Mutations that alter the timing and pattern of cubitus interruptus gene expression in Drosophila melanogaster.

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Copyright 0 1995 by the Genetics Society of America

Mutations that Alter the

Timing

and

Pattern of

cubitus interruptus

Gene

Expression

in

Drosophila melanogush

Diane

C. Sl~sarski,”~

Cynthia

Kelsey Motzny‘ and Robert Holmgren

Department of Biochemisty, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500

Manuscript received June 8, 1994

Accepted for publication September 15, 1994

ABSTRACT

The cubitus intenuptus ( c i ) gene is a member of the Drosophila segment polarity gene family and

encodes a protein with a zinc finger domain homologous to the vertebrate Gli genes and the nematode

tra-l gene. Three classes of existing mutations in the d locus alter the regulation of ci expression and can

be used to examine ci function during development. The first class of cimutations causes interruptions in

wing veins four and five due to inappropriate expression of the ci product in the posterior compartment

of imaginal discs. The second class of mutations eliminates ci protein early in embryogenesis and causes

the deletion of structures that are derived from the region including and adjacent to the engrailed

expressing cells. The third class of mutations eliminates ci protein later in embryogenesis and blocks

the formation of the ventral naked cuticle. The loss of d expression at these two different stages

in embryonic development correlates with the subsequent elimination of wingless expression. Adults

heterozygous for the unique

ci“

mutation have deletions between wing veins three and four. A similar

wing defect is present in animals mutant for the segment polarity gene fused that encodes a putative

serine/threonine kinase. In cia/+ and fused mutants, the deletions between wing veins three and four

correlate with increased ci protein levels in the anterior compartment. Thus, proper regulation of both

the ci mRNA and protein appears to be critical for normal Drosophila development.

D

URING Drosophila embryogenesis the antero-posterior body axis is divided into a series of segments that are the basic units for establishing the pattern of structures within the developing embryo. The segment polarity genes play a critical role in specifymg

pattern along the antero-posterior axis of each segment

(NUSSLEIN-VOLHARD and WIESCHAUS 1980). The initial

expression of several segment polarity genes is con-

trolled by pair rule genes, but continued expression

depends on interactions among the segment polarity

genes and their protein products (reviewed in INCHAM

1991; HOOPER and Scorn 1992).

Central to the pattern formation process is the main-

tenance of wingless (wg) gene expression in the cells

just anterior to the parasegmental boundary (MARTI-

NEZ-ARIAZ and LAWRENCE 1985; BAKER 1987, 1988; VAN

DEN HEUVEL et al. 1989). wgencodes a Drosophila mem-

ber of the Wnt family of secreted growth factors and

may act as a morphogen to establish pattern within

segments [VAN DEN HEUVEL et al. 1989; BEJSOVEC and

MARTINEZ-ARIAS 1991; GONZALEZ et al. 1991; BEJSOVEC

and WIESCHAUS 1993; SAMPEDRO et al. 1993 (who sug-

Corresponding author: Robert Holmgren, Department of Biochemis- try, Molecular Biology and Cell Biology, Northwestern University,

2153 Sheridan Rd., Evanston, IL 60208-3500,

E-mail: [email protected]

Baltimore, MD 21218-2685.

’

Present address: Department of Biology, Johns Hopkins University,

Co-first authors.

Genetics 139: 229-240 (Januav, 1995)

gest that wg is not a morphogen)]. In animals lacking

the wg protein, most of the pattern elements within a

segment are eliminated and replaced with a lawn of

denticles. In animals deficient for the ci region of the

fourth chromosome, wg expression in the epidermis is

eliminated during germ band extension (HIDALGO and

INCHAM 1990; C. MOTZNY and R. HOLMGREN, u n p u b lished results) and the ventral cuticle is covered with

denticles (NUSSLEIN-VOLHARD and WIESCHAUS 1980;

ORENIC et al. 1987).

The cubitus i n t m p u s ( c i ) region of the fourth chro-

mosome contains three genetically distinct classes of

mutations with a complex pattern of complementation

(Table 1) (HOCHMAN 1973; ORENIC et al. 1987). In this

work it is shown that all of these mutations alter the expression of a single transcription unit, which is re- ferred to as ci. Conceptual translation of the ci transcript

predicts a protein with five zinc fingers (ORENIC et al.

1990). The zinc finger domain has a high degree of

amino acid sequence identity with the vertebrate Gli

genes (84-93%) (KINZLER et al. 1987; RUPPERT et al.

1990) and a lower degree of identity with the tra-1 gene

of Caenorhabditis elegans (63%) (ZARKOWER and HODG

KIN 1992). Because ci was the first mutation to be isolated in this locus, we have given all of the mutations ci allele designations.

