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The ken and barbie gene encoding a putative transcription factor with a BTB domain and three zinc finger motifs functions in terminalia development of Drosophila.

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Archives of Insect Biochemistry and Physiology 54:77–94(2003)
The ken and barbie Gene Encoding A Putative
Transcription Factor With a BTB Domain and
Three Zinc Finger Motifs Functions in Terminalia
Development of Drosophila
Tamas Lukacsovich,1 Kazuya Yuge,2 Wakae Awano,1,3 Zoltan Asztalos,1 Shunzo Kondo,3
Naoto Juni,1,4 and Daisuke Yamamoto1,4*
Mutations in the ken and barbie locus are accompanied by the malformation of terminalia in adult Drosophila. Male and
female genitalia often remain inside the body, and the same portions of genitalia and analia are missing in a fraction of
homozygous flies. Rotated and/or duplicated terminalia are also observed. Terminalia phenotypes are enhanced by mutations
in the gap gene tailless, the homeobox gene caudal, and the decapentaplegic gene that encodes a TGFb-like morphogen. The
ken and barbie gene encodes a protein with three CCHH-type zinc finger motifs that are conserved in several transcription
factors such as Krüppel and BCL-6. All defects in ken and barbie mutants are fully rescued by the expression of a wild-type
genomic construct, which establishes the causality between phenotypes and the gene. Arch. Insect Biochem. Physiol. 54:77–
94, 2003. © 2003 Wiley-Liss, Inc.
KEYWORDS: genital disc; TGF-b; Tailless; Caudal; morphogensis
INTRODUCTION
The body plan of Drosophila is sexually dimorphic as in the case of the body plans of many other
animals. The most obvious sex difference occurs
in adult terminalia, i.e., the genitalia and analia.
Both the adult genitalia and analia arise from the
genital disc, a saclike cluster of primordial cells set
aside during mid-embryogenesis for the later development of imaginal terminalia (Jürgens and
Hartenstein, 1993). The embryonic primordium of
the genital disc comprises 15–20 cells derived from
several adbominal segments posterior to A8 and
the nonsegmented telson. Thus, it represents a
composite imaginal disc just as the eye-antennal
disc (Jürgens and Hartenstein, 1993).
1
ERATO Yamamoto Behavior Genes Project at Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan
2
BASE, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, Japan
3
Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan
4
School of Science and Engineering, Waseda University, Nishi-Tokyo, Tokyo Japan
Contract grant sponsor: Ministry of Education, Culture, Sports, Science and Technology of Japan; Contract grant sponsor: Waseda University; Contract grant
number: 2002B-031.
Tamas Lukacsovich’s present address is Department of Developmental and Cell Biology, University of California at Irvine, Irvine, CA 92697.
Zoltan Asztalos’s present address is Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.
*Correspondence to: Dr. Daisuke Yamamoto, School of Science and Engineering, Waseda University, 2-7-5 Higashi-Fushimi, Nishi-Tokyo 202-0021, Japan.
E-mail: daichan@waseda.jp
Received 10 April 2003; Accepted 26 June 2003
© 2003 Wiley-Liss, Inc.
DOI: 10.1002/arch.10105
Published online in Wiley InterScience (www.interscience.wiley.com)
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Lukacsovich et al.
The differences in the genital disc between
males and females become apparent near the end
of the second-instar larval stage: three primordia,
i.e., the female genital primordium, comprising a
single genital disc, the male genital primordium,
and the anal primordium develop differently depending on sex (Düberdorfer and Nöthiger, 1982;
Epper and Nöthiger, 1982; Nöthiger et al. 1977).
The female genital primordium derived from embryonic A8 grows whereas the male genital primordium does not. In males, the male genital
primordium derived from the A9 segment expands,
whereas the female counterpart is repressed. The
anal primordium consists of cells in A10, possibly
in A11 and the telson, and it develops into the
imaginal anus in both sexes despite the sexually
dimorphic anal structure (Nöthiger et al., 1977;
Schüpbach et al., 1978; Epper and Nöthiger, 1982).
The sex-specific differentiation of the genital disc
is under the control of the sex-determination cascade, which involves the Sexlethal (Sxl), transformer
(tra), transformer-2 (tra-2) and doublesex (dsx) genes
(Baker, 1989; MacDougall et al., 1995; Yamamoto
et al., 1996, 1998). For example, a clone of cells
without the tra gene produced in the genital disc
of a female second-instar larva may give rise to both
male and female genitalia in adults (Wieschaus and
Nöthiger, 1982). Moreover, chromosomally female
diplo-X flies bearing the temperature-sensitive tra2 mutation, tra-2ts, may develop male genitalia
when they experience temperature upshifts during
the larval stage or female genitalia when exposed
to temperature downshifts at the same stage
(Sánchez and Granadino, 1992). The sexual types
of the anal plate are also affected: temperature upshift leads to the formation of a male anal plate
whereas temperature downshift results in that of a
female anal plate (Sánchez and Granadino, 1992).
