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White as a reporter gene to detect transcriptional silencers specifying position-specific gene expression during Drosophila melanogaster eye development.


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Copyright 0 1995 hy the Genetics Society of America




Reporter Gene to Detect Transcriptional Silencers Specifying

Position-Specific Gene Expression During

Drosophila melanogaster Eye Development

Y. Henry Sun,*9t Ching-Jeng T ~ a i , ~ M. M. Green,* Ju-Lan Chao,* Chan Tze Yu,+ Thomas J. Jaw: Jih-Yun Yeh* and Viatcheslav




*Institute of Molecular Biology, Academia Sinica, Nankang Taipei, tGraduate Institute of Genetics, National Yang Ming University, Shipai Taipei, Taiwan, Republic of China, $Section of Molecular and Cellular Biology, Division of Biological Sciences, University of California, Davis, California 9561 6, and $Institute of Molecular Biology and Biotechnology-Forth, GR-71110, Heraklion, Crete, Greece

Manuscript received April 4, 1995 Accepted for publication August 4, 1995


The white+ gene was used as a reporter to detect transcriptional silencer activity in the Drosophila genome. Changes in the spatial expression pattern of white were scored in the adult eye as nonuniform patterns of pigmentation. Thirty-six independent P[lacw] transposant lines were collected. These repre- sent 12 distinct pigmentation patterns and probably 21 loci. The spatial pigmentation pattern is due to &acting suppression of white+ expression, and the suppression probably depends on cell position rather than cell type. The mechanism of suppression differs from inactivation by heterochromatin. In addition, activation of lac2 in P[lacw] occurs also in specific patterns in imaginal discs and embryos in many of the lines. The expression patterns of white’ and lac2 may reflect the activity of regulatory elements belonging to an endogenous gene near each P[lacw] insertion site. We speculate that these putative POSE (=sitionspecific expression) genes may have a role in pattern formation of the eye as well as other imaginal structures. Three of the loci identified are optomotur-blind, engrailed and invected. teashirt is also implicated as a candidate gene. We propose that this “silencer trap” may be an efficient way of

identifying genes involved in imaginal pattern formation.


ENES specifying positional information may be ex- pected to have spatially restricted expression dur- ing development. For spatial regulation of gene expres- sion, negative regulatory interactions are often involved in setting the spatial boundaries of gene expression (see

DAVIDSON 1993). For example, negative regulation by

hunchback and tailless sets the boundaries of the poste- rior expression domain of knirps in the embryo and is mediated via multiple binding sites for these negative transcription factors (PANKRATZ et al. 1992). The ventral repression of decapentaplegtc ( d p p ) , zerknullt (Zen) and

tolloid ( t l d ) is due to binding of the dorsal morphogen to a silencer element in each of the three genes (IP et al. 1991; HUANC et al. 1993; E R O V et al. 1994).

Enhancer elements are short DNA segments capable of acting in cis to enhance or confer specificity to the transcription of a nearby gene (see review by MUL.LER et al. 1988). The enhancer can exert its influence up- stream or downstream of a gene, in either orientation, and often over a distance of tens of kilobases. These special properties form the basis of the enhancer trap method, which uses random integration into the ge- nome of a reporter gene, most commonly the Escherichia

Corresponding author: Y . Henry Sun, Institute of Molecular Biology, Academia Sinica, Nankang, Taipei 11529, Taiwan, Republic of China. E-mail: rnbyhsun@ccvdx.sinica.edu.tw

‘Present address: European Molecular Biology Laboratory, Meyerhof- strasse 1 , D-69012 Heidelberg, Germany.

Genetics 141: 1075-1086 (November, 1995)

coli /3-galactosidase gene ( l a d ) fused to a weak constitu- tive promoter, to detect enhancer activities that confer expression specificity (O’KANE and GEHRING 1987). Si- lencer elements are similar to enhancers in properties (orientation independent and long-range effect) but with a negative effect on transcription (see RENKAWITZ

1990). Silencer elements, however, have not been sys- tematically searched for.

The white+ gene is an ideal reporter gene to detect transcriptional silencer elements in the Drosophila ge- nome. The pigmentation of the adult eye is a sensitive indicator of white+ expression. If white’ is inserted near a silencer element, its expression will be suppressed. In particular, this study reports the identification and characterization of 21 loci in which the expression of the inserted white’ gene is suppressed in nonuniform patterns, indicating the activity of silencers (operation- ally defined) specifying position-specific expression. This “silencer trap” may be an efficient way to identify genes involved in imaginal pattern formation.


Drosophila stocks: C y 0 P [ l a c w ] / S p was obtained from T.


1076 Y. H. Sun et al.

were from the Bloomington Stock Center, Bloomington, IN. Genetic markers are described in LINDSLEY and ZIMM (1992).

Analysis of eye pigmentation patterns: Unless otherwise stated, flies were cultured at 22-25", Adult flies were aged for 2 3 days before photographs of eyes were taken.