The

ci’

class of mutations causes breaks in the fourth

and fifth wing veins and includes the recessive mutation

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230 D. C. Slusarski, C. Kelsey Motzny and R. Holmgren

TABLE 1

Classes of ci mutations

Complementation

Class Phenotype properties

ci'

Disruptions in wing Complements the veins 4 and 5 segmentation defects

of the ~ 2 " ~ " ~ and

d1(4)17

classes

#) 13

Deletion of ventral Complements the naked cuticle in segmentation defects embryos of the ciL(4j17 class

and the wing vein defects of the

ci'

class

&4j 1 7 _{Deletion of the first row} _{Complements the}

of ventral denticles segmentation defects and variable deletions of the ~ i ' ( ~ j ' j class of ventral naked and the wing vein

cuticle defects of the ci'

class

mutation ci".

c

'

i

is an unusual allele because it is also

a member of the c2zf41z3 class of mutations. This class

includes the

c

'

i

and l(4) 13 ( ci1f4113) mutations and causes

recessive segmentation defects in which the ventral naked cuticle of each segment is deleted and replaced with a mirror image duplication of the denticle belt. The ci"4'17cla~~contains the

Cz

(ci") and Z ( 4 ) 1 7 ( ~ i ' ( ~ ) . ' ~ )

mutations. These mutants also have recessive segmenta-

tion defects. In ~ i ' ' ~ ) . ' ' mutants, the first row of denticles is removed from the abdominal segments, and there

are variable deletions of naked cuticle. The ci" mutants

are more extreme, and the naked cuticle is removed in

every segment. The ci" mutation also causes variable

dominant adult defects, including partial fusions be-

tween wing veins three and four and the deletion of

the ocelli.

The mutations were placed into these three groups

on the basis of their complementation properties.

~ i " ~ ' . ' ~

mutations fail to complement the ci" recessive segmen-

tation defect but complement the ci.' wing vein defect.

Mutations in the ciLf4"jr class and the ~i"".'~ class comple-

ment each other's segmentation defect and are viable in certain combinations. Deletions of the locus have a

phenotype similar to that of cia and fail to complement

both the and ci1'4'17 classes.

Here it is shown that mutations in the ci locus cause

a variety of alterations in the expression of the ci pro-

tein. The two classes of embryonic mutations eliminate

expression of the ci protein at different developmental

stages. The adult wing vein defects of the

ci'

class muta-

tions are caused by misexpression of the ci protein in

the posterior compartment of imaginal discs. The

tic'

mutation produces a truncated ci protein with an altered distribution pattern in the anterior compartment.

The distribution of the ci protein in

ci""/+

mutant

imaginal discs is similar to that of the wild-type ci protein in imaginal discs mutant for the putative serine/

threonine kinase gene fused

vu)

(PREAT et al. 1990).

MATERIALS AND METHODS

Stocks: The ci" and alleles were obtained from B. HOCHMAN, the ciu, Mhz<

ci'

and ciw alleles were provided by the Bowling Green Drosophila stock center, the tip'"" allele was provided by T. KORNBERG and the su(Hd) and su(Hw")

alleles were from V. CORCES. The gamma-ray revertant

gRS0

is a partial revertant of c i D (ORENIC et al. 1987).fu" is a strong

EMS mutation of f u generated by R. HOLMGREN.

Generating c i P h revertants: y q tip'"' females were crossed to P[q+A2-3] Sb/TM3; spaFi males. y q P[q+A2-3] S b / + ; ciphc/spupoL males were crossed to yw, spaPo' females. Each indi- vidual excision event was crossed to yw; s p P l flies and the y q sPaP"'+/sPuP0' sibling progeny crossed to identify both embryonic and adult mutant phenotypes. Each lethal line was characterized by examining cuticle pheno pes and by testing complementation with ci", ci1(4'17 and ci

2

.

Mapping ci mutants: Mutations were mapped by per- forming a series of restriction digests with DNA isolated from appropriate mutants, blotting the gel fractionated fragments onto nitrocellulose and probing with various cloned fragments that spanned the ci gene region. Fragments from ci cDNA clones were particularly useful because they did not contain repetitive sequences that are present in flanking genomic regions.

Cuticle preparations: The procedure of VAN DER MEER (1977) was used. The cuticles were mounted in a 1:l solution of Hoyers:lactic acid and photographed with phase-contrast microscopy.

Wing blade preparations: Wings were removed, dehydrated with propanol and mounted in euparal.

Antibody stainings: Antibody staining of embryos was performed according to PATEL et al. (1989) using the anti-ci rat monoclonal antibody 2A1 (C. MOTZNY and R. HOLMGREN, unpublished data), the anti-engrailed (en) mouse monclonal antibody 4D9 (gift from N. PATEL) and an anti-wg rabbit antibody (gift from R. NUSSE). With ci" it is possible to unambigu- ously identify mutant embryos because the ci" allele is viable and fertile over ciplnc. Progeny from c i D / c i p l " " parents were double labeled with anti-ci antibodies and anti-@galactosidase antibodies; embryos that fail to stain with the anti-p-galactosidase antibody are homozygous for the

c

'

i

mutation. The other ci embryonic lethal mutations are not fertile over tip"'. Mutant embryos were identified by the distinct staining patterns present in approximately one quarter of the embryos. For the anti-en stainings, the ci" mutant embryos were identified by double labeling with anti-ci antibodies. Embryos were staged according to CAMPOSORTECA and HARTENSTEIN (1985).

Imaginal discs were stained in a similar manner. Wandering third instar larvae were collected, rinsed in BSS (CHm and GEHNNG 1971), cut in half and turned inside out. Animals were fixed for 20 min in phosphate-buffered saline containing

2% paraformaldehyde. Stained discs were removed from the larval carcass and dehydrated for mounting.