Pattern formation in the genital disc is accomplished by cell-to-cell communication mediated by
diffusible signals such as decapentalegic (dpp), wingless (wg), and hedgehog (hh) gene products, in a
manner analogous to that in other imaginal discs
(Casares et al., 1997; Chen and Baker, 1997;
Freeland and Kuhn, 1996; Sánchez et al., 1997).
The imaginal structures that are eventually formed
by each primordium of the genital disc are different from each other. The fact that the male, female,
and anal primordia are the derivatives of different
embryonic (para) segments implies that the distinct developmental profile of each primordium is
ultimately specified by homeotic gene expression.
Indeed, mutations that result in the loss of AbdominalB (AbdB) or spalt (sal) homeotic gene functions transform the tail structures (including
genitalia) into those of the more anterior abdominal segments (Lewis, 1978). The homeobox gene
caudal (cad) is required for the proper formation
of A10 and the telson, including the anus (MacDonald and Struhl, 1986; Mlodzik et al., 1985,
1990; Mlodzik and Gehring, 1987), and is considered to function as a homeotic gene that directs
the pathway of anal development (Moreno and
Morata, 1999). Among the gap genes, tailless (tll)
is known to be involved in terminalia development
(Pignoni et al., 1990).
Thus, it is conceivable that morphogenetic cellto-cell interactions mediated by Dpp, Hh, Wg, and
related signals are instructed by homeotic gene
products that provide positional cues as well as by
the products of sex-determination genes that convey information on which sexual fate to adopt. Although this view provides a general framework for
understanding how terminalia differentiate in the
genital disc, the molecular mechanism of the system coordinating such complex regulatory networks is poorly understood.
Recent studies revealed, however, that the dachshund (dac) gene is differentially expressed in the
male and female genital discs through the sexually different actions of wg and dpp (Sánchez et
al., 2001; Keisman and Baker, 2001). FGF (fibroblast growth factor) signaling is also crucial for the
sexually dimorphic development of the genital disc:
FGF expressed only in the ectoderm-derived cells
of the male genital disc stimulates FGF-receptorexpressing mesodermal cells to migrate into the
male discs (Ahmad and Baker, 2002). These studies have unraveled some aspects of the complex
molecular network underlying the sexually dimorphic development of the genital disc.
Archives of Insect Biochemistry and Physiology
ken in dpp Signaling
79
In an attempt to elucidate the mechanism of
copulation control, we succeeded in isolating a
mutant, okina (ok, a name given by Ryu Ueda;
Yamamoto et al., 1997), the homozygotes of which
are occasionally devoid of external genitalia and/
or analia. The aristae of the mutant flies are unpigmented, thus the name okina, which in Japanese means a respectable old man with a white
beard. In subsequent studies, ok was found to be
an allele of ken and barbie (ken; Castrillon et al.,
1993), and is referred to as kenok in this study. As
inferred from their phenotypic similarity, ken was
found to interact synergistically with cad, tll, and
dpp. The ken gene encodes a putative transcription
factor with three zinc finger motifs, two of which
are aligned side by side similar to those found in
Krüppel and some other transcription factors. Finally, we demonstrated that a genomic fragment
containing only the ken transcription unit fully rescues all the deficits associated with the ken mutants, establishing the causality between the ken
gene and the phenotypes of these mutants.
genotypes of the flies. Through introduction of the
P(ry+D2-3) chromosome into the kenok line, the
mutator element was remobilized, resulting in approximately 30 lines with white eyes. The ken alleles used in this study had been outcrossed with
the w1118 strain with the CS genetic background for
5 generations. The mutant phenotypes did not
change following this treatment.
For scanning electron microscopy (SEM), the
flies were prepared for critical-point drying and
coated with a 2-nm-thick layer of gold. Images were
taken with a low-voltage prototype SEM.
MATERIALS AND METHODS
Total RNA was isolated in a single step using
trizol reagent (Gibco BRL), as described by Chomczynski and Sacchi (1987). Poly(A)+RNA was prepared using oligo(dT)-latex (manufactured by
Roche, distributed by Takara) following the protocol given by the supplier. Briefly, less than 150 mg
of total RNA was dissolved in 100 ml of elution
buffer (10 mM Tris-HCl (pH 7.5), 1 mM EDTA,
0.1% SDS, made with DEPC treated water). After
100 ml of Oligotex-dT30 was added, the mixture
was vortexed and heated to 65°C for 10 min, then
chilled on ice. Twenty microliters of 5M NaCl was
added to the mixture, which was then incubated
for 10 min at 37°C and then centrifuged at 15
Krpm for 15 min at room temperature. The supernatant was discarded and the pellet resuspended
in 100 ml of DEPC-treated water and incubated at
65°C for 5 min. The suspension was then again
centrifuged at 15 Krpm for 15 min at room temperature. Poly(A)+RNA contained in the supernatant was precipitated by ethanol.
