishing the anterior border of stripe 3 and the posteriorborder of stripe 7. knirps, a member of the nuclear receptor family of transcription factors, appears to establish the posteriorborder of stripe 3 and the anterior border of stripe 7. Activator and repressor proteins bind in vitro to several sites withinthe enhancer. These findings suggest a general model for the regulation of segmentation stripes, whereby enhancers integratepositional information provided by broadly distributed activators and spatially restricted repressors. q 1996 Academic Press, Inc.

INTRODUCTION and P-transformation experiments (Stanojevic et al., 1991;Small et al., 1992, 1993; Arnosti et al., 1996) has led to adetailed model of stripe 2 regulation. The most salient fea-Enhancers integrate both positive and negative regulatoryture of this model is that the stripe borders are establishedinformation to direct sharp, on/off patterns of gene expres-by transcriptional repressors. The maternal bicoid (bcd) gra-sion in the Drosophila embryo. Such enhancers, or regula-dient triggers the expression of hunchback (hb); the twotory modules, are typically 300–500 bp in length and con-proteins work synergistically to activate the stripe 2 en-tain tightly clustered binding sites for both transcriptionalhancer (Small et al., 1991). bcd also coordinates the expres-activators and repressors (reviewed by Ip et al., 1992; Jacklesion of two repressors, giant (gt; Kraut and Levine, 1991;et al., 1992). Modular promoters contain a series of en-Eldon and Pirrotta, 1991) and Kruppel (Kr; Hoch et al., 1990),hancers that can work independently of one another to gen-which define the anterior and posterior stripe borders, re-erate composite patterns of gene expression (Goto et al.,spectively (Stanojevic et al., 1991; Small et al., 1992). Direct1989; Harding et al., 1989; Howard and Struhl, 1990; Riddi-binding of the identified activator and repressor proteins tohough and Ish-Horowciz, 1991; Edgar et al., 1994). For ex-sites in the enhancer is thought to be required for regulationample, the segmentation gene even-skipped (eve) is ex-of stripe 2. The present study was undertaken to determinepressed in a series of seven transverse stripes in precellularwhether these general rules also apply to the regulation ofembryos (Frasch et al., 1987; Macdonald et al., 1986). Theother eve stripes.eve promoter contains a series of enhancers that control the

Previous promoter fusion studies and genetic analysesexpression of individual stripes (Goto et al., 1989; Hardingprovide a framework for the characterization of stripe 3et al., 1989; Stanojevic et al., 1991; Small et al., 1992, 1993).regulation (Frasch and Levine, 1987; Goto et al., 1989; SmallThe best characterized pair-rule enhancer controls the ex-et al., 1993). eve–lacZ fusion genes containing the first 8 kbpression of eve stripe 2.of the eve promoter region are expressed within the limits ofA combination of DNA binding assays (Stanojevic et al.,stripes 2, 3, and 7 (Goto et al., 1989; Harding et al., 1989).1989), transient cotransfection assays (Small et al., 1991),The eve stripe 2 enhancer is located between 01.6 and01.1kb upstream of the transcription start site. A 900-bp region,located between 03.8 and 02.9 kb, is essential for stripe 31 Present address: Department of Biology, 405 Hilgard Ave, Uni-

versity of California, Los Angeles, Los Angeles, CA 90036. expression. eve–lacZ fusion genes that contain deletions in

314

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315Regulation of Two Pair-Rule Stripes by a Single Enhancer

this interval show defective patterns of stripe 3 expression sites for a Drosophila Stat protein (D-Stat), which is ubiqui-tously distributed in the early embryo, and may be involved(Goto et al., 1989; Harding et al., 1989). A 500-bp sequence,

located between 03.8 and 03.3 kb, may be sufficient for in activation of the enhancer (Yan et al., 1996). These stud-ies suggest a general model for the regulation of pair-rulestripe 3 expression (Small et al., 1993). eve–lacZ fusion

genes containing this 500-bp sequence, along with the 500- stripes, whereby positional information provided by broadlydistributed activators and localized repressors is integratedbp stripe 2 enhancer, are expressed within the limits of

stripes 2, 3, and 7. However, if the two enhancers are di- by modular enhancers that direct sharp on/off borders ofgene expression.rectly attached without being separated by a ‘‘spacer’’ se-

quence, then either stripe 2 or stripe 3 expression is defec-tive, depending on the orientation of the two enhancers(Small et al., 1993). A normal staining pattern is restored MATERIALS AND METHODSwhen the two enhancers are separated by a 160-bp spacersequence derived from the native eve promoter region. P-Element Constructs and TransformationsThese observations prompted the proposal that the tran-

The eve –lacZ gene fusions used in this study were constructedscriptional repressors which define the stripe 2 and stripeby fusing various fragments from the regulatory region of the eve