In situ hybridizations: In situ hybridization to ci mRNA was performed using DIGlabeled RNA probes (TAUTZ and PFEIFLE 1989).

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Regulation of ci Expression 23 1

were fractionated on a 7.5% acrylamide gel and blotted onto nitrocellulose. Protein levels were examined by staining the

nitrocellulose with Ponceau S solution (Serva). ci protein was

visualized using the 2A1 monoclonal antibody, an HRPcou-

pled goat anti-rat IgG secondary (Sigma) and the electro-

chemiluminescence (ECL) detection system from Amersham.

RNA analysis: Late third instar larvae were homogenized

in an SDSurea solution, phenol extracted and ethanol precip

itated (MCKENZIE et al. 1975). PolyA+ RNAwas selected using

oligo dT cellulose

.

Equal amounts of RNA were loaded onto

a formaldehyde gel. After blotting, the RNAs were probed

with the 5' end of a n' cDNA. The hybridization intensities

were quantified relative to an rp49 standard using a Molecular

Dynamics phosphorimager.

RESULTS

Molecular analysis of the

ci',

ciw, c i D ,

dLC

and c i C e

mutations: Genetic analysis of the

ci',

c i D and

cite

muta-

tions did not distinguish whether they represented distinct genes or partially complementing alleles of a single locus. Previous work showed that each of these

mutations is associated with alterations in a 6-kb BglII

fragment that contains the 5' end of the ci transcript

(ORENIC et al. 1990) (Figure 1). These alterations could be due to polymorphisms, but the lack of recombina- tion on the fourth chromosome makes that possibility

unlikely (BERRY et al. 1991). Therefore, we mapped

these mutations in more detail and assessed changes in

the expression pattern of the ci protein.

Sequences

5'

to the ci transcript were conveniently

divided into four regions using the restriction enzymes

Nsd and BglII (Figure 1). The

ci'

mutation is caused by

the insertion of a gypsy element into region 3. The ci"

mutation is associated with the insertion of foreign DNA

into region

2.

The insertion appears to be an I element

(FINNEGAN 1989), because an I element probe hybrid-

izes in situ to the ci region (101F) in

ci"

mutant polytene

chromosomes and region 2 DNA hybridizes to a band

that comigrates with an I element containing fragment

from

tic" mutants (data not shown). The

c i D mutation

results from a small inversion (ORENIC et al. 1990). We

have mapped the breakpoints to two regions: region 1

and 10 kb 3' to the ci transcription unit (not shown in

Figure 1). The breakpoint is represented by a dashed

line because the exact position has not been deter-

mined. The spontaneous mutation c i w also has alter-

ations in these same two regions, but the nature of the

alterations has not been characterized. The ciphc line

contains an insertion of a whitei (w') enhancer-trap

(EATON and KORNBERC 1990) into region 3.

Analysis

of ci protein expression in ci mutant em- bryos: Expression of the cigene was assessed in mutants

using an anti-ci rat monoclonal antibody, 2A1 (C.

MOTZNY and R. HOLMGREN, unpublished results). The

antibody is specific to the ci protein product because

there is no labeling of A f Z f mutant embryos in which

the ci region is deleted (data not shown).

-

1 kb

E E X 5' E X

v

E l

94

183

-

217

-

228

-

FIGURE 1.-Map of the 5' end of the ci gene. The long

horizontal line represents the DNA from the 5' region of the

ci gene. The 6kb BgnI (B) fragment has been divided into

four segments using the restriction enzyme NstI (N). The

locations of insertions (V) and DNA alterations (-

-

-) associated with each mutation are indicated. The positions of these aberrations have been determined by Southern blot analysis

and are mapped to the resolution of the restriction map (with

the exception of the c i P l n C insertion, which has been precisely

mapped). The exon pattern of the ci transcript is shown below

the map. The 5' end corresponds to the end of the longest

ci cDNA clone. The extent of deletion mutations generated by imprecise excision of the ciPlac element is also shown below

the map of the region. The endpoints of the excision events

were determined by probing Southern blots of DNA from the

appropriate mutants and are mapped relative to the indicated

restriction sites. The distal break in mutation 183 is in fragment 4, whereas the distal breaks for deletions 94, 21 7 and

228are all within fragment 3. Deletions 183, 21 7and 228end

within the element, whereas deletion 94 eliminates the entire

P element and has a proximal breakpoint in fragment 1. The

extent of the deletions within the P element have not been

indicated on this map. The NsiI sites are only shown within

the 6kb BgZII fragment. B, BgZII; E, EcoRI; N, NsiI; X, XhoI.

In wild-type animals, expression of the ci protein initi-

ates during stage

5

and rapidly becomes expressed

throughout the embryo. During stage 10 ci mRNA is

eliminated from the posterior compartment of each

segment (EATON and KORNBERG 1990; ORENIC et al.

1990) and there is a corresponding decay of the protein

(Figure 2B). During stage 11 the wide stripe of ci pro-

tein expression begins to split, with higher levels of

antibody staining on the margins of the stripe and lower

level staining in the middle (Figure 2C).