P[Zacw] mobilization and isolation of POSE lines: All ex-

periments were done with flies carrying a null mutation (w"" or w551) in the endogenous white gene. Pigmentation in the eye is dependent on the mini-white+ (m-w+) gene in P[lacw]. Appropriate fly stocks were crossed to generate 7u/ ECyO

P[lacw]/Sp;Sb n A 2 - 3 ] / + male progeny, which were then mated to C(I)A, y attached-X females. This mating was done in multiple batches, so that flies with the same phenotype but isolated from different batches must be independently derived. In the progeny, Cy', Sb+ males with colored eyes, or Cy, Sb+ males with an eye color different from the original orange color were examined. We screened -382,000 flies, of which 12,000 clearly have the p[lacw] transposed to a new site. From these, 21 flies with nonuniform eye pigmentation were isolated. Secondary transposition were induced by rein- troduction of P[AZ-?] through mating. A(2)-2 and A(2)-6 were derived from secondary transposition induced in the Pol-1 line.

A( 1) was isolated as a P[ lucw] insertion into a small frag- ment of Xchromosome (y' sc) that was translocated to the Y chromosome (FRASER and GREEN 1964). Subsequently, the A( 1) males were mated to C ( l ) R M , zd" splfemales, and female progeny irradiated with y-ray to promote detachment. Female progeny carrying the y+ sc w spl A ( 1 ) fragment on a free X chromosome were recovered.

VG(2)A and VG(3)B were from a collection of 200 ran- domly selected lines carrying independent P[ lucw insertion established by D. L. LINDSLEY and obtained through the Bloomington Stock Center. Five lines [Eq-2, P-2, A(1), A(2)- 7 and A(2)-81 were isolated independently by M. M. GREEN. Number 65 was originally isolated as a taste mutant and mapped to 48A by V. RODRIGLIES (Molecular Biology Unit, Tata Institute of Fundamental Research, Bombay, India) and kindly provided by Dr. W. CHIA (Institute of Molecular and Cell Biology, National University of Singapore, Singapore). Eq-3 was kindly provided and mapped to 69C by W. GELBAR?' (The Biological Labs, Harvard University). DH-1 was kindly provided by Y.-W. LEU (Pennsylvania State University). AuI was kindly provided and mapped to 48A by C . IAMBT (Insti- tut fur Entwicklungsbiologie, University of Koln, Germany). wxba21 was kindly provided by C . HAMA (National Institute of Neurosciences, Tokyo, Japan).

The small fragment of Xcarrying the A( 1) insert was geneti- cally determined to extend from polytene chromosome bands 1Al to 1E1,2. The other POSE loci were mapped by in situ hybridization to salivary gland polytene chromosomes. The plasmid pC4PLZ (kindly provided by Dr. S. CREWS, UCLA) was used as hybridization probe. It contains lacZ and m-w+, so can be used to detect the P[lacw] insert.

Testing for the effect of position effect variegation (PEV) modifiers: Males from the POSE lines were mated to FM7/y w

f . Yfemales to generate progeny with an extra Ychromosome ( X X Y females and XYY males). Females from the POSE lines were mated to y w f . Y males to generate XXY female and X 0 male progeny. Su(var)325, Su(vur)327, Su(var)??O, and

E(var)?O3 stocks used were provided by T. GRICLIATTI (De- partment of Zoology, University of British Columbia, Can- ada). Their effect on PEV was confirmed on w"'.

Xgal staining: Imaginal discs were dissected from late third instar (climbing stage) larvae in 0.1 M NaPi (pH 7.2), fixed for 10-12 min with 0.3% glutaraldehyde in 0.1 M NaPi (pH

7.2), and stained with 0.3% Xgal in Fe/NaP solution (HIROMI et nl. 1985) for 1-12 hr at 37". After staining, the discs were

washed once with 70% ethanol, twice with 0.1 M NaPi, and stored at 4" in 25% glycerol, 0.05% sodium cyanide in 0.1 M NaPi. Embryos were stained according to the method of HI- ROMI et al. (1985) but without devitellination.


Isolation of P[Zacm transposants with spatially re-

stricted pigmentation in the eye: The transposable ele- ment P[Zacw] (BIER et al. 1989) was mobilized in the Drosophila genome to search for position-specific si- lencer and enhancer elements. P[Zacw] contains the

m-w+ gene (PIRROTTA 1988). When P[ Zacw] is inserted into different sites in the chromosomes, the eye color varies from very light yellow to very dark brownish red, reflecting the sensitivity of m-w+ expression to chromo- somal position effects. In general, the pigmentation level is uniform over the entire eye. We screened -12,000 flies that carried the P[Zacw] transposed to a new position and isolated 21 flies with nonuniform eye pigmentation. Several others were identified in similar experiments or as secondary transpositions derived from the original set. A few were kindly provided by other labs (see MATERIAL AND METHODS). In total, we

have collected 36 independent lines with spatially re- stricted pigmentation pattern in the eye, representing 12 distinct patterns (Figure 1, Table 1).

Some of the lines show pigmentation restricted to a specific region of the eye, some show a gradient of pigmentation level. There is no morphological diversi- fication in the compound eye that correlates with these domains of pigmentation. Patterned eye pigmentation derived from P[w'] insertions or chromosomal re- arrangements involving w+ have been reported (JACK

andJuDD 1979; GEHRINC et al. 1984; GREEN 1984; HA-

ZELRIGG et aZ. 1984; LEVIS et aZ. 1985; PIRROTTA et UL.