3 borders function over short distances, within the limits gene upstream of the PstI site that lies 42 bp upstream of the eveof the enhancer to which they are bound (Small et al., 1993). transcription initiation site. All gene fusions contain the eve TATAIf the two enhancers are coupled without a spacer sequence, box and basal promoter, as well as the 100-bp eve 5* untranslatedthen it is possible for a given repressor to influence both leader, and the first 22 codons of the eve protein-coding sequenceenhancers. fused to codon 5 of the lacZ coding sequence (Lawrence et al.,

1987). The promoter lacZ fusions were cloned into the P-elementGenetic studies suggest that eve stripes 2 and 3 are regu-transformation vector pCaSpeR (Thummel et al., 1988), which con-lated by different sets of genes (Frasch and Levine, 1987).tains the white gene as a selectable marker. The P-element vectorsFor example, mutations in the gap gene gt cause a severewere introduced into the w67 white0 strain by injection (Rubin andanterior expansion of the stripe 2 pattern, but have no effectSpradling, 1982) using the helper plasmid D 2,3. For each construct,on stripe 3 expression. Mutations in the gap genes hb andbetween four and seven lines were analyzed for lacZ expression in

knirps (kni) appear to expand the initial limits of the stripe 3 wild-type embryos. For analysis of the 500-bp stripe 3 / 7 enhancerpattern, suggesting that one or both encoded proteins might in various mutants, the seven lines generated were balanced torepress the stripe 3 enhancer. kni mutants have no effect determine which chromosome contained the transgene. Two sepa-on stripe 2 expression, while hb functions as an activator rate lines were tested in each experiment.of this stripe (Small et al., 1991). While kni and hb wereidentified as potential repressors of stripe 3, the previous

Analysis of Transgene Expression in Mutantgenetic studies failed to identify putative activators. How-Embryosever, recent studies have implicated a putative JAK kinase,

hopscotch (hop), in the activation of stripe 3 (Binari and The mutant alleles used in this study were: bicoid, bcdE1; nanos:Perrimon, 1994). Embryos lacking maternally deposited hop nosL7; torso, torPM51; torsolike, tslD89; hunchback, hb14F21; Kruppel,products exhibit segmentation defects that include a reduc- Kr1, Kr2; knirps, kniIID48; giant, gtYA82; tailless, tllPGX; huckebein,tion in the expression levels of eve stripe 3. This observation hkb2; even-skipped, Df(2R)eve1.27; hairy, hK1. Most of the stocks

were obtained from the national stock centers. For the maternalhas led to the proposal that the stripe 3 enhancer may bemutants, embryos were collected from crosses between males bear-activated by one or more Drosophila Stat proteins whiching the 500-bp stripe 3 / 7– lacZ transgene and virgin femalesare targets of JAK kinase activity (reviewed by Darnell ethomozygous for various mutant alleles. As expected, all progenyal., 1994).from these crosses exhibited the mutant phenotypes.Here we present a detailed characterization of a 500-bp

For zygotic mutants, embryos were collected from inter seenhancer element that regulates the expression of stripe 3.crosses among flies that contained a single copy of a given mutation

This enhancer also drives the expression of stripe 7, and and the transgene. For combinations between maternal and zygoticthus will be referred to as the stripe 3 / 7 enhancer. eve– mutants, males carrying the transgene and a single copy of thelacZ fusion genes containing this enhancer were examined zygotic mutation were crossed to virgin females homozygous forin a variety of segmentation mutants. These results suggest the maternal mutation, which also contained a single copy of the

zygotic mutation. In most cases, the mutant embryos were identi-the following model for the regulation of two stripes by afied by the observed change in the lacZ expression pattern, whichsingle enhancer. Activation is mediated by a ubiquitiouslyoccurred at the expected frequency. In some cases, especially whendistributed activator(s), which can switch on the enhancerno effect was observed on the lacZ expression pattern, the embryosalong the entire length of the early embryo. The two-stripewere double-stained with an anti-eve antibody to positively iden-pattern is defined by multiple tiers of repression mediatedtify mutant embryos.by the terminal system, the anterior morphogen bcd, and

Embryos were collected for 2 hr, and allowed to age an additionalthe gap proteins hb and kni. DNA binding assays suggest 2 hr before fixing and staining. Hybridizations using an anti-sensethat at least some of these regulatory interactions are direct. lacZ RNA probe were performed exactly as previously describedThere are 5 kni protein binding sites in the 500-bp stripe 3 (Jiang et al., 1991). The double staining protocol for simultaneous/ 7 enhancer. Previous studies identified 11 hb sites. These detection of eve protein and lacZ RNA was previously described

(Small et al., 1992).16 putative repressor sites are closely linked to two binding

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316 Small, Blair, and Levine