In

ci"

mutants, low level uniform expression of ci

protein is observed at stage 5 and this expression peaks

at or shortly after gastrulation (Figure 2D). With the initiation of germ band extension, the ci protein level begins to decrease relative to wild type (Figure 2E; com- pare the mutant embryo in the center with the adjacent

wild type siblings) and is barely visible by the end of

stage 11 (Figure 2F).

mutant embryos fail to express the

ci protein until stage 7, but as development proceeds,

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232 D. C. Slusarski, C. Kelsey Motzny and R. Holmgren

C

FIGURE 2.-Pattern of ci protein expression in embryonic development. Anterior is to the left and dorsal is up. Embryos were

stained with the 2A1 rat antici monoclonal. (A-C) Stages 8, 10 and 11 wild-type embryos. Initial expression of ci protein is

uniform throughout the segment; during stage 10 ci expression is eliminated in the en expressing cells and during stage 11 the

cells bracketing the m expressing cells show a higher level of antici antibodv labeling. (D-F) Stages 7, 8 and early stage 11 n'"

mutant embryos. The stage 7 embryo shows uniform expression of ci protein though the levels are somewhat lower than wild

type. With the initiation of germ band extension, protein levels begin to decrease, and bv stage 11 staining for the ci protein

has decreased to nearly background levels. (The embryo collections were specifically overstained to allow visualization of the

low ci protein levels in the mutants.) ( G I ) Stages 8, 10 and early stage 11 c;''~''' mutant embryos. At stage 7 expression of the

ci protein is not detected, but during stage 8 cells begin expressing the ci protein, and by stage 11 the pattern of expression,

though not wild type, is quite robust.

clusters of cells initiate ci protein expression. During

stage 10 (Figure 2H), the clusters begin to coalesce

into stripes, a n d by stage 11 the pattern of expression

approximates that of wild type, but at lower protein

levels (Figure 21). A distinction between the pattern of

ci expression in

cif'

and

~i"~"'

embryos is observed in

stage 11 embryos. In ci'(4)f7embryos the wide stripe splits

as it does in wild type. In ci" mutants the stripe remains

uniform (data not shown). In ci"/cirM embryos the pat-

tern of ci protein expression is a combination of the

two mutant patterns. Athough the level of protein ex-

pression is lower than that of wild-type, ci protein is

expressed throughout embryonic development and the

pattern resembles that of wild-type embryos.

In

n"

and c i " ' mutants, which generally have normal

embryonic development, the ci protein continues to

be expressed throughout the segment and fails to be

repressed in the posterior compartment (Figure 3, A

and

R).

Expression of wg

and

en in ci mutant embryos: T h e

consequences of eliminating ci protein expression a t

different times in development were assayed by follow- ing the patterns of wg and en protein expression. In

ci"

mutants, expression of the wg protein decays during

stage 12 (Figure

a),

whereas expression of t h e e n

pro-

tein appears normal (data not shown). By stage 10,

wg

protein expression is eliminated in regions of the ven-

tral epidermis of ciN4)" mutant embryos (Figure 4B).

In these mutants, ci protein levels rise during stages 10

a n d 11 and epidermal expression of the wg protein

returns in late stage 12 (Figure 4C). In c i N 4 ) ' i mutants,

en protein expression is normal through stage 10 but

during stages 11 a n d 12 breaks in the stripes of e n

expression become evident (Figure 4D).

Generation of deletion mutations withii the ci

pro-

moter region: To define the promoter elements re-

quired for proper n'expression, we generated deletions

of a marked P element insertion. This approach seemed

particularly appropriate given the complex nature of the existing ci alleles. T h e

n'/''"'

line carries a zu" en-

hancer trap and has a normal pattern of ci protein

expression during embryogenesis. Excision events were

induced by crossing this line to P[ly'A2-3](99R), a P

element transposase expressing strain (ROBERTSON d

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Regulation of ci Expression 233

patterns of these mutations placed them in the ci"""

group (Figure 5). On the ventral surface the first row

of denticles is missing from most o f the abdominal seg-

ments, and there are variable deletions of naked cuticle

including the Keilin's organs of the thoracic segments

(Figure 5, C and D). Dorsally the isolated row of large

triangular hairs (visible in abdominal segments 2-8,

see arrowhead in Figure 5E) and flanking naked cuticle

are eliminated from most segments, and there is a corresponding expansion of the region of socketed hairs

and fine hairs (Figure 5, G and H ) . T h e molecular

nature of the excision events was examined by Southern blot analysis and the approximate extent of the dele-

tions mapped (Figure 1). In all cases sequences around

the distal inverted-repeat of the transposable element

were altered.

ci protein expression in imaginal discs: Two types of

wing vein defects are observed in a' mutants. Mutations

of the

n"

class all cause disruptions in veins 4 and 5. In

n'"/+,

n", cri"'and

ci'"''"/+

mutants (h""5''is a revertant

of b"), we find defects ranging from minor interrup-

tions of vein 5 to fusions of veins 4 and 5 (Figure 6,

R-

FIGLIRE S.-ci expression in n" and ci'"mr1tants. Anterior E).

ci';./+

mutants have deletions between veins 3 and

is to the left and dorsal is up. (A) Stage 10

n"

mutant embryo. 4 (Figure 6F). The ci protein distrihutions in these mu- (€3) Stage 1 1 ci"' mutant embryo. In both mutants ci protein

expression persists in the posterior compartment. tants were examined in the developing imaginal discs.