1985; KASSIS et al. 1991; HAZELRICC and PETERSEN 1992; FAUVARQUE and DURA 1993; PETERSON et al. 1994).

Position-specific &acting suppression of white+ ex- pression: In these flies, the eye pigmentation patterns are dominantly heritable and reproducible, unlike the classical PEV, in which patches of white cells occur ran- domly among a background of pigmented eye cells (see review by SPOFFORD 1976). Upon introduction of the

A2-? transposase gene into these lines (28 lines were tested), P[Zacw] can transpose somatically and results in eyes mosaic in pigmentation, and also transpose in the germline and results in progeny with uniformly pig- mented eyes (data not shown). These results indicate that the m-w+ gene in these lines is intact and the spa- tially restricted expression pattern is due to chromo- somal position effect.


Silencer Detection in Drosophila 1077



Equatorial Posterior Dot Dorsal Half V-D Gradient D-V Gadient

Anterior Posterior A-P Gradient A-V Sector Post. Rim Clear Post. Mottle FIGURE 1.-Spatially restricted eye pigmentation in the POSE flies. Eye pigmentation patterns representative of each phenotypic group are shown. Orientation of the eyes is anterior to the right, and dorsal to the top.

nant over the m-w+ regulatory region. In several cases, when a fly carries two P[Zucw] copies, one that gives a spatial pattern and one that gives a light uniform color, or when the two P[ Zucw] each specify a distinct pattern, its eyes show a superimposition of each pigmentation patterns. This superimposition indicates that the s u p pressive effect is cipacting. In some of these lines, the pigmented region of the eye is quite dark, suggesting the effect of some transcriptional enhancer activity (see next section). Expression of the m-w+ gene may thus be enhanced in certain cells and suppressed in other cells.

Pigment granules are present in several types of pig- ment cells and photoreceptor cells (see review by PHIL LIPS and FORREST 1980). The lack of pigment in all cells in a region of the retina suggests that the suppressive effect is not dependent on cell type, but is dependent on the position of the cells in the retina. For simplicity, we call these lines the POSE (Esition-specific - expres- sion) lines.

All of the POSE lines reported here carry the P[ Zucw] construct, except for wxba2l and Eq-3. In wxba21, 3.4 kb of engruiled 5' flanking fragment was fused to ZucZ in the pw8 vector (HAMA et uZ. 1990). In Eq-3, a dpp-BMP4

fusion gene was cloned into pCaSpeR (PADCETT et uZ. 1993). In both cases, only one out of multiple trans- formants had the patterned eye pigmentation, indicat- ing that the eye pigmentation pattern is due to position effect and not due to the regulatory fragment in the P constructs. The fact that m-w+ in other P constructs can also respond to silencer indicates that the phenomenon is not peculiar to the P[ZucWl construct.

Because the m-w+ gene does not contain the regula-


tory region necessary for expression in the larval Mal- pighian tubule and adult testis sheath (LEVIS et ul. 1985; PIRROTTA et uZ. 1985), pigment formation in these tis- sues cannot be examined. The m-w+ gene also does not contain the region required for zeste interaction, and shows no zeste effect in several cases tested.

lacZ is activated by position-speciiic enhancer in the

eye and other imaginal discs: If the m-w" is under the control of a nearby silencer/enhancer, the enhancer- detecting ZucZin P[ Zucw] is also expected to be activated by the same enhancer. We did find that ZucZis expressed in the eye disc in many of the POSE lines. ZucZ expres- sion does not seem to be affected by the adjacent m-

w" gene, because the different lines showed different ZucZ expression patterns. In 17 of 32 lines examined, the pattern of ZucZ expression in the eye disc (Table 1,


Figures 2 and 3) is similar to the adult eye pigment pattern, suggesting that these two reporter genes are regulated by the same set of regulatory elements. It is possible that the silencer and enhancer acts in comple- mentary domains. Alternatively, the silencer may act uniformly over the eye, while a stronger enhancer acts in a position-specific manner.

In 15 POSE lines, the ZucZ expression in the eye disc of late third instar larva is either undetectable or differ- ent from the adult pigmentation pattern (Table 1, Fig- ure 2). There may be several explanations for such dis- crepancy. First, the expression patterns compared are from different developmental stages. The phenocritical period for white' function is in the early pupal stage



1078 Y. H. Sun et al.


S u m m a r y of the POSE lines

POSE lacZ expression

Eye pattern line Map E A W L Em Gene

Polar Pol-1 (1) 4C3-6 P P P P P qbtomotor-blind

Equatorial Eq-1 (3L) 69C1-2 P P P - P

Pol-2 (1) near Pol-1" P P P P P

Eq-2 (3) near Eq-1" P P P - P

Eq-3 (3L) 69C no lac%

Posterior P-1 (3R) 8661-4 - P P P P

Anterior A(2)-1 (2L) 40A1-4 P P P P

P-2 (3) near P-2"

A(2)-2 (2L) 40A1-4 P P P P

A(2)-3-5 (2L) 40A1-4 P P P P

A(2)-41 (2L) 4OA1-4 P

A(2)-5 (2L) 4OA1-4 P P P P

A(2)-6 (2L) 40A1-4 P P P P P (teashirt)