Five micrograms of Poly(A)+RNA was separated
on a 1% agarose gel containing formaldehyde as
Mutagenesis, Mutant Screening, and
Phenotype Analysis
Mutagenesis was performed by the jump-start
method with the BmDw element as a mutator and
the P(ry+D2-3) transposon as a jump starter. All flies
subjected to mutagenesis had a white– (w–) background, whereas the BmDw element carried a copy
of w+, enabling the recovery of chromosomes with
BmDw insertions by selecting flies with nonwhite
eye. After the establishment of fly lines with new
insertions, homozygous virgin males and females
were collected at eclosion, placed singly in food
vials, and grown for 3 days. For behavioral screening, single male/female pairs were introduced into
disposable plastic syringes (volume, 1 cm3). At least
10 pairs per strain were observed with the naked
eye for 1 h, and the time taken until copulation,
the duration of copulation, and the percentage of
pairs copulating were recorded. The observations
were performed by experimenters blind to the
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Molecular Cloning and Sequencing
Standard DNA and RNA in vitro molecular techniques were used as described (Sambrook et al.,
1989). Determination of nucleotide sequence was
carried out by using a 377 DNA sequencer (Applied Biosystems Prism).
Northern Blot Analysis
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described by Sambrook et al. (1989). Following
transfer of the separated products onto a Biodyneplus nylon membrane (Paul), the filters were hybridized with digoxigenin-labeled specific DNA
probes prepared using a DIG DNA Labeling kit
(Boehringer Mannheim). The probe on the filter was
detected with an anti-DIG antibody that recognizes
digoxigenin coupled to alkaline phosphatase, according to the given by the manufacturer’s protocol (Boehringer Mannheim).
Rescue of ken Mutants With Transgenes
For the rescue experiment with the genomic ken
gene, the DNA fragment between the PstI site at
position 18,011 and the HindIII site at position
8,959 was inserted into the pCasper3 shuttle vector, and injected into w1118 embryos with a phsB
helper plasmid to generate transgenic flies. The
nucleotide numbers given here correspond to those
of the sequence submitted to the DDBJ database
(Lukacsovich et al., 1999). The cloning of this genomic fragment was accomplished in two steps due
to the fact that it contains additional PstI and
HindIII restriction sites. First, a 6,075-bp PstI-EcoRI
restriction fragment, representing the 5¢ part of the
ken gene, was cloned into the corresponding restriction sites of the pCasper3 vector and then the
second part of the gene, a 2,977-bp EcoRI-HindIII
restriction fragment, was inserted into the EcoRIXbaI double digested vector obtained in the first
step (the HindIII site of the fragment was converted
previously into the SpeI site by ligation of a synthetic adapter sequence to this end of the fragment). Transgenic fly lines carrying ken wild-type
cDNA driven by either UAS or the hsp70 promoter
were also generated.
RESULTS
Phenotypic Characterization of ken Mutants
The kenok allele was isolated during screening
for sexual-behavior mutants as well as for those
showing low mating success and reduced copulatory duration (Yamamoto et al., 1997; Yamamoto
and Nakano, 1998). ken1was isolated based on its
abnormal genitalia (Castrillon et al., 1993). Careful examination of external structures revealed that
8% (n = 131) of male flies homozygous for kenok
have aberrant terminalia: some of them appear to
lack external genitalia (Fig. 1H), which actually
remain inside the abdominal cuticle. On the contrary, there are a few ken mutant flies with duplicated genitalia arranged side by side in a mirror
image (Fig. 1C,G). There are also flies whose orientation of genitalia is aberrant. Female terminalia
(Fig. 1B,C) is similarly affected by ken mutations
at a lower frequency (2%; n = 52) than male
terminalia (Fig. 1G, H). Aside from genital anomalies, all ken homozygotes had unpigmented aristae that are somewhat frail physically (Fig. 1J; see
also Fig. 1I). Several stronger alleles were identified by genetic complementation tests for reduced
viability when placed in trans to kenok or by molecular mapping (see below). These include kenP1244
(l(2)02970), kenP942 (l(2)00628), ken2 (l(2)k11035),
and ken3 (l(2)03907). The transallelic combination
kenok/kenP942 or kenok/kenP1244 yields flies that display
the terminalia phenotype. The terminalia phenotype
is 100% penetrant in a few ken1/kenP1244 escapers.
The level of expression of the arista phenotype is
not increased in these transallelic mutants.