In normal imaginal development, ci transcript and pro-

tein are restricted to the anterior compartment (Figure

Six independent embryonic lethal mutations were iso-

7,

A and B). T h e ci mRNA is uniformly expressed in the

lated. The nature of the segmentation defects, the pat- anterior compartment (Figure 7A), whereas antibody

terns of ci protein expression and the complementation labeling of the ci protein is elevated along the compart-

FICCRE 4.-Expression of en and wg proteins in ci mutants. Anterior is to the left. In a stage 12 Yj" mutant embryo, expression

of wg is fading (A). In n"'.""animals, wg expression begins to be eliminated in the epidermis (*) bv stage I O (B), but expression

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234 D. C . Slusarski, C. Kelsey M o t m y a n d R. Holmgren

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Regulation of n' Expression 2.73

x.

. ..

-

'8"-

. " .

. _._- ~. - . . -

em.+,/

.

- _."_ . -

F1c.t IU;. (i.--M'ing defects in ci mrmnts. Anterior is up. (A) A wild-type wing. From antcrior t o posterior t h r veins are n a m h c w d 1 through 3 , o f ~ v h i c h 3 t o .5 arc indic;ltctl. The I X ) U I I ~ ; I I ~ separating t h c anterior ;mcl posterior coI111);1rtl?lrnts runs.just anterior

t o \ving vein 4. 111 r i " / + , ci', c.i"md d ' " 5 "

/+

mutants there is a range o f vein t I c f c ~ t s in the posterior conl MI tmcnt. I n c.i"/+ mutants. the f o u r t h and filih veins do not reach t h c margin o f t h c wing black ( B ) . n" at 18' ( C ) anti r i h ' ( h ) have :I 111019' estrclne phrnotye i n which w i n s 4 and .5 fuse t o make a singlr w i n t h a t angles toward t h c posterior margin o f ' the wing. vi""5"/

+

(E) is a g ; ~ m n ~ : ~ - ~ x y rcvcrtant o f ri" ;and h a s t h c we;tkcst m u t a n t phenotype in which thcrc is a slight tliswption in vein 3. <if"/

+

n l ~ ~ t ; ~ n t s h:~vc a diffcwmt alteration that consists of deletions hecwccn w i n s 3 and 4 (F).

ment boundary (Figure 7B). I n c i ' / c i ' , e?'/+, r i L ' / +

and r i " / r i " ' mutants, inappropriate expression o f the

ci pt-otein is found i n the posterior compartmrnt (Fig-

~11-e

7,

C and D ) . T h e level o f inappropriate expression

correlates well with the extent of the wing vein disrup-

tions. r?"'5"/+ has the lowest level of ectopic expression (data not shown) and the least extreme defect. n " ' j c i " '

mutants have the highest level of ectopic expression

and the most extreme mutant phenotype. The c i ' / r i '

phenotype is temperature sensitive and more extreme

at lower temperatures; a correspondingly higher lrvel

of ectopic ci expression is obsened a t lower tempera-

tures. A s mentioned above, the ci' mutation is caused

by the insertion of ;I p p s y element. A number of muta-

tions caused by the insertion of gypsy elements can be

suppressed by mutations in the S I ~ / I / I ~ P . S . S ~ ~

or

Huily roing

( n l (H70)) gene (reviewed in COK(:I<S and G I W X 1991 ).

In .su(HuJ);

n"

animals the wing vein defect is sup-

pressed, and the ectopic expression ofci protein is rlirn-

inated (data not shown).

mRNA was isolated from ri' mrltants and . s ? r ( I f 7 ~ ) ; r i '

mlltants at third instar, pupal and adult stages. The

mRNA was fractionated by gel electrophoresis, blotted

onto nitrocellulose and probed for the ci transcript.

T h e level of the ei transcript was quantified o n a phos-

phorimager relative t o an r/)49 svantlard ( O ' < : O S X ~ . I . I .

and ROSBASII 1984). At the third instar l a ~ ~ a l stage, but

not in pupae or adults, the level of the ci transcript i n

ri' mutants is increased IO-fold relative t o that in

SN(H~O);

ri' mutants (data n o t shown). The size of the

ci transcript in these mutant backgrounds is i t l c ~ ~ t i ~ ; ~ l

to that ofwild-type, arguing that the ei' mutation causrs

higher levels o f expression from the normal c.i pro-

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236 D. C. Slusarski, C . Kelsev M o t m y and R. Holmgren

- B

C'

-

FIGURE 7.-ci expression in wing imaginal discs. Ventral is up and anterior is to thc Icft. I n wandering third instar larvae ci

mRNA is restricted to the anterior compartment (A). ci protein is also restricted to the anterior compartment of imaginal discs, but a4jacent to the compartment borlndary the level of antibody staining is greater ( R ) . In homozygous n"" mutants (C) and

n"'/+

mutants (D) expression of the ci protein is observed in the posterior compartment. The amount of ectopic ci protein correlates with the severity of the mutant phenotype. The highest level of ectopic expression is seen in homozygous n'"'mutants, which have the most severe defects. In d'/+ mutants (E), the level of antibody staining in the anterior Compartment is much higher than normal, and a distinction in levels between the region along the compartment boundary and the rest of the compartment is not seen. In Jc mutants (F) there is a similar change in the pattern of antibody staining to the ci protein; higher levels are seen across the entire segment.