FIG. 1. Identification of a minimal stripe 3 / 7 enhancer. (A) Summary of the eve promoter region. Previous studies have identifiedthree cis regulatory elements (black rectangles above the line; Goto et al., 1989; Harding et al., 1989). The autoregulatory element, locatedbetween 05.9 and 05.2 kb upstream of the transcription start site, is required for the maintenance and refinement of the eve expressionpattern during gastrulation and germ band elongation (Jiang et al., 1991). The 480-bp stripe 2 enhancer is located between 01.6 and 01.1kb (Small et al., 1992). The limits of the stripe 3 / 7 enhancer, which initiates the expression of stripe 3 and a weak stripe 7, were definedin this study, as described below. Sequences that contribute to wild-type levels of stripe 7 expression seem to be scattered over a regionat least 2 kb in length that includes the stripe 3 enhancer and the stripe 2 enhancer (gray rectangle below the line). (B) Summary of theeve promoter sequences used to identify the minimal stripe 3 / 7 enhancer. The regions indicated by the horizontal lines were attachedto a minimal eve– lacZ fusion gene and analyzed in transgenic embryos via P-transformation. A 2.3-kb region (fragment ‘‘A’’) extendingfrom a PstI site at 05.2 kb to an AflII site at 02.9 kb mediates expression of the lacZ fusion gene within the limits of stripes 3 and 7.Similar patterns were obtained with an 800-bp fragment that extends from a BamHI site at 03.8 kb to the AflII site at 02.9 kb (fragment‘‘B’’) and a 500-bp fragment that extends from the BamHI site at 03.8 kb to a SacI site at 03.3 kb (fragment ‘‘C’’). Smaller fragments, Dand E, gave markedly weaker patterns of expression, but did not uncouple stripe 3 expression from stripe 7 expression.

Protein Expression and DNA Binding Assays Identification of a Minimal Enhancer That DrivesExpression of eve Stripes 3 and 7

A glutathione-S transferase–knirps (GST–kni) expression con-struct was prepared by blunt-ending and ligating a 1090-bp NruI– Several DNA fragments from the interval from 03.8 toSacI fragment from the kni cDNA (Nauber et al., 1988) into the 02.9 kb were placed upstream of a minimal eve–lacZ fusionXhoI site of the pGEX-KG vector. This fragment encodes the first

gene (Fig. 1B), which contains the proximal-most 42 bp of342 amino acids of the kni protein, including the two putative zincthe eve promoter, as well as the entire 100-bp untranslatedfinger domains that are involved in DNA-binding (Hoch et al.,leader sequence and the first 22 codons of the eve protein1990). The fusion protein was expressed in bacteria according tocoding region (Lawrence et al., 1987). lacZ fusion genesstandard methods and purified by column chromatography with

glutathione–Sepharose 4B (Pharmacia Biotech, Uppsala, Sweden) were inserted into the CaSpeR P-transformation vector andusing methods suggested by the manufacturer. DNase I foot- expressed in transgenic embryos following P-element-medi-printing assays were performed exactly as previously described ated germ line transfer. Expression patterns were visualized(Hoey and Levine, 1988; Ip et al., 1992). by hybridizing transgenic embryos with a digoxigenin-la-

beled lacZ antisense RNA probe (Tautz and Peifle, 1989;Jiang et al., 1991).

RESULTS A 500-bp BamHI–SacI fragment, located between 03.8and 03.3 kb, directs expression of a strong stripe near thecenter of the embryo, and a weaker stripe in posterior re-The first 8 kb of the eve promoter region controls thegions (Fig. 2C). Colocalization of endogenous eve proteininitiation of stripes 2, 3, and 7 in early embryos (Goto etand lacZ RNA indicates that the stripes produced by theal., 1989; Harding et al., 1989; summarized in Fig. 1A). Ittransgene coincide with the endogenous stripes 3 and 7 (Fig.also contains an autoregulatory element that is essential2D). The expression of stripe 7 is considerably weaker thanfor the refinement and maintenance of the eve expressionstripe 3, suggesting that other regions of the promoter arepattern during gastrulation and germ band elongation (Jiangrequired for optimal stripe 7 expression (Fig. 1A). This iset al., 1991). Previous studies have implicated the 900-bpconsistent with previous promoter fusion assays, whichinterval between 03.8 and 02.9 kb as playing an essentialdemonstrated that other regions of the eve promoter canrole in the regulation of eve stripe 3 (Goto et al., 1989;

Harding et al., 1989). also direct weak expression of stripe 7 (Goto et al., 1989;


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Harding et al., 1989). Thus, sequences that control stripe 7 Mutations in (nanos) nos resulted in a broad band of stain-ing in abdominal regions of the embryo (Fig. 3D). In princi-seem to be scattered throughout a region that spans at least

2 kb of contiguous sequence. An essentially identical pat- ple, this broad staining pattern could be caused by an expan-sion of the stripe 7 response toward the anterior and/or antern was obtained with a 900-bp fragment spanning the en-

tire region from03.8 to02.9 kb (Small et al., 1993; data not expansion of the stripe 3 response toward the posterior.Thus, it is possible that nos mutants cause the removal ofshown). The weak, sporadic staining observed in anterior,

dorsal regions is due to vector sequences (Small et al., 1992; a repressor(s) that sets the posterior border of eve stripe 3,the anterior border of stripe 7, or both (see below).Gray et al., 1994). Ectopic staining at this position is ob-

served with other fusion genes inserted in this vector, in- In summary, the analysis of maternal mutants suggeststhat bcd and tor directly or indirectly repress the stripe 3 /cluding stripe 2–lacZ transgenes (e.g., Fig. 2B).