In accordance with the adult genital phenotype,
the genital disc was malformed in the ken mutants
(Fig. 2C). There were mutant discs that were split
into two subdivisions (Fig. 2D), which presumably
developed into duplicated genitalia as observed in
adults (Fig. 1C,G). In these experiments, flies carrying a dpp-lacZ reporter construct were used to examine the possible effect of ken mutation on dpp
expression. Even in the discs severely distorted by
ken mutation, strong dpp expression was observed
(Fig. 2C,D). In such discs, large changes in the overall disc structure prevented us from determining
whether the pattern of dpp expression is affected
by ken mutation. In accord with the variable phenotypic severity in the ken mutant adults, some
mutant discs appear to have a normal structure
(data not shown). dpp-reporter expression in these
mutant discs is indistinguishable from that in wildtype discs. This result indicates that either ken muArchives of Insect Biochemistry and Physiology
Fig. 1. Phenotypes of ken mutants. A–E: Examples of female terminalia of wild-type (A), ken1 (B, C), cadmd509 kenok (D), and
cadmd509 kenok; genomic-ken+ (E). The ken1 terminalia shown in B lacks a vulva and is characterized by a rotated anal plate, while that
in C represents duplicated organization. The cadmd509 kenok double-mutant female in D lacks an anal plate and vulva. The genomicken+ transgene completely rescues the phenotype of cadmd509 kenok (E). F–H: Examples of male terminalia of wild-type (F), ken1 (G),
and kenok (H). In G, the terminalia is duplicated whereas in H, it is absent. Dorsal to top. I,J: The antennae from wild-type and ken1,
respectively. Ap, anal plate; P, penis apparatus; Vp, vaginal plate.
ken in dpp Signaling
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Fig. 2. ken phenotype in the genital disc (ventral view).
A,B: Wild-type female (A) and male (B) discs. C,D: ken1/
kenok mutant male discs. The disc illustrated in D has a
“border” between the left and right halves. The dpp expression was determined using a reporter, dpp-lacZ, which
labels genital discs blue by conventional X-Gal staining.
tation does not affect dpp transcription at least in
this developmental stage, or the dpp reporter construct lacks ken-responsive sequences present in the
genomic dpp gene.
carries a GAL4-enhancer trap insertion in the cad
locus does not exhibit any discernible phenotypes
in external structures including aristae and terminalia as its own (Calleja et al., 1996), in contrast
to cad1, which leads to the loss of anal plates
(Jürgens and Hartenstein, 1993; Macdonald and
Struhl, 1986). The proportions of kenok homozygotes with the terminalia defect were 8% in the
cad wild-type background, 30% in the cadmd509 heterozygous background, and 87% in the cadmd509
homozygous background (Fig. 3).
Among the gap genes, tll appears to play an important role in terminalia formation, as a weak allele (tllle3) is known to lack portions of the anal
Mutations That Interact With ken Genetically
To define the developmental role of ken genetically, we examined the possible genetic interactions
of ken alleles with mutations known to affect genital structures. We found that a hypomorphic allele
of the homeobox gene cad (cadmd509) markedly increases the frequency of male terminal defects in
kenok homozygotes (Fig. 1D). The cadmd509 allele that
Archives of Insect Biochemistry and Physiology
ken in dpp Signaling
83
Fig. 3. Quantitative comparisons of phenotypic severity
between different genotypes.
The percentage of flies having
the terminalia defect is shown.
pads and hindgut. Interestingly, halving the gene
dosage in the tll locus increased the proportion of
kenok homozygous flies with the terminalia defect
(Fig. 3). Thus, tll and ken appear to function synergistically in genital disc development. Another
locus that we found to interact with ken is dpp,
which encodes a TGFb family secretory protein. The
frequency of observing the terminalia phenotype
in kenokhomozygotes is increased from 8 to 31%
(n = 116) by replacing a wild-type copy of dpp with
a mutant allele, dppd5 (Fig. 3).
Thus, kenok mutation created a sensitized genetic
condition, where a small reduction in tll and dpp
activities to levels that do not produce any discernible phenotypic effect as theirs results in explicit
abnormality in development. The observed interactions between ken, tll and dpp imply that these
three genes function in the same transduction pathOctober 2003
way or in different signaling pathways that cross
talk to each other.
Molecular Characterization of the ken Gene
The ken locus has been mapped at 60A in the
right arm of chromosome 2 (Lukacsovich et al.,
1999). We confirmed that both the arista and
terminalia phenotypes of kenok result from the Pelement insertion at 60A because its excision yields
revertants. The genomic DNA fragment flanking the
P-element insertion was recovered by the inverse
PCR method (Silver, 1991), and used to identify
recombinant P1 phages with the sequence identical with that flanking the P-element insertion. The
genomic sequences of two P1 phages designated
as DS00692 and DS06090 hybridized with the genomic sequence of the putative ken region. The in-
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Fig. 4. Legend on next page.
Archives of Insect Biochemistry and Physiology
ken in dpp Signaling
serts of the two P1 phages covered the genomic
region of more than 20 kb surrounding the kenok
P-element insertion. The insertion sites of the Pelement in kenok, ken1, kenP942, kenP1244, and ken2 were
determined by sequencing by vectorette PCR after
the amplification of genomic DNA flanking the respective insertions (Arnold and Hodgson, 1991).