In

CY''/+

imaginal discs, the ci protein is properly body staining is elevated throughout the anterior com-

restricted to the anterior compartment, but its level of _{partment (Figure}

_7E).

(9)

Regulation of n' Expression 237

A

B

C

D

E

-

_b

-

FIGCRE 8.-ci protein isolated from wing imaginal discs. Wild-type imaginal discs have a low level of ci protein (lane

A). In j l l mutants, ci protein levels are higher (lane R). In

n'"/+ the wild-type protein band 180 X 10'' M, can be seen

aswell as a mutant form at -120 X IO3 M, (lane C). The level of the mutant form is higher than that of the wild type.

In n'"/+ mutants the wild-typr form can be seen as well a s a

hint of a higher molecular weight form (lane D). A longer exposure shows that the ci protcin generated by the n " ) allcle

is -20 X 10'' M, larger than wild-type (lane E). The staining levels with ECL. detection give a general indication of relative protein concentrations hut the response is not linear and accentuates dimerences. Positions of molecular weight mark- ers are indicatrd a1 the left; sizcs correspond to 200, 116, 97,

66 and 45 (xIO" M , ) from top to bottom.

wing veins three and four. To determine whether there is a corresponding increase of ci protein in Jit mutant discs, we stained discs with antibodies for ci protein. A.

shown in Figure 7F, the level and distribution of the ci protein in J I L discs is nearly identical to

ci"/+

discs.

c i D and cirr mutations are associated with the genera-

tion of altered protein products: Western blotting was

used to examine the protein products of the ci locus from wild-type, )L,

d'/+

and

d'/+

imaginal discs.

Equal amount5 of protein were loaded onto an SDS polyacrylamide gel, blotted onto nitrocellulose and probed with the 'LA1 antibody. Figure 8. A and B, shows that the level of ci protein is greatly increased in J ~ L

mutants. In kc'/+ discs the wild-type protein band of

180 X 10'

M,

is present along with a second band at

-120 X IO3

M,

(Figure 8C). T h e lower band is present

at much higher levels than the wild-type band and presumably corresponds to a truncated ci protein product from the n'" gene. In

ci"/+

discs, a longer exposure (Figure 8E) shows a protein band -20 X 10"

M,

larger than the wild-type protein. This product presumably corresponds to a fusion protein generated by the ci"

inversion.

DISCUSSION

&I,

C;", C;",

me and CIJV)I7 are allelic and alter the

expression pattern of the ci protein: We have shown

that the h1, n'\!: n./>, ,.ti and c j / l q l 1 7 mutations all alter

the expression of the same transcription unit and are associated with DNA alterations that map at or 5' to the start of transcription. T h e observed alterations are most likely the cause of the corresponding mutations because polymorphisms are very rare on the fourth chromosome. This is a consequence of the lack of re- combination that causes the fourth chromosome to be selected as a single genetic unit (BERRY Pt nl. 1991).

Natural Drosophiln mplnnofinstprpoprllations appear to be isogenic for the fourth chromosome. Therefore, anv polymorphism present on mutant fourth chromosomes would have to have been generated since the initial isolation of that parental laboratory stock.

ci function is required to maintain wg protein ex-

pression: Each mutant phenotype can be explained bv

an alteration in the expression of the ci transcription unit. T h e embryonic defects are recessive and result from a loss of ci protein expression. T h e n5(4'1i class of mutations fail to express the ci protein through early germ band extension. Loss of early ci expression results in the elimination of wg protein expression in the ventral epidermis during stage 10, and presumably as a

consequence, causes the variable loss of en protein expression during stage 11 (BF.ISO\'EC and MARTINEZ- ARIAS 1991; HEKMSKKRK ~l nl. 1991). The consequences of these alterations include the elimination of the structures derived from the m expressing cells (HAMA ~t nl.

1990) and of structures derived from either side of the m expressing domain. Deletions anterior to the m expressing domain include the ventral black dots of the thoracic segments and deletions posterior to the pn ex-

pressing domain include the region of dorsal naked cuticle that follows the isolated row of triangular hairs.

late stage 12 and takes part in the formation of ventral naked cuticle (DFJSOVKC and MARTINEEARIA. 1991).

Elimination of r q f ~ ~ n c t i o n t h r o u g h stage 11 by temperature shifting a 71!g"allele from the nonpermissive to the permissive temperature 6 hr after egg laying generates a

phenotype similar to that of ci""li. T h e first row of

ventral denticles is eliminated and breaks in the stripes

of en protein expression are observed by stage 1 1 (R?l-

sow.<:

and MARTINEZ-ARIAS 1991). The phenotype of on the dorsal surface resembles that of temperature sensitive h ~ d g ~ h o g (hit) mutants that have been shifted to the nonpermissive temperature during stage

1 1 ( HEEMSKERK and DINARDO 1994). This suggests that just as m expression is disrupted in stage I 1 n'""" mutants, there may be a corresponding change in hh expression.