A(2)-7 (2L) 40A1-4 - - - - P

A(2)-8 (2L) 40A1-4 P - P P P

A(2)-9 (2L) 40A1-4 P

4 1 ) (1) 1A-B - - P

Ventrodorsal gradient VG(2)A (2R) 55C1-5 P P P P P

VG(2)B (2L) 26A1-2 - - - - -

VG(2)C (2L) 26A5-6 P

VG(3)A (3R) 85C1-3 P P P P P

VG(3)B (3R) 84El-6 P P P P P

Dorsoventral gradient DG(1)-1 (1) 7A6-8 P P P P P

DG(1)-2 (1) near DG(1)-2' P P P P P

Dorsal half DH-1 (3L) 6968-1 1 P P P P P

Anteroposterior gradient AG (3R) 86E6-8 - - - - P

Anteroventral sector AVSl (2R) 45A1-2 - - - -

- -

- - - -

DG(3) (3L) 67D8-12 P - - - P

DH-2 (3) P P P P P

#65 (2R) 48A - P P P P invected

AuI (2R) 48A - P P P P engrailed

Posterior-rim clear wxba2 1 (2R) 48A - P P P P invected

Posterior dot PD (1) 17A1-6 P - - W P

Posterior mottle PM(1) (1) 5D3-6 - - - -


P -


PM(3) (3L) 67El-4 - -

(2L) 33F-34A


P - -

- -

A total of 36 independent lines represent 12 distinct pigmentation patterns and probably 21 loci. The POSE lines were named based on their eye pigmentation pattern. When multiple POSE lines with the same eye pattern have the insertion in the same region, they are designated as -1, -2, etc. Where multiple loci gave the same eye pattern, the number in parenthesis indicates the chromosome the P[lacw] is located, and a letter following it distinguishes the loci. Map positions were determined by in situ hybridization to polytene chromosomes. Patterned expression of ZacZin the eye ( E ) , antenna (A), wing (W), leg (I,) imaginal discs and in embryo (Em) are indicated by P. Bold-faced P indicates that the lacZ expression pattern in the eye disc was similar to the adult eye pigmentation pattern. W indicates weak expression.

a Only one line in each sibling group (see text) of' the A(2) group was mapped by polytene in situ hybridization and listed. "Tight linkage based on recombination (<0.05 cM between Pol-1 and Pol-2, Eq-1 and Eq-2, P-1 and P-2; 0.09 cM between DG1 and DG2).

pattern at the early pupal stage, and not the pattern in late third instar larva. Consistent with this explanation,

lucZ expression has not been detected in the adult reti- nal cells in all lines, although can be detected in some other cells in the adults of a few lines (data not shown). Second, the domain of m-w" suppression and the do- main of lucZ activation may not exactly complement. One example is the case of Pol-1 (Figures 1 and 2). In Pol-1, although lucZ expression is detected in cells in


Silencer Detection in Drosophila 1079

Pol-1 Eq-1

FIGURE 2.--lacZ expression pattern m the eye-antennal disc of some POSE lines. Eye-antennal discs were isolated from late third instar larvae and stained with Xgal. The discs are all oriented with anterior to the right and dorsal to the top.

The expression patterns do not show variegation. Spe- cific expression patterns were also detected in some other larval tissues and in embryos of most lines (Ta- ble l ) .

Mapping and mutant phenotypes: The chromosomal position of the p[ZucwJ insert in many of the POSE lines has been determined by in situ hybridization to polytene chromosomes (Table 1 ) . Because the eye pig- mentation pattern is a dominant phenotype, comple- mentation test cannot be used to determine allelism. In the absence of mutant phenotype, the following criteria suggest that two inserts maybe in or near the same gene: mapping to the same site on polytene chromosome by

in situ hybridization, similar Xgal staining patterns, and

tight linkage by recombination test. Based on these cri- teria, the two members of the polar, equatorial, poste- rior, and dorsal-ventral gradient groups are assigned as putative pairs (Table 1). True allelism awaits comple- mentation of mutant phenotypes (see below).

Thirteen POSE lines showed either homozygous le- thality or sterility. The lethality in P-2, DH-1, DH-2, AuI and A(2)3-5 was shown to be due to P[ lucw] insertion (Table 2). The A2-3 transposase gene was used to in- duce the P[ h c w ] to transpose. Excision of P[ Zucw] was scored by loss of eye color. Of the multiple excision lines established, some are viable and show no apparent morphological defect, suggesting that the lethality was not due to a separate mutation on the chromosome, and that these viable lines probably represent precise

excision events. Both DH-1 and DH-2 are recessive le- thal and failed to complement each other, indicating that the P[Zucw] in DH-1 and DH-2 is disrupting the same gene. Based on similar reasoning, the lethality in AG, PM(2) and A ( 2 ) 3 1 was found to be independent of the


insertion (Table 2 ) . The sterility in A(2)2 and A(2)6 was also found to be independent of

the P[Zucw] insertion (data not shown).