All five insertions are clustered within a region of
less than 1 kb (Fig. 4). The nucleotide sequence
flanking the ken3 insertion, which was provided by
the Spradling laboratory, allowed us to map it in
close proximity to this region. The fact that the
four P-element insertions that do not complement
the ken phenotypes are all mapped within the 1kb genomic segment indicates that the transcription unit spanning this region is the most likely
candidate ken gene.
Fig. 4. Genomic structure of ken gene. A: Restriction sites
in the ken and adjacent genes, CK00282, thiolase, TM4SF,
and LD24471. The details of these neighboring genes are
described by Lukacsovich et al. (1999). The probe used
for Northern blotting is also indicated. B: P-element insertion sites in ken gene. C: Exon-intron organization of
ken gene. D: The sequences at the integration sites of the
P-element in kenP942, kenok, ken1, kenP1244, ken2, and ken3
alleles are boxed. The arrow below the kenok insertion site
represents the putative transcription start site. The arrow
beneath the first exon of the ken gene (shown with Gothic
letters) indicates the translation start site. Larger capitals
(except for the portion shown in Gothic letters) indicate
5¢ noncoding sequence. Small letters indicates intronic sequences. The nucleotide is numbered starting from the
PstI site at the 3¢ untranslated region of the adjacent thiolase
gene. The genomic rescue construct contains the DNA segment downstream from this PstI site. A: Restriction sites
are shown as B = BamHI, E = EcoRI, P = PstI, and H =
HindIII. All existing sites are shown for BamHI and EcoRI.
For PstI and HindIII, only sites that are used in either in
generating the genomic rescue construct or preparing DNA
probes for Northern blotting are shown. B: The P-element
insertion sites in kenok and other known allelic mutants
are indicated by triangles, and the direction of transcription of genes in this region is shown by arrows. LTR represents the long terminal repeat of a Copia-like element,
297. E1, E2, and E3 in C are the exons of the ken gene.
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Using the genomic fragments of the cloned 20kb region as probes, we isolated cDNA clones representing five different transcription units. Thus,
we are definite that at least five genes are tightly
packed in this 20-kb genomic segment. Extended
search for transcription units in subsequent experiments revealed that at least 10 genes are nested
within the 50-kb genomic region (Lukacsovich et
al., 1999).
To determine the transcription unit affected by
mutagenic P-element insertions, we performed
Northern blotting with relevant cDNA probes to
compare mRNA expression between the Canton-S
(CS) wild-type, kenok, and kenok-Rev1, a revertant in
which the kenok P-element is removed by precise
excision (see Materials and Methods). Only one
of the five aforementioned cDNA species detected
a significant difference in mRNA expression between kenok and CS or kenok-Rev1. This cDNA probe
recognized a single band of approximately 3.0 kb
length in CS and kenok-Rev1 flies (Fig. 5B). In kenok,
by contrast, only a trace amount of the transcript
is detected with the same probe. The level of mRNA
expression from this transcription unit in ken1 is
even lower than that in kenok. The transcription of
the neighboring gene is completely unchanged in
the ken1 homozygotes (Fig. 5A; see also Lukacsovich et al., 1999). Taken together, these results
indicate that the 3-kb transcript corresponds to the
ken gene.
Indeed, the ken1 and other P-element insertions
are localized at or near the 5¢ end of this transcription unit. The ken3 insertion is located in the first
intron. The P-elements in ken2, kenP1244, ken1, and
kenok are inserted between the translation start site
and the transcription start site of the ken gene. kenP942
bears the P-element insertion 375 nucleotides upstream of the transcription start site (Fig. 4).
The conceptual translation of cDNA nucleotide
sequence revealed that the open reading frame of
the ken gene can encode a protein of 601 amino
acids (Fig. 6A). After our submission of the ken
DNA and protein sequences to the Gen Bank database (accession numbers: AB010260-61), an identical sequence was published by Kühnlein et al.
(1998). Blast search revealed that the Ken protein
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Fig. 5. Northern blot analysis. A: mRNA blots hybridized with a genomic fragment that spans the ken and adjacent thiolase genes. The 1.6-kb transcript of thiolase is
intact while the 3-kb transcript is almost absent in ken1
mutant flies. mRNA were prepared from adult flies of the
indicated genotype. B: The 3-kb transcript (detected by
ken cDNA) in adults compared between wild-type, kenok
and kenok-Rev1. C: Developmental Northern analysis. E, embryos; 2nd, 2nd-instar larvae; 3rd, 3rd-instar larvae; P, pupae; A/m, male adults; A/f, female adults. The expression
level increases in the embryonic and pupal stages. In
adults, the mRNA expression level is higher in females
than in males.
has three C2H2-type zinc finger motifs (Miller et
al., 1985) in its C terminus (Fig. 6A). The region
composed of 47 amino acids from a.a. 500 to 546
contains the first zinc finger motif and part of the
second zinc finger motif. Interestingly, this region
is highly homologous to the zinc finger motifs in
humans, rather than to those in Drosophila, Krüppel
and BCL6 proteins (Fig. 6B; Chang and Ye, 1997;
Fukuda et al., 1995; Han et al., 1999). The identities of this region in Ken with those in the human
Krüppel and BCL6 are 55 and 57%, respectively.