In

n"'

mutants low levels of ci protein are expressed through the beginning of germ band extension but then decay. T h e expression of the en protein appears normal, but wg protein expression decays during stage

12. In n'" mutants, at least some of the structures de-

In n.l14)!7 mutant5 wg protein expression returns during

(10)

238 D. C. Slusarski, C. Kelsey Motzny and R. Holmgren

rived from the en expressing cells are still generated. On

the ventral surface the first row of denticles is generally retained, and on the dorsal surface the isolated row of triangular hairs is present. The ventral naked cuticle that would be generated by the anterior en expressing cells is eliminated along with the rest of the ventral

naked cuticle. The phenotype of ci" mutants is very

similar to that of wg" mutants that have been shifted to

the nonpermissive temperature during stages 11 and/

or 12 (BAKER 1988;

BE~sowc

and MARTINEZ-ARIAS

1991), suggesting that the ci" mutant phenotype can

be explained by the elimination of wg function. The

actual situation may be more complicated. SAMPEDRO

et 01. (1993) have shown that heat-shock expressed wg

is not able to cause naked cuticle formation in a cz"

mutant background. This result suggests that ci is epi-

static to wg in naked cuticle formation. The protein

product of the ci" mutation is actually a fusion protein.

The fusion protein may be missing up to 16 amino acids

from the N-terminus of ci and contains 2 2 0

x

103 M,.

from a different protein. The ci" fusion-protein should

contain the entire zinc finger domain and the re-

maining C-terminal sequences. Clearly, the protein

product of the ci" allele is in part functional, and it is

possible that the fusion protein may have some novel gain of function properties.

The complementation observed between and

ci1(4"7 class mutations is probably due to their different effects on ci gene expression. In the ~ i ' " ' ' ~ class muta-

tions a promoter element required for early ci expres-

sion is disrupted, and the inversion responsible for the

ci" mutation fortuitously causes the expression of a functional ci fusion protein early in embryogenesis. In

trans-heterozygotes between ci" and the

~i"~"'

class mu-

tants, a near wild-type pattern of expression is generated, and embryonic development proceeds relatively normally.

Deletion analysis of the promoter defines a region

required for early ci expression: A series of deletions

were generated that eliminate the early embryonic expression of ci protein. All such deletions affected se-

quences distal to the d'"" insertion site, suggesting that

an element required for early expression is located in this region. The position of this element is adjacent to

the gypsy insertion in ci' and distal to the putative I

element insertion in

ci'".

With gypsy insertions in the

yellow locus it has been shown that regulatory elements between a gypsy element and the promoter are functional, whereas elements beyond a gypsy element inser-

tion are inactivated (GEYER and GORGES 1992). In a

modi& of mdg4 (mod) mutant background, gypsy can

inhibit all the elements that activate yellow expression

independent of their position (GEORGIEV and GERASI-

MOVA 1989). This is not the case with the gypsy insertion

in

ci.

mod; ci' animals have a phenotype that appears

to be identical to that of ci alone (data not shown) ;

therefore, the promoter elements required for embry-

onic ci expression do not appear to respond to su(Hw)/

mod regulation. The elimination of early ci expression

by the insertion in ci" can probably be explained by its

location between the promoter and the element re-

quired for early expression.

All of the c2"nr deletions complement the ci" muta-

tion. Thus, the element required for late embryonic

expression of the ci gene was not removed by any of

the deletions generated by excision of the ci'""' element.

It is tempting to suggest that the late embryonic element may be proximal to the insertion in citr because late embryonic expression is not altered in these mutants.

ci

wing

vein defects are due to inappropriate protein

expression: In wild-type animals, ci mRNA and protein

expression are restricted to the anterior compartment of imaginal discs. The compartment boundary runs be-

tween wing veins three and four, so it was somewhat

surprising that a set of ci alleles cause defects in wing

veins four and five, which are derived from the posterior

compartment. This issue was resolved by finding that these mutations result in inappropriate expression of

the ci protein in the posterior compartment of the wing

imaginal disc. Ectopic expression in the posterior compartment is also observed in the developing embryo.

In particular, ci" mutant embryos have high protein

expression levels throughout the embryo that do not appear to compromise embryonic development. This

suggests that the elimination of ci protein from the

posterior compartment is more critical for subsequent adult development.

In the ci' mutation, a gypsy element is responsible

for the ectopic expression in the posterior compart-

ment. The gypsy element contains binding sites for the

su(Hw) protein (SPANA et al. 1988). It appears that bind-

ing of the su(Hw) protein to the gypsy element medi- ates the dramatic increase in the level of ci mRNA be-

cause this increase is absent in a su(Hw) mutant

background.

Position effects at the ci locus: The ci locus is particu-

larly sensitive to chromosomal position effects. Many

translocations involving the fourth chromosome give

rise to ci mutant phenotypes (both recessive and domi-

nant) that have disruptions in wing veins four and five

(STERN and KODANI 1955). As we have shown, this type

of phenotype is due to inappropriate expression of the ci protein in the posterior compartment. Although position effects could cause inappropriate expression in sev-

eral different manners, two seem particularly attractive.