We induced excision of P[ Zucw] in several POSE lines and recovered recessive mutations (Table 3). These al- lowed complementation tests to be performed. For ex- ample, four lethal alleles derived from excision in P-1 failed to complement lethality in P-2 and P-2 derived lethal alleles, suggesting that P-1 and P-2 are inserted in or near the same gene.



Pol-1 Eq-1

Y. H. Sun et al.

DH-1 VG(2)A VG(3)A



FIGURE S.-Examples of spatially restricted lac2 expression in imaginal wing disc. b c Z expression is detected by Xgal staining wing disc from late third instar larvae.

3 transposase gene, homozygous lethality and sterility, level of lac2 expression in imaginal discs, pattern of Xgal expression in embryo (data not shown), and sensi- tivity to heterochromatin (discussed later). Our work- ing hypothesis is that these represent premeiotic local transposition of the initial P[Zacw] insert. That P ele- ment transposes to nearby sites with high frequency


been reported (TOWER et al. 1993). The positions of P[Zacw] relative to the regulators may determine their


Reversion of lethality by P[laeVV1 excision

Excision lines

POSE line Total wt Lethal

P-2 DH-1 DH-2


A( 2) 3-5


PM(2) A( 2) 3-1

12 30 16 6 16 29 31 9


24 5 4 15 0 0 0


6 11 2


29 31 9

P [


was induced to transpose by A2-3. Excision events were selected by loss of eye color. The excision chromosome were individually balanced by Cy0 or TM3, Ser and scored for

viable homozygotes. wt, wild-type.

phenotypes. Molecular analysis of these insertion sites and correlation with the expression patterns will help to dissect the regulatory region.

Identification of several POSE genes: The silencer and enhancer activity detected by the m-w" and lac2 reporters suggests that there may be a nearby endoge- nous gene under their regulation. Because of their spa- tially restricted expression, such putative POSE genes may be involved in pattern formation during imaginal development. Based on chromosomal location and ex- pression pattern, we searched for such putative POSE genes.



lacw] insert in the two polar lines map to 4C3-

6 (Table 1). The gene optomotor-blind (omb) maps to 4C5-

6 (HEISENBERG et aZ. 1978), and its expression pattern in embryo, larval eye disc and brain, as detected by in

situ RNA hybridization (POECK et al. 1993), is nearly


Silencer Detection in Drosophila 1081

sion from Pol-1 have wing and leg abnormality. These defects are allelic to known omb alleles by complementa- tion tests


H. SUN and C.-J. TSAI, unpublished re- sults).

By in situ hybridization to polytene chromosomes,

the A(2) inserts map to 40A1-4 (Table 1). The high frequency of insertions at 40A14 (nine independent lines out of 35 POSE lines) suggests that this region is a hot spot for P insertion. The AR43 line of H A Z E ~ G G

et al. (1984) has the P[w"] insert mapped to 39E4OF

and has similar anterior eye pigmentation phenotype, so may have the insert in the same region. The gene

teashirt ( tsh) maps to 40A (FASANO et al. 1991).



insertions in the tsh locus also exhibit anterior pigmen- tation in the eye (S. KERRIDGE, personal communica- tion). The embryonic Xgal staining pattern of A(2)-6 (data not shown) is highly similar to the tsh expression pattern (FASANO et aZ. 1991). It is likely that the A(2)-6 insert is in or near the tsh locus. However, other A(2) lines have embryonic Xgal staining patterns different from the tsh gene, indicating that their lac23 are re- sponding to different regulatory elements, possibly of other genes in 40A14. Several of the A(2) 1' mes are homozygous lethal or sterile. However, these mutations complement a tsh lethal mutation (S. KERRIDGE, per- sonal communication).

The wxba21 line, which gives the posterior rim clear eye color pattern, carries a P insert at the -6 position of the invected (inv) gene (W et al. 1990). In wxba21

lacZ is expressed in a striped pattern in embryo and in

the posterior compartment in the antenna, wing, halter and leg discs. These patterns are characteristic of the

engrailed (en) and inv genes (COLEMAN et al. 1987; DREES

et al. 1987). The lacZ expression pattern was shown to be due to the insertion position at 48A and presumably reflects the enhancer activity of invected or shared by

invected and engrailed.

The AuI line has a P[Zacw] insert mapped at 48A and is homozygous lethal. Based on complementation test with deficiencies in the 48A region


Df(ZR))a28, Df(ZR)en307 Df(2R)enX31]

and with


the lethality is allelic to en. Molecular analysis also show that the P[lacw] is inserted 16 bp upstream of the en transcription start site (data not shown). However, the Xgal staining pattern in the embryo (not shown) and in the imaginal discs (Figure 3) is different from the en pattern, revealing the complexity of en regulation.

In the No. 65 line, the P[Zacw] insert was mapped to 48A. Its lacZ expression pattern in the embryo (not shown), the antenna, wing, haltere and leg discs (Fig- ures


and 3) are indistinguishable from the en-inv ex- pression patterns. Molecular analysis showed that the

P[lacw] is inserted between +95 and +96 in the inv

transcription unit (data not shown). The line originally had rough and small eye and defects in thoracic bristles. However, these have been segregated from the



insert. It is possible that en and inv are functionally

Heterozygotes Homozygotes

(C) A(2)-3-7

FIGURE 4.-Dosage effect of p[Zucw] insertions on eye pig- mentation. Three examples of the eye pigmentation patterns of flies heterozygous and homozygous for the fllacw] insert are shown.

redundant so that disruption of the inv gene caused no obvious morphological defect.