The conserved domain analysis database (Pfam,
protein family database of alignments and HMMs
at Sanger Center : http://www.sanger.ac.uk/Pfam/)
indicates that the N-terminus of the Ken protein
has a putative BTB/POZ damain, which is an evolutionarily conserved protein-protein interaction
domain known to affect the chromatin structure
(Katsani et al., 1999). The alignment of sequences
of the BTB domain (Fig. 6C) revealed that the
amino acid residues critical for its function are conserved among the Ken and other BTB proteins.
Thus, Ken is a putative transcription factor that has
three zinc finger motifs and a BTB domain.
ken Expression in Development
Developmental Northern blot analysis revealed
that ken transcription is developmentally regulated, i.e., elevated expression level in embryos
Archives of Insect Biochemistry and Physiology
ken in dpp Signaling
Fig. 6. Ken protein sequence and domains. A: Protein
sequence of Ken. Three zinc finger motifs and a BTB domain are indicated by gray and black backgrounds, respectively. Zinc finger motifs are abbreviated as ZF in the
schematic representation of the Ken protein shown. B: Sequence alignment of zinc finger motifs of Ken, BCL-6, and
Krüppel. Identical residues are indicated by a black background. BCL-6 and Krüppel are those of humans. In the
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illustrated region, the identities of the Ken sequence with
those of Krüppel and BCL-6 are 55 and 57%, respectively.
In particular cysteine and histidine residues are well conserved. C: Sequence alignment of selected BTB domains.
Black background indicates residues conserved in all proteins shown. Gray background indicates residues that are
conserved in at least half of the protein species illustrated.
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and pupae and much lower expression level in
larvae (Fig. 5C). In adult flies, ken expression is
sexually dimorphic, i.e., its expression level is
much higher in females than in males (Fig. 5C).
This high expression level in females likely reflects
its accumulation in oocytes because two cDNAs
corresponding to ken have been (BDGP library:
GM12839, GM01621) obtained by the screening
of an ovary cDNA library. Thus ken mRNA is maternally transmitted to embryos.
Since the ken gene is required for the normal
development of terminalia and antennae, it may
be expressed in the genital and eye-antennal discs.
We, therefore, carried out in situ hybridization experiments in order to examine the expression pattern of the ken gene in imaginal discs. In Drosophila,
the genital disc forms the genitalia, analia, and
hindgut of adults (Jürgens and Hartenstein,1993).
The former two structures are different between the
sexes. The eye-antennal disc develops into an eye,
an antenna, a maximal plap, and a hemilateral
head capsule (Jürgens and Hartenstein, 1993).
In situ hybridization experiments were performed with a single-strand digoxigenin-labeled
DNA probe specific to ken mRNA. ken mRNA was
found to be expressed ubiquitously in both male
and female genital discs (Fig. 7A,B) and eye-antennal discs (Fig. 7E) at low levels, but no signal
was detected in the brain (data not shown).
We also stained the genital disc of an enhancertrap-ken allele for b-galactosidase activity and confirmed the ubiquitous expression of ken at a low
level (Fig. 7C,D). In males, ken expression is intensified along the margin of the anterior bulbus
(Fig. 7D). In females, ken is expressed in the posterior compartment along the anterior-posterior
border, with medial expansion in the posteriormost
region (Fig. 7C). In contrast to its low expression
level in the imaginal discs, a high expression level
of ken was reported in the embryo by Kühnlein et
al. (1998). Our in situ hybridization analysis of
ken expression in the embryo confirmed their result. The ken transcript is expressed in a distinct
spatiotemporal pattern during embryogenesis (Fig.
8). At stage 5 (cellular blastderm), two rather faint
stripes can be detected at positions of 64% (ante-
rior domain expression; AD) and 17% (posterior
domain expression; PD) egg length (EL; 0%EL is
the posterior most position) (Fig. 8A). At stage 6
(early gastrulation), these two stripes become more
evident and detectable at the region posterior to
the cephalic furrow (CF) and in the hindgut primordium (Fig. 8B). AD is lost as gastrulation proceeds (stage 6–8), while PD remains (Fig. 8D). At
stage 15, AD appears again in the foregut and PD
in the hindgut and anal pad (Fig. 8E).