Subfragments from this 500-bp regulatory element direct 7 enhancer and participate in the formation of the anteriorborder of eve stripe 3. In contrast, nos may be required forsubstantially weaker staining patterns (Fig. 1B; data not

shown). Neither fragment D nor E uncouples the expression repression between the posterior border of stripe 3 and theanterior border of stripe 7.of stripes 3 and 7. Transgenic lines carrying these fragments

either express both stripes, albeit weakly, or neither stripe.In the following analyses, we examine the regulation of the Zygotic Regulation‘‘full-length’’ 500-bp eve stripe 3 / 7 enhancer.

To identify zygotic genes involved in the regulation ofstripes 3 and 7, the expression of the 500-bp stripe 3 / 7–

Maternal Regulation of the Stripe 3 / 7 Enhancer lacZ transgene was examined in a number of gap mutants,pair-rule mutants, and mutant combinations. Mutations inAs a first step towards identifying potential trans-acting

factors that control the expression of stripes 3 and 7, the three gap genes, tailless (tll), kni, and hb, caused dramaticchanges in the expression of the transgene at the positionfusion gene was expressed in mutants that disrupt each of

the three maternal anteroposterior determinants (reviewed of stripe 3, stripe 7, or both. Transgene expression was alsoexamined in embryos mutant for other gap genes includingby St. Johnston and Nusslein-Volhard, 1992). Mutations in

the torso (tor) gene abolish stripe 7 expression, but have no Kr, gt, and huckebein (hkb), and the pair-rule genes eveand hairy (h) (data not shown). The two-stripe pattern waseffect on stripe 3 (Fig. 3B; compare with 3A). This result

suggests that the two stripes might be regulated by distinct unaffected in each of these mutants, suggesting that thesegenes are not important for the regulation of this enhancer.activators. Alternatively, a common activator might be in-

dependently modulated by the torso signaling pathway at Stripe 7 expression is abolished in embryos that lack tll/

function (Fig. 4D), similar to the situation observed for torthe embryonic termini (see Discussion).Expanded patterns of expression were observed in mu- mutants (Fig. 3B). Stripe 3 expression is unaffected in this

background. This loss of stripe 7 is consistent with thetants lacking bcd/ function (Fig. 3C). bcd mutants causedan anterior expansion and shift in the stripe 3 pattern; stripe previous genetic analysis of eve regulation (Frasch and Lev-

ine, 1987).7 staining was reduced. This observation suggests that bcdeither directly or indirectly represses stripe 3 expression in There is a dramatic derepression of the staining pattern in

the presumptive abdomen of kni mutants (Fig. 4B; compareanterior regions, and thereby helps establish the anteriorstripe border. It is not clear why the level of stripe 7 is with Fig. 4A), similar to the situation observed in nos em-

bryos (Fig. 3D). This similarity is not surprising since previ-reduced in bcd mutants.The expanded stripe 3 pattern in bcd mutants does not ous studies have shown that the loss of nos gene function

results in ectopic expression of hb in abdominal regions,extend into the anterior-most regions of the embryo (Fig.3C). This might result from the tor pathway, which is active and a concomitant loss of kni expression (Hulskamp et al.,

1990; Struhl et al., 1992). As discussed previously, it is con-at both the anterior and posterior poles (reviewed by St.Johnston and Nusslein-Volhard, 1992; Perrimon, 1994). Evi- ceivable that nos mutations cause the removal of a repres-

sor(s) that establishes the posterior border of stripe 3, thedence for this possibility was obtained by analyzing thestripe 3 / 7– lacZ fusion gene in embryos derived from anterior border of stripe 7, or both. The continuous lacZ

staining staining observed in kni embryos suggests that knimutant females lacking both bcd/ and tor/ function (Fig.3E). The double mutants show a severe expansion of the protein may function as a repressor to form both borders.