Dosage effect and dosage compensation: All lines exhibit normal dosage effect, i.e., eye pigmentation in flies with two copies of the same q l a c w ] insert are darker than those with one copy of P[lacw] (data not shown). The m-w" gene contains -300 bp upstream sequences of white' (PIRROTTA 1988), which includes the region required for dosage compensation (PIR- ROTTA et al. 1985). Indeed, males have darker eye pig- mentation than females with the same copy number of P[ lacw] (data not shown).

In most POSE lines, although flies homozygous for

the pCZacw] insert have darker eye pigmentation, the


1082 Y. H. Sun et al.

of A(2)-3-51, heterozygotes have only scanty pigmented spots in the anterior region, while homozygotes have full pigmentation in the anterior third of the eye (Fig- ure 4B). In A(2)-3-7 [another sibling line of A(2)-3-51, the pigmented region in homozygotes has expanded to three-quarters of the eye (Figure 4C). The patterns in heterozygotes do not change when they become older and eye color becomes darker, suggesting that the expansion effect in homozygotes is not due to a twofold increase in pigmentation level. Rather, it seems that the extent of m-w+ repression is reduced in homozygotes. One possible explanation is that there is a pairing ef- fect. However, the effect is in contrast with the pairing effect reported previously (KASSIS et ul. 1991; HA-


1993), where homozygotes have a more suppresive ef- fect on W' expression. Because other POSE lines

showed no pairing effect, this effect is likely due to the genomic sequence near the P[lacw] site in these few A(2) lines. Another possibility is that the P insertion affects an endogenous gene, and that the gene is auto- regulatory. We hypothesize that the gene represses its own expression, directly or indirectly, in the posterior half of the eye, and homozygous mutation decreases the strength of its repression on itself.

Effect of PEV modifiers: To find out whether the suppressive effect on m-w+ expression involves local for- mation of heterochromatin-like structure, we tested the effect of several factors affecting PEV on the POSE eye pigmentation pattern.

In PEV, variegation decreases (fewer white spots) when flies were raised at 29", increases (more white spots) when flies were raised at 18", decreases with an extra Ychromosome, increases in males with no Ychro- mosome, and is suppressed by Su(uur) mutations, and enhanced by E(uur) mutations. The pattern of eye pig- mentation in most POSE lines is not affected by devel- opment at 29" or 18", nor by variation in the number of Y chromosomes. Su(uar)325, Su(uar)327, and Su-

(uur)330, and E(uur)303 also had no effect. In many

POSE lines, the pigment intensity was lower when flies were raised at 29", while in a few POSE lines [VG(2)A, VG(3)A, PM(l)], the pigment intensity was slightly higher when raised at 18". This temperature depen- dence on pigment level may be due to an effect on pigment synthesis and not due to effect on m-wf expres- sion.

The only POSE lines that were affected by PEV mod- ifiers are PM(2), PM(3), A(l), and many of the A(2) series. In A ( l ) and most of the A(2) lines, an extra Y

chromosome (in XYYmale and =female) enhances

pigmentation, while the absence of a Ychromosome in males (XO) suppresses pigmentation (Figure 5). When the pigmentation is suppressed, the pigmented area becomes mottled. In the lines that exhibit a variegated phenotype [e+, A ( l ) , A(2)3-4, A(2)41], enhancement of pigmentation results in a more uniformly pigmented


FIGURE 5.-Effect of Y chromosome number on some

POSE eye pigmentation. Heterozygous males of PM(2) and

A(2) with variation of Y chromosome number are shown.

anterior region, but the pigmentation does not extend into the posterior half of the eye. The response to varia- tion in the number of Y chromosomes is typical of posi- tion effect variegation (see review by SPOFFORD 1976), consistent with the mapping of these


inserts to 40A14 near the centromeric heterochromatin. How- ever, temperature (29" and 18") does not change the spatial pattern of pigmentation, and only the pigment amount is affected as described above. Su(uur)325,

Su(uur)327, Su(uar)330, and E(vur)303 also have no dis-

cernible effect. The fact that the posterior of the eye remains white even when the anterior pigmentation is enhanced in XXYfemales and XYYmales suggests that the suppression of m-w+ in the posterior region is by a mechanism unrelated to heterochromatin.


Silencer Detection in Drosophila 1083



FIGURL ,.-Maternal effect on PM(1) eye Pigmentation.

Eye color of female heterozygous for PM(1) with the PM( 1) X

chromosome inherited from (A) father and from (B) mother.

The mottling in PM(2) and PM(3) show enhanced variegation in X 0 male, XYY male and X X Y female, a behavior different from PEV. However, lacZ can be expressed in a spatially restricted pattern in their imagi- nal discs and did not show variegation (data not shown). Expression of PM(2) and PM(3) may be regu- lated by a mechanism related to, but different from, centromeric heterochromatin formation. The PM( 1) pattern is not affected by variation in Y chromosome number. However, there is a maternal effect: in flies heterozygous for PM( 1), eye color is nearly white if the PM(1) X chromosome was inherited from the father, and is darker with posterior mottling if inherited from the mother (Figure 6).