Functional Rescue of the ken Phenotypes by the
Cloned Wild-Type Gene
To demonstrate unequivocally that mutation in
the cloned gene is responsible for ken phenotypes,
we employed a functional rescue experiment in
which an artificially constructed wild-type transgene was tested for its ability to restore ken mutant abnormalities. The relatively simple structure
of the ken gene enabled us to perform the rescue
experiment with a genomic DNA construct. The
nucleotide sequence of a much larger genomic region surrounding the putative ken gene has already
been determined (Lukacsovich et al., 1999), and
it was found that the coding sequences of two adjacent genes, a fly homologue of the mammalian
mitochondrial trifunctional enzyme (thiolase) and
a new member of the transmembrane 4 superfamily, are situated less than 2 kb from the 5¢ and 3¢
ends of the candidate ken gene, respectively (Lukacsovich et al., 1999). In addition, the genomic ken
gene is relatively small. Therefore, we could insert
the entire, 9,052-bp genomic DNA fragment between two restriction sites, PstI at position 18011
(the number corresponds to that of genomic DNA
sequence submitted to DDBJ Database, accession
numbers: AB0 10260, 10261) and HindIII at position 8959, into the pCasper3 shuttle vector. This
DNA fragment extends from the 5¢ UTR of the
thiolase gene to the 3¢ end beyond the polyadenylation site of ken mRNA. Thus, it should contain the complete ken gene, presumably together
with all of its transcriptional regulatory sequences.
The integrated genomic DNA construct completely rescued the ken phenotypes. The viability
Archives of Insect Biochemistry and Physiology
ken in dpp Signaling
89
Fig. 7. ken expression pattern in genital disc. A,B: ken
mRNA expressions in the wild-type female (A) and male
(B) genital discs as detected by in situ hybridization with
a ken antisense probe. No signal was detected with a sense
probe (not shown). C,D: Genital discs of ken1/+ female
(C) and male (D) stained for b-galactosidase activity. It is
expressed widely at low levels. In the female disc, ken re-
porter expression is higher in the posterior compartment
along the anterior-posterior border than in other areas. In
the male disc, the b-galactosidase activity is higher in the
margin of the anterior bulbi than in other areas. E: ken
mRNA detected by in situ hybridization in eye-antennal
disc. Anterior to the left.
of kenok homozygous flies recovered to normal, and
no flies lacking the genitalia or with aberrant pigmentation of antennae were observed. The genomic ken+ transgene restored the viability of the
lethal allele kenP942, yielding adult flies homozygous for kenP942 with a proportion that was expected. These kenP942 homozygotes have normal
antennae, genitalia and analia, indicating that perfect rescue is attained by expression of the inserted
genomic ken+ transgene. Furthermore, the kenok phenotypes in terminalia that resulted from synergistic interaction with cadmd509, tllI49, or dpp5 are all
restored by introducing the genomic ken+ transgene
into the mutant flies (for an example see Fig. 1E).
Rescue of the kenok phenotypes was also achieved
by insertion of a full-length wild-type cDNA driven
by the heat shock promoter (hs-ken+). In this case,
the rescue effect was limited such that there were
October 2003
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Lukacsovich et al.
Fig. 8. ken expression in embryo. Wild-type embryos
at stages 5 (cellular blastoderm) (A), 6 (early gastrulation) (B), 7 (C), 9 (D), and 15 (E) were stained with
an antisense DNA probe for ken mRNA. No signal was
detected with a sense probe (not shown).
some kenok-homozygous flies with the transgene
that exhibited a mild antennal phenotype. Although the rescuing ability of hs-ken+ was incomplete, its effect was apparent without any heat
shock treatment, presumably due to “leaky” expression of hs-ken+ at 25°C. Because of this limitation,
we were unable to determine the developmental
stage at which the wild-type-ken function is required for the normal development of terminal and
antennal structures. Taken all these observations
together, the cloned gene encoding the zinc finger
protein is indeed confirmed to be the ken gene.
DISCUSSION
dpp plays a major organizer role in both embryonic and imaginal developments (Lawrence and
Struhl, 1996; Lecuit et al., 1996). It would also be
true for the genital disc, where dpp is expressed
along the border between the en-expressing posterior compartment and the cubitus interruptus (ci)expressing anterior compartment (Chen and Baker,
1997; Casares et al., 1997; Freeland and Kuhn,
1996). As in the case of leg and wing discs (Lepage
et al., 1995; Li et al., 1995; Pan and Rubin, 1995),
the loss of protein kinase A activity in a group of
cells in the anterior compartment by means of somatic mosaicism induces the ectopic dpp expression in these cells, which ultimately leads to the
duplication or overgrowth of some genital disc derivatives in adults. Conversely, the loss of the dpp
function results in the partial ablation of these
structures (Chen and Baker, 1997). We documented that hypomorphic mutations in the ken locus cause similar defects in genital-disc derivatives.