As discussed above, the gap gene tll is required for stripestripe 3 pattern, whereby staining extends to the anteriortip of the embryo. Stripe 7 expression is lost due to the 7 expression driven by the endogenous eve gene, and the

stripe 3 / 7– lacZ transgene. To test whether tll/ functionabsence of tor/ activity (see Fig. 3B). In these experiments,the bcd and tor pathways were simultaneously blocked by contributes to the staining pattern in kni mutants, double

mutants were analyzed which lack both kni/ and tor/ activ-using a recombinant chromosome containing mutations inthe bcd and torso-like (tsl) genes (Struhl et al., 1992). tsl is ity (Fig. 4E). tor mutants contain no tll/ activity. Surpris-

ingly, the staining pattern in tor kni double mutants encom-required, along with the product of the trunk (trk) gene, forthe activation of the tor receptor at the anterior and poste- passed the entire posterior half of the embryo, extending all

the way to the posterior pole. This abnormal staining pat-rior poles (Stevens et al., 1990; Savant-Bhousale and Mon-tell, 1994; Martin et al. 1994; Casanova et al., 1995). tern demonstrates that the stripe 3 / 7 enhancer can be


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FIG. 2. The eve stripe 3 / 7 enhancer directs an authentic pattern of expression. Embryos are oriented with anterior to the left anddorsal up. Staining patterns were visualized by in situ hybridization using digoxigenin-labeled antisense RNA probes. (A) Wild-type,cellularizing embryo hybridized with an eve antisense RNA probe. The staining pattern consists of seven transverse stripes along theanteroposterior axis. (B) Transgenic, cellularized embryo carrying a minimal eve stripe 2–lacZ fusion gene. Staining was visualized witha lacZ antisense RNA probe to detect the expression mediated by the transgene. Staining is observed within the limits of stripe 2. Ananterior, dorsal patch of expression is due to vector sequences in the P-transposon used in this analysis (Small et al., 1992). (C) Transgenic,cellularized embryo carrying the 500-bp eve stripe 3 / 7 enhancer attached to the eve– lacZ fusion gene. Staining is observed in central

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FIG. 4. Expression of the stripe 3 / 7 enhancer in zygotic segmentation mutants. Embryos are oriented with anterior to the left and dorsalup. The 500-bp stripe 3 / 7– lacZ transgene was introduced into various genetic backgrounds (see Materials and Methods). The staining patternswere visualized by in situ hybridization using a lacZ antisense RNA probe. (A) Staining pattern in a wild-type, cellularizing embryo. (B) Stainingpattern in a kni embryo. A broad band of staining is observed in the presumptive abdomen, similar to that observed in nos mutants (Fig. 3D).(C) Staining pattern in mutants lacking the zygotic component of hb gene activity. There is an anterior shift and expansion of stripe 3, similarto that observed in bcd mutants (see Fig. 3C). In addition, stripe 7 staining is augmented and expanded toward posterior regions. (D) Stainingpattern in a tll embryo. Stripe 7 staining is abolished, while stripe 3 is unaffected. (E) Staining pattern in a tor kni double mutant. Continuousintense staining extends from stripe 3 to the posterior tip of the embryo. This pattern is probably generated by a ubiquitous activator, sincetor and tll mutants lack stripe 7 (Figs. 3B and 4D). (F) Staining pattern in tor kni hb triple mutants. As for (C) the hb mutant affects onlyzygotic activity. There is a dramatic expansion of the stripe 3 pattern, which extends from the posterior tip of the embryo to cephalic regionsnear the anterior pole. The residual repression observed in the anterior-most regions might be mediated by bcd target genes or bcd itself.

regions and near the posterior pole. As discussed above, the anterior patch of expression is due to vector sequences in the P-transposon.(D) Transgenic embryo that was double stained with anti-eve antibodies and a lacZ antisense RNA probe. The endogenous eve proteinis stained brown, while the RNA expressed by the stripe 3 / 7–lacZ transgene is blue. The two lacZ stripes are expressed within thelimits of the endogenous stripes 3 and 7.FIG. 3. Expression of the stripe 3 / 7 enhancer in maternal mutants. Embryos are oriented with anterior to the left and dorsal up. In allcases, males carrying the 500-bp stripe 3 / 7–lacZ transgene were mated with females homozygous for the indicated maternal mutations.Transgene staining patterns were visualized by in situ hybridization using a lacZ antisense RNA probe. (A) Staining pattern in a wild-typecellularizing embryo. Intense expression is observed within the limits of stripe 3. A somewhat weaker stripe 7 pattern is also observed (seeFig. 2C). (B) Staining pattern in an embryo derived from a tor female. Expression of stripe 7 is abolished, while the stripe 3 pattern appearsnormal. (C) Staining pattern in an embryo derived from a bcd female. There is an anterior expansion and shift of the stripe 3 pattern. Stripe 7staining appears reduced. (D) Staining pattern in a nos embryo. A broad band of staining is observed in the presumptive abdomen, which isreminiscent of the normal kni expression pattern. (E) Staining pattern in a bcd tsl double mutant. Expression is restricted to anterior regions.These mutants also exhibit a marked anterior shift in the kni expression pattern (data not shown).


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scriptional repression are required for establishing the two-stripe pattern directed by the stripe 3 / 7 enhancer. Thisconclusion is underscored by the analysis of triple mutants,which are completely devoid of tor/ and kni/ activities andalso lack the zygotic component of hb/ function (Fig. 4F).Continuous staining is observed throughout the posterior75% of the embryo, extending from presumptive cephalicregions to the posterior pole. The residual repression seenin the anterior-most regions is probably due to bcd, sinceremoving bcd and the terminal system expands the patternall the way to the anterior pole (Fig. 3E).