Use of white as a reporter gene for silencer/enhancer detection: Mini-white', with its modest expression level, can be used as a reporter for the detection of both enhancer and silencer activity. Its expression pattern is manifested as pigmentation pattern in the adult eye, an easily scorable phenotype. In contrast, the popular ZacZreporter requires a staining step to reveal its expres- sion, hence a propagating stock has to be established before some individual can be sacrificed for staining. Although m-w' expression can only be seen in the adult eye, the advantage is that regulatory activity in an ear- lier, less accessible developmental (early pupal) stage, when some of the crucial developmental decisions are being made, can be easily detected. Our use of a consti- tutively expressed reporter gene provides a general ap- proach to screen for silencer elements, which may play important roles in developmental gene regulation. Other reporter genes, e.g., yellow or an ubiquitously ex- pressed lacZ, can be used in such "silencer trap" to

examine silencer activities in other tissues.

Screening for POSE genes: The above method was

used to screen for regulatory elements specifying posi- tion-dependent expression during eye development. We report here the identification of 36 independent isolates representing 21 loci that exhibit such regulatory

activities. The expression patterns of m-w' and ZucZmay mimic those of certain adjacent endogenous genes. For simplicity, we call these genes the POSE genes.


in enhancer trapping, there is the possibility that the c k acting regulation seen by the reporter genes may not faithfully reflect that of any endogenous gene, either because the enhancer or silencer are not used by an endogenous gene or because the reporter genes only respond to a subset of regulatory elements, whose activi- ties are different from the complete set acting on the endogenous gene. However, of the 21 loci we found, three have been identified to be optornotor-blind, en- grailed, and invected, another one very likely corresponds to teashirt. All four genes are known, or suspected (inv), to be involved in developmental regulation, providing strong support for the usefulness of this approach. Us- ing the same approach, BHOJWANI et al. (1995) had also found that P[Zucw] insertion in wingless, teashirt and engrailed give patterned eye pigmentation. The finding that in many of the POSE lines the lac2 expression pattern in the eye disc is similar to the adult eye pigmen- tation (Table 1;


Figures 1 and 2) further suggests that the patterned expressions are not artifacts. Our data also suggest that many of the putative POSE genes are also expressed in other imaginal discs and in em- bryos, in specific patterns. Thus this silencer trap screen would be an efficient method to identify genes involved in pattern formation in a variety of tissue and develop- mental stages.

Of the 21 loci defined by the 36 POSE lines, seven loci are represented by more than one line (Table 1). The high frequency of repeated occurrence suggest that the number of loci that exhibit a patterned ex- pression, at least in the eye disc in early pupa, may be limited. Our collection of 21 loci, however, does not include all possible genes with spatially restricted ex- pression pattern in the eye. Spatially restricted, white' dependent eye pigmentation patterns due to position effect on P[w"] insert or on translocated w" have been reported. The patterns include halo (darker pigmenta- tion in the periphery, lighter in the center), posterior crescent, ventral half, A-P gradient, and P-A gradient

(JACK and JuDD 1979; GEHRING et al. 1984; GREEN 1984; HAZELRIGG et al. 1984; LEVIS et al. 1985; PIRROTTA et al. 1985; HAZELRIGG and PETERSEN 1992; PETERSON et al. 1994). P constructs containing a fragment of the regula- tory region of engrailed or poijhomeotic, in addition to m- w' and ZucZ, also gave some transformants exhibiting patterned eye pigmentation (KASSIS et al. 1991; FAU- VARQUE and DURA 1993). Spatially restricted expression patterns in the eye disc have also been identified by enhancer trap method (BROOK et al. 1993).


1084 Y. H. Sun et ul.


Excision mutants derived from the POSE lines

POSE line Alleles Phenotype Allelism

Pol-1 Eq-1 Eq-2

P-1 P-2 DH-1 DH-2



7 5 4 1 15 12

wing/leg defects lethal, embryonic lethal"

lethal" lethal lethal

lethal, semi-lethal 9 lethal

3 viable with bristle defects

allelic to omb

complementation group I

complementation group I1

allelic to P-2 allelic to P-2 allelic to DH-1 allelic to DH-2 allelic to DH-2

P[lucw] was induced to transpose. Both excisions (white eye) and transpositions (change of eye color) were selected and balanced. Balanced lines with homozygous phenotype were tested for complementation with an existing lethal (P-2, DH-1 or DH-2) or with each other. The major complementation group probably represent the gene near the P[lucw] insertion site; only these are listed. Other ungrouped lethals were assumed to be due to unrelated mutation elsewhere on the same chromosome.