The penetrance of the phenotype was improved by
reducing the activity of the wild-type ken locus or
the dpp activity.
Archives of Insect Biochemistry and Physiology
ken in dpp Signaling
An important finding in this study is that ken
alleles also interact genetically with cad and tll mutations in terminalia formation. cad is a homeobox
gene that has been implicated to play a role in the
determination of the terminal segment of embryos.
It has been reported that cad is selectively expressed
in the A10-equivalent portion of the genital disc
during postembryonic development, and that hypomorphic cad1 mutant flies are devoid of anal
plates that originate from A10 (Jürgens and Hartenstein, 1993). We demonstrated that a weaker hypomorph, cadmd509, which does not exhibit any
abnormalities in terminalia, dominantly enhances
the terminalia ken phenotype, including the loss
of anal plates. Similarly, the expression of the terminalia ken phenotype was intensified by a reduction in the activity of tll. tll is the gene encoding a
steroid receptor-like protein (Pignoni et al., 1990)
that likely regulates the transcription of downstream genes required for the formation of terminal structures (Steingrimsson et al., 1991). These
results led us to presume that the malformation
of terminalia in the ken, cad, and tll mutants is associated with impairment of dpp signaling.
Interestingly, the expressions of cad, ken, and dpp
all converge in the border between parasegments
15 and 16 (i.e., A10) in the developing genital disc
(this study and Freeland and Kuhn, 1996), with a
partial overlap in their expression domains. However, dpp expression in the genital disc was not discernibly affected by the ken mutation, as examined
using the dpp-reporter. Furthermore, some of the
genital discs were already structurally distorted at
the third-instar larval stage (Fig. 2). In addition, the
ken expression level in the genital disc was quite
low (Fig. 7). These observations are in favor of the
hypothesis that the ken gene plays a role in terminalia formation at a developmental stage earlier than
the third larval instar, and indeed the ken gene is
strongly expressed in the embryo (Fig. 8). Kühnlein
et al. (1998) demonstrated that the AD of ken expression is eliminated by a mutation in the hunchback (hb) gene, which is one of the targets of Bicoid
(Bcd) (Tautz, 1998). They also showed that, in tll
mutant embryos, the AD level of ken is reduced and
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91
the PD is lost (Kühnlein et al., 1998). Thus, it is
conceivable that ken interacts with cad, tll, and dpp
during embryogenesis for the normal development
of the genital premordium.
The amino acid sequence of the ken gene product deduced from the nucleotide sequence of cDNA
suggests that it is a transcription factor with zinc
finger motifs similar to those found in the Krüppel
and BCL-6 products. We favor the idea that Ken is
downstream of cad because the cad activity is predominantly regulated at the translational level at
least in oocytes, as a result of Bicoid binding to
cad mRNA (Dubnau and Struhl, 1996; RiveraPomar et al., 1996). Furthermore, the ken gene has
putative sequence motifs in its 5¢ regulatory region
that may serve as a Cad-binding site, although we
have not demonstrated such binding experimentally. Thus far, Cad is known to directly activate
fushi tarazu (ftz) transcription (Dearolf et al., 1989;
Hayashi and Scott, 1990).
On the other hand, there is sufficient evidence
supporting the idea that tll functions upstream of
ken. First, the ken mRNA expression level in the
embryo is decreased by tllI49 mutation, as mentioned above (Kühnlein et al., 1998). Second, the
5¢ region of the ken gene contains sequences that
are likely bound by Tll. Third, Tll protein expressed
in bacteria binds to the putative promoter region
of the ken gene, as shown in a mobility shift assay
(Lukacsovich, unpublished data).
More data are required to determine how ken
interacts with dpp. The lack of effect of ken mutation on dpp reporter expression in the genital disc
does not necessarily mean that ken is downstream
of dpp, since the regulatory genomic sequences that
are responsive to Ken might be omitted from the
dpp-reporter minigene. Likewise, our data do not exclude the possibility that ken is not directly involved
in the dpp pathway, but that it acts through wingless
(wg) signaling, which is known to function antagonistically against dpp in the genital disc (Tsuda et
al., 1999). These facts indicate that cell-by-cell analysis of the transcription control of the ken gene in
association with Dpp signaling is inevitable for determining the role of ken in this pathway.
92
Lukacsovich et al.
ACKNOWLEDGMENTS
We thank the members of Yamamoto laboratory for their comments on the manuscript, H.
Nakato, M. Okabe, R. Murakami, H. Jäckle, G.
Morata, S. Goto, and the Umeå stock center for fly
stocks, cDNAs, and plasmids for protein expression, and Sachiko Kondo and Yuka Kai for secretarial assistance. This study was supported in part
by Special Cooperation Funds for Promoting Science and Technology from the Ministry of Education, Culture, Sports, Science and Technology of
Japan to D. Y. and by Waseda University grant no.
2002B-031 to D. Y.
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