The simplest model that emerges from the genetic experi-ments is that eve stripe 3 is regulated by one or more ubiqui-tously distributed activators, while the anterior and poste-rior stripe borders are formed primarily by the hb and knirepressors, respectively. However, a previous study (Fraschand Levine, 1987) appears to be inconsistent with a role forkni in the specification of the posterior border of stripe 3(Reinitz and Sharp, 1995). The earlier analysis employedpolyclonal antibodies to visualize the distribution of eveprotein in various segmentation mutants. The study wascomplicated by the simultaneous detection of all seven eve

FIG. 5. kni is essential for establishing the posterior stripe 3 bor- stripes and by the lag between the time of appearance ofder. kni embryos are oriented with anterior to the left and dorsal RNAs and proteins. In fact, the use of histochemical in situup. The staining pattern was visualized by in situ hybridization hybridization assays (not available at the time of the earlierusing an eve antisense RNA probe. (A) eve pattern in a kni mutant work) is consistent with a role for the kni repressor in theat the midpoint of nuclear cleavage cycle 14. Stripes 1 and 2 appear specification of the posterior stripe 3 border (Fig. 5). Whennormal, but there is a broad band of staining, which encompasses

the transcription stripes are first formed during early phasesstripes 3 through 7. (B) The same as (A), except that the embryoof nuclear cleavage cycle 14, there is a continuous band ofis about 20 –30 min older and has completed cellularization. Thestaining extending from stripe 3 through stripe 7 in kniposterior stripe 3 border has partially formed, although stainingmutants (Fig. 5A). A posterior border appears at slightlypersists in the region between stripes 3 and 7.later stages, possibly due to pair-rule repressors which actdirectly or indirectly on the autoregulatory element (Fig.5B). These results suggest that kni plays an essential rolein the establishment of the posterior stripe border; theactivated in posterior regions even in the absense of tll/

function. We propose that this activation is mediated by a maintenance of the border might depend on repressors en-coded by other pair-rule genes such as runt or hairy (Paroushubiquitous factor, possibly the same factor that activates

eve stripe 3 (see Discussion). It is conceivable that this ubiq- et al., 1994; Tsai and Gergen, 1994).uitous factor acts with tll to initiate stripe 7 expression,but is normally repressed at the posterior pole of wild-type

The Stripe 3 / 7 Enhancer Contains kni-Bindingembryos by other components of the terminal system.SitesThe formation of the anterior stripe 3 border also appears

to involve multiple tiers of transcriptional repression. Mu- Previous studies have identified 11 hb-binding sites thatlie within the 500-bp stripe 3/ 7 enhancer (Stanojevic et al.,tants lacking zygotic hb/ function exhibit both an anterior

shift and expansion of stripe 3 (Fig. 4C). The posterior border 1989; summarized in Fig. 7). In this study, DNaseI footprintassays were performed to determine whether kni might di-of stripe 7 is also expanded. These results suggest that hb

either directly or indirectly defines the anterior stripe 3 rectly repress the stripe 3 enhancer. These experiments in-volved the use of a bacterial GST–kni fusion protein thatborder, as well as the posterior border of stripe 7. This latter

activity probably involves the posterior stripe of hb expres- contains the N-terminal 342 amino acid (AA) residues ofkni (Nauber et al., 1988; see Materials and Methods). Thission, which appears in response to the torso pathway (Mar-

golis et al., 1995). The anterior shift and expansion of stripe region includes both of the putative cys/cys zinc fingersthat mediate DNA binding (Hoch et al., 1990).3 in hb mutants is not quite as severe as that observed for

bcd mutants (Fig. 3C), suggesting that bcd might influence Binding assays were performed with two overlapping frag-ments from the 500-bp enhancer (fragment C, Fig. 1B) thatthe anterior stripe 3 border beyond its regulation of hb. bcd

is known to regulate the expression of additional putative directs expression of stripes 3 and 7 in transgenic embryos. Atotal of five kni-binding sites were identified, as shown inrepressors, such as gt and orthodenticle (otd) (Finkelstein et

al., 1990; Eldon and Pirrotta, 1991; Kraut and Levine, 1991). Fig. 6. These sites are all located within a 330-bp region thatencompasses the 5* two-thirds of the enhancer. The core rec-The preceding results suggest that multiple tiers of tran-