'I Weaker alleles gave rise to pharate adults with severely malformed legs, which die upon eclosion.

cause the retinal cells differentiate in a posterior to anterior progression, one possible interpretation for the patterns in the anterior-posterior axis may be a change in the relative timing of w" expression (PIR-

R O m A et al. 1985). It has been found that for eye pig-

ment formation, w" expression is required primarily in the first 2 days of pupal development (EPHRUSSI and HEROLD 1945; STELLER and PIRROTTA 1985). If w+ ex- pression stops earlier than normal, then only the more mature cells in the posterior side of the eye will be pigmented. If w" expression is turned on later than normal, then the posterior cells have already past the short critical period for w+ and will have no pigment and only anterior cells will be pigmented. The temporal regulation would thus be transformed into spatial pat- tern. However, although this explanation may be true for some cases, it cannot apply to patterns other than the anterior-posterior type. Our preliminary results also show that ZacZexpression in the eye disc of some POSE lines precedes and is independent of the movement of morphogenetic furrow (J.-L. CHAO and Y . H. SUN, unpublished result). We favor the explanation that the

m-w+ gene has come under the control of a cisacting regulatory element, which is responding to cell position within the eye. Screening for mutations affecting the eye pigmentation pattern will help to dissect the cis acting regulatory region and to find genes that regulate the POSE gene expression.

Developmental functions: Genes with spatially re- stricted expression pattern in imaginal discs have been reported. Some encode metabolic enzymes (e.g., NADP- malic enzyme) of unknown developmental role (KUHN and SPREY 1987). Some encode cell surface proteins

(e.g., the integrins) and may be involved in morpho- genic movement or cell adhesion (FRISTROM et al.

1993). Some (e.g., wingless, hedgehog, Distal-less) have or may have a regulatory role in imaginal development (BRYANT 1993; COHEN 1993).

Based on their spatial expression patterns, we specu- late that the putative POSE genes may be involved in pattern formation in imaginal development, either in the specification of, or are responding to, the positional information. optornotor-blind encodes a DNA-binding protein and affects brain and wing development (BRUN- NER et al. 1992; PFLUGFELDER et al. 1992a,b). It also affects eye morphology (G. PFLUGFELDER, personal communication). teashirt is a homeotic gene required for the determination of segmental identity in the em- bryonic trunk region (RODER et al. 1992; DE ZULUETA

et al. 1994). Because exisiting tsh mutants are embryonic lethal, its role in imaginal development awaits further analysis. en encodes a homeo-box containing protein and is essential for the specification of the posterior compartments (GARCIA-BELLJDO and SANTAMARIA 1972;

MORATA and LAWRENCE 1975). inv is expressed in pat- terns very similar to engrailed and encodes a homeo-box protein with significant homology to engrailed protein. Although the AuI line with P[lacw] in en showed no lacZ expression in the eye disc, en was reported to be expressed weakly in the region behind the morphogen- etic furrow in the eye disc (BROWER 1986; HAMA et al.

1990). The wxba21 line (with P[ lacw] in inv) does show lacZ expression in the eye disc. Clonal analysis of lethal

en alleles did not reveal any effect on eye morphology

(LAWRENCE and STRUHL 1982). It is possible that inv

can compensate for loss of en function in the eye. If the putative POSE genes are involved in pattern formation in imaginal development, then the P[lacw]


Silencer Detection in Drosophila 1085

be published elsewhere). For each of these POSE lines, multiple mutations were generated and were deter- mined to be in the same complementation group, sug- gesting that the mutations are in a gene near the origi- nal P[ZucwJ insertion site. Most of these mutations caused lethality before the adult stage. Clonal analysis may be required to examine their effect on imaginal development.

The fact that lac2 is also expressed in imaginal discs other than the eye disc suggests that at least part of the genetic system used in pattern formation is shared among the different body parts. It had been shown that the limbs (wing, haltere, and legs) and even antenna may share part of the same set of positional informa- tion, although interpreted differently depending on the specific disc (e.g., see review by WILLIAMS and CARROLL

1993). Our finding suggests that the eye also shares part of the same set of positional information.

Early pattern formation in eye development had not been extensively studied, partly due to the lack of posi- tional markers. Our collection of POSE lines provide such cell-autonomous markers for positional identity, and should greatly facilitate future studies.

We are very grateful to Drs. K. MATTHEWS (Bloomington Stock Center), T. TANIMURA, W. CHIA, V. RODRIGUES, Y.-W. LEU, W. GEL

BART, C. K I A M B T , and C. HAMA for generously providing the fly stocks, S . CREWS for providing the pC4PLZ plasmid, GORDON CHANG and YELJ-LOONG CHANG for maintaining the fly cultures, WEIGUANG Fu for performing Xgal staining of embryos, Yr H o for helping in Xgal staining of the Pol-1 imaginal discs, and SHUN-HWEI LIN for helping in fly screening. We also thank JYCHIAN CHEN, GERT PFLUGFELDER,

S1’ANI.Y COHEN and DER-HWA HUANG for valuable comments on the manuscript, and P. SINHA, G. PFI.ucFELDER and S . KERRIDGE for pro- viding information prior to publication. This work was supported by grants from the National Science Council (NSC 81-0412-B-001-12, 82- 0203-Bo01-076, 82-0203-B001-98) and the Academia Sinica of the Republic of China.


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domain shared between Drosophila optomotor-blind and mouse



FIGURE Anterior 1.-Spatially restricted eye pigmentation in the
FIGURE 2.--lacZ third  instar  larvae  and  stained  with expression  pattern m the  eye-antennal  disc of some  POSE  lines


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