The translocation of sequences onto the fourth chro-

mosome might disrupt the normal en mediated repres-

sion of ci expression in the posterior compartment, or

the position effect could change chromosomal struc-

(11)

Regulation of ci Expression 239

promoter and nearby enhancer elements which are not subject to en regulation.

n' function is regulated posttranscriptionally The ci

mRNA is uniformly distributed in the anterior compartment as is Pgalactosidase expression from an enhancer

trap in the ci locus (EATON and KORNBERG 1990). The

pattern of ci protein expression in imaginal discs is

distinct from that of the mRNA. The ci protein levels, as visualized by antibody staining, are higher in regions adjacent to the compartment boundary. This is true for both the monoclonal antibody 2A1 and a rat polyclonal serum (data not shown). Therefore, it is unlikely that this distinction is due to the masking of a specific epi- tope. These results suggest that the rate of ci protein synthesis and/or turnover are regulated.

The product of the fu gene appears to play a role in

the posttranscriptional regulation of

ci.

In wing discs

elimination of the fu kinase elevates ci protein levels

throughout the anterior compartment. This is particu-

larly striking away from the compartment boundary

where the quantity of ci protein is low. The elevation

of ci protein levels is also seen in the cicx/+ wing discs.

Interestingly, alterations away from the compartment boundary in these mutants may not be relevant to the

wing vein defect. Mosaic analysis with fu has shown that

i t is only the region just anterior to the compartment

boundary that requires fu activity (FAUSTO-STERLING

1978).

The position of the ci gene in the segmental pat-

terning pathway: In the patterning of embryonic seg-

ments both ci and fu are epistatic to patched (ptc)

(FORBES et al. 1993) and ptc is epistatic to hh (INGHAM

et al. 1991). If a simple linear pathway exists from the

hh signal through the generation of naked cuticle, the

gene order would be hh, ptc, fu, ci, wg (Figure 9A)

(FORBES et al. 1993; PERRIMON 1994). Imaginal discs

develop at the compartmental boundaries of the em-

bryo and the patterns of hh, ptc, fu and ci expression in

imaginal discs closely reflect those established by stage

1 1 of embryonic development. hh is expressed in the

posterior compartment (LEE et al. 1992; MOHLER and

VANI 1992; TABATA et al. 1992), ptc (HOOPER and SCOTT

1989; NAKANO et al. 1989) and ci (EATON and KORNBERG

1990; ORENIC et al. 1990) are restricted to the anterior

compartment and fu (PREAT et al. 1990) is expressed in

all cells. The hh protein is secreted from cells in the posterior compartment and is thought to initiate a signal transduction cascade in the cells adjacent to the compartment boundary. Therefore, it is of interest to

consider whether the linear pathway proposed above

can explain the pattern of ci protein expression in fu

mutant imaginal discs, If fu kinase activity is negatively regulated by ptc and ptc repression is relieved in response to the hh signal, then fu activity should be repressed in cells that are beyond the range of the hh

signal. In imaginal discs the fu kinase has a demon-

B

hh

4

ptc

4

ci -t wg

4

iU

C

hh

+

ci+ wg

PtC

"I

fu

FIGURE 9.-Models for the regulation of wgexpression and

naked cuticle formation within segments. (A) A linear model based on the epistasis relationships of hh, ptc, fu and ci. Cells receiving the hh signal will repress ptc protein function and activate the expression of wg. Cells at a distance from the hh signal will have active ptc protein, inactive fu kinase and no wgexpression. (B) A branched model in which the activity of the fu kinase is not regulated by the hh signal. In this model the activity of the ptc protein is regulated by hh. (C) An

alternative branched model in which the ptc protein is not regdated by the hh signal and instead ptc regulates the activity of the fu kinase.

strated effect on ci protein levels far away from any potential hh signal, suggesting that branched or parallel pathways may be present.

A linear pathway is also inconsistent with the results

of SAMPEDRO et ai. (1993). As described above, the ex-

pression of wg from a heat-shock promoter is able to

cause naked cuticle formation in an hh mutant back-

ground but not in a c i D mutant background. Therefore,

there is a distinction between the roles of ci and hh.

This suggests that ci functions downstream of wg in

naked cuticle formation or that more complicated in-

teractions between wgand ci exist. Figures 9, B and C,

presents two branched models that are consistent with the observations. Additional models are possible, and a combination of genetic and biochemical experiments will be necessary to determine which best reflects the actual signal transduction pathway.

We thank DAVID WRIGHT for many stimulating discussions and for

his invaluable help in working out the relationship between ci" and

ci". We thank VICTOR CORCES, JOAN HOOPER, YU ZHANG, DARLENE

BUENZOW,JENNIFER KENNEDY, ANNA MARTI SUBIRANA and AMY BElso-

VK for critically reading the manuscript. R.H. thanks ANGUS LAMOND

and a Fogarty fellowship for giving him a desk, a bench and no telephone for his sabbatical at the EMBL in Heidelberg. This work was supported by National Science Foundation grant DCB 89-01394 and National Institutes of Health grant NS28472 to R.H.

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