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FIG. 7. Factor binding sites in the stripe 3 / 7 enhancer. (A) Thecomplete sequence of the enhancer is presented. This region mapsbetween 03880 and 03373 bp upstream of the eve transcriptionstart site. Previous studies identified 11 hb-binding sites. These areindicated by the original nomenclature used by Stanojevic et al.(1989). The locations of the five kni-binding sites identified in Fig.6 are also shown. Two putative activator sites, DSB1 and DSB2(Yan et al., 1996), are located near the middle of the enhancer. (B)Summary of factor binding sites. The 16 putative hb and kni repres-sor sites are shown, along with the two D-Stat activator sites. Thisarrangement of binding sites, with closely linked transcriptionalFIG. 6. The eve stripe 3 / 7 enhancer contains kni-binding sites.activators and repressors, is similar to the organization of the eveA 330-bp BamHI–KpnI fragment, which contains the 5* two-thirdsstripe 2 enhancer (Small et al., 1992).of the stripe 3 enhancer, was labeled with 32P on each strand, incu-

bated with increasing amounts of a GST-knirps fusion protein, di-gested with DNaseI, and then fractionated on a polyacrylamide/urea gel. (A) Footprint pattern obtained on the coding strand of theDNA fragment. Three binding sites can be observed, as indicated The complete stripe 3/ 7 enhancer sequence is presentedto the right of the autoradiogram. Note DNase I hypersensitive in Fig. 7. As discussed above, it includes 11 hb-binding sitessites that flank the kni2 and kni3 sites. Lane ‘‘0’’ shows the DNaseI (Stanojevic et al., 1989) and 5 kni sites. Recent studies sug-digestion pattern without addition of the GST–knirps fusion pro- gest that optimal expression of this enhancer might dependtein. Lanes 1–3 contain twofold increases in the amount of the

on a putative janus tyrosine kinase (JAK), hop (Binari andfusion protein. The lane labeled ‘‘GA’’ contains the G /A reactionPerrimon, 1994). Embryos lacking maternal hop productsfrom a Maxam–Gilbert sequencing mix. (B) Footprint pattern ob-exhibit reduced levels of eve stripe 3 expression. In mamma-tained on the noncoding strand. Two binding sites (kni4 and kni5)lian systems, activation by JAKs results in the nuclear trans-can be observed. An additional weak kni-binding site that lies be-location of specific transcriptional activators, known astween these two sites is also visible. The autoradiogram is labeled

as in (A). (C) Alignment of the five knirps protein binding sites Stats (reviewed by Darnell et al., 1994). A Drosophila mem-identified within the stripe 3 / 7 enhancer. A consensus sequence ber of the Stat family (D-Stat) has been recently identifiedfor these five sites is shown below, which is somewhat similar to (Yan et al., 1996). After activation in cultured cells, D-Stata previous consensus sequence reported for kni (Hoch et al., 1992). binds to two potential activator sites (DSB1 and DSB2)

within the stripe 3 / 7 enhancer (Yan et al., 1996; Fig.7A). Thus, the eve stripe 3 / 7 enhancer contains tightlyognition sequences of these binding sites show some similar-

ity to a previously identified consensus sequence for kni-bind- clustered activator and repressor sites (Fig. 7B), similar tothe organization of a number of enhancers that are activeing sites: A-a/t-C-T-A-a/g-A-T-C (Fig. 6C; Hoch et al., 1990).


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in the early embryo such as the eve stripe 2 enhancer (Small stripe 5. The central Kr pattern extends from the posteriorborder of stripe 2 to the anterior border of stripe 5, while theet al., 1992) and the rhomboid NEE (Ip et al., 1992).abdominal band of gt expression extends from the posteriorborder of stripe 5 to the anterior border of stripe 7 (Stano-jevic et al., 1989; Kraut and Levine, 1991). It is conceivableDISCUSSIONthat ubiquitous expression of the stripe 2 activators, bcdand hb, would produce two stripes (2 and 5), similar to theWe have characterized a minimal enhancer that initiates

the expression of eve stripes 3 and 7 in precellular embryos. situation observed for the stripe 3 / 7 enhancer.Evidence is presented that this enhancer is regulated byone or more ubiquitously distributed activators and the gap

Ubiquitous Activationprotein tll. The two stripes are formed by repression medi-ated by the terminal system, the maternal morphogen bcd, The identities of activators that regulate gene expressionand the gap proteins hb and kni. Identified activators and in middle and posterior regions of the embryo have beenrepressors bind closely linked sites within the stripe 3 / 7 elusive. Recent studies suggest that the homeodomain pro-enhancer, suggesting that the borders of both stripes are tein caudal (cad) might be important for establishing seg-formed through a short-range mechanism of transcriptional mentation gene expression in the presumptive abdomenrepression such as competition or quenching (reviewed by (Rivera-Pomar et al., 1995). The genetic analyses presentedLevine and Manley, 1989; Johnson, 1995). Thus it appears here are consistent with the notion that the stripe 3 / 7that the general rules governing the regulation of eve stripe enhancer is regulated by one or more ubiquitously distrib-2 also apply to stripes 3 and 7. In all three cases, repressors uted activators. Most notably, triple mutants lacking theplay the decisive role in defining the stripe borders. Also, hb and kni repressors, as well as the tor pathway, exhibiteach stripe enhancer integrates both positive and negative continous expression that extends from presumptive ce-regulatory information to generate sharp borders of gene phalic regions to the posterior tip of the embryo (Fig. 4F).expression. As discussed above, recent studies suggest that a JAK-Stat

system (hop-D-Stat) is required for optimal expression ofstripe 3 (Binari and Perrimon, 1994). The identification ofA Common Strategy for Stripesa Drosophila Stat homolog that binds specifically to the

The regulation of the stripe 3/ 7 enhancer is summarized stripe 3 / 7 enhancer supports this hypothesis (Yan et al.,in Fig. 8. The key feature of this model is that spatially 1996). However, it is conceivable that the stripe 3 / 7 en-localized repressors define the anterior and posterior bordersof stripes 3 and 7. For example, the abdominal band of kniexpression extends from the posterior border of eve stripe3 to the anterior border of stripe 7 in precellular embryos(Kraut and Levine, 1991; Pankratz et al., 1992). Moreover,we have shown that the stripe 3 / 7 enhancer containsfive kni-binding sites. These observations suggest that knifunctions as a repressor to directly define the posterior bor-der of stripe 3 and the anterior border of stripe 7. Similararguments pertain to hb (Stanojevic et al., 1989; Pankratzet al., 1992). The preponderance of the evidence suggeststhat hb might directly define the anterior border of stripe 3and the posterior border of stripe 7 (Margolis et al., 1995).

FIG. 8. A model for transcriptional regulation of the eve stripe 3The demonstration that repression mediated by gap pro- / 7 enhancer. A schematic representation of a cellularizing embryoteins forms the stripe 3 and stripe 7 borders is reminiscent is oriented with anterior to the left and dorsal up. Genetic studies,of the situation previously described for the eve stripe 2 promoter fusion assays, and DNA binding experiments suggest thatenhancer. The stripe 2 borders are defined by the gap repres- the stripe 3 / 7 enhancer is regulated by one or more ubiquitouslysors gt and Kr (reviewed by Small and Levine, 1992). How- distributed activators, including D-Stat (top). Stripe 7 expression

also depends on tll. The anterior and posterior borders of stripe 3ever, the two enhancers are distinct in that the stripe 3 /are established by the hb and kni repressors, respectively. The same7 enhancer generates two stripes (3 and 7), while the striperepressors also appear to form the borders of stripe 7. Since hb and2 enhancer primarily directs just a single stripe (althoughkni proteins bind to specific sites in the enhancer, these repressivethere is occasionally a very weak and variable stripe 7; seeinteractions may be direct. Genetic experiments suggest that theSmall et al., 1992). This difference, one vs two stripes, canstripe 3 / 7 enhancer is also regulated by additional repressors,be explained on the basis of the spatial distribution of thewhich probably act indirectly. For example, tor/ function is re-

stripe activators. bcd and hb are the primary activators of quired for repression in the posterior-most regions of the embryo.stripe 2; both proteins are spatially restricted to the anterior Similarly, tor/ and bcd/ functions are required for repression inhalf of precellular embryos (Small et al., 1991). In principle, the anterior-most regions. Presumably, bcd and tor regulate targetthe combined limits of the gt and Kr repressors could permit genes that function as sequence-specific repressors of the stripe 3

/ 7 enhancer at the anterior and posterior poles.expression of the stripe 2 enhancer within the limits of


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hancer is regulated by additional activators. For example, might quench nearby bcd activators that are already boundto DNA (Arnosti et al., 1996).the tll protein might participate in the activation of stripe

7 since this stripe is essentially abolished in tll mutants(Frasch and Levine, 1987; Kraut and Levine, 1991).

ACKNOWLEDGMENTS

Multiple Tiers of Repression We are grateful to Riqiang Yan and James Darnell for sharingresults prior to publication. We thank Rachel Kraut, Gary Struhl,Both the stripe 2 and stripe 3 / 7 enhancers appear to beJon Margolis, and Jessica Treisman for mutant fly stocks and/orregulated by multiple repressors. For example, the anteriorDNA constructs, Manfred Frasch for the anti-eve antibody, Ron

border of stripe 3 may be established by a combination of Park and Tim Hoey for advice on DNase I footprinting, and Rajeshrepressors under the control of the bcd and tor patterning Vakani for assistance in resequencing the stripe 3 / 7 enhancer.systems. In zygotic hb embryos, stripe 3 expression shifts We also thank past and present members of the Levine lab, espe-and expands into anterior regions (Fig. 4C). In bcd mutants, cially John Reinitz, David Arnosti, and Susan Gray for stimulatingwhich lack both bcd and zygotic hb products, there is a discussions. This work was supported by a grant from the NIH

(GM34431) to M.L. and by a Shannon Award from the NIHmore dramatic expansion into anterior regions, but the pat-(GM51946) to S.S.tern does not extend all the way to the anterior pole (Fig.

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Regulation of Two Pair-Rule Stripes by a Single Enhancer in theDrosophilaEmbryo